Gene Expression and Regulation

*Gene Expression and Phenotypic Traits*

s00360-015-0897-5

*B*. 2015;**185**(5):539-546. DOI: 10.1007/

[39] Peng Y, Chang L, Wang Y, et al. Genome-wide differential expression of long noncoding RNAs and mRNAs in ovarian follicles of two different chicken breeds. Genomics. 2019;**111**(6):1395- 1403. DOI: 10.1016/j.ygeno.2018.09.012

[32] Tagirov M, Rutkowska J. Chimeric embryos—Potential mechanism of avian offspring sex manipulation. Behavioral Ecology. 2013;**24**(4):802-805.

[33] Wrobel ER, Molina E, Khan NY, et al. Androgen and mineralocorticoid receptors are present on the germinal disc region in laying hens: Potential mediators of sex ratio adjustment in birds? General and Comparative Endocrinology. 2019;**287**:113353. DOI:

[34] Bruggeman V, Van AP, Decuypere E. Developmental endocrinology of the reproductive axis in the chicken embryo.

DOI: 10.1093/beheco/art007

10.1016/j.ygcen.2019.113353

Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology.

s1095-6433(02)00022-3

[36] Weber C, Capel B. Sex reversal. Current Biology. 2018;**28**(21):R1234-R1236. DOI: 10.1016/j.cub.2018.09.043

[37] Huang S, Ye L, Chen H. Sex determination and maintenance: The role of DMRT1 and FOXL2. Asian Journal of Andrology. 2017;**19**(6):619- 624. DOI: 10.4103/1008-682X.194420

[38] Balashenko NA, Dromashko SE. Long non-coding RNAs and their functions. Vestsi Natsyyanal'nai akademii navuk Belarusi. Seryya biyalagichnych navuk. Proceedings of the National Academy of Sciences of Belarus. 2017;**4**:110-119. (in Russian)

2002;**131**(4):839-846. DOI: 10.1016/

[35] Aslam ML, Woelders H. Steroid hormones and female energy balance: Relation to offspring primary sex ratio. In: Hester PY, editor. Egg Innovations and Strategies for Improvements. United States: Academic Press; 2017. pp. 47-54. DOI: 10.1016/ B978-0-12-800879-9.00005-6

**52**

**55**

many diseases.

**Chapter 5**

*Morgan Salmon*

ous disease processes.

**1. Introduction**

modification, epigenetics, RNA, promoters

**Abstract**

Transcriptional and Epigenetic

Krüppel-like factors (KLFs) are a family of zinc finger transcription factors (ZF-TF) that are now known to be involved in complex biological processes including cancer, proliferation, and cardiovascular disease as well as developmental processes. KLFs first gained notoriety when it became known that they are crucial for promoting and maintenance of stem cell pluripotency. Over the past 20 years since the discovery of Krüppel-like factor 1 (KLF1), this transcription factor family has grown to include 18 members and 7 closely related members of the specificity protein 1 (Sp1) family. In the present study, we review the mechanisms related to regulation of KLFs by direct promoter activation or repression. We will also review and discuss some mechanisms of posttranslational modifications that could affect KLF function. We seek to understand how these transcriptional regulators are themselves regulated and how that regulation could become aberrant during vari-

**Keywords:** Krüppel-like zinc finger proteins, transcription, posttranslational

The specificity protein 1 (Sp1)/Krüppel-like factor (KLF) proteins are a family of highly conserved transcription factors that are characterized by the presence of three highly homologous Cys2/His2-type zinc fingers near the C-terminus that bind GC/CACCC box. Amino acid sequences in the transcription activation/ repression domains are less conserved among family members; however, there are subfamilies based on sequence similarities within this group. These subfamilies tend to share co-activators or co-repressors to aid in how they regulate genes. So far, seven members in the specificity protein (Sp) subgroup and 18 members in the KLF subgroup have been identified in mammalian cells [1]. This family of transcription factors is able to function as both transcriptional activators and repressors based on the gene and cellular contexts. KLFs gained notoriety as Krüppel-like factor 4 (KLF4), Krüppel-like factor 2 (KLF2), and Krüppel-like factor 5 (KLF5) were suggested to be important for embryonic stem cells and stem cell reprogramming [2–7] alongside Oct4, Sox2, and Nanog. However, we have only begun to touch the surface of the transcriptional control these factors exert during embryonic development, maintenance of normal function, and the breakdown of normal processes seen in

Regulation of Krüppel-Like

Transcription Factors

## **Chapter 5**

## Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors

*Morgan Salmon*

## **Abstract**

Krüppel-like factors (KLFs) are a family of zinc finger transcription factors (ZF-TF) that are now known to be involved in complex biological processes including cancer, proliferation, and cardiovascular disease as well as developmental processes. KLFs first gained notoriety when it became known that they are crucial for promoting and maintenance of stem cell pluripotency. Over the past 20 years since the discovery of Krüppel-like factor 1 (KLF1), this transcription factor family has grown to include 18 members and 7 closely related members of the specificity protein 1 (Sp1) family. In the present study, we review the mechanisms related to regulation of KLFs by direct promoter activation or repression. We will also review and discuss some mechanisms of posttranslational modifications that could affect KLF function. We seek to understand how these transcriptional regulators are themselves regulated and how that regulation could become aberrant during various disease processes.

**Keywords:** Krüppel-like zinc finger proteins, transcription, posttranslational modification, epigenetics, RNA, promoters

## **1. Introduction**

The specificity protein 1 (Sp1)/Krüppel-like factor (KLF) proteins are a family of highly conserved transcription factors that are characterized by the presence of three highly homologous Cys2/His2-type zinc fingers near the C-terminus that bind GC/CACCC box. Amino acid sequences in the transcription activation/ repression domains are less conserved among family members; however, there are subfamilies based on sequence similarities within this group. These subfamilies tend to share co-activators or co-repressors to aid in how they regulate genes. So far, seven members in the specificity protein (Sp) subgroup and 18 members in the KLF subgroup have been identified in mammalian cells [1]. This family of transcription factors is able to function as both transcriptional activators and repressors based on the gene and cellular contexts. KLFs gained notoriety as Krüppel-like factor 4 (KLF4), Krüppel-like factor 2 (KLF2), and Krüppel-like factor 5 (KLF5) were suggested to be important for embryonic stem cells and stem cell reprogramming [2–7] alongside Oct4, Sox2, and Nanog. However, we have only begun to touch the surface of the transcriptional control these factors exert during embryonic development, maintenance of normal function, and the breakdown of normal processes seen in many diseases.

The goal of this chapter is to begin to describe our current knowledge of how the KLFs are regulated during development or disease. We seek to begin to understand the ways cells either promote or repress the presence of the KLFs through a variety of transcriptional and translational mechanisms.

## **2. Regulation by and of KLFs**

## **2.1 Krüppel-like factor 1**

Krüppel-like factor 1 (KLF1) or erythroid Krüppel-like factor is an essential transcription factor for erythroid development and was found to be key in the regulation of many facets of blood development. KLF1 is expressed in the developing blood as well as being weakly expressed in mast cells [1]. KLF1 is key to blood development as Klf1−/− mice die around E14 due to severe anemia [8]. Several studies also showed KLF1 is able to directly bind to the β-globin promoter to activate the gene's transcription as part of fetal hematopoiesis in the liver [9, 10]. The null embryos provided a wealth of knowledge about KLF1 early on, suggesting that β-thalassemia could be linked with KLF1 deletions [11]. More recent studies have also shown that KLF1 is able to either directly or indirectly repress the transcription of the -globin gene to promote the expression of β-globin during blood development [12].

In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA-binding domain, causes hemolytic anemia and is dominant over wildtype KLF1 [13]. This variant in the developing liver demonstrates defects in erythroid maturation that resemble those seen with the KLF1−/−, again demonstrating the importance of KLF1 in blood development. Furthermore, recent studies suggest that there is an enhancer element in the KLF1 gene that is susceptible to methylation and that elevated levels of methylation in that region correlate with patients with juvenile myelomonocytic leukemia (JMML) [14]. KLF1 was also found to play a role in the inhibition of megakaryocytes while also stimulating erythroid lineages at the same time [15].

## **2.2 Krüppel-like factor 2**

Krüppel-like factor 2 or lung Krüppel-like factor (LKLF) was isolated in humans in 1999 and found to be 85% similar in nucleotide identity and 90% similar in its amino acids to mouse and located on chromosome 19p13.1 [16]. Of special interest, a region of 75 nucleotides within its proximal promoter was found to be identical between human and mouse [16]. This identical region in the mouse and human promoters for KLF2 has been found to be critical for its regulation in lung, blood, endothelial cell, and T lymphocyte development [15–22]. KLF2 was shown to be essential for normal development within mice, and knockout embryos were lethal around day 12.5 and lung function was also severely impaired in KLF2−/− chimeras [22]. KLF2 expression appears to also be important for the maintenance of normal lung function, as methylation of KLF2 was associated with metastasis and worsening prognosis in non-small-cell lung cancer [23].

KLF2 was also shown to be essential for early erythropoiesis and regulation of the β-globin gene, and klf2−/− mice also exhibited hemorrhage in developing blood cells [17]. In mature T cells, KLF2 is required for T-cell trafficking, and elimination of KLF2 in T cells affects the expression of sphingosine-1-phosphate receptor and CD2L and beta7 integrins, receptors all important in T-cell trafficking [18, 24].

**57**

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors*

stress, and activation via nucleolin was also PI3K dependent [33].

ERK5 was also shown to be important in T-cell activation, and ERK5−/− cells were

KLF2 is also an important regulator of heart and aorta development and normal maintenance of endothelial cells [27–29]. KLF2 has been shown to be activated by shear stress through the conserved 75-base pair region in the human and mouse promoters [30]. This region was shown to requite PI3K for activation and PCAF (p300/CAMP-response element-binding protein-associated factor) and heterogeneous nuclear riboprotein D to induce acetylation of H3 and H4 histones [31]. Additional riboproteins and acetyltransferases such as HnRNP-U, hnRNP-D, and p300 were also found to bind via this conserved region in the KLF2 promoter [32]. KLF2 was also found to be activated by nucleolin in endothelial cells following shear

In terms of a negative regulation of KLF2 in endothelial cells, KLF2 was shown to be negatively regulated by p53, which bound to the KLF2 promoter to induce deacetylation of the KLF2 histone H3 [34]. Tumor necrosis factor alpha (TNF-α) was shown to activate NF-Кβ p65 to complex with histone deacetylase 4 to prevent MEF2 binding to the KLF2 promoter, demonstrating a possible additional mechanism of the downregulation of KLF2 in endothelial cells in response to injury. Finally, low-density lipoprotein (LDL) cholesterol was found to stimulate the methylation of both DNA and histones on the KLF2 promoter and to contribute to the downregulation of KLF2 in response to LDL cholesterol. These mechanisms suggest there are a number of complex pathways that control the expression of KLF2 in a

Krüppel-like factor 3 (KLF3) or basic Krüppel-like factor (BKLF) is widely expressed and abundant in erythroid cells. KLF3 is believed to regulate adipogenesis, erythropoiesis, and B-cell development [35, 36]. KLF3 is able to interact with the co-repressor CtBP to repress gene transcription much like Krüppel-like factor 8 (KLF8) and Krüppel-like factor 12 (KLF12), and the N-terminal repression domain is important for this interaction in KLF3 [37–39]. KLF3 has been found to be sumoylated and that this sumoylation also affects its interaction with CtBP [37]. KLF3 has been shown to have a role in adipogenesis as forced expression of KLF3 was shown to block adipocyte differentiation [40]. Recent methylation data from endothelial cells demonstrates that KLF3 is highly methylated in flow-dependent conditions but can be reversed with 5-aza-2′-deoxycytidine treatments [41]

Krüppel-like factor 4 or gut-enriched Krüppel-like factor (GKLF) or endothelial

zinc finger (EZF) protein is most similar to KLF2 and functions in the regulation of the epithelial of the gut and skin, endothelial cells, smooth muscle cells in vascular disease, and induced pluripotent stem cells (iPSC) [1, 42]. KLF4−/− mice died shortly after birth due to epithelial barrier defects in skin and gut barriers [43]. KLF4 is regulated by AP-2alpha during early and mid-embryogenesis to help

KLF4 became well-known after the discovery that it was one of the regulating factors along with Oct4, Sox2, and Nanog of induced pluripotent stem cells [4–7]. Oct4 was later found to regulate the expression of KLF2, while LIF/Stat3 was thought to regulate the activation of KLF4 in embryonic stem cells [45, 46]. Additional studies have suggested that posttranslational modifications increase or

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

number of different tissue types.

**2.3 Krüppel-like factor 3**

**2.4 Krüppel-like factor 4**

regulate proliferation [44].

unable to activate genes for T-cell function [25, 26].

### *Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

ERK5 was also shown to be important in T-cell activation, and ERK5−/− cells were unable to activate genes for T-cell function [25, 26].

KLF2 is also an important regulator of heart and aorta development and normal maintenance of endothelial cells [27–29]. KLF2 has been shown to be activated by shear stress through the conserved 75-base pair region in the human and mouse promoters [30]. This region was shown to requite PI3K for activation and PCAF (p300/CAMP-response element-binding protein-associated factor) and heterogeneous nuclear riboprotein D to induce acetylation of H3 and H4 histones [31]. Additional riboproteins and acetyltransferases such as HnRNP-U, hnRNP-D, and p300 were also found to bind via this conserved region in the KLF2 promoter [32]. KLF2 was also found to be activated by nucleolin in endothelial cells following shear stress, and activation via nucleolin was also PI3K dependent [33].

In terms of a negative regulation of KLF2 in endothelial cells, KLF2 was shown to be negatively regulated by p53, which bound to the KLF2 promoter to induce deacetylation of the KLF2 histone H3 [34]. Tumor necrosis factor alpha (TNF-α) was shown to activate NF-Кβ p65 to complex with histone deacetylase 4 to prevent MEF2 binding to the KLF2 promoter, demonstrating a possible additional mechanism of the downregulation of KLF2 in endothelial cells in response to injury. Finally, low-density lipoprotein (LDL) cholesterol was found to stimulate the methylation of both DNA and histones on the KLF2 promoter and to contribute to the downregulation of KLF2 in response to LDL cholesterol. These mechanisms suggest there are a number of complex pathways that control the expression of KLF2 in a number of different tissue types.

## **2.3 Krüppel-like factor 3**

*Gene Expression and Phenotypic Traits*

**2. Regulation by and of KLFs**

**2.1 Krüppel-like factor 1**

same time [15].

**2.2 Krüppel-like factor 2**

ing prognosis in non-small-cell lung cancer [23].

of transcriptional and translational mechanisms.

The goal of this chapter is to begin to describe our current knowledge of how the KLFs are regulated during development or disease. We seek to begin to understand the ways cells either promote or repress the presence of the KLFs through a variety

Krüppel-like factor 1 (KLF1) or erythroid Krüppel-like factor is an essential transcription factor for erythroid development and was found to be key in the regulation of many facets of blood development. KLF1 is expressed in the developing blood as well as being weakly expressed in mast cells [1]. KLF1 is key to blood development as Klf1−/− mice die around E14 due to severe anemia [8]. Several studies also showed KLF1 is able to directly bind to the β-globin promoter to activate the gene's transcription as part of fetal hematopoiesis in the liver [9, 10]. The null embryos provided a wealth of knowledge about KLF1 early on, suggesting that β-thalassemia could be linked with KLF1 deletions [11]. More recent studies have also shown that KLF1 is able to either directly or indirectly repress the transcription of the -globin gene to

In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA-binding domain, causes hemolytic anemia and is dominant over wildtype KLF1 [13]. This variant in the developing liver demonstrates defects in erythroid maturation that resemble those seen with the KLF1−/−, again demonstrating the importance of KLF1 in blood development. Furthermore, recent studies suggest that there is an enhancer element in the KLF1 gene that is susceptible to methylation and that elevated levels of methylation in that region correlate with patients with juvenile myelomonocytic leukemia (JMML) [14]. KLF1 was also found to play a role in the inhibition of megakaryocytes while also stimulating erythroid lineages at the

Krüppel-like factor 2 or lung Krüppel-like factor (LKLF) was isolated in humans in 1999 and found to be 85% similar in nucleotide identity and 90% similar in its amino acids to mouse and located on chromosome 19p13.1 [16]. Of special interest, a region of 75 nucleotides within its proximal promoter was found to be identical between human and mouse [16]. This identical region in the mouse and human promoters for KLF2 has been found to be critical for its regulation in lung, blood, endothelial cell, and T lymphocyte development [15–22]. KLF2 was shown to be essential for normal development within mice, and knockout embryos were lethal around day 12.5 and lung function was also severely impaired in KLF2−/− chimeras [22]. KLF2 expression appears to also be important for the maintenance of normal lung function, as methylation of KLF2 was associated with metastasis and worsen-

KLF2 was also shown to be essential for early erythropoiesis and regulation of the β-globin gene, and klf2−/− mice also exhibited hemorrhage in developing blood cells [17]. In mature T cells, KLF2 is required for T-cell trafficking, and elimination of KLF2 in T cells affects the expression of sphingosine-1-phosphate receptor and CD2L and beta7 integrins, receptors all important in T-cell trafficking [18, 24].

promote the expression of β-globin during blood development [12].

**56**

Krüppel-like factor 3 (KLF3) or basic Krüppel-like factor (BKLF) is widely expressed and abundant in erythroid cells. KLF3 is believed to regulate adipogenesis, erythropoiesis, and B-cell development [35, 36]. KLF3 is able to interact with the co-repressor CtBP to repress gene transcription much like Krüppel-like factor 8 (KLF8) and Krüppel-like factor 12 (KLF12), and the N-terminal repression domain is important for this interaction in KLF3 [37–39]. KLF3 has been found to be sumoylated and that this sumoylation also affects its interaction with CtBP [37]. KLF3 has been shown to have a role in adipogenesis as forced expression of KLF3 was shown to block adipocyte differentiation [40]. Recent methylation data from endothelial cells demonstrates that KLF3 is highly methylated in flow-dependent conditions but can be reversed with 5-aza-2′-deoxycytidine treatments [41]

## **2.4 Krüppel-like factor 4**

Krüppel-like factor 4 or gut-enriched Krüppel-like factor (GKLF) or endothelial zinc finger (EZF) protein is most similar to KLF2 and functions in the regulation of the epithelial of the gut and skin, endothelial cells, smooth muscle cells in vascular disease, and induced pluripotent stem cells (iPSC) [1, 42]. KLF4−/− mice died shortly after birth due to epithelial barrier defects in skin and gut barriers [43]. KLF4 is regulated by AP-2alpha during early and mid-embryogenesis to help regulate proliferation [44].

KLF4 became well-known after the discovery that it was one of the regulating factors along with Oct4, Sox2, and Nanog of induced pluripotent stem cells [4–7]. Oct4 was later found to regulate the expression of KLF2, while LIF/Stat3 was thought to regulate the activation of KLF4 in embryonic stem cells [45, 46]. Additional studies have suggested that posttranslational modifications increase or decrease the stability of KLF4 mRNA and these modifications control the exit from pluripotency [47]. Furthermore, these modifications mediate the ability of KLF4 to complex with other pluripotency transcription factors and bind DNA. Finally, Oct4 has been shown to contain a linker region that is important for loosening chromatin, complexing with Brg1, and allowing for KLF4 to bind during cellular reprogramming [2]. Clearly, the interactions and mechanisms of pluripotency factors in stem cells are complex and require further investigation.

KLF4 is required for normal functioning of the gut epithelial as deletion of KLF4 resulted in altered proliferation [48]. KLF4 and KLF5 are often found in the same types of tissues, bind to similar or identical DNA elements, and often exert opposing affects in different tissue types. KLF4 has been found to bind with p53 on the p21 genes in epithelial cells and in smooth muscle cells to inhibit proliferation [42, 49, 50].

In the case of smooth muscle cell proliferation, sumoylation of KLF4 causes it to fall off the p21 promoter and decreases p21 transcription following PDGF-BB treatments [51]. Sumoylation is also believed to affect binding of KLF4 to smooth muscle marker genes in TGFβ treatment [52, 53]. In smooth muscle cells in vascular disease, KLf4 has been shown to be activated by Sp1 and Oct4 binding to the KLF4 promoter [54, 55]. Separately, in macrophages KLF4 sumoylation promotes an IL-4-induced macrophage polarization to an M2 state, suggesting KLF4 plays a role in inflammation and macrophage polarization states [56]. However, in endothelial cells KLF4 is important along with KLF2 for the maintenance of endothelial cell integrity and normal endothelial barrier function [29]. KLF4 function in vascular disease could fill chapters of books investigating its many roles and functions; however, our goal is to highlight some of the mechanisms of its regulation in these processes.

Finally, KLF4 is also regulated by DNA methylation in several different types of cancers. KLF4 was found to be hypermethylated in renal cell carcinomas [57] and endometrial cancers [58]. However, a surprising discovery was KLF4 can bind to methylated regions of chromatin to mediate activation of transcription without the need for demethylation of the DNA in some types of cancer cells [59, 60]. These studies demonstrate a new role for some transcription factors as methylation readers in the transcription process.

## **2.5 Krüppel-like factor 5**

Krüppel-like factor 5 or intestinal-enriched Krüppel-like factor (IKLF) or basic transcription element-binding protein 2 (BTEB2) is located on chromosome 13q22.1 and is important in the expression of the gut epithelia, vascular smooth muscle cells, and white adipose tissues [1, 61]. KLF5 is important in epithelial cells as it is located in the base of the crypts where cells are proliferating toward the villi. In general, KLF4 and KLF5 have been shown to compete to the same sites on DNA [62] and have also been suggested to be involved in their own regulation [42]. KLF5 has been shown to be important in gastric tumor progression and initiation and often correlate with KRAS mutations [63, 64].

KLF5 has also demonstrated to be important in the development and maintenance of the heart, aorta, and lung systems [20, 65–69]. Following angiotensin II induction, KLF5 was shown to bind to PDGF-A and activate it. KLF5 was also shown to be activated by RARα binding site in the KLF5 promoter [65, 70]. KLF5 has been shown to be regulated by acetylation. When KLF5 is associated with p300, it is acetylated and able to activate gene expression. Conversely, when SET is bound to KLF5, it prevents acetylation of KLF5 and its transcriptional activity [71]. These studies suggest that KLF5 can be regulated directly by modifications to control its transcriptional activity.

**59**

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors*

Expression of KLF5 in breast cancers was found to be correlated with a negative prognosis and decreased survival [72], while in clear cell renal cell carcinoma, hypermethylation and decreased expression of KLF5 were associated with a poorer prognosis [73]. Hypermethylation of KLF5 in acute myeloid leukemia was also associated with a poorer prognosis [74]. These studies suggest that KLF5 function in cancer is cell and perhaps even cell lineage specific. Within various cancers, KLF5 has also been demonstrated to be regulated by micro-RNAs. In gastric cancer, miR-145-5p directly targets KLF5 and promotes the differentiation of gastric cancer via KLF5 downregulation [75]. Separately, in hepatocellular carcinoma miR-214-5p acted as a tumor suppressor that could directly target and promote the downregulation of KLF5 [76]. These data demonstrate complex regulatory pathways involved in

Krüppel-like factor 6 (KLF6) or zinc finger transcription factor 9 (ZF9) has been shown to be important for endothelial biology, adipogenesis, and tumor suppression in a wide variety of cancers. During embryogenesis, it is expressed in a timesensitive manner in the kidney, cornea, gut, and yolk sac [77–80]. KLF6−/− mice are embryonic lethal due to yolk sac abnormalities [77–80]. KLF6 has been suggested to have a role in endothelial vascular remodeling following injury as it binds and activated urokinase plasminogen activator 1, endoglin, and matrix metalloproteinase 9 [81]. Interestingly, KLF6 has an alternative form of regulation because the gene produces at least four different isoforms that are able to affect DNA binding and transcription [82]. The full-length isoform of KLF6 is believed to function as a tumor suppressor and can be regulated by loss of heterozygosity, mutation, or decreased expression in different cancer types. The full-length KLf6 was found to have one deleted allele in prostate cancer, and the leftover allele was mutated 71% of the time, preventing KLF6 from functioning to activate p21 [83]. Of the isoforms of KLF6, the Krüppel-like factor 6 splice variant 1 (KLF6-SV1) was found to be oncogenic and upregulated in prostate, lung, and breast cancers and inhibits the activity of the full-length KLF6 [82]. This is the first KLF to be regulated in part by alternative splicing and suggests that directed targeting of the splice variants of

KLF6 could represent a potential target for elimination therapy.

methylation of its promoter during adipocyte formation [87].

KLF6 can be regulated by methylation both to downregulate its expression and to prevent its binding to certain sites in cancer. Studies have suggested a possible role for methylation of KLF6 in hepatocellular carcinoma and in colorectal cancer [84, 85]. Separately, KLF6 can be prevented from binding on the SIRT5 promoter by the presence of DNA methylation during adipocyte differentiation [86]. KLF6 also could not bind the tissue factor pathway inhibitor-2 promoter following hyper-

Krüppel-like factor 7 (KLF7) or ubiquitous Krüppel-like factor (UKLF) has high expression in the brain and spinal cord and is important in the developing brain and nervous system [88]. KLF7 was identified originally in 1998, located on chromosome 2, and was believed to share a strong similarity with KLF6 [89]. Studies by Laub et al. found that KLF7 was important for upregulation of p21, repression of cyclin D1, and growth arrest in neuronal cells, thereby helping to lead to their differentiation and maturation [88]. In separate but related studies, the same laboratory found that elimination of KLF7 leads to neonatal lethality and the elimination affected areas of the olfactory, visual system, cerebral cortex, and hippocampus [90].

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

KLF5 regulation in cancer progression.

**2.6 Krüppel-like factor 6**

**2.7 Krüppel-like factor 7**

### *Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

Expression of KLF5 in breast cancers was found to be correlated with a negative prognosis and decreased survival [72], while in clear cell renal cell carcinoma, hypermethylation and decreased expression of KLF5 were associated with a poorer prognosis [73]. Hypermethylation of KLF5 in acute myeloid leukemia was also associated with a poorer prognosis [74]. These studies suggest that KLF5 function in cancer is cell and perhaps even cell lineage specific. Within various cancers, KLF5 has also been demonstrated to be regulated by micro-RNAs. In gastric cancer, miR-145-5p directly targets KLF5 and promotes the differentiation of gastric cancer via KLF5 downregulation [75]. Separately, in hepatocellular carcinoma miR-214-5p acted as a tumor suppressor that could directly target and promote the downregulation of KLF5 [76]. These data demonstrate complex regulatory pathways involved in KLF5 regulation in cancer progression.

## **2.6 Krüppel-like factor 6**

*Gene Expression and Phenotypic Traits*

ers in the transcription process.

correlate with KRAS mutations [63, 64].

**2.5 Krüppel-like factor 5**

transcriptional activity.

[42, 49, 50].

cells are complex and require further investigation.

decrease the stability of KLF4 mRNA and these modifications control the exit from pluripotency [47]. Furthermore, these modifications mediate the ability of KLF4 to complex with other pluripotency transcription factors and bind DNA. Finally, Oct4 has been shown to contain a linker region that is important for loosening chromatin, complexing with Brg1, and allowing for KLF4 to bind during cellular reprogramming [2]. Clearly, the interactions and mechanisms of pluripotency factors in stem

KLF4 is required for normal functioning of the gut epithelial as deletion of KLF4 resulted in altered proliferation [48]. KLF4 and KLF5 are often found in the same types of tissues, bind to similar or identical DNA elements, and often exert opposing affects in different tissue types. KLF4 has been found to bind with p53 on the p21 genes in epithelial cells and in smooth muscle cells to inhibit proliferation

In the case of smooth muscle cell proliferation, sumoylation of KLF4 causes it to fall off the p21 promoter and decreases p21 transcription following PDGF-BB treatments [51]. Sumoylation is also believed to affect binding of KLF4 to smooth muscle marker genes in TGFβ treatment [52, 53]. In smooth muscle cells in vascular disease, KLf4 has been shown to be activated by Sp1 and Oct4 binding to the KLF4 promoter [54, 55]. Separately, in macrophages KLF4 sumoylation promotes an IL-4-induced macrophage polarization to an M2 state, suggesting KLF4 plays a role in inflammation and macrophage polarization states [56]. However, in endothelial cells KLF4 is important along with KLF2 for the maintenance of endothelial cell integrity and normal endothelial barrier function [29]. KLF4 function in vascular disease could fill chapters of books investigating its many roles and functions; however, our goal

is to highlight some of the mechanisms of its regulation in these processes.

Finally, KLF4 is also regulated by DNA methylation in several different types of cancers. KLF4 was found to be hypermethylated in renal cell carcinomas [57] and endometrial cancers [58]. However, a surprising discovery was KLF4 can bind to methylated regions of chromatin to mediate activation of transcription without the need for demethylation of the DNA in some types of cancer cells [59, 60]. These studies demonstrate a new role for some transcription factors as methylation read-

Krüppel-like factor 5 or intestinal-enriched Krüppel-like factor (IKLF) or basic transcription element-binding protein 2 (BTEB2) is located on chromosome 13q22.1 and is important in the expression of the gut epithelia, vascular smooth muscle cells, and white adipose tissues [1, 61]. KLF5 is important in epithelial cells as it is located in the base of the crypts where cells are proliferating toward the villi. In general, KLF4 and KLF5 have been shown to compete to the same sites on DNA [62] and have also been suggested to be involved in their own regulation [42]. KLF5 has been shown to be important in gastric tumor progression and initiation and often

KLF5 has also demonstrated to be important in the development and maintenance of the heart, aorta, and lung systems [20, 65–69]. Following angiotensin II induction, KLF5 was shown to bind to PDGF-A and activate it. KLF5 was also shown to be activated by RARα binding site in the KLF5 promoter [65, 70]. KLF5 has been shown to be regulated by acetylation. When KLF5 is associated with p300, it is acetylated and able to activate gene expression. Conversely, when SET is bound to KLF5, it prevents acetylation of KLF5 and its transcriptional activity [71]. These studies suggest that KLF5 can be regulated directly by modifications to control its

**58**

Krüppel-like factor 6 (KLF6) or zinc finger transcription factor 9 (ZF9) has been shown to be important for endothelial biology, adipogenesis, and tumor suppression in a wide variety of cancers. During embryogenesis, it is expressed in a timesensitive manner in the kidney, cornea, gut, and yolk sac [77–80]. KLF6−/− mice are embryonic lethal due to yolk sac abnormalities [77–80]. KLF6 has been suggested to have a role in endothelial vascular remodeling following injury as it binds and activated urokinase plasminogen activator 1, endoglin, and matrix metalloproteinase 9 [81]. Interestingly, KLF6 has an alternative form of regulation because the gene produces at least four different isoforms that are able to affect DNA binding and transcription [82]. The full-length isoform of KLF6 is believed to function as a tumor suppressor and can be regulated by loss of heterozygosity, mutation, or decreased expression in different cancer types. The full-length KLf6 was found to have one deleted allele in prostate cancer, and the leftover allele was mutated 71% of the time, preventing KLF6 from functioning to activate p21 [83]. Of the isoforms of KLF6, the Krüppel-like factor 6 splice variant 1 (KLF6-SV1) was found to be oncogenic and upregulated in prostate, lung, and breast cancers and inhibits the activity of the full-length KLF6 [82]. This is the first KLF to be regulated in part by alternative splicing and suggests that directed targeting of the splice variants of KLF6 could represent a potential target for elimination therapy.

KLF6 can be regulated by methylation both to downregulate its expression and to prevent its binding to certain sites in cancer. Studies have suggested a possible role for methylation of KLF6 in hepatocellular carcinoma and in colorectal cancer [84, 85]. Separately, KLF6 can be prevented from binding on the SIRT5 promoter by the presence of DNA methylation during adipocyte differentiation [86]. KLF6 also could not bind the tissue factor pathway inhibitor-2 promoter following hypermethylation of its promoter during adipocyte formation [87].

## **2.7 Krüppel-like factor 7**

Krüppel-like factor 7 (KLF7) or ubiquitous Krüppel-like factor (UKLF) has high expression in the brain and spinal cord and is important in the developing brain and nervous system [88]. KLF7 was identified originally in 1998, located on chromosome 2, and was believed to share a strong similarity with KLF6 [89]. Studies by Laub et al. found that KLF7 was important for upregulation of p21, repression of cyclin D1, and growth arrest in neuronal cells, thereby helping to lead to their differentiation and maturation [88]. In separate but related studies, the same laboratory found that elimination of KLF7 leads to neonatal lethality and the elimination affected areas of the olfactory, visual system, cerebral cortex, and hippocampus [90].

They also further investigated the roles of p21 and p27 and found KLF7 affected their expression in these areas during development [90]. Additional studies suggest that KLF7 regulates a number of genes in olfactory neuron development and axonal growth [91, 92]. In corneal epithelial differentiation, KLF7 was found by ChIPsequencing to inhibit the activity of KLF4 to promote a corneal "progenitor"-like state [93].

KLF7 has also been suggested to play a role in type 2 diabetes. Studies have suggested that there are single nucleotide polymorphisms (SNPs) in the KLF7 gene that are associated with increased type 2 diabetes in Asian populations [94]. The same group further investigated the role of KLF7 and found that overexpression of KLF7 impaired the insulin production system and secretion in pancreatic beta cells while also inhibiting insulin sensitivity in the peripheral tissues [95]. KLF7 was also found to activate the TLR4/NF-kB/IL-6 pathway in adipocytes [96]. Finally, KLF7 has recently been also been found to be elevated in gastric cancers in patient samples in some populations and has been suggested to be a possible biomarker for the disease [97].

## **2.8 Krüppel-like factor 8**

Krüppel-like factor 8 is expressed at low level in most tissue types [1]. KLF8 is a member of the same subfamily of Krüppel-like factors that includes KLF3 and KLF12 as all three KLFs recruit CtBP to repress transcription [37–39, 98]. These data also demonstrated that KLF8 needs its own DNA-binding domain to bind DNA but needs its repression domain for interaction with CtBP. KLF8 has been shown to be upregulated and activated during several types of cancers including those from ovarian, breast, and renal carcinomas [99–101]. KLF8 was also shown to activate the FHL2 gene in pancreatic cancer cells and to promote metastasis and epithelial-to-mesenchymal (EMT) transitions in pancreatic tumor cells [100, 101]. Furthermore, KLF8 was shown in gastric cancer to induce HIF-1 expression and promote epithelial-to-mesenchymal transitions in gastric cancer [102]. Finally, KLF8 methylation levels were also tested in prostate cancer cell lines but did not prove to be causally related to the progression of prostate tumors [103].

## **2.9 Krüppel-like factor 9**

Krüppel-like factor 9 (KLF9) or basic transcription element-binding protein (BTEB) is broadly expressed, but its expression is especially high in the developing brain and thymus and in the smooth muscle of the gut and bladder [1, 104]. Interestingly, it has been demonstrated that although the mRNA for KLF9 is transcribed in many areas, the brain is the main organ where it is translated into protein [105]. The zinc fingers of the KLF9 gene are commonly now thought to be very closely related to Sp1 as they have a high sequence similarity. However, beyond their DNA-binding domains, these proteins share little sequence similarity [105]. In the brain expression, there is a thyroid hormone response element in the promoter of the KLF9 gene that accounts for its transcription and expression in the postnatal brain [105, 106]. KLF9 was also found to bind to a number of proximal promoter regions on genes important for brain function to repress transcription in hippocampal neurons [106, 107].

KLF9 expression has been noted in cancers of the mammary glands and uterus because of its ability to interact with the progesterone response elements to stimulate progesterone response elements [108, 109]. KLF9 is also required for the development of fertility in females as KLF9−/− mice were subfertile and were unable to differentiate their reproductive tissue without KLF9 [109]. KLF9−/− mice also were

**61**

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors*

found to have aberrant regulation of their intestinal crypt cell proliferation and villus migration [110]. These data suggest that KLF9 also regulates the smooth muscle

Finally, in follicular lymphoma, KLF9 was found to be hypermethylated and silenced in tumors along with a number of polycomb genes [111]. Separately, in breast cancer hypermethylation of KLF9 was correlated with a favorable cancer

Krüppel-like factor 10 (KLF10) or transforming growth factor-inducible early

KLF10 has been cited to be important in bone development and osteoporosis, adipocyte development, and heart, lung, brain, and T-cell activation [1, 116]. In adipocyte differentiation, C/EBPβ was found to bind and activate the KLF10 promoter, while KLF10 bound to the C-EBPα promoter to inhibit its activation [117]. In bone development, SNP analysis revealed that variants in the KLF10 gene were associated with bone loss in older men [118]. Conversely, studies in KLF10 null mice suggest a gender-specific role of KLF10 in the maintenance of bone density [19]. KLF10 null osteoblasts were also found to be defective in mineralization and in osteoblast support of osteoclast differentiation [119]. Finally, KLF10 null mice had impaired tendon function as adults with corresponding difficulty in tendon function [120]. In heart development, KLF10−/− mice developed cardiac hypertrophy and an increase in ventricle size and an increase in wall thickness, suggesting the importance of KLF10 to the maintenance of normal heart function [121]. KLF10 is also important in T-cell and Treg development along with TGFβ as deletion of KLF10 in

T cells augmented atherosclerosis and led to impaired T-cell function [122].

KLF10 has been shown to be methylated in pancreatic cancers by DNMT1 with a correlation between methylation status and tumor grade [123]. The more the methylation and repression of the KLF10 promoter, the worse the tumor grade. These studies suggest that an important regulatory mechanism for KLF10 is also via

Krüppel-like factor 11 (KLF11) or transforming growth factor-inducible early gene 2 (TIEG2) or FKLF is known to be expressed in the pancreas and in erythroid cells in the fetal liver. KLF11 is located in humans at chromosome 2p25 [1, 124–126]. KLF11 shares 91% homology with KLF10 in the zinc finger domain and 44% homology with the N-terminus of KLF10 [127]. These studies also demonstrated that overexpression of KLF11 inhibits cell proliferation [127] and is induced by

KLF11 contains three repression domains that are believed to be important for its repressor activities [128]. TGFβ signaling pathway induction means that KLF11

gene 1 (TIEG1) is known as a TGFβ-inducible gene as it is rapidly induced by TGFβ treatments and then quickly returns back to basal levels [113, 114]. KLF10 is induced by multiple members of the TGFβ superfamily and then goes on to suppress Smad7 and co-activate together with Smad2. It is believed that KLF10 plays a major role in the mediation of TGFβ inhibition of cell proliferation and inflammation and induction of apoptosis [113, 115]. The rapid induction and then degradation of KLF10 are believed to be accounted for by SIAH proteasomal degradation [113]. In these studies, KLF10 was found to interact directly with SIAH which then mediates its degradation [113]. These studies suggest a protein degradation method

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

and the turnover of intestinal cells.

**2.10 Krüppel-like factor 10**

methylation of its promoter.

**2.11 Krüppel-like factor 11**

TGFβ signaling pathways.

prognosis [112].

of regulation.

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

found to have aberrant regulation of their intestinal crypt cell proliferation and villus migration [110]. These data suggest that KLF9 also regulates the smooth muscle and the turnover of intestinal cells.

Finally, in follicular lymphoma, KLF9 was found to be hypermethylated and silenced in tumors along with a number of polycomb genes [111]. Separately, in breast cancer hypermethylation of KLF9 was correlated with a favorable cancer prognosis [112].

## **2.10 Krüppel-like factor 10**

*Gene Expression and Phenotypic Traits*

state [93].

the disease [97].

**2.8 Krüppel-like factor 8**

**2.9 Krüppel-like factor 9**

pal neurons [106, 107].

They also further investigated the roles of p21 and p27 and found KLF7 affected their expression in these areas during development [90]. Additional studies suggest that KLF7 regulates a number of genes in olfactory neuron development and axonal growth [91, 92]. In corneal epithelial differentiation, KLF7 was found by ChIPsequencing to inhibit the activity of KLF4 to promote a corneal "progenitor"-like

KLF7 has also been suggested to play a role in type 2 diabetes. Studies have suggested that there are single nucleotide polymorphisms (SNPs) in the KLF7 gene that are associated with increased type 2 diabetes in Asian populations [94]. The same group further investigated the role of KLF7 and found that overexpression of KLF7 impaired the insulin production system and secretion in pancreatic beta cells while also inhibiting insulin sensitivity in the peripheral tissues [95]. KLF7 was also found to activate the TLR4/NF-kB/IL-6 pathway in adipocytes [96]. Finally, KLF7 has recently been also been found to be elevated in gastric cancers in patient samples in some populations and has been suggested to be a possible biomarker for

Krüppel-like factor 8 is expressed at low level in most tissue types [1]. KLF8 is a member of the same subfamily of Krüppel-like factors that includes KLF3 and KLF12 as all three KLFs recruit CtBP to repress transcription [37–39, 98]. These data also demonstrated that KLF8 needs its own DNA-binding domain to bind DNA but needs its repression domain for interaction with CtBP. KLF8 has been shown to be upregulated and activated during several types of cancers including those from ovarian, breast, and renal carcinomas [99–101]. KLF8 was also shown to activate the FHL2 gene in pancreatic cancer cells and to promote metastasis and epithelial-to-mesenchymal (EMT) transitions in pancreatic tumor cells [100, 101]. Furthermore, KLF8 was shown in gastric cancer to induce HIF-1 expression and promote epithelial-to-mesenchymal transitions in gastric cancer [102]. Finally, KLF8 methylation levels were also tested in prostate cancer cell lines but did not

prove to be causally related to the progression of prostate tumors [103].

Krüppel-like factor 9 (KLF9) or basic transcription element-binding protein (BTEB) is broadly expressed, but its expression is especially high in the developing brain and thymus and in the smooth muscle of the gut and bladder [1, 104]. Interestingly, it has been demonstrated that although the mRNA for KLF9 is transcribed in many areas, the brain is the main organ where it is translated into protein [105]. The zinc fingers of the KLF9 gene are commonly now thought to be very closely related to Sp1 as they have a high sequence similarity. However, beyond their DNA-binding domains, these proteins share little sequence similarity [105]. In the brain expression, there is a thyroid hormone response element in the promoter of the KLF9 gene that accounts for its transcription and expression in the postnatal brain [105, 106]. KLF9 was also found to bind to a number of proximal promoter regions on genes important for brain function to repress transcription in hippocam-

KLF9 expression has been noted in cancers of the mammary glands and uterus because of its ability to interact with the progesterone response elements to stimulate progesterone response elements [108, 109]. KLF9 is also required for the development of fertility in females as KLF9−/− mice were subfertile and were unable to differentiate their reproductive tissue without KLF9 [109]. KLF9−/− mice also were

**60**

Krüppel-like factor 10 (KLF10) or transforming growth factor-inducible early gene 1 (TIEG1) is known as a TGFβ-inducible gene as it is rapidly induced by TGFβ treatments and then quickly returns back to basal levels [113, 114]. KLF10 is induced by multiple members of the TGFβ superfamily and then goes on to suppress Smad7 and co-activate together with Smad2. It is believed that KLF10 plays a major role in the mediation of TGFβ inhibition of cell proliferation and inflammation and induction of apoptosis [113, 115]. The rapid induction and then degradation of KLF10 are believed to be accounted for by SIAH proteasomal degradation [113]. In these studies, KLF10 was found to interact directly with SIAH which then mediates its degradation [113]. These studies suggest a protein degradation method of regulation.

KLF10 has been cited to be important in bone development and osteoporosis, adipocyte development, and heart, lung, brain, and T-cell activation [1, 116]. In adipocyte differentiation, C/EBPβ was found to bind and activate the KLF10 promoter, while KLF10 bound to the C-EBPα promoter to inhibit its activation [117]. In bone development, SNP analysis revealed that variants in the KLF10 gene were associated with bone loss in older men [118]. Conversely, studies in KLF10 null mice suggest a gender-specific role of KLF10 in the maintenance of bone density [19]. KLF10 null osteoblasts were also found to be defective in mineralization and in osteoblast support of osteoclast differentiation [119]. Finally, KLF10 null mice had impaired tendon function as adults with corresponding difficulty in tendon function [120].

In heart development, KLF10−/− mice developed cardiac hypertrophy and an increase in ventricle size and an increase in wall thickness, suggesting the importance of KLF10 to the maintenance of normal heart function [121]. KLF10 is also important in T-cell and Treg development along with TGFβ as deletion of KLF10 in T cells augmented atherosclerosis and led to impaired T-cell function [122].

KLF10 has been shown to be methylated in pancreatic cancers by DNMT1 with a correlation between methylation status and tumor grade [123]. The more the methylation and repression of the KLF10 promoter, the worse the tumor grade. These studies suggest that an important regulatory mechanism for KLF10 is also via methylation of its promoter.

#### **2.11 Krüppel-like factor 11**

Krüppel-like factor 11 (KLF11) or transforming growth factor-inducible early gene 2 (TIEG2) or FKLF is known to be expressed in the pancreas and in erythroid cells in the fetal liver. KLF11 is located in humans at chromosome 2p25 [1, 124–126]. KLF11 shares 91% homology with KLF10 in the zinc finger domain and 44% homology with the N-terminus of KLF10 [127]. These studies also demonstrated that overexpression of KLF11 inhibits cell proliferation [127] and is induced by TGFβ signaling pathways.

KLF11 contains three repression domains that are believed to be important for its repressor activities [128]. TGFβ signaling pathway induction means that KLF11 often cooperates with Smads to induce changes in transcription following TGFβ treatment. KLF11 later was found to be activated by several members of the TGFβ superfamily and not just by TGFβ treatment alone [114]. Studies have shown in neuronal cells that KLF11 regulates the transcription of the dopamine D2 receptor by complexing with p300, a histone acetylase, to promoter transcription [129]. KLF11 was also found to regulate collagen gene expression through the heterochromatin protein 1 gene-silencing pathway, as mutants defective for coupling to this epigenetic modifier lose the ability to repress COL1A2 and to prevent fibrosis in KLF11−/− mice [130]. As part of the TGFβ induction of KLF11, TGFβ induction allows KLF11 to interact with Smad3 and to repress certain promoters. In the case of pancreatic cancer, KLF11 was found to bind with Smad3 to the c-myc promoter following TFG-β treatment [131].

KLF11 is important not only for its TGFβ response but also for its associations with diabetes and obesity [132, 133]. A variant of KLF11 was found that could lead to type 2 diabetes and obesity [134]. Further studies revealed additional variants that may affect KLF11 regulation of the insulin promoter and type 2 diabetes [133]. KLF11 was also found to interact with p300 in maturity-onset diabetes of the young to induce transcriptional changes in the pancreas [135]. In converse, KLF11 can also interact with mSin3a in pancreatic cancer by repression of the Smad7 promoter [136]. Ectopic expression of KLF11 increased the sensitivity of cells to oxidative drugs [137]. Methylation of KLF11 has been suggested to be one mechanism of its downregulation in several types of cancers [138, 139].

## **2.12 Krüppel-like factor 12**

Krüppel-like factor 12 or BETB1 was first identified in the regulation of the AP-2α gene and is located on chromosome 13q21-13q22 [140]. In the case of the AP-2α gene, KLF12 functions as a transcriptional activator and appears to relate back to KLF12's function as a marker of tumor development [141–143]. KLF12 is a marker for gastric cancer progression, and overexpression of KLF12 promotes tumor cell invasion and progression [142]. However, in lung cancer cell lines, it was shown that KLF12 was important for the regulation of anoikis and the progression through the S phase of cell cycle [141]. These data suggest that KLF12 may have multiple different roles in cancer beyond what was previously identified. KLF12 is also one of the KLF factors to interact with the mSin3a repressor complex via an alpha-helical motif in a repression domain of the transcription factor [144].

KLF12 not only plays roles in tumor progression but is also believed to play a role in the developing kidney after birth. KLF12 was shown to be expressed in the collecting ducts of the kidney after birth and could directly regulate the UT-A1 but not the ENaC promoters, two genes important for the development of the collecting ducts [145]. A recent study suggests that KLF12 might in part be regulated in cancer by the methylation of miR-205 by long noncoding RNA ELF3-antisense RNA 1. These data suggest that miR-205 and RNA ELF3-antisense RNA 1 exist in a complex regulatory loop involving KLF12 [146].

## **2.13 Krüppel-like factor 13**

Krüppel-like factor 13 (KLF13) or BTEB3, FKLF2, or RFLAT-1 was first discovered along with Krüppel-like factor 14 (KLF14) using an expressed sequence tag database to search for additional conserved KLF DNA-binding domains [129]. KLF13−/− mice are one of the few KLF mice that are viable and fertile; however, they display abnormal blood cell development [147, 148] suggesting that KLF13 is

**63**

regions.

**2.14 Krüppel-like factor 14**

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors*

upper limbs and heart, via its interaction with the TBX5 promoter [153].

critical for both B- and T-cell developments [148–150]. One part of this developmental process is KLF13's interaction with PPAR4 [151] to regulate CCL5. Not only is KLF13 important for blood cell development, it has also been shown to be important for the developing heart [104, 152]. To this end, KLF14 can also be linked to Holt-Oram syndrome, an inherited disorder characterized by abnormalities of the

KLF13 has also recently been suggested to be a tumor suppressor in glioma cells [154]. These studies found that KLF13 was downregulated by hypomethylation across the gene to promote its silencing; however, decreases in DNMT1 expression or decreases in hypomethylation patterns of KLF13 decreased proliferation and migration of glioma cells [154]. Another example of KLF13 methylation is the methylation of the obesity-related variant of KLF13: cg07814318. The methylation of this particular SNP appears to be related to increased childhood obesity [155]. These studies suggest that methylation of promoters could be one possible mechanism of

Another possible mechanism of regulation of KLF13 is through the co-repressor

Krüppel-like factor 14 was first discovered using expressed sequence tag databases to search for the presence of additional conserved KLF DNA-binding domains [129]. KLF14 has 72% similarity with the human Sp2; however, the majority of its similarity exists within its DNA-binding domain [129]. Most reports suggest that its expression is ubiquitous [1]. Interestingly, KLF14 is intron-less and exists on chromosome 7q32. KLF14 is a mono-allelic expression pattern and shown to be hypomethylated in many tissues, further suggesting a pattern of ubiquitous expression [156]. Further evidence also suggests that KLF14 could be derived from a retro-transposed copy of Krüppel-like factor 16 (KLF16) [156] and could be an example of accelerated evolution. KLF14 deletion has recently been linked with centrosome amplification, aneuploidy, and spontaneous tumorigenesis because KLF14 functions as a repressor of polo-like kinase 4 (PLK4). Without the repressive activities of KLF14 on PLK-14, PLK-14 can cause chromosomal abnormalities and promote tumorigenesis in cancer cells. The KLF14 gene has been linked to genomic

variants that are highly correlative with basal cell carcinoma [157].

Genome-wide association studies not only revealed that KLF14 was linked with basal cell carcinoma, it also has revealed that KLF14 is linked with cholesterol metabolism, metabolic disease, and coronary artery disease. These studies suggest that KLF14 might function as an imprinted master regulator of metabolic function and that mutation of certain SNPs within the KLF14 gene can lead to a large-scale deregulation of metabolic gene function [158]. KLF14 was also found to regulate levels of HDL-C and hepatic ApoA-I production [159]. Guo et al. were able to find evidence that perhexiline was able to activate KLF14 and to reduce lesions in ApoE−/− atherosclerotic mice [159]. Separate but related studies suggest that this activity is related to the phosphorylation of KLF14 by both p38 MAPK and ERK kinase [160]. However, KLF14 was found to be decreased in endothelial cells in atherosclerosis, and overexpression of KLF14 actually inhibited NF-KB signaling by suppressing p65 [161]. KLF14 has also been shown to interact with p300 to promote sphingosine kinase activation and to enhance sphingosine production [162].

complex mSin3a [144]. In this instance, KLF13 was found to interact with the mSin3a repressor complex via an alpha-helical motif in a repression domain [144]. Additional studies from this group suggest that multiple KLF factors (BTEB1, BTEB3, BTEB4) could also contain this alpha-helical domain in their repression

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

regulation of KLFs in development or disease.

### *Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

critical for both B- and T-cell developments [148–150]. One part of this developmental process is KLF13's interaction with PPAR4 [151] to regulate CCL5. Not only is KLF13 important for blood cell development, it has also been shown to be important for the developing heart [104, 152]. To this end, KLF14 can also be linked to Holt-Oram syndrome, an inherited disorder characterized by abnormalities of the upper limbs and heart, via its interaction with the TBX5 promoter [153].

KLF13 has also recently been suggested to be a tumor suppressor in glioma cells [154]. These studies found that KLF13 was downregulated by hypomethylation across the gene to promote its silencing; however, decreases in DNMT1 expression or decreases in hypomethylation patterns of KLF13 decreased proliferation and migration of glioma cells [154]. Another example of KLF13 methylation is the methylation of the obesity-related variant of KLF13: cg07814318. The methylation of this particular SNP appears to be related to increased childhood obesity [155]. These studies suggest that methylation of promoters could be one possible mechanism of regulation of KLFs in development or disease.

Another possible mechanism of regulation of KLF13 is through the co-repressor complex mSin3a [144]. In this instance, KLF13 was found to interact with the mSin3a repressor complex via an alpha-helical motif in a repression domain [144]. Additional studies from this group suggest that multiple KLF factors (BTEB1, BTEB3, BTEB4) could also contain this alpha-helical domain in their repression regions.

## **2.14 Krüppel-like factor 14**

*Gene Expression and Phenotypic Traits*

following TFG-β treatment [131].

**2.12 Krüppel-like factor 12**

regulatory loop involving KLF12 [146].

**2.13 Krüppel-like factor 13**

factor [144].

downregulation in several types of cancers [138, 139].

often cooperates with Smads to induce changes in transcription following TGFβ treatment. KLF11 later was found to be activated by several members of the TGFβ superfamily and not just by TGFβ treatment alone [114]. Studies have shown in neuronal cells that KLF11 regulates the transcription of the dopamine D2 receptor by complexing with p300, a histone acetylase, to promoter transcription [129]. KLF11 was also found to regulate collagen gene expression through the heterochromatin protein 1 gene-silencing pathway, as mutants defective for coupling to this epigenetic modifier lose the ability to repress COL1A2 and to prevent fibrosis in KLF11−/− mice [130]. As part of the TGFβ induction of KLF11, TGFβ induction allows KLF11 to interact with Smad3 and to repress certain promoters. In the case of pancreatic cancer, KLF11 was found to bind with Smad3 to the c-myc promoter

KLF11 is important not only for its TGFβ response but also for its associations with diabetes and obesity [132, 133]. A variant of KLF11 was found that could lead to type 2 diabetes and obesity [134]. Further studies revealed additional variants that may affect KLF11 regulation of the insulin promoter and type 2 diabetes [133]. KLF11 was also found to interact with p300 in maturity-onset diabetes of the young to induce transcriptional changes in the pancreas [135]. In converse, KLF11 can also interact with mSin3a in pancreatic cancer by repression of the Smad7 promoter [136]. Ectopic expression of KLF11 increased the sensitivity of cells to oxidative drugs [137]. Methylation of KLF11 has been suggested to be one mechanism of its

Krüppel-like factor 12 or BETB1 was first identified in the regulation of the AP-2α gene and is located on chromosome 13q21-13q22 [140]. In the case of the AP-2α gene, KLF12 functions as a transcriptional activator and appears to relate back to KLF12's function as a marker of tumor development [141–143]. KLF12 is a marker for gastric cancer progression, and overexpression of KLF12 promotes tumor cell invasion and progression [142]. However, in lung cancer cell lines, it was shown that KLF12 was important for the regulation of anoikis and the progression through the S phase of cell cycle [141]. These data suggest that KLF12 may have multiple different roles in cancer beyond what was previously identified. KLF12 is also one of the KLF factors to interact with the mSin3a repressor complex via an alpha-helical motif in a repression domain of the transcription

KLF12 not only plays roles in tumor progression but is also believed to play a role in the developing kidney after birth. KLF12 was shown to be expressed in the collecting ducts of the kidney after birth and could directly regulate the UT-A1 but not the ENaC promoters, two genes important for the development of the collecting ducts [145]. A recent study suggests that KLF12 might in part be regulated in cancer by the methylation of miR-205 by long noncoding RNA ELF3-antisense RNA 1. These data suggest that miR-205 and RNA ELF3-antisense RNA 1 exist in a complex

Krüppel-like factor 13 (KLF13) or BTEB3, FKLF2, or RFLAT-1 was first discovered along with Krüppel-like factor 14 (KLF14) using an expressed sequence tag database to search for additional conserved KLF DNA-binding domains [129]. KLF13−/− mice are one of the few KLF mice that are viable and fertile; however, they display abnormal blood cell development [147, 148] suggesting that KLF13 is

**62**

Krüppel-like factor 14 was first discovered using expressed sequence tag databases to search for the presence of additional conserved KLF DNA-binding domains [129]. KLF14 has 72% similarity with the human Sp2; however, the majority of its similarity exists within its DNA-binding domain [129]. Most reports suggest that its expression is ubiquitous [1]. Interestingly, KLF14 is intron-less and exists on chromosome 7q32. KLF14 is a mono-allelic expression pattern and shown to be hypomethylated in many tissues, further suggesting a pattern of ubiquitous expression [156]. Further evidence also suggests that KLF14 could be derived from a retro-transposed copy of Krüppel-like factor 16 (KLF16) [156] and could be an example of accelerated evolution. KLF14 deletion has recently been linked with centrosome amplification, aneuploidy, and spontaneous tumorigenesis because KLF14 functions as a repressor of polo-like kinase 4 (PLK4). Without the repressive activities of KLF14 on PLK-14, PLK-14 can cause chromosomal abnormalities and promote tumorigenesis in cancer cells. The KLF14 gene has been linked to genomic variants that are highly correlative with basal cell carcinoma [157].

Genome-wide association studies not only revealed that KLF14 was linked with basal cell carcinoma, it also has revealed that KLF14 is linked with cholesterol metabolism, metabolic disease, and coronary artery disease. These studies suggest that KLF14 might function as an imprinted master regulator of metabolic function and that mutation of certain SNPs within the KLF14 gene can lead to a large-scale deregulation of metabolic gene function [158]. KLF14 was also found to regulate levels of HDL-C and hepatic ApoA-I production [159]. Guo et al. were able to find evidence that perhexiline was able to activate KLF14 and to reduce lesions in ApoE−/− atherosclerotic mice [159]. Separate but related studies suggest that this activity is related to the phosphorylation of KLF14 by both p38 MAPK and ERK kinase [160]. However, KLF14 was found to be decreased in endothelial cells in atherosclerosis, and overexpression of KLF14 actually inhibited NF-KB signaling by suppressing p65 [161]. KLF14 has also been shown to interact with p300 to promote sphingosine kinase activation and to enhance sphingosine production [162].

These data suggest a complicated pattern of expression for a ubiquitous transcription factor that could produce paradoxical effects in inflammatory disease such as cardiovascular disease or cancer. Interestingly, there still appears to be less known about how KLF14 itself is regulated.

### **2.15 Krüppel-like factor 15**

KLF15 or kidney-enriched Krüppel-like factor (KKLF) demonstrates low levels of cardiac-specific expression during development but then exhibits adult expression in the kidney, liver, pancreas, heart, skeletal muscle, lung, and ovary. KLF15 was originally thought to be important for the regulation of different cell types in the kidney and repressed genes such as CLC-K1 and CLC-K2 [163]. However, its regulatory effects can be seen in the heart, skeletal muscle, gluconeogenesis, and circadian rhythms. In terms of the heart, KLF15 was demonstrated to be an inhibitor of cardiac fibrosis by repression of connective tissue growth factor (CTGF) [164]. In this mechanism, KLF15 inhibits the recruitment of the co-activator P-CAF but does not prevent SMAD3 from binding to the promoter [164]. Additional studies by the same group demonstrated that KLF15 was a negative regulator of cardiac hypertrophy via inhibition of GATA4 and MEF2 functions [165]. Recent studies further suggest that KLF15 was identified as a putative upstream regulator of metabolic gene expression in the heart via RNA-Seq and methylation sequencing and that KLF15 was itself regulated by EZH2 in a SET domain-dependent manner [166]. KLF15 was demonstrated to be silenced via methylation in ischemic cardiomyopathy which in turn leads to the silencing of many cardiac-specific genes.

KLF15 has been shown to also be important for metabolism [167]. In terms of the skeletal muscle, overnight fasting and endurance exercise induce KLF15 expression, while knockout of KLF15 induces abnormal energy flux, excessive muscle fatigue, and impaired endurance capacity [168]. KLF15 was later shown to complex in the liver with liver X receptor (LXR) to inhibit SREBF1 during fasting by recruiting the co-repressor RIP140 [169]. Finally, KLF15 is also important for nitrogen homeostasis and the maintenance of circadian rhythm as KLF15 knockout mice had no amino acid rhythm and no rhythm of the production of urea from ammonia [170]. These studies suggest the importance of KLF15 and suggest that investigations into how it is regulated by chromatin readers and writers will become important to these metabolic diseases.

## **2.16 Krüppel-like factor 16**

Krüppel-like factor 16 or dopamine receptor regulating factor (DRRF) was first discovered in its regulation of the dopamine receptors in the developing brain and eye [171]. It is now known that KLF16 is expressed not only in the developing brain but also in the thymus, intestine, kidney, liver, heart, and bladder. KLF16 has recently been shown to not only regulate the dopamine receptor but also to regulate the ephrin receptor A5 (EphA5), but this regulation was methylation specific as methylation of the EphA5 promoter prevented KLF16 from binding [171]. These data suggest that one possible epigenetic mechanism regulating KLF16 is methylation of regions near its binding site.

KLF16 was found by Daftary et al. to bind to all three types of KLF binding site, the GC, CA, and BTE boxes using electromobility shift assays but prefers binding to the BTE box in cells and to mediate its effects via mSin3a, a transcriptional co-repressor complex but suggests that this function is both promoter and cell context dependent [172]. To further study this interaction, site-directed mutagenesis was performed of all of the serine, threonine, and tyrosine residues

**65**

dation by micro-RNAs.

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors*

believed to be possible targets for kinase phosphorylation signaling and found that mutation of tyrosine-10 altered the ability of KLF16 to interact with mSin3a [172]. Finally, KLF16 was also found to be regulated by nuclear localization and to be excluded from heterochromatin within the nucleus [172]. These studies suggest complex posttranslational regulatory mechanisms for KLF16 function in a cell- and

Krüppel-like factor 17 (KLF17) was first discovered in mouse as zinc finger protein 393 (ZFP393) or ZNF393 where it was shown to be expressed in the testis and ovaries, and the gene spans 8 kb in the distal portion of chromosome 4 in the mouse [173]. In humans KLF17 maps to chromosome 1p34.1. When it was discovered back in 2002, it was believed to be the first C2H2 germ cell-specific zinc finger protein. Identification of KLF17 in the human revealed that KLF17 was expressed not only in the testis but also in the brain and bone, albeit at relatively low amounts [174]. KLF17 also contains low sequence similarity between the human and mouse orthologues; however, a detailed transcriptional binding analysis by van Vliet et al. was able to demonstrate that KLF17 was a Krüppel-like transcription factor rather than being more closely linked to the specificity protein

KLF17 is hypothesized to be a tumor suppressor in multiple types of cancers, and a decrease in its expression has become correlated with a poor cancer prognosis [175]. KLF17 was demonstrated to be a tumor suppressor gene in metastatic breast cancer lines whose downregulation promotes the epithelial-to-mesenchymal transition in cancer cells [176]. These studies also suggested that KLF17 is a direct negative regulator of inhibitor of DNA binding 1 (ID1). Sadly, they do not offer a direct mechanism for the downregulation of KLF17 during breast cancer metastasis, but they do provide compelling data to suggest that KLF17 might have multiple functions in the male and female sex organs and that suppression of this factor could

Further evidence in non-small-cell lung cancer also suggests that KLF17 could function as a tumor suppressor [177]. These studies suggested that p53 recruits p300 to the KLF17 promoter to acetylate and turn on transcription [177]. In addition, p53 also physically interacts with KLF17 and promotes binding of KLF17 to certain gene promoters and promotes transcription of p53, p21, and pRB [177]. These data suggest an intricate cross-talk between KLF17 and p53 in tumorigenesis. Another way KLF17 is believed to inhibit cancer progression is through inhibition of proliferation via repression of UPAI-1 [178], which Cai et al. proposed inhibited the invasive properties of small-cell lung cancer cells. KLF17 was also suggested to be a tumor suppressor through a TGFB-/SMAD-dependent mechanism where KLF17 physically interacts with SMAD3 to target genes to prevent metastases [179]. MiR-9, a micro-RNA important for tumor invasion and metastasis, has been shown to inhibit the activation of KLF17 by directly binding to the 3′-untranslated region (3′-UTR) [175]. These pathways suggest that KLF17 can be regulated both by direct promoter activation and by posttranscriptional modifications such as RNA degra-

In converse, in endometrial cancer KLF17 was found to be an inducer of epithelial-to-mesenchymal transition and resulted in activation of TWIST1 [180]. This finding demonstrated that KLF17 bound directly to the TWIST promoter to activate its transcription [180]. KLF17 was also shown to bind directly to estrogen receptor alpha (ERα) to prevent it from being able to bind directly to chromatin [181]. ERα then also contributed to the suppression of KLF17 using the

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

promoter-dependent manner.

factor family (Sp family) [173].

lead to increased tumorigenic potential [176].

**2.17 Krüppel-like factor 17**

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

believed to be possible targets for kinase phosphorylation signaling and found that mutation of tyrosine-10 altered the ability of KLF16 to interact with mSin3a [172]. Finally, KLF16 was also found to be regulated by nuclear localization and to be excluded from heterochromatin within the nucleus [172]. These studies suggest complex posttranslational regulatory mechanisms for KLF16 function in a cell- and promoter-dependent manner.

## **2.17 Krüppel-like factor 17**

*Gene Expression and Phenotypic Traits*

about how KLF14 itself is regulated.

important to these metabolic diseases.

tion of regions near its binding site.

**2.16 Krüppel-like factor 16**

**2.15 Krüppel-like factor 15**

These data suggest a complicated pattern of expression for a ubiquitous transcription factor that could produce paradoxical effects in inflammatory disease such as cardiovascular disease or cancer. Interestingly, there still appears to be less known

KLF15 or kidney-enriched Krüppel-like factor (KKLF) demonstrates low levels of cardiac-specific expression during development but then exhibits adult expression in the kidney, liver, pancreas, heart, skeletal muscle, lung, and ovary. KLF15 was originally thought to be important for the regulation of different cell types in the kidney and repressed genes such as CLC-K1 and CLC-K2 [163]. However, its regulatory effects can be seen in the heart, skeletal muscle, gluconeogenesis, and circadian rhythms. In terms of the heart, KLF15 was demonstrated to be an inhibitor of cardiac fibrosis by repression of connective tissue growth factor (CTGF) [164]. In this mechanism, KLF15 inhibits the recruitment of the co-activator P-CAF but does not prevent SMAD3 from binding to the promoter [164]. Additional studies by the same group demonstrated that KLF15 was a negative regulator of cardiac hypertrophy via inhibition of GATA4 and MEF2 functions [165]. Recent studies further suggest that KLF15 was identified as a putative upstream regulator of metabolic gene expression in the heart via RNA-Seq and methylation sequencing and that KLF15 was itself regulated by EZH2 in a SET domain-dependent manner [166]. KLF15 was demonstrated to be silenced via methylation in ischemic cardiomyopathy which in turn leads to the silencing of many cardiac-specific genes. KLF15 has been shown to also be important for metabolism [167]. In terms of the skeletal muscle, overnight fasting and endurance exercise induce KLF15 expression, while knockout of KLF15 induces abnormal energy flux, excessive muscle fatigue, and impaired endurance capacity [168]. KLF15 was later shown to complex in the liver with liver X receptor (LXR) to inhibit SREBF1 during fasting by recruiting the co-repressor RIP140 [169]. Finally, KLF15 is also important for nitrogen homeostasis and the maintenance of circadian rhythm as KLF15 knockout mice had no amino acid rhythm and no rhythm of the production of urea from ammonia [170]. These studies suggest the importance of KLF15 and suggest that investigations into how it is regulated by chromatin readers and writers will become

Krüppel-like factor 16 or dopamine receptor regulating factor (DRRF) was first discovered in its regulation of the dopamine receptors in the developing brain and eye [171]. It is now known that KLF16 is expressed not only in the developing brain but also in the thymus, intestine, kidney, liver, heart, and bladder. KLF16 has recently been shown to not only regulate the dopamine receptor but also to regulate the ephrin receptor A5 (EphA5), but this regulation was methylation specific as methylation of the EphA5 promoter prevented KLF16 from binding [171]. These data suggest that one possible epigenetic mechanism regulating KLF16 is methyla-

KLF16 was found by Daftary et al. to bind to all three types of KLF binding site, the GC, CA, and BTE boxes using electromobility shift assays but prefers binding to the BTE box in cells and to mediate its effects via mSin3a, a transcriptional co-repressor complex but suggests that this function is both promoter and cell context dependent [172]. To further study this interaction, site-directed mutagenesis was performed of all of the serine, threonine, and tyrosine residues

**64**

Krüppel-like factor 17 (KLF17) was first discovered in mouse as zinc finger protein 393 (ZFP393) or ZNF393 where it was shown to be expressed in the testis and ovaries, and the gene spans 8 kb in the distal portion of chromosome 4 in the mouse [173]. In humans KLF17 maps to chromosome 1p34.1. When it was discovered back in 2002, it was believed to be the first C2H2 germ cell-specific zinc finger protein. Identification of KLF17 in the human revealed that KLF17 was expressed not only in the testis but also in the brain and bone, albeit at relatively low amounts [174]. KLF17 also contains low sequence similarity between the human and mouse orthologues; however, a detailed transcriptional binding analysis by van Vliet et al. was able to demonstrate that KLF17 was a Krüppel-like transcription factor rather than being more closely linked to the specificity protein factor family (Sp family) [173].

KLF17 is hypothesized to be a tumor suppressor in multiple types of cancers, and a decrease in its expression has become correlated with a poor cancer prognosis [175]. KLF17 was demonstrated to be a tumor suppressor gene in metastatic breast cancer lines whose downregulation promotes the epithelial-to-mesenchymal transition in cancer cells [176]. These studies also suggested that KLF17 is a direct negative regulator of inhibitor of DNA binding 1 (ID1). Sadly, they do not offer a direct mechanism for the downregulation of KLF17 during breast cancer metastasis, but they do provide compelling data to suggest that KLF17 might have multiple functions in the male and female sex organs and that suppression of this factor could lead to increased tumorigenic potential [176].

Further evidence in non-small-cell lung cancer also suggests that KLF17 could function as a tumor suppressor [177]. These studies suggested that p53 recruits p300 to the KLF17 promoter to acetylate and turn on transcription [177]. In addition, p53 also physically interacts with KLF17 and promotes binding of KLF17 to certain gene promoters and promotes transcription of p53, p21, and pRB [177]. These data suggest an intricate cross-talk between KLF17 and p53 in tumorigenesis. Another way KLF17 is believed to inhibit cancer progression is through inhibition of proliferation via repression of UPAI-1 [178], which Cai et al. proposed inhibited the invasive properties of small-cell lung cancer cells. KLF17 was also suggested to be a tumor suppressor through a TGFB-/SMAD-dependent mechanism where KLF17 physically interacts with SMAD3 to target genes to prevent metastases [179]. MiR-9, a micro-RNA important for tumor invasion and metastasis, has been shown to inhibit the activation of KLF17 by directly binding to the 3′-untranslated region (3′-UTR) [175]. These pathways suggest that KLF17 can be regulated both by direct promoter activation and by posttranscriptional modifications such as RNA degradation by micro-RNAs.

In converse, in endometrial cancer KLF17 was found to be an inducer of epithelial-to-mesenchymal transition and resulted in activation of TWIST1 [180]. This finding demonstrated that KLF17 bound directly to the TWIST promoter to activate its transcription [180]. KLF17 was also shown to bind directly to estrogen receptor alpha (ERα) to prevent it from being able to bind directly to chromatin [181]. ERα then also contributed to the suppression of KLF17 using the

co-repressor histone deacetylase 1 (HDAC1) to promote KLF17 deacetylation and chromatin condensation [181].

## **2.18 Krüppel-like factor 18**

Krüppel-like factor 18 (KLF18) was identified in 2013 from sequence similarity searches and gene synteny analyses and was shown at that time to be highly related to KLF17 [182]. Like KLF17, it is believed to be expressed in the developing testis and restricted to that area. Little data currently exists examining its function; however, a detailed analysis of its structure and phylogenic tree in placental mammals has been investigated in detail by Pei et al. [182]. This group also suggested that KLF18 might be a pseudogene of KLF17 since its expression pattern is restricted and it is similar in sequence to KLF17. Despite this hypothesis, three genes in mouse and rat were identified that closely resemble KLF18: Zfp352, Zfp352-like, and Zfp353 [182]. The promoter and/or details into the transcriptional activation of this KLF are currently unknown. A more detailed analysis of the functions and regulations of KLF18 would provide more insight into this transcription factor's function.

## **3. Concluding remarks**

Over the past 20 years since the discovery of the first KLF transcription factor, there continues to be a growing body of evidence to suggest that KLFs are important to tumor progression, cardiovascular disease, metabolism, and even circadian rhythm [1]. While much of the work has focused on the functions of these factors and their roles in various disease processes, there still remains additional needed work to explain how the various KLFs become activated and/or repressed during diseased states. There is a growing body of evidence, which we have attempted to discuss in some detail in this chapter, in the more extensively studied KLFs such as KLF4, KLF5, and KLF2 that suggest that the KLFs are regulated extensively by posttranslational modifications such as phosphorylation, acetylation, ubiquitination, and sumoylation. These modifications appear to be critical for co-factor recruitment and determination of whether KLFs interact with either activators or repressors of transcription. It has been interesting to see the wealth of information that has developed over the past 20 years investigating the roles of these various factors in various diseases; however, relatively speaking, we still know little about how these factors are activated and/or repressed transcriptionally during diseased states.

Since the onset of the era of big data, more of the KLF field has come to focus on the roles of pathway analysis following genetic ablation of a KLF in a cell-specific manner. These studies have yielded enormous amounts of data that offer valuable insight into the overlap between various KLF factors in diseases [183]. It will be of interest in the future to see how the integration of single-cell genomics will come into play with various different roles of the same KLF in various cell types in diseased states [184]. For example, the integration of single-cell RNA-Seq [184] with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) [185, 186] in cells where a single KLF bear separate functions could offer deeper insight of the role of the niche environment on KLF function and/or on the roles of KLFs in downstream activations of different types of pathways during disease. Cardiovascular diseases have recently begun to investigate single-cell sequencing with other factors, such as Tcf21, and were able to use these innovative studies to investigate the role of this factor in smooth muscle cell to fibroblast transitions during atherosclerosis [184]. It will be exciting to see how KLF biology will use this technology to further investigate how these transcription factors regulate disease.

**67**

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors*

Not only will the integration of single-cell studies with KLF function give us greater insight into KLF function in development and disease, but the study of the role of RNA posttranscriptional modifications will most likely play an emerging role in the KLF field in the near future [184]. Since the sequencing of the human genome and the growing realization of the stronger role of RNA in transcriptional and translational control, there has been a re-emergence of interest in the field of RNA posttranscriptional modifications [187]. There are over 100 different types

trated in the 3′-UTR of many messenger RNAs and that micro-RNAs are capable of mediating this modification via a sequence pairing mechanism to help promote stem cell pluripotency [187–192]. This new role for RNA modification and stem cell maintenance has immense implications for KLFs involved in induced pluripotent stem maintenance like KLF4. Therefore, it will be of interest to determine whether RNA modifications affect other disease processes by similar sequence pairing

In conclusion, the KLF field has offered many insights to different disease processes since the discovery of the first KLF over the past 20 years. New insights into the regulation of these factors will hopefully grant novel methods to directly and properly target these factors to inhibit diseased states that currently have no medical treatment therapy. Perhaps the newly emerging CRISP technology will be able to directly target KLFs in a cell-specific manner as many KLFs have opposing functions in many different cell types. In any case, this transcription factor family has offered much excitement since its discovery and hopefully will offer new

insights as the field studies these factors in more depth in the future.

does not necessarily represent the views of the AHA.

The authors declare no conflict of interest.

**Notes/thanks/other declarations**

DNMT1 DNA methyltransferase 1 EphA5 Ephrin receptor A5

ID1 Inhibitor of DNA binding 1

ER Estrogen receptor HDAC Histone deacetylase

EMT Epithelial-to-mesenchymal transition

This work was supported by the AHA Scientist Development Grant 14SDG18730000 (MS). The content is solely the responsibility of the authors and

We thank Anthony Herring and Cindy Dodson for their knowledge and techni-


A) modification is

A has recently been shown to be concen-

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

of RNA modifications of which the N6

mechanisms.

**Acknowledgements**

**Conflict of interest**

cal expertise.

**Nomenclatures**

the most common [187]. Interestingly, m6

## *Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

Not only will the integration of single-cell studies with KLF function give us greater insight into KLF function in development and disease, but the study of the role of RNA posttranscriptional modifications will most likely play an emerging role in the KLF field in the near future [184]. Since the sequencing of the human genome and the growing realization of the stronger role of RNA in transcriptional and translational control, there has been a re-emergence of interest in the field of RNA posttranscriptional modifications [187]. There are over 100 different types of RNA modifications of which the N6 -methyladenosine (m6 A) modification is the most common [187]. Interestingly, m6 A has recently been shown to be concentrated in the 3′-UTR of many messenger RNAs and that micro-RNAs are capable of mediating this modification via a sequence pairing mechanism to help promote stem cell pluripotency [187–192]. This new role for RNA modification and stem cell maintenance has immense implications for KLFs involved in induced pluripotent stem maintenance like KLF4. Therefore, it will be of interest to determine whether RNA modifications affect other disease processes by similar sequence pairing mechanisms.

In conclusion, the KLF field has offered many insights to different disease processes since the discovery of the first KLF over the past 20 years. New insights into the regulation of these factors will hopefully grant novel methods to directly and properly target these factors to inhibit diseased states that currently have no medical treatment therapy. Perhaps the newly emerging CRISP technology will be able to directly target KLFs in a cell-specific manner as many KLFs have opposing functions in many different cell types. In any case, this transcription factor family has offered much excitement since its discovery and hopefully will offer new insights as the field studies these factors in more depth in the future.

## **Acknowledgements**

*Gene Expression and Phenotypic Traits*

chromatin condensation [181].

**2.18 Krüppel-like factor 18**

**3. Concluding remarks**

co-repressor histone deacetylase 1 (HDAC1) to promote KLF17 deacetylation and

Krüppel-like factor 18 (KLF18) was identified in 2013 from sequence similarity searches and gene synteny analyses and was shown at that time to be highly related to KLF17 [182]. Like KLF17, it is believed to be expressed in the developing testis and restricted to that area. Little data currently exists examining its function; however, a detailed analysis of its structure and phylogenic tree in placental mammals has been investigated in detail by Pei et al. [182]. This group also suggested that KLF18 might be a pseudogene of KLF17 since its expression pattern is restricted and it is similar in sequence to KLF17. Despite this hypothesis, three genes in mouse and rat were identified that closely resemble KLF18: Zfp352, Zfp352-like, and Zfp353 [182]. The promoter and/or details into the transcriptional activation of this KLF are currently unknown. A more detailed analysis of the functions and regulations of KLF18 would provide more insight into this transcription factor's function.

Over the past 20 years since the discovery of the first KLF transcription factor, there continues to be a growing body of evidence to suggest that KLFs are important to tumor progression, cardiovascular disease, metabolism, and even circadian rhythm [1]. While much of the work has focused on the functions of these factors and their roles in various disease processes, there still remains additional needed work to explain how the various KLFs become activated and/or repressed during diseased states. There is a growing body of evidence, which we have attempted to discuss in some detail in this chapter, in the more extensively studied KLFs such as KLF4, KLF5, and KLF2 that suggest that the KLFs are regulated extensively by posttranslational modifications such as phosphorylation, acetylation, ubiquitination, and sumoylation. These modifications appear to be critical for co-factor recruitment and determination of whether KLFs interact with either activators or repressors of transcription. It has been interesting to see the wealth of information that has developed over the past 20 years investigating the roles of these various factors in various diseases; however, relatively speaking, we still know little about how these factors are activated and/or repressed transcriptionally during diseased states. Since the onset of the era of big data, more of the KLF field has come to focus on the roles of pathway analysis following genetic ablation of a KLF in a cell-specific manner. These studies have yielded enormous amounts of data that offer valuable insight into the overlap between various KLF factors in diseases [183]. It will be of interest in the future to see how the integration of single-cell genomics will come into play with various different roles of the same KLF in various cell types in diseased states [184]. For example, the integration of single-cell RNA-Seq [184] with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) [185, 186] in cells where a single KLF bear separate functions could offer deeper insight of the role of the niche environment on KLF function and/or on the roles of KLFs in downstream activations of different types of pathways during disease. Cardiovascular diseases have recently begun to investigate single-cell sequencing with other factors, such as Tcf21, and were able to use these innovative studies to investigate the role of this factor in smooth muscle cell to fibroblast transitions during atherosclerosis [184]. It will be exciting to see how KLF biology will use this technology to further investigate how these transcription factors regulate disease.

**66**

This work was supported by the AHA Scientist Development Grant 14SDG18730000 (MS). The content is solely the responsibility of the authors and does not necessarily represent the views of the AHA.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Notes/thanks/other declarations**

We thank Anthony Herring and Cindy Dodson for their knowledge and technical expertise.

## **Nomenclatures**



## **Author details**

Morgan Salmon Department of Surgery, University of Virginia School of Medicine, Charlottesville, VA, USA

\*Address all correspondence to: msa5m@virginia.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**69**

2006;**126**:663-676

Nature. 1995;**375**:316-318

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors*

[9] Asano H, Stamatoyannopoulos G. Activation of beta-globin promoter by erythroid Krüppel-like factor. Molecular and Cellular Biology. 1998;**18**:102-109

Stamatoyannopoulos G, Witkowska HE, Orkin SH. Fetal expression of a human Agamma globin transgene rescues globin chain imbalance but not

hemolysis in EKLF null mouse embryos.

[11] Perkins AC, Sharpe AH, Orkin SH. Lethal beta-thalassaemia in mice lacking the erythroid CACCCtranscription factor EKLF. Nature.

[12] Tallack MR, Perkins AC. Three fingers on the switch: Krüppellike factor 1 regulation of γ-globin to β-globin gene switching. Current Opinion in Hematology.

[13] Cantú I, van de Werken HJG,

Gillemans N, Stadhouders R, Heshusius S, Maas A, et al. The mouse KLF1 Nan variant impairs nuclear condensation and erythroid maturation. PLoS One.

[14] Fluhr S, Krombholz CF, Meier A, Epting T, Mücke O, Plass C, et al. Epigenetic dysregulation of the

erythropoietic transcription factor KLF1 and the β-like globin locus in juvenile myelomonocytic leukemia. Epigenetics.

[15] Frontelo P, Manwani D, Galdass M, Karsunky H, Lohmann F, Gallagher PG,

megakaryocyte lineage commitment.

[16] Wani MA, Conkright MD, Jeffries S, Hughes MJ, Lingrel JB. cDNA isolation, genomic structure, regulation, and chromosomal localization of human

et al. Novel role for EKLF in

Blood. 2007;**110**:3871-3880

[10] Perkins AC, Peterson KR,

Blood. 2000;**95**:1827-1833

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2013;**20**:193-200

2019;**14**:e0208659-e

2017;**12**:715-723

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

[2] Chen K, Long Q, Xing G, Wang T, Wu Y, Li L, et al. Heterochromatin loosening by the Oct4 linker region facilitates Klf4 binding and iPSC reprogramming. The EMBO Journal.

[3] Homma K, Sone M, Taura D, Yamahara K, Suzuki Y, Takahashi K, et al. Sirt1 plays an important role in mediating greater functionality of human ES/iPS-derived vascular endothelial cells. Atherosclerosis.

[4] Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnology. 2008;**26**:101-106

[5] Narazaki G, Uosaki H, Teranishi M, Okita K, Kim B, Matsuoka S, et al. Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. Circulation. 2008;**118**:498-506

[6] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;**131**:861-872

[7] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell.

[8] Nuez B, Michalovich D, Bygrave A, Ploemacher R, Grosveld F. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.

[1] McConnell BB, Yang VW. Mammalian Krüppel-like factors in health and diseases. Physiological Reviews. 2010;**90**:1337-1381

**References**

2020;**39**:e99165

2010;**212**:42-47

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

## **References**

*Gene Expression and Phenotypic Traits*

IL-4 Interleukin-4 IL-6 Interleukin-6

M**6**A N6

KLF Krüppel-like factor

p300 Histone acetylase P53 TP53 or tumor protein P50 Subunit of NF-KB

pRB Phosphorylated RB SMC Smooth muscle cells

TWIST TWIST1-protein

ZFP Zinc finger protein

P65 Subunit of NF-KB signaling

SM-actin Smooth muscle alpha actin Sp Specificity proteins

PDGF-BB Platelet-derived growth factor BB

TFG-β Transforming growth factor beta TNF-α Tumor necrosis factor alpha

ZF-TF Zinc finger transcription factor


P21 p21CIP1, cyclin-dependent protein inhibitor

NF-KB Nuclear Factor kappa-light-chain-enhancer of activated B cells

Smad Proteins transduce signals from transforming growth factor beta

**68**

**Author details**

Morgan Salmon

Charlottesville, VA, USA

Department of Surgery, University of Virginia School of Medicine,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: msa5m@virginia.edu

provided the original work is properly cited.

[1] McConnell BB, Yang VW. Mammalian Krüppel-like factors in health and diseases. Physiological Reviews. 2010;**90**:1337-1381

[2] Chen K, Long Q, Xing G, Wang T, Wu Y, Li L, et al. Heterochromatin loosening by the Oct4 linker region facilitates Klf4 binding and iPSC reprogramming. The EMBO Journal. 2020;**39**:e99165

[3] Homma K, Sone M, Taura D, Yamahara K, Suzuki Y, Takahashi K, et al. Sirt1 plays an important role in mediating greater functionality of human ES/iPS-derived vascular endothelial cells. Atherosclerosis. 2010;**212**:42-47

[4] Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnology. 2008;**26**:101-106

[5] Narazaki G, Uosaki H, Teranishi M, Okita K, Kim B, Matsuoka S, et al. Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. Circulation. 2008;**118**:498-506

[6] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;**131**:861-872

[7] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;**126**:663-676

[8] Nuez B, Michalovich D, Bygrave A, Ploemacher R, Grosveld F. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature. 1995;**375**:316-318

[9] Asano H, Stamatoyannopoulos G. Activation of beta-globin promoter by erythroid Krüppel-like factor. Molecular and Cellular Biology. 1998;**18**:102-109

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2015;**35**:1562-1569

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Krüppel-like factor 2. Arteriosclerosis, Thrombosis, and Vascular Biology.

[35] Crossley M, Whitelaw E, Perkins A, Williams G, Fujiwara Y, Orkin SH. Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells. Molecular and Cellular Biology.

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regulation involves heterogeneous nuclear ribonucleoproteins and acetyltransferases. Biochemistry.

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2011;**31**:133-141

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*Gene Expression and Phenotypic Traits*

[17] Basu P, Morris PE, Haar JL, Wani MA, Lingrel JB, Gaensler KML, et al. KLF2 is essential for primitive erythropoiesis and regulates the human and murine embryonic beta-like globin genes in vivo. Blood.

1999;**60**:78-86

2005;**106**:2566-2571

2006;**442**:299-302

3953-3963

1998;**7**(4):229-238

1999;**274**:21180-21185

2017;**9**:2024-2037

[19] Hawse JR, Iwaniec UT,

lung Krüppel-like factor. Genomics.

survival. Science (New York, NY).

[26] Sohn SJ, Li D, Lee LK, Winoto A. Transcriptional regulation of tissuespecific genes by the ERK5 mitogenactivated protein kinase. Molecular and Cellular Biology. 2005;**25**:8553-8566

Salloum FN, Kukreja RC, et al. Krüppellike factor 2 is required for normal mouse cardiac development. PLoS One.

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Developmental Cell. 2006;**11**:845-857

[29] Sangwung P, Zhou G, Nayak L, Chan ER, Kumar S, Kang DW, et al. KLF2 and KLF4 control endothelial identity and vascular integrity. JCI

[30] Huddleson JP, Srinivasan S,

induces endothelial KLF2 gene

region. Biological Chemistry.

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[32] Ahmad N, Lingrel JB. Krüppellike factor 2 transcriptional

Ahmad N, Lingrel JB. Fluid shear stress

expression through a defined promoter

Insight. 2017;**2**:e91700

2004;**385**:723-729

[27] Chiplunkar AR, Lung TK, Alhashem Y, Koppenhaver BA,

2013;**8**:e54891

[25] Ohnesorge N, Viemann D, Schmidt N, Czymai T, Spiering D, Schmolke M, et al. Erk5 activation elicits a vasoprotective endothelial phenotype via induction of Krüppel-like factor 4 (KLF4). The Journal of Biological Chemistry. 2010;**285**:26199-26210

1997;**277**:1986-1990

[18] Carlson CM, Endrizzi BT, Wu J, Ding X, Weinreich MA, Walsh ER, et al. Krüppel-like factor 2 regulates thymocyte and T-cell migration. Nature.

Bensamoun SF, Monroe DG, Peters KD, Ilharreborde B, et al. TIEG-null mice display an osteopenic gender-specific phenotype. Bone. 2008;**42**:1025-1031

[20] Lin S-CJ, Wani MA, Whitsett JA, Wells JM. Klf5 regulates lineage formation in the pre-implantation mouse embryo. Development (Cambridge, England). 2010;**137**:

[21] Wani M, Means R, Lingrel J. Loss of LKLF function results in embryonic lethality in mice. Transgenic Research.

[22] Wani MA, Wert SE, Lingrel JB. Lung Krüppel-like factor, a zinc finger transcription factor, is essential for normal lung development. The Journal of Biological Chemistry.

[23] Jiang W, Xu X, Deng S, Luo J, Xu H, Wang C, et al. Methylation of krüppel-like factor 2 (KLF2) associates with its expression and non-small cell lung cancer progression. American Journal of Translational Research.

[24] Kuo CT, Veselits ML, Leiden JM. LKLF: A transcriptional regulator of single-positive T cell quiescence and

**70**

[33] Huddleson JP, Ahmad N, Lingrel JB. Up-regulation of the KLF2 transcription factor by fluid shear stress requires nucleolin. The Journal of Biological Chemistry. 2006;**281**:15121-15128

[34] Kumar A, Kim C-S, Hoffman TA, Naqvi A, Dericco J, Jung S-B, et al. p53 impairs endothelial function by transcriptionally repressing Krüppel-like factor 2. Arteriosclerosis, Thrombosis, and Vascular Biology. 2011;**31**:133-141

[35] Crossley M, Whitelaw E, Perkins A, Williams G, Fujiwara Y, Orkin SH. Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells. Molecular and Cellular Biology. 1996;**16**:1695-1705

[36] Vu T, Gatto D, Turner V, Funnell A, Mak KS, Norton L, et al. Impaired B Cell Development in the Absence of Krüppel-like factor 3. Journal of Immunology (Baltimore, MD: 1950). 2011;**187**:5032-5042

[37] Pearson RCM, Funnell APW, Crossley M. The mammalian zinc finger transcription factor Krüppel-like factor 3 (KLF3/BKLF). IUBMB Life. 2011;**63**:86-93

[38] Turner J, Crossley M. Cloning and characterization of mCtBP2, a co-repressor that associates with basic Krüppel-like factor and other mammalian transcriptional regulators. The EMBO Journal. 1998;**17**:5129-5140

[39] Turner J, Nicholas H, Bishop D, Matthews JM, Crossley M. The LIM protein FHL3 binds basic Krüppellike factor/Krüppel-like factor 3 and its co-repressor C-terminal-binding

protein 2. The Journal of Biological Chemistry. 2003;**278**:12786-12795

[40] Sue N, Jack BHA, Eaton SA, Pearson RCM, Funnell APW, Turner J, et al. Targeted disruption of the basic Krüppel-like factor gene (Klf3) reveals a role in adipogenesis. Molecular and Cellular Biology. 2008;**28**:3967-3978

[41] Dunn J, Thabet S, Jo H. Flowdependent epigenetic DNA methylation in endothelial gene expression and atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;**35**:1562-1569

[42] McConnell BB, Ghaleb AM, Nandan MO, Yang VW. The diverse functions of Krüppel-like factors 4 and 5 in epithelial biology and pathobiology. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology. 2007;**29**:549-557

[43] Segre J, Bauer C, Fuchs E. KLF4 is a transcription factor required for establishing the barrier function of the skin. Nature Genetics. 1999;**22**:356-60

[44] Ehlermann J, Pfisterer P, Schorle H. Dynamic expression of Krüppel-like factor 4 (Klf4), a target of transcription factor AP-2alpha during murine midembryogenesis. The Anatomical Record Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology. 2003;**273**:677-680

[45] Hall J, Guo G, Wray J, Eyres I, Nichols J, Grotewold L, et al. Oct4 and LIF/Stat3 additively induce Krüppel factors to sustain embryonic stem cell self-renewal. Cell Stem Cell. 2009;**5**:597-609

[46] Jiang J, Chan Y-S, Loh Y-H, Cai J, Tong G-Q, Lim C-A, et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nature Cell Biology. 2008;**10**:353-360

[47] Dhaliwal NK, Abatti LE, Mitchell JA. KLF4 protein stability regulated by interaction with pluripotency transcription factors overrides transcriptional control. Genes and Development. 2019:**33**(15-16):1069-1082

[48] Katz JP, Perreault N, Goldstein BG, Actman L, McNally SR, Silberg DG, et al. Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the adult stomach. Gastroenterology. 2005;**128**:935-945

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[50] Yoshida T, Kaestner KH, Owens GK. Conditional deletion of Krüppel-like factor 4 delays downregulation of smooth muscle cell differentiation markers but accelerates neointimal formation following vascular injury. Circulation Research. 2008;**102**:1548-1557

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[52] Liu Y, Sinha S, Owens G. A transforming growth factor-b control element required for SM a-actin expression in vivo also partially mediates GKLF-dependent transcriptional repression. The Journal of Biological Chemistry. 2003;**278**:48004-48011

[53] Liu Y, Sinha S, McDonald OG, Shang Y, Hoofnagle MH, Owens GK. Kruppel-like factor 4 abrogates myocardin-induced activation of smooth muscle gene expression. The Journal of Biological Chemistry. 2005;**280**(10):9719-9727

[54] Cherepanova OA, Gomez D, Shankman LS, Swiatlowska P, Williams J, Sarmento OF, et al. Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective. Nature Medicine. 2016;**22**:657-665

[55] Deaton RA, Gan Q, Owens GK. Sp1 dependent activation of KLF4 is required for PDGF-BB-induced phenotypic modulation of smooth muscle. American Journal of Physiology—Heart and Circulatory Physiology. 2009;**296**:H1027-H1H37

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[59] Oyinlade O, Wei S, Kammers K, Liu S, Wang S, Ma D, et al. Analysis of KLF4 regulated genes in cancer cells reveals a role of DNA methylation in promoter- enhancer interactions. Epigenetics. 2018;**13**:751-768

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2008;**135**:2563-72

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[63] Nandan MO, Ghaleb AM, McConnell BB, Patel NV, Robine S, Yang VW. Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMin and KRASV12 mutations. Molecular Cancer. 2010;**9**:63

[64] Nandan MO, McConnell BB, Ghaleb AM, Bialkowska AB, Sheng H, Shao J, et al. Krüppel-like factor 5 mediates

cellular transformation during oncogenic KRAS-induced intestinal tumorigenesis. Gastroenterology.

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[66] Nagai R, Suzuki T, Aizawa A, Shindo T, Manabe I. Significance of the transcription factor KLF5 in cardiovascular remodeling. Journal of Thrombosis and Haemostasis. Aug

a Krüppel-like zinc-finger type transcription factor, mediates both smooth muscle cell activation and cardiac hypertrophy. Advances in Experimental Medicine and Biology.

2008;**134**:120-130

2003;**538**:57-66

2005;**3**(8):1569-1576

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Cell Metabolism. 2005;**1**:27-39

[68] Suzuki T, Sawaki D, Aizawa K, Munemasa Y, Matsumura T,

encoding an intestinal-enriched member of the Krüppel-like factor family expressed in intestinal

1999;**27**:1263-1270

[61] Conkright MD, Wani MA, Anderson KP, Lingrel JB. A gene

## *Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

encoding an intestinal-enriched member of the Krüppel-like factor family expressed in intestinal epithelial cells. Nucleic Acids Research. 1999;**27**:1263-1270

*Gene Expression and Phenotypic Traits*

regulated by interaction with pluripotency transcription factors overrides transcriptional control. Genes and Development.

2019:**33**(15-16):1069-1082

2005;**128**:935-945

[49] Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW. Krüppellike factors 4 and 5: the yin and yang regulators of cellular proliferation. Cell

[50] Yoshida T, Kaestner KH, Owens GK. Conditional deletion of Krüppel-like factor 4 delays downregulation of smooth muscle cell differentiation markers but accelerates neointimal formation following vascular injury. Circulation

Research. 2005;**15**:92-96

Research. 2008;**102**:1548-1557

Research. 2016;**342**:20-31

2003;**278**:48004-48011

2005;**280**(10):9719-9727

[52] Liu Y, Sinha S, Owens G. A transforming growth factor-b control element required for SM a-actin expression in vivo also partially mediates GKLF-dependent transcriptional repression. The Journal of Biological Chemistry.

[53] Liu Y, Sinha S, McDonald OG, Shang Y, Hoofnagle MH, Owens GK. Kruppel-like factor 4 abrogates myocardin-induced activation of smooth muscle gene expression. The Journal of Biological Chemistry.

[51] Nie C-J, Li YH, Zhang X-H, Wang Z-P, Jiang W, Zhang Y, et al. SUMOylation of KLF4 acts as a switch in transcriptional programs that control VSMC proliferation. Experimental Cell

[48] Katz JP, Perreault N, Goldstein BG, Actman L, McNally SR, Silberg DG, et al. Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the adult stomach. Gastroenterology.

[54] Cherepanova OA, Gomez D, Shankman LS, Swiatlowska P, Williams J, Sarmento OF, et al. Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective. Nature Medicine.

[55] Deaton RA, Gan Q, Owens GK. Sp1-

[56] Wang K, Zhou W, Cai Q, Cheng J, Cai R, Xing R. SUMOylation of KLF4 promotes IL-4 induced macrophage M2 polarization. Cell Cycle (Georgetown,

[57] Li H, Wang J, Xiao W, Xia D, Lang B, Yu G, et al. Epigenetic alterations of Krüppel-like factor 4 and its tumor suppressor function in renal cell carcinoma. Carcinogenesis.

[58] Danková Z, Braný D, Dvorská D, Ňachajová M, Fiolka R, Grendár M, et al. Methylation status of KLF4 and HS3ST2 genes as predictors of endometrial cancer and hyperplastic endometrial lesions. International Journal of Molecular Medicine.

[59] Oyinlade O, Wei S, Kammers K, Liu S, Wang S, Ma D, et al. Analysis of KLF4 regulated genes in cancer cells reveals a role of DNA methylation in promoter- enhancer interactions.

Epigenetics. 2018;**13**:751-768

[60] Wan J, Su Y, Song Q, Tung B, Oyinlade O, Liu S, et al. Methylated cis-regulatory elements mediate KLF4 dependent gene transactivation and cell

migration. eLife. 2017;**6**:e20068

[61] Conkright MD, Wani MA, Anderson KP, Lingrel JB. A gene

dependent activation of KLF4 is required for PDGF-BB-induced phenotypic modulation of smooth muscle. American Journal of Physiology—Heart and Circulatory Physiology. 2009;**296**:H1027-H1H37

2016;**22**:657-665

Tex). 2017;**16**:374-81

2013;**34**:2262-2270

2018;**42**:3318-3328

**72**

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[63] Nandan MO, Ghaleb AM, McConnell BB, Patel NV, Robine S, Yang VW. Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMin and KRASV12 mutations. Molecular Cancer. 2010;**9**:63

[64] Nandan MO, McConnell BB, Ghaleb AM, Bialkowska AB, Sheng H, Shao J, et al. Krüppel-like factor 5 mediates cellular transformation during oncogenic KRAS-induced intestinal tumorigenesis. Gastroenterology. 2008;**134**:120-130

[65] Nagai R, Shindo T, Manabe I, Suzuki T, Kurabayashi M. KLF5/BTEB2, a Krüppel-like zinc-finger type transcription factor, mediates both smooth muscle cell activation and cardiac hypertrophy. Advances in Experimental Medicine and Biology. 2003;**538**:57-66

[66] Nagai R, Suzuki T, Aizawa A, Shindo T, Manabe I. Significance of the transcription factor KLF5 in cardiovascular remodeling. Journal of Thrombosis and Haemostasis. Aug 2005;**3**(8):1569-1576

[67] Oishi Y, Manabe I, Tobe K, Tsushima K, Shindo T, Fujiu K, et al. Krüppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation. Cell Metabolism. 2005;**1**:27-39

[68] Suzuki T, Sawaki D, Aizawa K, Munemasa Y, Matsumura T,

Ishida J, et al. Krüppel-like factor 5 shows proliferation-specific roles in vascular remodeling, direct stimulation of cell growth, and inhibition of apoptosis. The Journal of Biological Chemistry. 2009;**284**:9549-9557

[69] Wan H, Luo F, Wert SE, Zhang L, Xu Y, Ikegami M, et al. Krüppel-like factor 5 is required for perinatal lung morphogenesis and function. Development (Cambridge, England). 2008;**135**:2563-72

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[71] Miyamoto S, Suzuki T, Muto S, Aizawa K, Kimura A, Mizuno Y, et al. Positive and negative regulation of the cardiovascular transcription factor KLF5 by p300 and the oncogenic regulator SET through interaction and acetylation on the DNA-binding domain. Molecular and Cellular Biology. 2003;**23**:8528-8541

[72] Tong D, Czerwenka K, Heinze G, Ryffel M, Schuster E, Witt A, et al. Expression of KLF5 is a prognostic factor for disease-free survival and overall survival in patients with breast cancer. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2006;**12**:2442-2448

[73] Fu R-J, He W, Wang X-B, Li L, Zhao H-B, Liu X-Y, et al. DNMT1-maintained hypermethylation of Krüppel-like factor 5 involves in the progression of clear cell renal cell carcinoma. Cell Death & Disease. 2017;**8**:e2952-e

[74] Diakiw SM, Perugini M, Kok CH, Engler GA, Cummings N, To LB, et al. Methylation of KLF5 contributes to reduced expression in acute myeloid

leukaemia and is associated with poor overall survival. British Journal of Haematology. 2013;**161**:884-888

[75] Zhou T, Chen S, Mao X. miR-145-5p affects the differentiation of gastric cancer by targeting KLF5 directly. Journal of Cellular Physiology. 2019;**234**:7634-7644

[76] Pang J, Li Z, Wang G, Li N, Gao Y, Wang S. miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells. Journal of Cellular Biochemistry. 2018. DOI: 10.1002/jcb.27498

[77] Fischer EA, Verpont MC, Garrett-Sinha LA, Ronco PM, Rossert JA. Klf6 is a zinc finger protein expressed in a cell-specific manner during kidney development. Journal of the American Society of Nephrology. 2001;**12**(4):726-735

[78] Laub F, Aldabe R, Ramirez F, Friedman S. Embryonic expression of Krüppel-like factor 6 in neural and non-neural tissues. Mechanisms of Development. 2001;**106**:167-170

[79] Matsumoto N, Kubo A, Liu H, Akita K, Laub F, Ramirez F, et al. Developmental regulation of yolk sac hematopoiesis by Krüppel-like factor 6. Blood. 2006;**107**:1357-1365

[80] Nakamura H, dr Chiambaretta F, Sugar J, Sapin V, Yue BYJT. Developmentally regulated expression of KLF6 in the mouse cornea and lens. Investigative Ophthalmology & Visual Science. 2004;**45**:4327-32

[81] Atkins GB, Jain MK. Role of Krüppel-like transcription factors in endothelial biology. Circulation Research. 2007;**100**:1686-1695

[82] DiFeo A, Martignetti JA, Narla G. The role of KLF6 and its splice variants in cancer therapy. Drug Resistance Updates: Reviews and

Commentaries in Antimicrobial and Anticancer Chemotherapy. 2009;**12**:1-7

[83] Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, et al. KLF6, a candidate tumor suppressor gene mutated in prostate cancer. Science (New York, NY). 2001;**294**:2563-6

[84] Song J, Kim CJ, Cho YG, Kim SY, Nam SW, Lee SH, et al. Genetic and epigenetic alterations of the KLF6 gene in hepatocellular carcinoma. Journal of Gastroenterology and Hepatology. 2006;**21**:1286-1289

[85] Babaei K, Khaksar R, Zeinali T, Hemmati H, Bandegi A, Samidoust P, et al. Epigenetic profiling of MUTYH, KLF6, WNT1 and KLF4 genes in carcinogenesis and tumorigenesis of colorectal cancer. Biomedicine. 2019;**9**:22

[86] Hong J, Wang X, Mei C, Zan L. Competitive regulation by transcription factors and DNA methylation in the bovine SIRT5 promoter: Roles of E2F4 and KLF6. Gene. 2019;**684**:39-46

[87] Guo H, Lin Y, Zhang H, Liu J, Zhang N, Li Y, et al. Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells. BMC Molecular Biology. 2007;**8**:110

[88] Laub F, Aldabe R, Friedrich V Jr, Ohnishi S, Yoshida T, Ramirez F. Developmental expression of mouse Krüppel-like transcription factor KLF7 suggests a potential role in neurogenesis. Developmental Biology. 2001;**233**:305-318

[89] Matsumoto N, Laub F, Aldabe R, Zhang W, Ramirez F, Yoshida T, et al. Cloning the cDNA for a new human zinc finger protein defines a group of closely related Krüppel-like transcription factors. The Journal of Biological Chemistry. 1998;**273**:28229-28237

**75**

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[97] Jiang Z, Yu T, Fan Z, Yang H, Lin X. Krüppel-like factor 7 is a marker of aggressive gastric cancer and poor prognosis. Cellular Physiology and Biochemistry. 2017;**43**:1090-1099

[98] van Vliet J, Turner J, Crossley M. Human Krüppel-like factor 8: A CACCC-box binding protein that associates with CtBP and represses transcription. Nucleic Acids Research.

2000;**28**:1955-1962

2007;**26**:456-461

2017;**14**:4883-4889

2014;**32**:2397-2404

2017;**7**:40636

[99] Wang X, Zhao J. KLF8

tumorigenesis, invasion and metastasis of colorectal cancer cells by transcriptional activation of FHL2. Oncotarget. 2015;**6**:25402-25417

transcription factor participates in oncogenic transformation. Oncogene.

[100] Yan Q, Zhang W, Wu Y, Wu M, Zhang M, Shi X, et al. KLF8 promotes

[101] Yi X, Zai H, Long X, Wang X, Li W, Li Y. Krüppel-like factor 8 induces epithelial-to-mesenchymal transition and promotes invasion of pancreatic cancer cells through transcriptional activation of four and a half LIMonly protein 2. Oncology Letters.

[102] Liu N, Wang Y, Zhou Y, Pang H, Zhou J, Qian P, et al. Krüppel-like factor 8 involved in hypoxia promotes the invasion and metastasis of gastric cancer via epithelial to mesenchymal transition. Oncology Reports.

[103] Møller M, Strand SH, Mundbjerg K,

[104] Martin KM, Metcalfe JC, Kemp PR. Expression of Klf9 and Klf13 in mouse

Liang G, Gill I, Haldrup C, et al. Heterogeneous patterns of DNA methylation-based field effects in histologically normal prostate tissue from cancer patients. Scientific Reports.

development. Mechanisms of Development. 2001;**103**:149-151

*DOI: http://dx.doi.org/10.5772/intechopen.91652*

[91] Kajimura D, Dragomir C, Ramirez F, Laub F. Identification of genes regulated

by transcription factor KLF7 in differentiating olfactory sensory neurons. Gene. 2007;**388**:34-42

Experimental Cell Research.

[93] Klein RH, Hu W, Kashgari G, Lin Z, Nguyen T, Doan M, et al. Characterization of enhancers and the role of the transcription factor KLF7 in regulating corneal epithelial differentiation. The Journal of Biological

Chemistry. 2017;**292**:18937-18950

[94] Kanazawa A, Kawamura Y, Sekine A, Iida A, Tsunoda T,

Kashiwagi A, et al. Single nucleotide polymorphisms in the gene encoding Krüppel-like factor 7 are associated with type 2 diabetes. Diabetologia.

Kawamori R, Maeda S. Overexpression of Krüppel-like factor 7 regulates adipocytokine gene expressions in human adipocytes and inhibits glucoseinduced insulin secretion in pancreatic beta-cell line. Molecular endocrinology (Baltimore, MD). 2006;**20**:844-56

[96] Zhang M, Wang C, Wu J, Ha X, Deng Y, Zhang X, et al. The effect and mechanism of KLF7 in the TLR4/ NF-κB/IL-6 inflammatory signal pathway of adipocytes. Mediators of Inflammation. 2018;**2018**:1756494

2011;**317**:464-473

2005;**48**:1315-1322

[95] Kawamura Y, Tanaka Y,

[92] Caiazzo M, Colucci-D'Amato L, Volpicelli F, Speranza L, Petrone C, Pastore L, et al. Krüppel-like factor 7 is required for olfactory bulb dopaminergic neuron development.

[90] Laub F, Lei L, Sumiyoshi H, Kajimura D, Dragomir C, Smaldone S, et al. Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system. Molecular and Cellular Biology.

2005;**25**:5699-5711

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

[90] Laub F, Lei L, Sumiyoshi H, Kajimura D, Dragomir C, Smaldone S, et al. Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system. Molecular and Cellular Biology. 2005;**25**:5699-5711

*Gene Expression and Phenotypic Traits*

[75] Zhou T, Chen S, Mao X. miR-145-5p affects the differentiation of gastric cancer by targeting KLF5 directly. Journal of Cellular Physiology.

2019;**234**:7634-7644

10.1002/jcb.27498

2001;**12**(4):726-735

[77] Fischer EA, Verpont MC, Garrett-Sinha LA, Ronco PM,

leukaemia and is associated with poor overall survival. British Journal of Haematology. 2013;**161**:884-888

Commentaries in Antimicrobial and Anticancer Chemotherapy. 2009;**12**:1-7

[83] Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, et al. KLF6, a candidate tumor suppressor gene mutated in prostate cancer. Science (New York, NY). 2001;**294**:2563-6

[84] Song J, Kim CJ, Cho YG, Kim SY, Nam SW, Lee SH, et al. Genetic and epigenetic alterations of the KLF6 gene in hepatocellular carcinoma. Journal of Gastroenterology and Hepatology.

[85] Babaei K, Khaksar R, Zeinali T, Hemmati H, Bandegi A, Samidoust P, et al. Epigenetic profiling of MUTYH, KLF6, WNT1 and KLF4 genes in carcinogenesis and tumorigenesis of colorectal cancer. Biomedicine.

[86] Hong J, Wang X, Mei C, Zan L. Competitive regulation by transcription factors and DNA methylation in the bovine SIRT5 promoter: Roles of E2F4 and KLF6. Gene. 2019;**684**:39-46

[87] Guo H, Lin Y, Zhang H, Liu J, Zhang N, Li Y, et al. Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells. BMC Molecular Biology. 2007;**8**:110

[88] Laub F, Aldabe R, Friedrich V Jr, Ohnishi S, Yoshida T, Ramirez F. Developmental expression of mouse Krüppel-like transcription factor KLF7 suggests a potential role in neurogenesis. Developmental Biology.

[89] Matsumoto N, Laub F, Aldabe R, Zhang W, Ramirez F, Yoshida T, et al. Cloning the cDNA for a new human zinc finger protein defines a group of closely related Krüppel-like transcription factors. The Journal of Biological Chemistry. 1998;**273**:28229-28237

2001;**233**:305-318

2006;**21**:1286-1289

2019;**9**:22

[76] Pang J, Li Z, Wang G, Li N, Gao Y, Wang S. miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells. Journal of Cellular Biochemistry. 2018. DOI:

Rossert JA. Klf6 is a zinc finger protein expressed in a cell-specific manner during kidney development. Journal of the American Society of Nephrology.

[78] Laub F, Aldabe R, Ramirez F, Friedman S. Embryonic expression of Krüppel-like factor 6 in neural and non-neural tissues. Mechanisms of Development. 2001;**106**:167-170

[79] Matsumoto N, Kubo A, Liu H, Akita K, Laub F, Ramirez F, et al. Developmental regulation of yolk sac hematopoiesis by Krüppel-like factor 6.

[80] Nakamura H, dr Chiambaretta F,

Developmentally regulated expression of KLF6 in the mouse cornea and lens. Investigative Ophthalmology & Visual

Blood. 2006;**107**:1357-1365

Sugar J, Sapin V, Yue BYJT.

Science. 2004;**45**:4327-32

[81] Atkins GB, Jain MK. Role of Krüppel-like transcription factors in endothelial biology. Circulation Research. 2007;**100**:1686-1695

[82] DiFeo A, Martignetti JA, Narla G. The role of KLF6 and its splice variants in cancer therapy. Drug Resistance Updates: Reviews and

**74**

[91] Kajimura D, Dragomir C, Ramirez F, Laub F. Identification of genes regulated by transcription factor KLF7 in differentiating olfactory sensory neurons. Gene. 2007;**388**:34-42

[92] Caiazzo M, Colucci-D'Amato L, Volpicelli F, Speranza L, Petrone C, Pastore L, et al. Krüppel-like factor 7 is required for olfactory bulb dopaminergic neuron development. Experimental Cell Research. 2011;**317**:464-473

[93] Klein RH, Hu W, Kashgari G, Lin Z, Nguyen T, Doan M, et al. Characterization of enhancers and the role of the transcription factor KLF7 in regulating corneal epithelial differentiation. The Journal of Biological Chemistry. 2017;**292**:18937-18950

[94] Kanazawa A, Kawamura Y, Sekine A, Iida A, Tsunoda T, Kashiwagi A, et al. Single nucleotide polymorphisms in the gene encoding Krüppel-like factor 7 are associated with type 2 diabetes. Diabetologia. 2005;**48**:1315-1322

[95] Kawamura Y, Tanaka Y, Kawamori R, Maeda S. Overexpression of Krüppel-like factor 7 regulates adipocytokine gene expressions in human adipocytes and inhibits glucoseinduced insulin secretion in pancreatic beta-cell line. Molecular endocrinology (Baltimore, MD). 2006;**20**:844-56

[96] Zhang M, Wang C, Wu J, Ha X, Deng Y, Zhang X, et al. The effect and mechanism of KLF7 in the TLR4/ NF-κB/IL-6 inflammatory signal pathway of adipocytes. Mediators of Inflammation. 2018;**2018**:1756494

[97] Jiang Z, Yu T, Fan Z, Yang H, Lin X. Krüppel-like factor 7 is a marker of aggressive gastric cancer and poor prognosis. Cellular Physiology and Biochemistry. 2017;**43**:1090-1099

[98] van Vliet J, Turner J, Crossley M. Human Krüppel-like factor 8: A CACCC-box binding protein that associates with CtBP and represses transcription. Nucleic Acids Research. 2000;**28**:1955-1962

[99] Wang X, Zhao J. KLF8 transcription factor participates in oncogenic transformation. Oncogene. 2007;**26**:456-461

[100] Yan Q, Zhang W, Wu Y, Wu M, Zhang M, Shi X, et al. KLF8 promotes tumorigenesis, invasion and metastasis of colorectal cancer cells by transcriptional activation of FHL2. Oncotarget. 2015;**6**:25402-25417

[101] Yi X, Zai H, Long X, Wang X, Li W, Li Y. Krüppel-like factor 8 induces epithelial-to-mesenchymal transition and promotes invasion of pancreatic cancer cells through transcriptional activation of four and a half LIMonly protein 2. Oncology Letters. 2017;**14**:4883-4889

[102] Liu N, Wang Y, Zhou Y, Pang H, Zhou J, Qian P, et al. Krüppel-like factor 8 involved in hypoxia promotes the invasion and metastasis of gastric cancer via epithelial to mesenchymal transition. Oncology Reports. 2014;**32**:2397-2404

[103] Møller M, Strand SH, Mundbjerg K, Liang G, Gill I, Haldrup C, et al. Heterogeneous patterns of DNA methylation-based field effects in histologically normal prostate tissue from cancer patients. Scientific Reports. 2017;**7**:40636

[104] Martin KM, Metcalfe JC, Kemp PR. Expression of Klf9 and Klf13 in mouse development. Mechanisms of Development. 2001;**103**:149-151

[105] Morita M, Kobayashi A, Yamashita T, Shimanuki T, Nakajima O, Takahashi S, et al. Functional analysis of basic transcription element binding protein by gene targeting technology. Molecular and Cellular Biology. 2003;**23**:2489-2500

[106] Hu F, Knoedler J, Denver RJ. KrüPpel-like factor 9 enhances thyroid hormone receptor? Autoinduction in tadpole brain in vivo, increasing tissue sensitivity to thyroid hormone and accelerating metamorphosis. Frontiers in Endocrinology. DOI: 10.3389/conf. fendo.2011.03.00021

[107] Knoedler JR, Subramani A, Denver RJ. The Krüppel-like factor 9 cistrome in mouse hippocampal neurons reveals predominant transcriptional repression via proximal promoter binding. BMC Genomics. 2017;**18**:299

[108] Simmen RCM, Pabona JMP, Velarde MC, Simmons C, Rahal O, Simmen FA. The emerging role of Krüppel-like factors in endocrineresponsive cancers of female reproductive tissues. The Journal of Endocrinology. 2010;**204**:223-231

[109] Simmen RCM, Eason RR, McQuown JR, Linz AL, Kang T-J, Chatman L Jr, et al. Subfertility, uterine hypoplasia, and partial progesterone resistance in mice lacking the Krüppellike factor 9/basic transcription element-binding protein-1 (Bteb1) gene. The Journal of Biological Chemistry. 2004;**279**:29286-29294

[110] Simmen FA, Xiao R, Velarde MC, Nicholson RD, Bowman MT, Fujii-Kuriyama Y, et al. Dysregulation of intestinal crypt cell proliferation and villus cell migration in mice lacking Krüppel-like factor 9. American Journal of Physiology: Gastrointestinal and Liver Physiology. 2007;**292**:G1757-G1G69

[111] Bennett LB, Schnabel JL, Kelchen JM, Taylor KH, Guo J, Arthur GL, et al.

DNA hypermethylation accompanied by transcriptional repression in follicular lymphoma. Genes, Chromosomes and Cancer. 2009;**48**:828-841

[112] Kang L, Lai M-D. BTEB/KLF9 and its transcriptional regulation. Hereditas. 2007;**29**:515-522

[113] Subramaniam M, Hawse JR, Rajamannan NM, Ingle JN, Spelsberg TC. Functional role of KLF10 in multiple disease processes. BioFactors (Oxford, England). 2010;**36**:8-18

[114] Spittau B, Krieglstein K. Klf10 and Klf11 as mediators of TGF-beta superfamily signaling. Cell and Tissue Research. 2012;**347**:65-72

[115] Yajima S, Lammers CH, Lee SH, Hara Y, Mizuno K, Mouradian MM. Cloning and characterization of murine glial cell-derived neurotrophic factor inducible transcription factor (MGIF). The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 1997;**17**:8657-8666

[116] Subramaniam M, Harris SA, Oursler MJ, Rasmussen K, Riggs BL, Spelsberg TC. Identification of a novel TGF-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts. Nucleic Acids Research. 1995;**23**:4907-4912

[117] Liu Y, Peng W-Q, Guo Y-Y, Liu Y, Tang Q-Q, Guo L. Krüppel-like factor 10 (KLF10) is transactivated by the transcription factor C/EBPβ and involved in early 3T3-L1 preadipocyte differentiation. The Journal of Biological Chemistry. 2018;**293**(36):14012-14021

[118] Yerges LM, Klei L, Cauley JA, Roeder K, Kammerer CM, Ensrud KE, et al. Candidate gene analysis of femoral neck trabecular and cortical volumetric bone mineral density in older men. Journal of Bone and Mineral Research: The Official Journal of the American

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[125] D'Souza UM, Lammers C-H, Hwang CK, Yajima S, Mouradian MM. Developmental expression of the zinc finger transcription factor DRRF (dopamine receptor regulating factor). Mechanisms of Development.

[126] Song C-Z, Gavriilidis G, Asano H, Stamatoyannopoulos G. Functional study of transcription factor KLF11 by targeted gene inactivation. Blood Cells, Molecules, and Diseases. 2005;**34**:53-59

[127] Cook T, Gebelein B, Mesa K, Mladek A, Urrutia R. Molecular cloning and characterization of TIEG2 reveals a new subfamily of transforming growth factor-beta-inducible Sp1-like zinc finger-encoding genes involved in the regulation of cell growth. The Journal of Biological Chemistry.

[128] Cook T, Gebelein B, Belal M, Mesa K, Urrutia R. Three conserved transcriptional repressor domains are a defining feature of the TIEG subfamily of Sp1-like zinc finger proteins. The Journal of Biological Chemistry.

[129] Scohy S, Gabant P, Van Reeth T, Hertveldt V, Dreze PL, Van Vooren P, et al. Identification of KLF13 and KLF14 (SP6), novel members of the SP/XKLF transcription factor family. Genomics.

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1999;**274**:29500-29504

2000;**70**:93-101

2002;**110**:197-201

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Society for Bone and Mineral Research.

[119] Subramaniam M, Gorny G, Johnsen SA, Monroe DG, Evans GL, Fraser DG, et al. TIEG1 null mousederived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro. Molecular and Cellular Biology.

[120] Bensamoun SF, Tsubone T, Subramaniam M, Hawse JR, Boumediene E, Spelsberg TC, et al. Age-dependent changes in the mechanical properties of tail tendons in TGF-beta inducible early gene-1 knockout mice. Journal of Applied Physiology (Bethesda, MD: 1985).

[121] Rajamannan NM, Subramaniam M, Abraham TP, Vasile VC, Ackerman MJ, Monroe DG, et al. TGFbeta inducible early gene-1 (TIEG1) and cardiac hypertrophy: Discovery and

characterization of a novel signaling

[122] Cao Z, Wara AK, Icli B, Sun X, Packard RRS, Esen F, et al. Krüppellike factor KLF10 targets transforming growth factor-beta1 to regulate CD4(+) CD25(−) T cells and T regulatory cells. The Journal of Biological Chemistry.

[123] Chang VHS, Chu P-Y, Peng S-L, Mao T-L, Shan Y-S, Hsu C-F, et al. Krüppel-like factor 10 expression as a prognostic indicator for pancreatic adenocarcinoma. The American Journal

Stamatoyannopoulos G. FKLF, a novel Krüppel-like factor that activates human

embryonic and fetal β-like globin genes. Molecular and Cellular Biology.

of Pathology. 2012;**181**:423-430

pathway. Journal of Cellular Biochemistry. 2007;**100**:315-325

2009;**284**:24914-24924

[124] Asano H, Li XS,

1999;**19**:3571-3579

2010;**25**:330-338

2005;**25**:1191-1199

2006;**101**:1419-24

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

Society for Bone and Mineral Research. 2010;**25**:330-338

*Gene Expression and Phenotypic Traits*

[105] Morita M, Kobayashi A,

[106] Hu F, Knoedler J, Denver RJ. KrüPpel-like factor 9 enhances thyroid hormone receptor? Autoinduction in tadpole brain in vivo, increasing tissue sensitivity to thyroid hormone and accelerating metamorphosis. Frontiers in Endocrinology. DOI: 10.3389/conf.

[107] Knoedler JR, Subramani A, Denver RJ. The Krüppel-like factor 9 cistrome in mouse hippocampal neurons reveals predominant transcriptional repression via proximal promoter binding. BMC Genomics. 2017;**18**:299

[108] Simmen RCM, Pabona JMP, Velarde MC, Simmons C, Rahal O, Simmen FA. The emerging role of Krüppel-like factors in endocrineresponsive cancers of female reproductive tissues. The Journal of Endocrinology. 2010;**204**:223-231

[109] Simmen RCM, Eason RR, McQuown JR, Linz AL, Kang T-J, Chatman L Jr, et al. Subfertility, uterine hypoplasia, and partial progesterone resistance in mice lacking the Krüppellike factor 9/basic transcription

2004;**279**:29286-29294

Nicholson RD, Bowman MT,

element-binding protein-1 (Bteb1) gene. The Journal of Biological Chemistry.

[110] Simmen FA, Xiao R, Velarde MC,

Fujii-Kuriyama Y, et al. Dysregulation of intestinal crypt cell proliferation and villus cell migration in mice lacking Krüppel-like factor 9. American Journal of Physiology: Gastrointestinal and Liver Physiology. 2007;**292**:G1757-G1G69

[111] Bennett LB, Schnabel JL, Kelchen JM, Taylor KH, Guo J, Arthur GL, et al.

2003;**23**:2489-2500

fendo.2011.03.00021

Yamashita T, Shimanuki T, Nakajima O, Takahashi S, et al. Functional analysis of basic transcription element binding protein by gene targeting technology. Molecular and Cellular Biology.

DNA hypermethylation accompanied by transcriptional repression in follicular lymphoma. Genes, Chromosomes and

[112] Kang L, Lai M-D. BTEB/KLF9 and its transcriptional regulation. Hereditas.

[113] Subramaniam M, Hawse JR, Rajamannan NM, Ingle JN, Spelsberg TC. Functional role of KLF10 in multiple disease processes. BioFactors (Oxford, England).

[114] Spittau B, Krieglstein K. Klf10 and Klf11 as mediators of TGF-beta superfamily signaling. Cell and Tissue

[115] Yajima S, Lammers CH, Lee SH, Hara Y, Mizuno K, Mouradian MM. Cloning and characterization of murine glial cell-derived neurotrophic factor inducible transcription factor (MGIF). The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 1997;**17**:8657-8666

[116] Subramaniam M, Harris SA, Oursler MJ, Rasmussen K, Riggs BL, Spelsberg TC. Identification of a novel TGF-beta-regulated gene encoding a putative zinc finger protein in human osteoblasts. Nucleic Acids Research.

[117] Liu Y, Peng W-Q, Guo Y-Y, Liu Y, Tang Q-Q, Guo L. Krüppel-like factor 10 (KLF10) is transactivated by the transcription factor C/EBPβ and involved in early 3T3-L1 preadipocyte differentiation. The Journal of Biological Chemistry. 2018;**293**(36):14012-14021

[118] Yerges LM, Klei L, Cauley JA, Roeder K, Kammerer CM, Ensrud KE, et al. Candidate gene analysis of femoral neck trabecular and cortical volumetric bone mineral density in older men. Journal of Bone and Mineral Research: The Official Journal of the American

1995;**23**:4907-4912

Research. 2012;**347**:65-72

Cancer. 2009;**48**:828-841

2007;**29**:515-522

2010;**36**:8-18

**76**

[119] Subramaniam M, Gorny G, Johnsen SA, Monroe DG, Evans GL, Fraser DG, et al. TIEG1 null mousederived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro. Molecular and Cellular Biology. 2005;**25**:1191-1199

[120] Bensamoun SF, Tsubone T, Subramaniam M, Hawse JR, Boumediene E, Spelsberg TC, et al. Age-dependent changes in the mechanical properties of tail tendons in TGF-beta inducible early gene-1 knockout mice. Journal of Applied Physiology (Bethesda, MD: 1985). 2006;**101**:1419-24

[121] Rajamannan NM, Subramaniam M, Abraham TP, Vasile VC, Ackerman MJ, Monroe DG, et al. TGFbeta inducible early gene-1 (TIEG1) and cardiac hypertrophy: Discovery and characterization of a novel signaling pathway. Journal of Cellular Biochemistry. 2007;**100**:315-325

[122] Cao Z, Wara AK, Icli B, Sun X, Packard RRS, Esen F, et al. Krüppellike factor KLF10 targets transforming growth factor-beta1 to regulate CD4(+) CD25(−) T cells and T regulatory cells. The Journal of Biological Chemistry. 2009;**284**:24914-24924

[123] Chang VHS, Chu P-Y, Peng S-L, Mao T-L, Shan Y-S, Hsu C-F, et al. Krüppel-like factor 10 expression as a prognostic indicator for pancreatic adenocarcinoma. The American Journal of Pathology. 2012;**181**:423-430

[124] Asano H, Li XS,

Stamatoyannopoulos G. FKLF, a novel Krüppel-like factor that activates human embryonic and fetal β-like globin genes. Molecular and Cellular Biology. 1999;**19**:3571-3579

[125] D'Souza UM, Lammers C-H, Hwang CK, Yajima S, Mouradian MM. Developmental expression of the zinc finger transcription factor DRRF (dopamine receptor regulating factor). Mechanisms of Development. 2002;**110**:197-201

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[127] Cook T, Gebelein B, Mesa K, Mladek A, Urrutia R. Molecular cloning and characterization of TIEG2 reveals a new subfamily of transforming growth factor-beta-inducible Sp1-like zinc finger-encoding genes involved in the regulation of cell growth. The Journal of Biological Chemistry. 1998;**273**:25929-25936

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[133] Neve B, Fernandez-Zapico ME, Ashkenazi-Katalan V, Dina C, Hamid YH, Joly E, et al. Role of transcription factor KLF11 and its diabetesassociated gene variants in pancreatic beta cell function. Proceedings of the National Academy of Sciences of the United States of America. 2005;**102**:4807-4812

[134] Gutiérrez-Aguilar R, Froguel P, Hamid YH, Benmezroua Y, Jørgensen T, Borch-Johnsen K, et al. Genetic analysis of Krüppel-like zinc finger 11 variants in 5864 Danish individuals: Potential effect on insulin resistance and modified signal transducer and activator of transcription-3 binding by promoter variant -1659G>C. The Journal of Clinical Endocrinology and Metabolism. 2008;**93**:3128-3135

[135] Fernandez-Zapico ME, van Velkinburgh JC, Gutiérrez-Aguilar R, Neve B, Froguel P, Urrutia R, et al. MODY7 gene, KLF11, is a novel p300-dependent regulator of Pdx-1 (MODY4) transcription in pancreatic islet beta cells. The Journal of Biological Chemistry. 2009;**284**:36482-36490

[136] Ellenrieder V, Buck A, Harth A, Jungert K, Buchholz M, Adler G, et al. KLF11 mediates a critical mechanism in TGF-beta signaling that is inactivated by Erk-MAPK in pancreatic cancer cells. Gastroenterology. 2004;**127**:607-620

[137] Fernandez-Zapico ME, Mladek A, Ellenrieder V, Folch-Puy E, Miller L, Urrutia R. An mSin3A interaction domain links the transcriptional activity of KLF11 with its role in growth regulation. The EMBO Journal. 2003;**22**:4748-4758

[138] Potapova A, Hasemeier B, Römermann D, Metzig K, Göhring G, Schlegelberger B, et al. Epigenetic inactivation of tumour suppressor gene KLF11 in myelodysplastic syndromes\*. European Journal of Haematology. 2010;**84**:298-303

[139] Wang G, Li X, Tian W, Wang Y, Wu D, Sun Z, et al. Promoter DNA methylation is associated with KLF11 expression in epithelial ovarian cancer. Genes, Chromosomes and Cancer. 2015;**54**:453-462

[140] Imhof A, Schuierer M, Werner O, Moser M, Roth C, Bauer R, et al. Transcriptional regulation of the AP-2alpha promoter by BTEB-1 and AP-2rep, a novel wt-1/egr-related zinc finger repressor. Molecular and Cellular Biology. 1999;**19**:194-204

[141] Godin-Heymann N, Brabetz S, Murillo MM, Saponaro M, Santos CR, Lobley A, et al. Tumour-suppression function of KLF12 through regulation of anoikis. Oncogene. 2016;**35**:3324-3334

[142] Nakamura Y, Migita T, Hosoda F, Okada N, Gotoh M, Arai Y, et al. Krüppel-like factor 12 plays a significant role in poorly differentiated gastric cancer progression. International Journal of Cancer. 2009;**125**:1859-1867

[143] Rozenblum E, Vahteristo P, Sandberg T, Bergthorsson JT, Syrjakoski K, Weaver D, et al. A genomic map of a 6-Mb region at 13q21 q22 implicated in cancer development: Identification and characterization of candidate genes. Human Genetics. 2002;**110**:111-121

[144] Zhang J-S, Moncrieffe MC, Kaczynski J, Ellenrieder V, Prendergast FG, Urrutia R. A conserved α-helical motif mediates the interaction of Sp1-like transcriptional repressors

**79**

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13 regulates CCL5 transcription. The Journal of Immunology.

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[153] Darwich R, Li W, Yamak A, Komati H, Andelfinger G, Sun K, et al. KLF13 is a genetic modifier of the Holt-Oram syndrome gene TBX5. Human Molecular Genetics. 2017;**26**:942-954

[154] Wu R, Yun Q, Zhang J, Bao J. Downregulation of KLF13 through DNMT1-mediated hypermethylation promotes glioma cell proliferation and invasion. OncoTargets and Therapy.

[155] Koh I-U, Lee H-J, Hwang J-Y, Choi N-H, Lee S. Obesity-related CpG methylation (cg07814318) of Krüppel-like factor-13 (KLF13) gene with childhood obesity and its cismethylation quantitative loci. Scientific

[156] Parker-Katiraee L, Carson AR, Yamada T, Arnaud P, Feil R, Abu-Amero SN, et al. Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution. PLoS Genetics. 2007;**3**:e65

[157] Stacey SN, Sulem P, Masson G, Gudjonsson SA, Thorleifsson G, Jakobsdottir M, et al. New common variants affecting susceptibility to basal cell carcinoma. Nature Genetics.

2019;**12**:1509-1520

Reports. 2017;**7**:45368

2009;**41**:909-914

2011;**43**:561-564

[158] Small KS, Hedman ÅK,

Grundberg E, Nica AC, Thorleifsson G, Kong A, et al. Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes. Nature Genetics.

2007;**178**:7081-7087

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with the corepressor mSin3A. Molecular and Cellular Biology. 2001;**21**:5041-5049

Sasaki S, Uchida S. Postnatal expression

transporter promoter. Biochemical and Biophysical Research Communications.

[146] Yuan J, Kang J, Yang M. Long non-coding RNA ELF3-antisense RNA 1 promotes osteosarcoma cell proliferation by upregulating Krüppellike factor 12 potentially via methylation of the microRNA-205 gene. Oncology

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2008;**283**:11897-11904

[147] Gordon AR, Outram SV, Keramatipour M, Goddard CA, Colledge WH, Metcalfe JC, et al. Splenomegaly and modified erythropoiesis in KLF13−/− mice. The Journal of Biological Chemistry.

[148] Zhou M, McPherson L, Feng D, Song A, Dong C, Lyu SC, et al. Krüppellike transcription factor 13 regulates T lymphocyte survival in vivo. Journal of Immunology. 2007;**178**:5496-5504

[150] Song A, Patel A, Thamatrakoln K, Liu C, Feng D, Clayberger C, et al. Functional domains and DNAbinding sequences of RFLAT-1/ KLF13, a Krüppel-like transcription factor of activated T lymphocytes. The Journal of Biological Chemistry.

[151] Huang B, Ahn Y-T, McPherson L, Clayberger C, Krensky AM. Interaction of PRP4 with Krüppel-like factor

[149] Outram SV, Gordon AR, Hager-Theodorides AL, Metcalfe J, Crompton T, Kemp P. KLF13 influences multiple stages of both B and T cell development. Cell Cycle (Georgetown,

Tex). 2008;**7**:2047-55

2002;**277**:30055-30065

[145] Suda S, Rai T, Sohara E,

2006;**344**:246-252

of KLF12 in the inner medullary collecting ducts of kidney and its trans-activation of UT-A1 urea

*Transcriptional and Epigenetic Regulation of Krüppel-Like Transcription Factors DOI: http://dx.doi.org/10.5772/intechopen.91652*

with the corepressor mSin3A. Molecular and Cellular Biology. 2001;**21**:5041-5049

*Gene Expression and Phenotypic Traits*

[132] Lomberk G, Grzenda A, Mathison A, Escande C, Zhang J-S, Calvo E, et al. Krüppel-like factor 11 regulates the expression of metabolic genes via an evolutionarily conserved protein-interaction domain functionally disrupted in maturity onset diabetes of the young. The Journal of Biological Chemistry. 2013;**288**:17745-17758

its role in growth regulation. The EMBO

Journal. 2003;**22**:4748-4758

2010;**84**:298-303

2015;**54**:453-462

Biology. 1999;**19**:194-204

[142] Nakamura Y, Migita T,

[138] Potapova A, Hasemeier B, Römermann D, Metzig K, Göhring G, Schlegelberger B, et al. Epigenetic inactivation of tumour suppressor gene KLF11 in myelodysplastic syndromes\*. European Journal of Haematology.

[139] Wang G, Li X, Tian W, Wang Y, Wu D, Sun Z, et al. Promoter DNA methylation is associated with KLF11 expression in epithelial ovarian cancer. Genes, Chromosomes and Cancer.

[140] Imhof A, Schuierer M, Werner O, Moser M, Roth C, Bauer R, et al. Transcriptional regulation of the AP-2alpha promoter by BTEB-1 and AP-2rep, a novel wt-1/egr-related zinc finger repressor. Molecular and Cellular

[141] Godin-Heymann N, Brabetz S, Murillo MM, Saponaro M, Santos CR, Lobley A, et al. Tumour-suppression function of KLF12 through regulation of anoikis. Oncogene. 2016;**35**:3324-3334

Hosoda F, Okada N, Gotoh M, Arai Y, et al. Krüppel-like factor 12 plays a significant role in poorly differentiated gastric cancer progression. International Journal of Cancer. 2009;**125**:1859-1867

[143] Rozenblum E, Vahteristo P, Sandberg T, Bergthorsson JT, Syrjakoski K, Weaver D, et al. A

[144] Zhang J-S, Moncrieffe MC, Kaczynski J, Ellenrieder V,

2002;**110**:111-121

genomic map of a 6-Mb region at 13q21 q22 implicated in cancer development: Identification and characterization of candidate genes. Human Genetics.

Prendergast FG, Urrutia R. A conserved α-helical motif mediates the interaction of Sp1-like transcriptional repressors

[133] Neve B, Fernandez-Zapico ME, Ashkenazi-Katalan V, Dina C, Hamid YH, Joly E, et al. Role of transcription factor KLF11 and its diabetes-

associated gene variants in pancreatic beta cell function. Proceedings of the National Academy of Sciences of the United States of America.

[134] Gutiérrez-Aguilar R, Froguel P, Hamid YH, Benmezroua Y, Jørgensen T, Borch-Johnsen K, et al. Genetic analysis of Krüppel-like zinc finger 11 variants in 5864 Danish individuals: Potential effect on insulin resistance and modified signal transducer and activator of transcription-3 binding by promoter variant -1659G>C. The Journal of Clinical Endocrinology and Metabolism.

2005;**102**:4807-4812

2008;**93**:3128-3135

[135] Fernandez-Zapico ME, van Velkinburgh JC, Gutiérrez-Aguilar R, Neve B, Froguel P, Urrutia R, et al. MODY7 gene, KLF11, is a novel p300-dependent regulator of Pdx-1 (MODY4) transcription in pancreatic islet beta cells. The Journal of Biological Chemistry. 2009;**284**:36482-36490

[136] Ellenrieder V, Buck A, Harth A, Jungert K, Buchholz M, Adler G, et al. KLF11 mediates a critical mechanism in TGF-beta signaling that is inactivated by Erk-MAPK in pancreatic cancer cells. Gastroenterology. 2004;**127**:607-620

Folch-Puy E, Miller L, Urrutia R. An mSin3A interaction domain links the transcriptional activity of KLF11 with

[137] Fernandez-Zapico ME, Mladek A, Ellenrieder V,

**78**

[145] Suda S, Rai T, Sohara E, Sasaki S, Uchida S. Postnatal expression of KLF12 in the inner medullary collecting ducts of kidney and its trans-activation of UT-A1 urea transporter promoter. Biochemical and Biophysical Research Communications. 2006;**344**:246-252

[146] Yuan J, Kang J, Yang M. Long non-coding RNA ELF3-antisense RNA 1 promotes osteosarcoma cell proliferation by upregulating Krüppellike factor 12 potentially via methylation of the microRNA-205 gene. Oncology Letters. 2020;**19**:2475-2480

[147] Gordon AR, Outram SV, Keramatipour M, Goddard CA, Colledge WH, Metcalfe JC, et al. Splenomegaly and modified erythropoiesis in KLF13−/− mice. The Journal of Biological Chemistry. 2008;**283**:11897-11904

[148] Zhou M, McPherson L, Feng D, Song A, Dong C, Lyu SC, et al. Krüppellike transcription factor 13 regulates T lymphocyte survival in vivo. Journal of Immunology. 2007;**178**:5496-5504

[149] Outram SV, Gordon AR, Hager-Theodorides AL, Metcalfe J, Crompton T, Kemp P. KLF13 influences multiple stages of both B and T cell development. Cell Cycle (Georgetown, Tex). 2008;**7**:2047-55

[150] Song A, Patel A, Thamatrakoln K, Liu C, Feng D, Clayberger C, et al. Functional domains and DNAbinding sequences of RFLAT-1/ KLF13, a Krüppel-like transcription factor of activated T lymphocytes. The Journal of Biological Chemistry. 2002;**277**:30055-30065

[151] Huang B, Ahn Y-T, McPherson L, Clayberger C, Krensky AM. Interaction of PRP4 with Krüppel-like factor

13 regulates CCL5 transcription. The Journal of Immunology. 2007;**178**:7081-7087

[152] Lavallée G, Andelfinger G, Nadeau M, Lefebvre C, Nemer G, Horb ME, et al. The Krüppel-like transcription factor KLF13 is a novel regulator of heart development. The EMBO Journal. 2006;**25**:5201-5213

[153] Darwich R, Li W, Yamak A, Komati H, Andelfinger G, Sun K, et al. KLF13 is a genetic modifier of the Holt-Oram syndrome gene TBX5. Human Molecular Genetics. 2017;**26**:942-954

[154] Wu R, Yun Q, Zhang J, Bao J. Downregulation of KLF13 through DNMT1-mediated hypermethylation promotes glioma cell proliferation and invasion. OncoTargets and Therapy. 2019;**12**:1509-1520

[155] Koh I-U, Lee H-J, Hwang J-Y, Choi N-H, Lee S. Obesity-related CpG methylation (cg07814318) of Krüppel-like factor-13 (KLF13) gene with childhood obesity and its cismethylation quantitative loci. Scientific Reports. 2017;**7**:45368

[156] Parker-Katiraee L, Carson AR, Yamada T, Arnaud P, Feil R, Abu-Amero SN, et al. Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution. PLoS Genetics. 2007;**3**:e65

[157] Stacey SN, Sulem P, Masson G, Gudjonsson SA, Thorleifsson G, Jakobsdottir M, et al. New common variants affecting susceptibility to basal cell carcinoma. Nature Genetics. 2009;**41**:909-914

[158] Small KS, Hedman ÅK, Grundberg E, Nica AC, Thorleifsson G, Kong A, et al. Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes. Nature Genetics. 2011;**43**:561-564

[159] Guo Y, Fan Y, Zhang J, Lomberk GA, Zhou Z, Sun L, et al. Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production. The Journal of Clinical Investigation. 2015;**125**:3819-3830

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[161] Hu W, Lu H, Zhang J, Fan Y, Chang Z, Liang W, et al. Krüppel-like factor 14, a coronary artery disease associated transcription factor, inhibits endothelial inflammation via NF-kappaB signaling pathway. Atherosclerosis. 2018;**278**:39-48

[162] de Assuncao TM, Lomberk G, Cao S, Yaqoob U, Mathison A, Simonetto DA, et al. New role for Krüppel-like factor 14 as a transcriptional activator involved in the generation of signaling lipids. The Journal of Biological Chemistry. 2014;**289**:15798-15809

[163] Uchida S, Tanaka Y, Ito H, Saitoh-Ohara F, Inazawa J, Yokoyama KK, et al. Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Krüppel-like factor, a novel zinc finger repressor. Molecular and Cellular Biology. 2000;**20**:7319-7331

[164] Wang B, Haldar SM, Lu Y, Ibrahim OA, Fisch S, Gray S, et al. The Krüppel-like factor KLF15 inhibits connective tissue growth factor (CTGF) expression in cardiac fibroblasts. Journal of Molecular and Cellular Cardiology. 2008;**45**:193-197

[165] Fisch S, Gray S, Heymans S, Haldar SM, Wang B, Pfister O, et al. Krüppel-like factor 15 is a regulator of cardiomyocyte hypertrophy. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**:7074-7079

[166] Pepin ME, Ha C-M, Crossman DK, Litovsky SH, Varambally S, Barchue JP, et al. Genome-wide DNA methylation encodes cardiac transcriptional reprogramming in human ischemic heart failure. Laboratory Investigation: A Journal of Technical Methods and Pathology. 2019;**99**:371-86

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[168] Haldar SM, Jeyaraj D, Anand P, Zhu H, Lu Y, Prosdocimo DA, et al. Krüppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation. Proceedings of the National Academy of Sciences. 2012;**109**:6739-6744

[169] Takeuchi Y, Yahagi N, Aita Y, Murayama Y, Sawada Y, Piao X, et al. KLF15 enables rapid switching between lipogenesis and gluconeogenesis during fasting. Cell Reports. 2016;**16**:2373-2386

[170] Jeyaraj D, Scheer FAJL, Ripperger JA, Haldar SM, Lu Y, Prosdocimo DA, et al. Klf15 orchestrates circadian nitrogen homeostasis. Cell Metabolism. 2012;**15**:311-323

[171] Wang J, Galvao J, Beach KM, Luo W, Urrutia RA, Goldberg JL, et al. Novel roles and mechanism for Krüppellike factor 16 (KLF16) regulation of neurite outgrowth and ephrin receptor A5 (EphA5) expression in retinal ganglion cells. The Journal of Biological Chemistry. 2016;**291**:18084-18095

[172] Daftary GS, Lomberk GA, Buttar NS, Allen TW, Grzenda A, Zhang J, et al. Detailed structuralfunctional analysis of the Krüppellike factor 16 (KLF16) transcription factor reveals novel mechanisms

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[177] Ali A, Bhatti MZ, Shah AS, Duong HQ, Alkreathy HM, Mohammad SF, et al. Tumor-suppressive p53 signaling empowers metastatic inhibitor KLF17 dependent transcription to overcome tumorigenesis in non-small cell lung cancer. The Journal of Biological Chemistry. 2015;**290**:21336-21351

[178] Cai X-D, Che L, Lin J-X, Huang S, Li J, Liu X-Y, et al. Krüppel-like factor 17 inhibits urokinase plasminogen activator gene expression to suppress cell invasion through the Src/p38/ MAPK signaling pathway in human lung adenocarcionma. Oncotarget.

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Its low expression is involved in cancer metastasis. Tumour Biology.

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Genomics. 2006;**87**:474-482

[174] Yan W, Burns KH, Ma L,

for silencing Sp/KLF sites involved in metabolism and endocrinology. The Journal of Biological Chemistry.

2012;**287**:7010-7025

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for silencing Sp/KLF sites involved in metabolism and endocrinology. The Journal of Biological Chemistry. 2012;**287**:7010-7025

*Gene Expression and Phenotypic Traits*

Lomberk GA, Zhou Z, Sun L, et al. Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production. The Journal of Clinical Investigation. 2015;**125**:3819-3830

of Sciences of the United States of America. 2007;**104**:7074-7079

Pathology. 2019;**99**:371-86

2007;**5**:305-312

2012;**109**:6739-6744

[167] Gray S, Wang B, Orihuela Y, Hong E-G, Fisch S, Haldar S, et al. Regulation of gluconeogenesis by Krüppel-like factor 15. Cell Metabolism.

[168] Haldar SM, Jeyaraj D, Anand P, Zhu H, Lu Y, Prosdocimo DA, et al. Krüppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation. Proceedings of the National Academy of Sciences.

[169] Takeuchi Y, Yahagi N, Aita Y, Murayama Y, Sawada Y, Piao X, et al. KLF15 enables rapid switching between lipogenesis and gluconeogenesis during fasting. Cell Reports. 2016;**16**:2373-2386

[170] Jeyaraj D, Scheer FAJL, Ripperger JA, Haldar SM, Lu Y,

Metabolism. 2012;**15**:311-323

[171] Wang J, Galvao J, Beach KM, Luo W, Urrutia RA, Goldberg JL, et al. Novel roles and mechanism for Krüppellike factor 16 (KLF16) regulation of neurite outgrowth and ephrin receptor A5 (EphA5) expression in retinal ganglion cells. The Journal of Biological Chemistry. 2016;**291**:18084-18095

[172] Daftary GS, Lomberk GA, Buttar NS, Allen TW, Grzenda A, Zhang J, et al. Detailed structuralfunctional analysis of the Krüppellike factor 16 (KLF16) transcription factor reveals novel mechanisms

Prosdocimo DA, et al. Klf15 orchestrates circadian nitrogen homeostasis. Cell

[166] Pepin ME, Ha C-M, Crossman DK, Litovsky SH, Varambally S, Barchue JP, et al. Genome-wide DNA methylation encodes cardiac transcriptional reprogramming in human ischemic heart failure. Laboratory Investigation: A Journal of Technical Methods and

[159] Guo Y, Fan Y, Zhang J,

[160] Wei X, Yang R, Wang C, Jian X, Li L, Liu H, et al. A novel role for the Krüppel-like factor 14 on macrophage inflammatory response and atherosclerosis development. Cardiovascular Pathology. 2017;**27**:1-8

[161] Hu W, Lu H, Zhang J, Fan Y, Chang Z, Liang W, et al. Krüppel-like factor 14, a coronary artery disease associated transcription factor, inhibits endothelial inflammation via NF-kappaB signaling pathway. Atherosclerosis. 2018;**278**:39-48

[162] de Assuncao TM, Lomberk G, Cao S, Yaqoob U, Mathison A, Simonetto DA, et al. New role for Krüppel-like factor 14 as a transcriptional activator involved in the generation of signaling lipids. The Journal of Biological Chemistry.

2014;**289**:15798-15809

[163] Uchida S, Tanaka Y, Ito H,

[164] Wang B, Haldar SM, Lu Y, Ibrahim OA, Fisch S, Gray S, et al. The Krüppel-like factor KLF15 inhibits connective tissue growth factor (CTGF) expression in cardiac fibroblasts. Journal of Molecular and Cellular Cardiology.

[165] Fisch S, Gray S, Heymans S, Haldar SM, Wang B, Pfister O, et al. Krüppel-like factor 15 is a regulator of cardiomyocyte hypertrophy. Proceedings of the National Academy

2008;**45**:193-197

Saitoh-Ohara F, Inazawa J, Yokoyama KK, et al. Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Krüppel-like factor, a novel zinc finger repressor. Molecular and Cellular Biology. 2000;**20**:7319-7331

**80**

[173] van Vliet J, Crofts LA, Quinlan KG, Czolij R, Perkins AC, Crossley M. Human KLF17 is a new member of the Sp/ KLF family of transcription factors. Genomics. 2006;**87**:474-482

[174] Yan W, Burns KH, Ma L, Matzuk MM. Identification of Zfp393, a germ cell-specific gene encoding a novel zinc finger protein. Mechanisms of Development. 2002;**118**:233-239

[175] Zhou S, Tang X, Tang F. Krüppellike factor 17, a novel tumor suppressor: Its low expression is involved in cancer metastasis. Tumour Biology. 2016;**37**:1505-1513

[176] Gumireddy K, Li A, Gimotty PA, Klein-Szanto AJ, Showe LC, Katsaros D, et al. KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer. Nature Cell Biology. 2009;**11**:1297-1304

[177] Ali A, Bhatti MZ, Shah AS, Duong HQ, Alkreathy HM, Mohammad SF, et al. Tumor-suppressive p53 signaling empowers metastatic inhibitor KLF17 dependent transcription to overcome tumorigenesis in non-small cell lung cancer. The Journal of Biological Chemistry. 2015;**290**:21336-21351

[178] Cai X-D, Che L, Lin J-X, Huang S, Li J, Liu X-Y, et al. Krüppel-like factor 17 inhibits urokinase plasminogen activator gene expression to suppress cell invasion through the Src/p38/ MAPK signaling pathway in human lung adenocarcionma. Oncotarget. 2017;**8**:38743-38754

[179] Ali A, Zhang P, Liangfang Y, Wenshe S, Wang H, Lin X, et al. KLF17 empowers TGF-beta/Smad signaling by targeting Smad3-dependent pathway to suppress tumor growth and metastasis

during cancer progression. Cell Death & Disease. 2015;**6**:e1681

[180] Dong P, Kaneuchi M, Xiong Y, Cao L, Cai M, Liu X, et al. Identification of KLF17 as a novel epithelial to mesenchymal transition inducer via direct activation of TWIST1 in endometrioid endometrial cancer. Carcinogenesis. 2014;**35**:760-768

[181] Ali A, Ielciu I, Alkreathy HM, Khan AA. KLF17 attenuates estrogen receptor alpha-mediated signaling by impeding ERalpha function on chromatin and determines response to endocrine therapy. Biochimica et Biophysica Acta. 1859;**2016**:883-895

[182] Pei J, Grishin NV. A new family of predicted Krüppel-like factor genes and pseudogenes in placental mammals. PLoS One. 2013;**8**:e81109

[183] Shankman LS, Gomez D, Cherepanova OA, Salmon M, Alencar GF, Haskins RM, et al. KLF4 dependent phenotypic modulation of SMCs plays a key role in atherosclerotic plaque pathogenesis. Nature Medicine. 2015;**21**:628-637

[184] Wirka RC, Wagh D, Paik DT, Pjanic M, Nguyen T, Miller CL, et al. Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis. Nature Medicine. 2019;**25**:1280-1289

[185] Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. ATAC-seq: A method for assaying chromatin accessibility genome-wide. Current Protocols in Molecular Biology. 2015;**109**:21.9.1-9

[186] Shashikant T, Ettensohn CA. Genome-wide analysis of chromatin accessibility using ATAC-seq. Methods in Cell Biology. 2019;**151**:219-235

[187] Liu J, Jia G. Methylation modifications in eukaryotic messenger RNA. Journal of Genetics and Genomics. 2014;**41**:21-33

[188] Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, et al. Topology of the human and mouse m6 A RNA methylomes revealed by m6 A-seq. Nature. 2012;**485**:201-206

Chapter 6

Abstract

on existing knowledge.

1. Introduction

83

immune regulation, cancer

Circular RNAs and Its Biological

Functions in Health and Disease

Circular RNAs (circRNAs) belong to the family of long noncoding RNAs (lncRNA) that, unlike linear RNAs, are characterized by a covalently closed circular RNA structure lacking 5<sup>0</sup> cap and 3<sup>0</sup> poly-adenylated tails. circRNAs have a role in epigenetic regulation of downstream targets. circRNAs play a crucial role in regulating gene and protein expressions by acting as a microRNA (miRNA) sponge and RNA binding protein (RBP) sponge and interact with proteins to affect cell behavior. circRNA expression profiles differ between physiological and pathological states. Moreover, the expression patterns of circRNAs exhibit differences in a tissue-specific manner. Although investigations on circRNAs have been exploding nowadays, yet only a limited number of circRNAs are identified. Furthermore, further researches are needed to shed light on their functions and targets. Therefore, circRNAs are becoming vital as potential biomarkers that may be used for the

diagnosis and treatment of diseases. In this chapter, we review the current

Keywords: circular RNAs, cardiovascular diseases, neurological disorders,

advancement of cirRNAs with regard to their biogenesis, biological functions, gene regulatory mechanisms, and implications in human diseases and summarize the recent studies on circRNAs as potential diagnostic and prognostic biomarkers based

The ENCyclopedia Of DNA Elements (ENCODE) project reported that noncoding RNAs (ncRNAs) unexpectedly consist of more than 70% of the human genome [1]. After the data released by ENCODE project consortium, numerous studies have focused on the identification and function of these transcripts [2]. ncRNAs can be a group based on their different characteristic features [3]. Long noncoding RNAs (lncRNAs) are subclass of ncRNAs that have been recently proved to have a role in physiological and pathological processes [4]. lncRNAs are >200 nucleotides long, divergent class of RNA transcripts that coordinate expression of protein-coding genes. Yet, they have a lack of ability to encode proteins [5]. Circular RNAs (circRNAs) are a special subtype of lncRNAs [6]. circRNAs are characterized by a single-stranded covalently closed loop structure with neither a 5<sup>0</sup> cap nor a 3<sup>0</sup> poly (A) tail [7]. Due to their circular structure, circRNAs are more stable than the linear mRNA counterpart and not susceptible to RNA exonuclease cleavage [6, 7]. The presence of circRNA was first demonstrated in the cytoplasm of eukaryotic cells in

Atiye Seda Yar Saglam, Ebru Alp and Hacer Ilke Onen

[189] Fu Y, Dominissini D, Rechavi G, He C. Gene expression regulation mediated through reversible m6 A RNA methylation. Nature Reviews. Genetics. 2014;**15**:293

[190] Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, Jaffrey SR. Single-nucleotide-resolution mapping of m6 A and m6 Am throughout the transcriptome. Nature Methods. 2015;**12**:767-772

[191] Niu Y, Zhao X, Wu Y-S, Li M-M, Wang X-J, Yang Y-G. N6-methyladenosine (m6 A) in RNA: An old modification with A novel epigenetic function. Genomics, Proteomics & Bioinformatics. 2013;**11**:8-17

[192] Zhang Z, Chen L-Q, Zhao Y-L, Yang C-G, Roundtree IA, Zhang Z, et al. Single-base mapping of m6 A by an antibody-independent method. Science Advances. 2019;**5**:eaax0250

## Chapter 6

*Gene Expression and Phenotypic Traits*

RNA. Journal of Genetics and Genomics. 2014;**41**:21-33

[188] Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, et al. Topology of the human and mouse m6

RNA methylomes revealed by m6

[189] Fu Y, Dominissini D, Rechavi G, He C. Gene expression regulation mediated through reversible m6

methylation. Nature Reviews. Genetics.

[190] Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, Jaffrey SR. Single-nucleotide-resolution

A and m6

the transcriptome. Nature Methods.

[191] Niu Y, Zhao X, Wu Y-S, Li M-M, Wang X-J, Yang Y-G. N6-methyl-

modification with A novel epigenetic function. Genomics, Proteomics & Bioinformatics. 2013;**11**:8-17

[192] Zhang Z, Chen L-Q, Zhao Y-L, Yang C-G, Roundtree IA, Zhang Z, et al. Single-base mapping of m6

Advances. 2019;**5**:eaax0250

antibody-independent method. Science

A) in RNA: An old

Nature. 2012;**485**:201-206

2014;**15**:293

mapping of m6

2015;**12**:767-772

adenosine (m6

A

A-seq.

A RNA

Am throughout

A by an

**82**

## Circular RNAs and Its Biological Functions in Health and Disease

Atiye Seda Yar Saglam, Ebru Alp and Hacer Ilke Onen

## Abstract

Circular RNAs (circRNAs) belong to the family of long noncoding RNAs (lncRNA) that, unlike linear RNAs, are characterized by a covalently closed circular RNA structure lacking 5<sup>0</sup> cap and 3<sup>0</sup> poly-adenylated tails. circRNAs have a role in epigenetic regulation of downstream targets. circRNAs play a crucial role in regulating gene and protein expressions by acting as a microRNA (miRNA) sponge and RNA binding protein (RBP) sponge and interact with proteins to affect cell behavior. circRNA expression profiles differ between physiological and pathological states. Moreover, the expression patterns of circRNAs exhibit differences in a tissue-specific manner. Although investigations on circRNAs have been exploding nowadays, yet only a limited number of circRNAs are identified. Furthermore, further researches are needed to shed light on their functions and targets. Therefore, circRNAs are becoming vital as potential biomarkers that may be used for the diagnosis and treatment of diseases. In this chapter, we review the current advancement of cirRNAs with regard to their biogenesis, biological functions, gene regulatory mechanisms, and implications in human diseases and summarize the recent studies on circRNAs as potential diagnostic and prognostic biomarkers based on existing knowledge.

Keywords: circular RNAs, cardiovascular diseases, neurological disorders, immune regulation, cancer

## 1. Introduction

The ENCyclopedia Of DNA Elements (ENCODE) project reported that noncoding RNAs (ncRNAs) unexpectedly consist of more than 70% of the human genome [1]. After the data released by ENCODE project consortium, numerous studies have focused on the identification and function of these transcripts [2]. ncRNAs can be a group based on their different characteristic features [3]. Long noncoding RNAs (lncRNAs) are subclass of ncRNAs that have been recently proved to have a role in physiological and pathological processes [4]. lncRNAs are >200 nucleotides long, divergent class of RNA transcripts that coordinate expression of protein-coding genes. Yet, they have a lack of ability to encode proteins [5]. Circular RNAs (circRNAs) are a special subtype of lncRNAs [6]. circRNAs are characterized by a single-stranded covalently closed loop structure with neither a 5<sup>0</sup> cap nor a 3<sup>0</sup> poly (A) tail [7]. Due to their circular structure, circRNAs are more stable than the linear mRNA counterpart and not susceptible to RNA exonuclease cleavage [6, 7]. The presence of circRNA was first demonstrated in the cytoplasm of eukaryotic cells in

1979 [8]. It was thought that circRNAs were a by-product formed during splicing mechanism in the first year [9]. Numerous circRNAs have been predicted with the technical developments in high-throughput RNA sequencing (RNA-seq) and methodological innovations in bioinformatics. The presence and function of the predicted circRNAs in different tissues and cell lines are widely studied nowadays. After the determination of their role in the control of gene expression, circRNAs have gained great attention by researchers in this field. In this chapter, we will focus on circRNAs and their biological functions in health and disease.

## 2. Biogenesis of circRNAs

According to the gene structure they contain, circRNAs can be divided into three groups: exonic circRNA (ecircRNA), circRNAs from introns (ciRNAs), and exonintron circRNA (elciRNA) [10]. To date, many studies have shown that circRNAs mainly emerged during pre-mRNA splicing process of protein coding genes. Unlike canonical mRNA splicing mechanism, down-stream donor splice site is covalently joined with an upstream acceptor splice sites during circRNA formation. This splicing mechanism is called "back-splicing" [7]. The back-splicing mechanism is depicted in Figure 1. circRNAs can also be formed through the hybridization of complementary inverted sequences (such as human Alu repeats) in introns [10]. If Alu sequences are located in different introns of the same gene, this leads to

> generate circRNAs, which contains multiple exons [7]. Exon circularization is facilitated by cis-acting inverted repeat sequences as well as by trans-acting RNA-binding proteins (RBPs), which interact with unique sequences in introns [11, 12]. ecircRNA or elciRNA formation is promoted by either cis-acting elements or trans-acting factors as in Figure 2A. ecircRNAs can also be formed from elciRNAs by removal of intronic sequences [13]. Apart from the other circRNAs, ecircRNAs are transported into cytoplasm [14]. In human cells, the existence of ciRNAs is demonstrated by Zhang et al. [15]. ciRNAs are generated through a lariat-derived mechanism relying on mainly a consensus motif containing a 7-nt GU-rich element adjacent to the 5<sup>0</sup> splice site and an 11-nt C-rich element adjacent to the branchpoint site. After cleavage of 3<sup>0</sup> end, stable ciRNA is produced [15].

> Possible model for the formation of structurally different circRNA. (AI) Intron-driven circularization: circRNAs may form by hybridization of the introns with inverted repeats or Alu sequence. (AII) RBP-driven circularization: RBPs bind to specific sequence in introns that bring the exons close together and trigger the circularization. At the end of these two ways, elciRNAs or ecircRNAs are generated. Only ecircRNAs can be transported to cytoplasm. (B) Formation of ciRNA. The ciRNAs are generated from intron lariat by splicing reaction. Purple arrow represents 7-nt GU-rich element. Yellow arrow represents 11-nt C-rich element near to

The predicted biogenesis of ciRNA is shown Figure 2B.

Circular RNAs and Its Biological Functions in Health and Disease

DOI: http://dx.doi.org/10.5772/intechopen.88764

3.1 circRNAs can act as miRNA sponges

Figure 2.

85

the branchpoint site.

3. Gene regulation and biologic functions of circRNAs

properties of mRNA binding, scaffolding, and cellular translocation.

The expression patterns of circRNAs are specific to the cell type or phase of development [16]. Although the all-biological functions of circRNAs are not entirely defined, some are well studied in the literature. Biological functions of circRNAs include micro RNA (miRNA) sponge, regulation of gene expression and

As a major component of gene regulators, competing/competetive endogenous RNA (ceRNA) contains a micro RNA response element (MRE-competitively binds miRNA) and can affect the regulatory functions of miRNAs [17]. Growing evidence has indicated that circRNAs can act as ceRNA or miRNA sponge molecules. Because

#### Figure 1.

Schematic illustration of the circRNA formation by back splicing mechanism. Unlike canonical mRNA splicing mechanism, the 3<sup>0</sup> splice donor site of exon 1 binds to the 5<sup>0</sup> splice acceptor site of exon 4 during circRNA formation. The back-splicing results in a circRNA including exon 2 and 3 and linear mRNA with skipped exon 2 and 3. ss, splice site.

Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

#### Figure 2.

1979 [8]. It was thought that circRNAs were a by-product formed during splicing mechanism in the first year [9]. Numerous circRNAs have been predicted with the technical developments in high-throughput RNA sequencing (RNA-seq) and meth-

predicted circRNAs in different tissues and cell lines are widely studied nowadays. After the determination of their role in the control of gene expression, circRNAs have gained great attention by researchers in this field. In this chapter, we will focus

According to the gene structure they contain, circRNAs can be divided into three groups: exonic circRNA (ecircRNA), circRNAs from introns (ciRNAs), and exonintron circRNA (elciRNA) [10]. To date, many studies have shown that circRNAs mainly emerged during pre-mRNA splicing process of protein coding genes. Unlike canonical mRNA splicing mechanism, down-stream donor splice site is covalently joined with an upstream acceptor splice sites during circRNA formation. This splicing mechanism is called "back-splicing" [7]. The back-splicing mechanism is depicted in Figure 1. circRNAs can also be formed through the hybridization of complementary inverted sequences (such as human Alu repeats) in introns [10]. If Alu sequences are located in different introns of the same gene, this leads to

Schematic illustration of the circRNA formation by back splicing mechanism. Unlike canonical mRNA splicing mechanism, the 3<sup>0</sup> splice donor site of exon 1 binds to the 5<sup>0</sup> splice acceptor site of exon 4 during circRNA formation. The back-splicing results in a circRNA including exon 2 and 3 and linear mRNA with skipped exon

odological innovations in bioinformatics. The presence and function of the

on circRNAs and their biological functions in health and disease.

2. Biogenesis of circRNAs

Gene Expression and Phenotypic Traits

Figure 1.

84

2 and 3. ss, splice site.

Possible model for the formation of structurally different circRNA. (AI) Intron-driven circularization: circRNAs may form by hybridization of the introns with inverted repeats or Alu sequence. (AII) RBP-driven circularization: RBPs bind to specific sequence in introns that bring the exons close together and trigger the circularization. At the end of these two ways, elciRNAs or ecircRNAs are generated. Only ecircRNAs can be transported to cytoplasm. (B) Formation of ciRNA. The ciRNAs are generated from intron lariat by splicing reaction. Purple arrow represents 7-nt GU-rich element. Yellow arrow represents 11-nt C-rich element near to the branchpoint site.

generate circRNAs, which contains multiple exons [7]. Exon circularization is facilitated by cis-acting inverted repeat sequences as well as by trans-acting RNA-binding proteins (RBPs), which interact with unique sequences in introns [11, 12]. ecircRNA or elciRNA formation is promoted by either cis-acting elements or trans-acting factors as in Figure 2A. ecircRNAs can also be formed from elciRNAs by removal of intronic sequences [13]. Apart from the other circRNAs, ecircRNAs are transported into cytoplasm [14]. In human cells, the existence of ciRNAs is demonstrated by Zhang et al. [15]. ciRNAs are generated through a lariat-derived mechanism relying on mainly a consensus motif containing a 7-nt GU-rich element adjacent to the 5<sup>0</sup> splice site and an 11-nt C-rich element adjacent to the branchpoint site. After cleavage of 3<sup>0</sup> end, stable ciRNA is produced [15]. The predicted biogenesis of ciRNA is shown Figure 2B.

## 3. Gene regulation and biologic functions of circRNAs

The expression patterns of circRNAs are specific to the cell type or phase of development [16]. Although the all-biological functions of circRNAs are not entirely defined, some are well studied in the literature. Biological functions of circRNAs include micro RNA (miRNA) sponge, regulation of gene expression and properties of mRNA binding, scaffolding, and cellular translocation.

#### 3.1 circRNAs can act as miRNA sponges

As a major component of gene regulators, competing/competetive endogenous RNA (ceRNA) contains a micro RNA response element (MRE-competitively binds miRNA) and can affect the regulatory functions of miRNAs [17]. Growing evidence has indicated that circRNAs can act as ceRNA or miRNA sponge molecules. Because of containing plenty of MRE, circRNAs can competitively bind to miRNAs (generally several copies of miRNA) and adsorb them like a sponge [18]. As a result, miRNAs can no longer act on their target mRNA [19]. Therefore, circRNAs can regulate the gene expression and also give rise to decreasing of the functional miRNA [17, 18]. Compared to other ceRNAs, circRNA binds more effectively to miRNAs. Therefore, they are also called "super sponge" [20]. The most characteristic miRNA sponge "antisense to the cerebellar degeneration related protein 1 transcript" (CDR1as)/ciRS-7 includes approximately 70 conserved binding sites for miRNA-7 (miR-7) and forms a complex with Argonaute (AGO) proteins [21]. CDR1as-miR-7 complex co-localizes in the cytoplasm and supresses degradation of miR-7-target mRNAs [17]. Interestingly, circRNA has been reported that it is also displayed to be abundant in exosomes in serum [16]. Therefore, Li et al. suggested that sorting of circRNAs to exosomes was regulated by altering levels of associated miRNA in producer cells [16, 22]. In addition, researchers have found that CDR1as including exosomes inhibit miR-7-induced growth in recipient cells [22]. Testesspecific circRNA/circSry [the circular transcript of sex determining region Y (Sry) gene] can also serve as a sponge for miRNA-138 [23]. It contains 16 MREs of miRNA-138 and regulates the expression of miR-138-target genes, functioning similar to CDR1as [17]. Additionaly, many other circRNAs have been identified as miRNA sponges such as hsa\_circ\_001569, heart-related circRNA (HRCR), itchy E3 ubiquitin protein ligase circRNA (circITCH), forkhead box O3 circRNA (circfoxo3), homeodomain interacting protein kinase 3 circRNA (circHIPK3), mitochondrial tRNA translation optimization 1 circRNA (circMTO1), zinc finger protein 609 circRNA (cirZNF609), and baculoviral IAP repeat containing 6 circRNA (circBIRC6) [24]. Among them, cirITCH regulates the expression of ITCH by acting as a sponge for miR-214, miR-17, and miR-7 [22].

cognate DNA. The formation of R-loop (RNA:DNA hybrid) gives rise to termination of SEP3 gene transcription [13]. Moreover, circRNAs can be paired with DNA to generate DNA-RNA triple helixes. Therefore, this pairing may affect DNA

(circEIF3J) and poly(A)-binding protein-interacting protein 2 circRNA (circPAIP2) have been suggested to have cis-regulatory effects on parental genes and promote transcription of EIF3J and PAIP2. This cis-regulatory effect occurs by binding of its circRNAs to Pol II, U1 snRNP, and their parental gene promoters [18, 25]. When transcription is initiated, the production of eIciRNA can be increased so that this phenomenon generates a positive feedback loop [23]. Moreover, they have a function as positive regulators through their interactions with the elongating Pol II complex [24, 25, 31]. In addition, exonic circular antisense noncoding RNA in the INK4 (a family of cyclin-dependent kinase inhibitors) locus (circANRIL/cANRIL) reduces ANRIL that inhibits transcription of INK4/ARF gene by binding to the Polycomb Gene (PcG) complex. Thus, cANRIL regulates the transcription of

Some circRNAs may affect nuclear translocation of other proteins to nucleus and regulation of gene transcription. For example, CircAmotl1 may increase nuclear translocation of signal transducer and activator of transcription 3 (STAT3) to regu-

Another capability of circRNAs is to ensure that cellular proteins remain in their natural cellular position. It has been reported that circAmotl1 can enhance stability of c-myc by maintaining its nuclear retention and increase its binding affinity to several promoters. Therefore, it upregulates c-myc targets such as hypoxia inducible factor-1 alpha (HIF-1α), cell division cycle 25A (Cdc25a), ETS Like-1 (ELK-1) [24]. In another example, cytoplasmic circ-foxo3 interacts with differentiation-1 (ID-1), HIF-1α, focal adhesion kinase (FAK), the transcription factor E2 (E2F1) and

circRNAs also serve as scaffolding in the assembly of protein complexes [13]. It

has been reported that circ-foxo3 acts as an adaptor to bridge between cyclindependent kinase 2 (CDK2) and CDK inhibitor p21 (cyclin-dependent kinase inhibitor 1A). This interaction (circ-foxo3/CDK2/p21) inhibits cell-cycle progression within G1 to S-phase transition [18, 24]. However, downregulation of circfoxo3 leads to the release of CDK2 from p21 and CDK2 phosphorylates cyclin E and cyclin A for cell cycle progression. On the other hand, the circ-foxo3 connects the murine double minute 2 (MDM2) to tumor protein p53 (p53), and induces the degradation of p53 by ubiquitination. However, circ-foxo3 weakly interacts with foxo3 and suppresses foxo3 from MDM2-mediated polyubiquitination and

Some ciRNAs (e.g., ci-ankrd52, ci-sirt7) and eIciRNAs (e.g., circEIF3J, circPAIP2) regulate the transcription of their parental genes. eIciRNAs can regulate parental gene transcription in a cis-acting manner [18, 23, 24]. Recent studies have been indicated that nuclear elciRNAs (localized to the promoter of their parental genes) interact with U1 small nuclear ribonucleoproteins (snRNPs) and RNA pol II and promote the transcription initiation of their parental genes [28, 30]. For example, eukaryotic translation initiation factor 3J circRNA

Circular RNAs and Its Biological Functions in Health and Disease

DOI: http://dx.doi.org/10.5772/intechopen.88764

replication [29].

INK4/ARF [32].

3.3 Cellular translocation properties

3.4 Scaffolding properties

proteasome degradation [30].

87

late the expression of mitosis-related genes [24].

prevents their translocation from cytoplasm to other location [13].

Apart from common phenomenon, in some cases, the binding of circRNAs to miRNAs may not always lead to inhibition of miRNAs. Linearization and AGO2-mediated cleavage of CDR1as can occur when CDR1as interacts with miR-671. Thus, bound miR-7 is released from CDR1as [25]. On the other hand, in spite of the role of circRNA in gene regulation as a classical sponge effect, some recent studies have revealed that the number of circRNAs with miRNA sponge property is limited. Besides inhibition effect, it has been regarded that the interaction between circRNAs and miRNA is also related to sorting, storage and localization of miRNA [18].

#### 3.2 circRNAs regulate gene expression and interact with protein

In addition to their miRNA sponge function, circRNAs can also act as sponges for other components as RBPs. There are many proteins known as RBP such as AGO protein, RNA quaking, muscleblind (MBL) protein, RNA polymerase II (Pol II), eukaryotic initiation factor 4A-III [26]. RBPs bind specific sequences to their target genes and control all stages of mRNA lifecycle including splicing, nuclear export, stability and subcellular localisation [27]. A number of circRNAs contain a large amount of binding sites for a single or multiple RNA-binding proteins. For example, circRNA protein sponge derived from the MBL locus includes binding sites of mbl protein. Thus, mbl is prevented from binding to other targets. In a parallel study, circular RNA of polyadenylate-binding nuclear protein 1 (circPABPN1) derived from PABPN1 gene binds to HuR (enhance PABPN1 translation) and prohibits its binding to PABPN1 mRNA [28].

circRNAs also inhibits parental gene transcription in target genes via invading RNA binding sites. Strongly binding of circRNA, derived from sepallata3 (SEP3) gene, to its cognate DNA locus blocks the binding of its linear isoform to the

Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

of containing plenty of MRE, circRNAs can competitively bind to miRNAs (generally several copies of miRNA) and adsorb them like a sponge [18]. As a result, miRNAs can no longer act on their target mRNA [19]. Therefore, circRNAs can regulate the gene expression and also give rise to decreasing of the functional miRNA [17, 18]. Compared to other ceRNAs, circRNA binds more effectively to miRNAs. Therefore, they are also called "super sponge" [20]. The most characteristic miRNA sponge "antisense to the cerebellar degeneration related protein 1 transcript" (CDR1as)/ciRS-7 includes approximately 70 conserved binding sites for miRNA-7 (miR-7) and forms a complex with Argonaute (AGO) proteins [21]. CDR1as-miR-7 complex co-localizes in the cytoplasm and supresses degradation of miR-7-target mRNAs [17]. Interestingly, circRNA has been reported that it is also displayed to be abundant in exosomes in serum [16]. Therefore, Li et al. suggested that sorting of circRNAs to exosomes was regulated by altering levels of associated miRNA in producer cells [16, 22]. In addition, researchers have found that CDR1as including exosomes inhibit miR-7-induced growth in recipient cells [22]. Testesspecific circRNA/circSry [the circular transcript of sex determining region Y (Sry) gene] can also serve as a sponge for miRNA-138 [23]. It contains 16 MREs of miRNA-138 and regulates the expression of miR-138-target genes, functioning similar to CDR1as [17]. Additionaly, many other circRNAs have been identified as miRNA sponges such as hsa\_circ\_001569, heart-related circRNA (HRCR), itchy E3 ubiquitin protein ligase circRNA (circITCH), forkhead box O3 circRNA (circfoxo3), homeodomain interacting protein kinase 3 circRNA (circHIPK3), mitochondrial tRNA translation optimization 1 circRNA (circMTO1), zinc finger protein 609 circRNA (cirZNF609), and baculoviral IAP repeat containing 6 circRNA (circBIRC6) [24]. Among them, cirITCH regulates the expression of ITCH by acting

Apart from common phenomenon, in some cases, the binding of circRNAs to miRNAs may not always lead to inhibition of miRNAs. Linearization and AGO2-mediated cleavage of CDR1as can occur when CDR1as interacts with miR-671. Thus, bound miR-7 is released from CDR1as [25]. On the other hand, in spite of the role of circRNA in gene regulation as a classical sponge effect, some recent studies have revealed that the number of circRNAs with miRNA sponge property is limited. Besides inhibition effect, it has been regarded that the interaction between circRNAs and miRNA is also related to sorting, storage and localization

In addition to their miRNA sponge function, circRNAs can also act as sponges for other components as RBPs. There are many proteins known as RBP such as AGO protein, RNA quaking, muscleblind (MBL) protein, RNA polymerase II (Pol II), eukaryotic initiation factor 4A-III [26]. RBPs bind specific sequences to their target genes and control all stages of mRNA lifecycle including splicing, nuclear export, stability and subcellular localisation [27]. A number of circRNAs contain a large amount of binding sites for a single or multiple RNA-binding proteins. For example, circRNA protein sponge derived from the MBL locus includes binding sites of mbl protein. Thus, mbl is prevented from binding to other targets. In a parallel study, circular RNA of polyadenylate-binding nuclear protein 1 (circPABPN1) derived from PABPN1 gene binds to HuR (enhance PABPN1 translation) and prohibits its

circRNAs also inhibits parental gene transcription in target genes via invading RNA binding sites. Strongly binding of circRNA, derived from sepallata3 (SEP3) gene, to its cognate DNA locus blocks the binding of its linear isoform to the

3.2 circRNAs regulate gene expression and interact with protein

as a sponge for miR-214, miR-17, and miR-7 [22].

Gene Expression and Phenotypic Traits

of miRNA [18].

86

binding to PABPN1 mRNA [28].

cognate DNA. The formation of R-loop (RNA:DNA hybrid) gives rise to termination of SEP3 gene transcription [13]. Moreover, circRNAs can be paired with DNA to generate DNA-RNA triple helixes. Therefore, this pairing may affect DNA replication [29].

Some ciRNAs (e.g., ci-ankrd52, ci-sirt7) and eIciRNAs (e.g., circEIF3J, circPAIP2) regulate the transcription of their parental genes. eIciRNAs can regulate parental gene transcription in a cis-acting manner [18, 23, 24]. Recent studies have been indicated that nuclear elciRNAs (localized to the promoter of their parental genes) interact with U1 small nuclear ribonucleoproteins (snRNPs) and RNA pol II and promote the transcription initiation of their parental genes [28, 30]. For example, eukaryotic translation initiation factor 3J circRNA (circEIF3J) and poly(A)-binding protein-interacting protein 2 circRNA (circPAIP2) have been suggested to have cis-regulatory effects on parental genes and promote transcription of EIF3J and PAIP2. This cis-regulatory effect occurs by binding of its circRNAs to Pol II, U1 snRNP, and their parental gene promoters [18, 25]. When transcription is initiated, the production of eIciRNA can be increased so that this phenomenon generates a positive feedback loop [23]. Moreover, they have a function as positive regulators through their interactions with the elongating Pol II complex [24, 25, 31]. In addition, exonic circular antisense noncoding RNA in the INK4 (a family of cyclin-dependent kinase inhibitors) locus (circANRIL/cANRIL) reduces ANRIL that inhibits transcription of INK4/ARF gene by binding to the Polycomb Gene (PcG) complex. Thus, cANRIL regulates the transcription of INK4/ARF [32].

## 3.3 Cellular translocation properties

Some circRNAs may affect nuclear translocation of other proteins to nucleus and regulation of gene transcription. For example, CircAmotl1 may increase nuclear translocation of signal transducer and activator of transcription 3 (STAT3) to regulate the expression of mitosis-related genes [24].

Another capability of circRNAs is to ensure that cellular proteins remain in their natural cellular position. It has been reported that circAmotl1 can enhance stability of c-myc by maintaining its nuclear retention and increase its binding affinity to several promoters. Therefore, it upregulates c-myc targets such as hypoxia inducible factor-1 alpha (HIF-1α), cell division cycle 25A (Cdc25a), ETS Like-1 (ELK-1) [24]. In another example, cytoplasmic circ-foxo3 interacts with differentiation-1 (ID-1), HIF-1α, focal adhesion kinase (FAK), the transcription factor E2 (E2F1) and prevents their translocation from cytoplasm to other location [13].

#### 3.4 Scaffolding properties

circRNAs also serve as scaffolding in the assembly of protein complexes [13]. It has been reported that circ-foxo3 acts as an adaptor to bridge between cyclindependent kinase 2 (CDK2) and CDK inhibitor p21 (cyclin-dependent kinase inhibitor 1A). This interaction (circ-foxo3/CDK2/p21) inhibits cell-cycle progression within G1 to S-phase transition [18, 24]. However, downregulation of circfoxo3 leads to the release of CDK2 from p21 and CDK2 phosphorylates cyclin E and cyclin A for cell cycle progression. On the other hand, the circ-foxo3 connects the murine double minute 2 (MDM2) to tumor protein p53 (p53), and induces the degradation of p53 by ubiquitination. However, circ-foxo3 weakly interacts with foxo3 and suppresses foxo3 from MDM2-mediated polyubiquitination and proteasome degradation [30].

## 3.5 mRNA binding properties

Most circRNAs are capable of interaction with mRNAs. It has been reported that they can be able to regulate the stability of mRNAs as well. In addition to its miRNA sponge function, CDR1as is also proposed to form a duplex structure with CDR1 mRNA and stabilizes it. Similarly, stabilization of mature intercellular adhesion molecule 1 mRNAs in macrophages was found to be facilitated by RasGEF domain family member 1B circRNA (circRasGEF1B) [18].

circRNA Possible target CVD Biological function or

circANRIL (ex 5–7) PES-1 AS and CAD Impairs pre-rRNA maturation

circANRIL (ex 4–6) ASVD Neighboring gene regulation

Hsa\_circ\_0010729 mir-186 AS and CHD Regulates vascular endothelial

circWDR77 mir-124 AS Regulates VSMC proliferation

circ-SATB2 mir-939 AS Inhibits the expression of

hsa\_circ\_0124644 CAD Potential biomarker of

hsa\_-circ\_0001879 CAD Significant upregulated

CDR1as miR-7a MI Upregulates the expression of

MFACR miR-652-3p MI Upregulates apoptosis and

and LVD

hsa\_circ\_0004104 Dysregulation of

plaque rupture

circR-284 miR-221 AS and carotid

MICRA Acute MI, HF,

89

circACTA2 miR-548f-5p AS and CHD Maintains contractile

Hsa\_circ\_0003575 miR-199-3p, mir-

DOI: http://dx.doi.org/10.5772/intechopen.88764

9-5p, mir-377-3p, and miR-141-3p

Circular RNAs and Its Biological Functions in Health and Disease

description

apoptosis

AS Regulates endothelial cell

1α axis

in HASMCs

a miRNA sponge

a miRNA sponge Regulates cell phenotypic differentiation, proliferation, apoptosis, and migration in

VSMC

p27Kipi axis

patients

apoptosis

such as INK4a

and ribosome biogenesis and increases nucleolar stress and

proliferation and angiogenesis acting as a miRNA sponge

cell proliferation and apoptosis via targeting miR-186 and HIF-

phenotype of VSMC Mediates NRG-1-ICD regulation of α-SMA expression

and migration via targeting miR-124 and FGF2 Inhibits the expression of SM22a and STIM1 by acting as

SM22a and STIM1 by acting as

Reduces the proliferation of VSMCs by circR-284/mir-221/

Upregulated circR-284:miR-221 ratio in the early stage of carotid plaque rupture

coronary artery disease

expression levels in CAD

PARP and SP1 acting as a miRNA sponge and promotes

Potential biomarker of left ventricular dysfunction in the patients with acute MI

mitochondrial fission

overexpression of hsa\_circ\_0004104

atherosclerosis-related genes by

Ref

[32]

[48]

[49]

[50]

[51]

[52]

[53, 54]

[55]

[56]

[56]

[57]

[58, 59]

[60]

[32, 47]

#### 3.6 The effect of circRNA as a translator

Recent studies have shown that some circRNAs can be entered to translational process in spite of considering noncoding RNA [33, 34]. A limited number of studies have indicated the potential protein coding properties of circRNAs until now but the translational efficiency might be low [33]. circRNA containing internal ribosomal entry site (IRES) and open reading frame can be translated into protein or polypeptide. In eukaryotes, IRES is an alternative way of initiating translation, independent of 5<sup>0</sup> cap structure and 3<sup>0</sup> poly (A) tail recognition [19]. It has been demonstrated that the 40S subunit of the eukaryotic ribosome can interact with circRNA-containing IRES and then begin translation in in vivo and in vitro experiments. It has been shown that zinc finger protein 609 circRNA (circZNF609) can be translated into a novel ZNF609 protein isoform and potential function during myogenesis. Another study indicated that novel proteins have been translated from F-box and WD repeat domain containing 7 circRNA (circFBXW7) and SNF2 histone linker PHD ring helicase circRNA (circSHPRH) in glioblastoma cell lines. A new isoform protein encoded by circFBXW7 with open reading frame was found to inhibit glioma cell growth [18, 34].

A recent study reported that N6-methyladenosine (m6A), a most common base modification of RNA, can promote the protein translation of circRNA in human cells, even if one m6A motif can initiate circRNA translation [18, 19, 29]. m6Adriven circRNA translation is prevalent, and several endogenous circRNAs have the potential for translation and regulatory role in a cell against enviromental factors [29].

#### 3.7 The effect of circRNAs on splicing

Recent studies have shown that there is a competition between backsplicing and linear splicing. Thus, the biogenesis of circRNAs leads to loss of protein-coding mRNA levels and inhibits parental gene expression [13]. On the other hand, the level of circRNA is negatively correlated to the splicing efficiency of certain genes due to the competition between linear splicing and circRNA biogenesis [30].

## 4. circRNAs in cardiovascular diseases

Cardiovascular disease (CVD) is one of the most important health problems. It causes most of the deaths worldwide [35]. According to recent studies, a number of circRNAs may play a significant role during development of CVD or pathological conditions such as cardiac hypertrophy, coronary artery disease, atherosclerotic vascular disease, cardiomyopathy, cardiac fibrosis, heart failure (HF), ischemia, and myocardial infarction (MI) [36–38]. However, in development of heart disease, the regulatory mechanisms and functional importance of several circRNAs are not clear [38]. circRNAs are also concentrated in body fluids such as seminal fluid,


3.5 mRNA binding properties

Gene Expression and Phenotypic Traits

Most circRNAs are capable of interaction with mRNAs. It has been reported that they can be able to regulate the stability of mRNAs as well. In addition to its miRNA sponge function, CDR1as is also proposed to form a duplex structure with CDR1 mRNA and stabilizes it. Similarly, stabilization of mature intercellular adhesion molecule 1 mRNAs in macrophages was found to be facilitated by RasGEF

Recent studies have shown that some circRNAs can be entered to translational

A recent study reported that N6-methyladenosine (m6A), a most common base modification of RNA, can promote the protein translation of circRNA in human cells, even if one m6A motif can initiate circRNA translation [18, 19, 29]. m6Adriven circRNA translation is prevalent, and several endogenous circRNAs have the potential for translation and regulatory role in a cell against enviromental

Recent studies have shown that there is a competition between backsplicing and

Cardiovascular disease (CVD) is one of the most important health problems. It causes most of the deaths worldwide [35]. According to recent studies, a number of circRNAs may play a significant role during development of CVD or pathological conditions such as cardiac hypertrophy, coronary artery disease, atherosclerotic vascular disease, cardiomyopathy, cardiac fibrosis, heart failure (HF), ischemia, and myocardial infarction (MI) [36–38]. However, in development of heart disease, the regulatory mechanisms and functional importance of several circRNAs are not clear [38]. circRNAs are also concentrated in body fluids such as seminal fluid,

linear splicing. Thus, the biogenesis of circRNAs leads to loss of protein-coding mRNA levels and inhibits parental gene expression [13]. On the other hand, the level of circRNA is negatively correlated to the splicing efficiency of certain genes due to the competition between linear splicing and circRNA biogenesis [30].

process in spite of considering noncoding RNA [33, 34]. A limited number of studies have indicated the potential protein coding properties of circRNAs until now but the translational efficiency might be low [33]. circRNA containing internal ribosomal entry site (IRES) and open reading frame can be translated into protein or polypeptide. In eukaryotes, IRES is an alternative way of initiating translation, independent of 5<sup>0</sup> cap structure and 3<sup>0</sup> poly (A) tail recognition [19]. It has been demonstrated that the 40S subunit of the eukaryotic ribosome can interact with circRNA-containing IRES and then begin translation in in vivo and in vitro experiments. It has been shown that zinc finger protein 609 circRNA (circZNF609) can be translated into a novel ZNF609 protein isoform and potential function during myogenesis. Another study indicated that novel proteins have been translated from F-box and WD repeat domain containing 7 circRNA (circFBXW7) and SNF2 histone linker PHD ring helicase circRNA (circSHPRH) in glioblastoma cell lines. A new isoform protein encoded by circFBXW7 with open reading frame was found to

domain family member 1B circRNA (circRasGEF1B) [18].

3.6 The effect of circRNA as a translator

inhibit glioma cell growth [18, 34].

3.7 The effect of circRNAs on splicing

4. circRNAs in cardiovascular diseases

factors [29].

88


saliva, and blood. Thus, their potential usage as clinical biomarkers may be possible

Circular RNAs and Its Biological Functions in Health and Disease

DOI: http://dx.doi.org/10.5772/intechopen.88764

circANRIL is generated as an antisense transcript from the INK4A/ARF gene locus by alternative splicing [36]. SNPs localized within chromosome 9p21 are likely to affect the INK4/ARF locus. These SNPs can regulate ANRIL splicing and may lead to circANRIL production [39]. Interestingly, there is an association between 9p21 SNPs and the susceptibility to atherosclerosis [41]. circANRIL is also implicated in the pathogenesis of atherosclerosis [42]. In another study, Burd et al. suggested that 9p21 SNPs affect the coordination of ANRIL expression and splicing via interaction of different PcG complexes. Furthermore, PcG complexes are targeted to the INK4/ARF locus and that leads to inhibition of INK4/ARF transcription. Moreover, they also indicated that their study is the first to provide evidence for relationship between circRNA and atherosclerotic vascular disease

circANRIL also disrupts exonuclease-mediated pre-rRNA processing and ribosome biogenesis by binding to pescadillo homologue 1 (PES1). This leads to nuclear stress and p53 activation in cells [38, 39]. Therefore, it supresses cell proliferation

Consequenly, circANRIL acts as a protective factor against atherosclerosis [41, 43]. On the other hand, it has been indicated that a novel circular RNA product of ANRIL, cANRIL (exon4-6) also regulates the expression of INK4/ARF [32].

In addition, circRNA serves as a protein scaffold such as circAmotl1 in cardiac dysfunction [35, 43]. circAmotl1 facilitates phosphorylation of protein kinase B (AKT) and nuclear translocation of pAKT by forming ternary complexes with AKT and phosphoinositide-dependent protein kinase (PDK) [43–45]. Zeng et al. have suggested that pAKT translocation may be responsible for protection of heart cells

circ-foxo3 is another circRNA described to may have a role in the cardiovascular diseases. Stress-related proteins (HIF-1α and FAK) and senescence-related proteins [inhibitor of DNA-binding protein (ID1) and E2F1] are arrested in cytoplasm by circ-foxo3. Therefore, circ-foxo3 prevents translocation of these proteins into the nucleus. As a consequence, this mechanism promotes cardiac senecence through ectopic expression of circ-foxo3 [41, 46]. Besides these functions, circRNAs are reported to also show their effects as miRNA sponge in cardiovascular diseases. circRNAs and their function in cardiovascular diseases are indicated in Table 1. Although there is limited number of studies until today, CVD-related studies for circRNA are in progress. Therefore, it is still required the identification of circRNA as candidate biomarkers for CVDs. Moreover, biologic functions of circRNA in vascular endothelial cell and heart tissue should be validated in further studies.

Recent studies have shown that circRNAs are plentifully expressed in normal neuronal cells [73–75]. They may be found abundantly in neuronal cells for several reasons: (i) brain contains more host genes of circRNA such as neuronal genes,

and inhibits apoptosis in vascular smooth muscle cells and macrophages.

from cardiomyopathy caused by doxorubicin [45].

5. circRNAs in neurological disorders

91

Some heart specific RNA-splicing regulators are also important players for heart development. One of the RNA-splicing regulators is RBM20 that is required for splicing of cardiac-related genes such as titin [38]. Its mutation leads to exon retention in the region of I-band and results in larger titin isoforms [30]. According to RNA-seq researches in tissues from dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy, 80 different circRNAs are derived from the titin gene

in the future [39].

(TTN) [30, 40].

(ASVD) [32].

CVD, cardiovascular disease; AS, atherosclerosis; ASVD, atherosclerotic vascular disease; CHD, coronary heart disease; CAD, coronary artery disease; MI, myocardial infarction; HF, heart failure; LVD, left ventricular dysfunction; EH, essential hypertension; DCM, dilated cardiomyopathy; VSMC, vascular smooth muscle cell; ROS, reactive oxygen species; ANRIL, antisense noncoding RNA in the INK4 locus; PES1, pescadillo homologue 1; ACTA2, actin alpha 2; WDR77, WD repeat domain 77; STIM1, stromal interaction molecule 1; SATB2, special AT-rich sequence-binding protein 2; CDR1, cerebellar degeneration-related protein 1; MICRA, myocardial infarction associated circRNA; MFCAR, mitochondrial fission and apoptosis-related circRNA; HRCR, heart-related circRNA; ARC, apoptosis repressor with CARD domain; NCX1, sodium/ calcium exchanger 1; AMOTL1, angiomotin like 1; AKT, protein kinase B; PDK, phosphoinositide-dependent protein kinase; TTN, titin; RYR2, ryanodine receptor 2; Foxo3, forkhead box O3; ID1, inhibitor of DNA binding 1; E2F1, E2F transcription Factor 1; FAK, focal adhesion kinase; HIF-1α, hypoxia inducible factor-1; FGF2, fibroblast growth factor2; PARP, poly ADP-ribose polymerase; and MEF2A, myocyte enhancer factor 2A.

Table 1.

Summary of identified circRNA in the cardiovascular disease.

Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

circRNA Possible target CVD Biological function or

circ-081881 mir-548 Acute MI Positively regulates PPARγ

circRNA-010567 miR-141 MI May mediate fibrosis-

hypertrophy

myocardial injury

cardiomyopathy

circTTN DCM Dysregulated in disease model [65, 66] circRyr2 Cardiomyopathy [65]

> Hypertension and CAD

and

rno\_circRNA\_016002 Hypertension Upregulated in hypertensive

hsa\_circ\_0014243 hsa-miR-10a-5p EH Crucial role in the genesis and

hsa\_circ\_0037911 miR-637 EH Upregulated in hypertension

hsa\_circ\_0126991 EH May serve as a stable biomarker

Cardiac senescence

CVD, cardiovascular disease; AS, atherosclerosis; ASVD, atherosclerotic vascular disease; CHD, coronary heart disease; CAD, coronary artery disease; MI, myocardial infarction; HF, heart failure; LVD, left ventricular dysfunction; EH, essential hypertension; DCM, dilated cardiomyopathy; VSMC, vascular smooth muscle cell; ROS, reactive oxygen species; ANRIL, antisense noncoding RNA in the INK4 locus; PES1, pescadillo homologue 1; ACTA2, actin alpha 2; WDR77, WD repeat domain 77; STIM1, stromal interaction molecule 1; SATB2, special AT-rich sequence-binding protein 2; CDR1, cerebellar degeneration-related protein 1; MICRA, myocardial infarction associated circRNA; MFCAR, mitochondrial fission and apoptosis-related circRNA; HRCR, heart-related circRNA; ARC, apoptosis repressor with CARD domain; NCX1, sodium/ calcium exchanger 1; AMOTL1, angiomotin like 1; AKT, protein kinase B; PDK, phosphoinositide-dependent protein kinase; TTN, titin; RYR2, ryanodine receptor 2; Foxo3, forkhead box O3; ID1, inhibitor of DNA binding 1; E2F1, E2F transcription Factor 1; FAK, focal adhesion kinase; HIF-1α, hypoxia inducible factor-1; FGF2, fibroblast growth factor2; PARP, poly

HRCR mir-223 HF and cardiac

Gene Expression and Phenotypic Traits

circNCX1 miR-133a-3p Ischemic

circAmotl1 AKT and PDK Cardiac repair

circZNF609 miR-615-5p and miR-150-5p

Hsa-circ-0005870 hsa-miR-619-5p, hsa-

circ-foxo3 ID-1, E2F1, FAK, and HIF-1α

ADP-ribose polymerase; and MEF2A, myocyte enhancer factor 2A.

Summary of identified circRNA in the cardiovascular disease.

Table 1.

90

miR-5095, hsa-miR-1273 g-3p, and hsamiR-5096

description

hypertrophy

Increases the expression of ARC by acting as a miRNA sponge. Suppresses cardiac

acting as a miRNA sponge

associated protein resection

Promotes cardiomyocyte apoptosis by acting as a miRNA sponge and increased in response to ROS

Facilitates the nuclear translocation of AKT and PDK1 Improves survival and decreases apoptosis

Inhibits cell proliferation, migration, and tube formation and promotes cell apoptosis Acts as a miRNA sponge and leads to upregulation of MEF2A

expression

Hypertension Downregulated in hypertension patients

> rat strains compared to normotensive rats

development of EH and presents a certain diagnostic capability for EH

for early diagnosis of EH

Interacts with ID-1, E2F1, FAK, and HIF-1α and induces cellular senescence in aging hearts

patients

Ref

[61]

[62]

[63]

[64]

[45]

[67]

[68]

[69]

[70]

[65, 71]

[72]

[46]

saliva, and blood. Thus, their potential usage as clinical biomarkers may be possible in the future [39].

Some heart specific RNA-splicing regulators are also important players for heart development. One of the RNA-splicing regulators is RBM20 that is required for splicing of cardiac-related genes such as titin [38]. Its mutation leads to exon retention in the region of I-band and results in larger titin isoforms [30]. According to RNA-seq researches in tissues from dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy, 80 different circRNAs are derived from the titin gene (TTN) [30, 40].

circANRIL is generated as an antisense transcript from the INK4A/ARF gene locus by alternative splicing [36]. SNPs localized within chromosome 9p21 are likely to affect the INK4/ARF locus. These SNPs can regulate ANRIL splicing and may lead to circANRIL production [39]. Interestingly, there is an association between 9p21 SNPs and the susceptibility to atherosclerosis [41]. circANRIL is also implicated in the pathogenesis of atherosclerosis [42]. In another study, Burd et al. suggested that 9p21 SNPs affect the coordination of ANRIL expression and splicing via interaction of different PcG complexes. Furthermore, PcG complexes are targeted to the INK4/ARF locus and that leads to inhibition of INK4/ARF transcription. Moreover, they also indicated that their study is the first to provide evidence for relationship between circRNA and atherosclerotic vascular disease (ASVD) [32].

circANRIL also disrupts exonuclease-mediated pre-rRNA processing and ribosome biogenesis by binding to pescadillo homologue 1 (PES1). This leads to nuclear stress and p53 activation in cells [38, 39]. Therefore, it supresses cell proliferation and inhibits apoptosis in vascular smooth muscle cells and macrophages. Consequenly, circANRIL acts as a protective factor against atherosclerosis [41, 43]. On the other hand, it has been indicated that a novel circular RNA product of ANRIL, cANRIL (exon4-6) also regulates the expression of INK4/ARF [32].

In addition, circRNA serves as a protein scaffold such as circAmotl1 in cardiac dysfunction [35, 43]. circAmotl1 facilitates phosphorylation of protein kinase B (AKT) and nuclear translocation of pAKT by forming ternary complexes with AKT and phosphoinositide-dependent protein kinase (PDK) [43–45]. Zeng et al. have suggested that pAKT translocation may be responsible for protection of heart cells from cardiomyopathy caused by doxorubicin [45].

circ-foxo3 is another circRNA described to may have a role in the cardiovascular diseases. Stress-related proteins (HIF-1α and FAK) and senescence-related proteins [inhibitor of DNA-binding protein (ID1) and E2F1] are arrested in cytoplasm by circ-foxo3. Therefore, circ-foxo3 prevents translocation of these proteins into the nucleus. As a consequence, this mechanism promotes cardiac senecence through ectopic expression of circ-foxo3 [41, 46]. Besides these functions, circRNAs are reported to also show their effects as miRNA sponge in cardiovascular diseases. circRNAs and their function in cardiovascular diseases are indicated in Table 1.

Although there is limited number of studies until today, CVD-related studies for circRNA are in progress. Therefore, it is still required the identification of circRNA as candidate biomarkers for CVDs. Moreover, biologic functions of circRNA in vascular endothelial cell and heart tissue should be validated in further studies.

## 5. circRNAs in neurological disorders

Recent studies have shown that circRNAs are plentifully expressed in normal neuronal cells [73–75]. They may be found abundantly in neuronal cells for several reasons: (i) brain contains more host genes of circRNA such as neuronal genes,

which play roles in neurogenesis, neuronal development, and neuronal differentiation [11, 74], (ii) the expression levels of circRNAs are higher in brain than other tissues [75, 76], (iii) due to the slow division rates of neurons, circRNAs may accumulate more in the brain than other tissues [77], (iv) neuronal genes contain long introns (>10 kb) with inverted repeat sequences, thereby simplifying formation of circRNAs [10], and (v) circRNAs due to the absence of 5<sup>0</sup> and 3<sup>0</sup> ends result in greater stability than linear RNAs, leading to a relatively longer half-life [78]. The half-life of circRNAs is approximately 20 h, compared with corresponding linear isoforms (no more than 8 h) [79].

with multiple protein subunits, thus acting as "scaffolding" for RBPs [7, 98]. Thereby, it facilitates the interaction by potentially increasing the stability of the circRNA transcripts. Due to its multiple functions in brain, researchers have suggested that ciRS-7 can be a potential biomarker for neurodegenerative disorders

Circular RNAs and Its Biological Functions in Health and Disease

Alzheimer's disease is a chronic neurological disease. Lukiw et al. showed that the expression level of ciRS-7 is decreased in hippocampal CA1 region in sporadic AD [83]. Functional deficiency of ciRS-7 can lead to upregulation of miR-7 in AD brain and may cause the downregulation of several AD-relevant mRNA targets, including the ubiquitin conjugating enzyme E2A (UBE2A) [83, 84, 99, 100]. This autophagic protein, UBE2A, is a central effector in the ubiquitination cycle. UBE2A

amyloidogenesis [99]. In contrast to the previous studies, Shi et al. have shown that ciRS-7 promotes the degradation of amyloid precursor protein (APP) and betasecretase 1 (BACE1) in an nuclear factor kappa beta (NF-κB)-dependent manner [101]. Hence, future studies are needed to reveal ciRS-7 function/functions and its

CircSry can serve as a miRNA sponge in neural cells. CircSry inhibits miR-138 [53, 85], which is a potential molecular regulator of human memory function [102]. CircSry has multiple binding sites for miR-138 and promotes tau phosphorylation by targeting the "retinoic acid receptor alpha/glycogen synthase kinase-3β" (RARA/ GCK-3β) pathway [86]. Some studies have indicated that miR-138 influences learning and memory abilities by regulating acyl protein thioesterase 1 [87, 102]. Therefore, association of circSry and miR-138 in AD should be further investigated.

Parkinson disease, progressive age-related neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta [103, 104]. To date, five genes have been determined to cause PD, such as αsynuclein (SNCA), parkin, dj-1, PTEN-induced kinase 1 (pink1), and leucine-rich repeat kinase 2 (lrrk2) [105]. SNCA is the key player in the pathogenesis of PD based on neuropathologic, genetic, and cellular evidence [106]. The overexpression and aggregation of SNCA, a target gene of miR-7, is considered as a distinctive marker in PD [107, 108]. miR-7 has been proposed to play a role in PD by reducing expression of SNCA [88]. ciRS-7 plays a protective role by inhibiting miR-7 that directly regulates the expression of SNCA [109]. miR-7 alleviates SNCA expression dose-dependently and induces the degradation of SNCA mRNA levels [88]. These results suggest that ciRS-7 serves as a miR-7 sponge in vitro. Furthermore, the silencing of ciRS-7 increases miR-7 activity and decreases the expression of miR-7 target genes [110]. In addition, circSNCA, another circRNA, can sponge miR-7, thereby regulating expression of SNCA, resulting in decreased autophagy and increased apoptosis in cells [111]. These findings are in concordance with the results of a study, which showed that autophagy can prevent PD [112], and that of

the other study, which demonstrated that apoptosis is related to PD [113].

circRNAs may participate in inflammatory reactions that induce neuropathy. Some circRNAs may affect immune responses due to the fact that they contain virus miRNA binding sites. For instance, hsa-circRNA 2149 contains 13 unique, head to

5.3 circRNA and inflammatory neuropathy

93

is crucial for clearing amyloid peptides via phagocytosis and contributes to

including AD and PD [83].

exact role in AD pathology.

5.2 circRNA in Parkinson's disease

5.1 circRNA in Alzheimer's disease

DOI: http://dx.doi.org/10.5772/intechopen.88764

The latest studies have shown that circRNAs could attenuate cell senescence and cell survival and may be involved in the regulation of aging and age-related neurological diseases [80–82]. Thus, circRNAs are expected to be new potential biomarkers and target for aging and age-related neurological diseases (Table 2). These studies have suggested that circRNAs may play an important role in pathological mammalian brain function, which is implicated in disorders in central nervous system (CNS) including Alzheimer's disease (AD), Parkinson's disease (PD), neuropsychiatric disorders, prion disease, and inflammatory neuropathy.

CDR1as, a circRNA, is highly plentiful and specifically expressed in the mammalian brain [85]. Some studies have indicated that ciRS-7 contains multiple antimiR-7 sequences. This suggests that ciRS-7 may function as a sponge to sequester the normal functions of miR-7 [57, 95–97]. ciRS-7 can regulate the stability of mRNA targets in the brain by binding to miR-7 [78, 85]. Besides, ciRS-7 can interact


AD, Alzheimer's disease; PD, Parkinson disease; UBE2A, ubiquitin conjugating enzyme E2 A; RARA/GCK-3β, retinoic acid receptor alpha/glycogen synthase kinase-3β; and RTF3, runt-related transcription factor 3.

### Table 2.

Functional mechanism of cirRNAs in neurological disease.

with multiple protein subunits, thus acting as "scaffolding" for RBPs [7, 98]. Thereby, it facilitates the interaction by potentially increasing the stability of the circRNA transcripts. Due to its multiple functions in brain, researchers have suggested that ciRS-7 can be a potential biomarker for neurodegenerative disorders including AD and PD [83].

## 5.1 circRNA in Alzheimer's disease

which play roles in neurogenesis, neuronal development, and neuronal differentiation [11, 74], (ii) the expression levels of circRNAs are higher in brain than other tissues [75, 76], (iii) due to the slow division rates of neurons, circRNAs may accumulate more in the brain than other tissues [77], (iv) neuronal genes contain long introns (>10 kb) with inverted repeat sequences, thereby simplifying formation of circRNAs [10], and (v) circRNAs due to the absence of 5<sup>0</sup> and 3<sup>0</sup> ends result in greater stability than linear RNAs, leading to a relatively longer half-life [78]. The half-life of circRNAs is approximately 20 h, compared with corresponding linear

The latest studies have shown that circRNAs could attenuate cell senescence and cell survival and may be involved in the regulation of aging and age-related neurological diseases [80–82]. Thus, circRNAs are expected to be new potential biomarkers and target for aging and age-related neurological diseases (Table 2). These studies have suggested that circRNAs may play an important role in pathological mammalian brain function, which is implicated in disorders in central nervous system (CNS) including Alzheimer's disease (AD), Parkinson's disease (PD), neu-

CDR1as, a circRNA, is highly plentiful and specifically expressed in the mammalian brain [85]. Some studies have indicated that ciRS-7 contains multiple antimiR-7 sequences. This suggests that ciRS-7 may function as a sponge to sequester the normal functions of miR-7 [57, 95–97]. ciRS-7 can regulate the stability of mRNA targets in the brain by binding to miR-7 [78, 85]. Besides, ciRS-7 can interact

Possible mechanisms Ref

[83, 84]

[85–87]

[88]

[78, 89, 90]

[91, 92]

[93, 94]

[53]

downregulate AD relevant targets, such as ubiquitin conjugating enzyme UBE2A, which play an essential role in the clearance

memory ability and is increased in AD, and it promotes tau phosphorylation by targeting the RARA/GCK-3β pathway

expression, promotes the degradation of αsynuclein mRNA levels, and protects cells

miRNA deregulation and affects brain

Hsa-circRNA 2149 has been detected in

miR-138 can balance the ratio of Th1 and Th2 via suppressing the function of RTF3

chronic CD28-associated CD8 (+) T cell

of amyloid peptides

against oxidative stress

expression of ciRS-7

CD19+ leukocytes

function

circRNA100783 CircRNA100783 may be involved in

aging AD, Alzheimer's disease; PD, Parkinson disease; UBE2A, ubiquitin conjugating enzyme E2 A; RARA/GCK-3β, retinoic acid receptor alpha/glycogen synthase kinase-3β; and RTF3, runt-related transcription factor 3.

ropsychiatric disorders, prion disease, and inflammatory neuropathy.

ciRS-7 miR-7 AD ciRS-7 is reduced in AD, and miR-7 can

circSry miR-138 AD mir-138 participate in learning and

ciRS-7 miR-7 PD miR-7 may downregulate α-synuclein

Neuropsychiatric disorders

neuropathy

neuropathy

— Inflammatory

circSry miR-138 Inflammatory

Functional mechanism of cirRNAs in neurological disease.

ciRS-7 miR-7 Prion disease Prion protein PrPc can upregulate

isoforms (no more than 8 h) [79].

Gene Expression and Phenotypic Traits

CircRNA Target Neurological

ciRS-7 miR-7

hsacircRNA 2149

Table 2.

92

miR-671

disease

Alzheimer's disease is a chronic neurological disease. Lukiw et al. showed that the expression level of ciRS-7 is decreased in hippocampal CA1 region in sporadic AD [83]. Functional deficiency of ciRS-7 can lead to upregulation of miR-7 in AD brain and may cause the downregulation of several AD-relevant mRNA targets, including the ubiquitin conjugating enzyme E2A (UBE2A) [83, 84, 99, 100]. This autophagic protein, UBE2A, is a central effector in the ubiquitination cycle. UBE2A is crucial for clearing amyloid peptides via phagocytosis and contributes to amyloidogenesis [99]. In contrast to the previous studies, Shi et al. have shown that ciRS-7 promotes the degradation of amyloid precursor protein (APP) and betasecretase 1 (BACE1) in an nuclear factor kappa beta (NF-κB)-dependent manner [101]. Hence, future studies are needed to reveal ciRS-7 function/functions and its exact role in AD pathology.

CircSry can serve as a miRNA sponge in neural cells. CircSry inhibits miR-138 [53, 85], which is a potential molecular regulator of human memory function [102]. CircSry has multiple binding sites for miR-138 and promotes tau phosphorylation by targeting the "retinoic acid receptor alpha/glycogen synthase kinase-3β" (RARA/ GCK-3β) pathway [86]. Some studies have indicated that miR-138 influences learning and memory abilities by regulating acyl protein thioesterase 1 [87, 102]. Therefore, association of circSry and miR-138 in AD should be further investigated.

## 5.2 circRNA in Parkinson's disease

Parkinson disease, progressive age-related neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta [103, 104]. To date, five genes have been determined to cause PD, such as αsynuclein (SNCA), parkin, dj-1, PTEN-induced kinase 1 (pink1), and leucine-rich repeat kinase 2 (lrrk2) [105]. SNCA is the key player in the pathogenesis of PD based on neuropathologic, genetic, and cellular evidence [106]. The overexpression and aggregation of SNCA, a target gene of miR-7, is considered as a distinctive marker in PD [107, 108]. miR-7 has been proposed to play a role in PD by reducing expression of SNCA [88]. ciRS-7 plays a protective role by inhibiting miR-7 that directly regulates the expression of SNCA [109]. miR-7 alleviates SNCA expression dose-dependently and induces the degradation of SNCA mRNA levels [88]. These results suggest that ciRS-7 serves as a miR-7 sponge in vitro. Furthermore, the silencing of ciRS-7 increases miR-7 activity and decreases the expression of miR-7 target genes [110]. In addition, circSNCA, another circRNA, can sponge miR-7, thereby regulating expression of SNCA, resulting in decreased autophagy and increased apoptosis in cells [111]. These findings are in concordance with the results of a study, which showed that autophagy can prevent PD [112], and that of the other study, which demonstrated that apoptosis is related to PD [113].

## 5.3 circRNA and inflammatory neuropathy

circRNAs may participate in inflammatory reactions that induce neuropathy. Some circRNAs may affect immune responses due to the fact that they contain virus miRNA binding sites. For instance, hsa-circRNA 2149 contains 13 unique, head to

tail spanning reads. Researchers discovered hsa-circRNA 2149 in CD19+ leukocytes, but not CD341 leukocytes or neutrophils. On the other hand, circRNA100783 may be involved in chronic CD28-related CD8(+) T cell aging and for this reason could be a novel biomarker for this conditions [93]. Furthermore, circSry, another circRNA, can repress miR-138 activity, which could balance T helper 1 (Th1) and T helper 2 (Th2) expressions through suppressing the function of runt-related transcription factor 3 (RUNX3) [94].

Disease CircRNA Regulation miRNA

DOI: http://dx.doi.org/10.5772/intechopen.88764

SLE Hsa\_circ\_102584 ↑ miR-766-3p

Hsa\_circ\_400011 ↑ miR-296-3p

Circular RNAs and Its Biological Functions in Health and Disease

Hsa\_circ\_101471 ↑ miR-328-5p

Hsa\_circ\_100226 ↓ miR-30b-3p

Hsa\_circ\_003524 ↑ — Hsa\_circ\_101873 ↑ — Hsa\_circ\_103047 ↑ —

RA Hsa\_circ\_104871 ↑ — It serves as potential

sponge targets

miR-762 miR-412-3p let-7i-3p miR-431-3P

miR-146b-3p miR-181d-3p miR-504-3p

miR-136-5p miR-665 miR-486-3p miR-601

miR-138-5p miR-145-3p miR-24-3p miR-620 miR-875-3p CDR1as/ciRS-7 ↓ — It functions as the miR-7

Hsa\_circ\_0057980 ↓ miR-181d It functions as the miR-181d

Hsa\_circ\_0088088 ↓ miR-16 It functions as the miR-16

Hsa\_circ\_0001045 ↑ miR-30a It functions as the miR-30a

Hsa\_circ\_0035560 ↓ — It arranges negatively the

GSDMB ecircRNA ↑ miR-1275 It functions as the miR-1275

MS Hsa\_circ\_0005402 ↓ — It can be improved as MS

PBC Hsa\_circ\_402458 ↑ miR-522-3p It may be appropriate for

95

Potential functions Ref

[117]

[100, 118]

[119]

[86, 120]

[121–123]

[124]

[124, 125]

[121, 126]

[127]

It may be improved as novel noninvasive biomarkers for

sponge to increase expression of PTEN and restricts hyperresponsiveness of B cells

biomarkers for diagnosis and performs severity or pathological course of RA

sponge to suppress the development of RA

sponge to suppress the development of RA

sponge to promote the biogenesis of RA

biomarkers

induce MS

miR-149 Both circRNAs are derived from the ANXA2

PBC diagnosis

miR-943 It functions as the miR-522

biogenesis of MS

and miR-149 sponges to

and miR-943 sponges to counter chronic

SLE

#### 5.4 circRNA and prion diseases

Most prion diseases are infectious via transmissible particles composed of prion protein in scrapie (PrPSc), an isomer of noninfectious cellular prion protein (PrPc). Studies have discovered that ciRS-7 expression is induced by PrPc overexpression [91, 92]. ciRS-7 may suppress miR-7 activity and therefore ciRS-7 may be involved in the prion disease pathogenesis.

## 5.5 circRNA and neuropsychiatric disorders

Apart from in brain tumors, ciRS-7 may also play a role in neuropsychiatric disorders. Increased miR-7 levels have been determined in neuropsychiatric disorders, serving as a proof for ciRS-7-mediated deregulation of dendritic spine density via a miR-7-SHANK3 (SH3 and multiple ankyrin repeat domains 3) axis [89, 90]. In recent study, Piwecka et al. showed that ciRS-7 knockout mice display behavioural phenotypes related to neuropsychiatric disorders. Deleting of ciRS-7 locus in mice leads to synaptic transmission function disorder and unusual neuropsychiatric-like behavior [78]. Other than miR-7, ciRS-7 also has a binding site to miR-671, which is deregulated in all brain regions in ciRS-7 deficient mice; however, the direction of changes was opposite. It is designated that the binding site on ciRS-7 is completely complementary to miR-671, and the interaction of these two molecules could lead to AGO-mediated ciRS-7 slicing and miR-671 deterioration. On the contrary, the binding sites on ciRS-7 are partial complementaries to miR-7. For this reason, it is likely that circRNAs can serve as a platform to store and transport certain miRNAs [78, 89, 90].

Currently, circRNA studies in the CNS are in progress. To date, there is a limited number of circRNA identified in neurological disorders. Moreover, previous studies mainly focus on ciRS-7 function. Therefore, it is still needed to identify candidate circRNAs as a potential biomarker in neurological disease. In addition, their functional properties in neuronal cells should be also validated in further studies.

## 6. The role of circRNAs in immune regulation

Although many circRNAs are under survey, their roles in autoimmune diseases remain incomprehensible, and there are insufficient data to determine their exact role of circRNAs in such diseases [24, 114].

The connection between miRNAs and immunity has been well-studied, which has led to the hypothesis that circRNAs may contribute to immune regulation by interacting with miRNAs. In particular, due to their abilities to serve as miRNA and protein sponges, they can regulate gene expression and encode proteins. Therefore, circRNAs can participate in the development and progression of different immune responses and immune diseases [23, 24, 114]. On the basis of the current studies, the majority of circRNAs defined in autoimmune diseases are ecircRNAs, and a few are


tail spanning reads. Researchers discovered hsa-circRNA 2149 in CD19+ leukocytes, but not CD341 leukocytes or neutrophils. On the other hand, circRNA100783 may be involved in chronic CD28-related CD8(+) T cell aging and for this reason could be a novel biomarker for this conditions [93]. Furthermore, circSry, another circRNA, can repress miR-138 activity, which could balance T helper 1 (Th1) and T helper 2 (Th2) expressions through suppressing the function of runt-related tran-

Most prion diseases are infectious via transmissible particles composed of prion protein in scrapie (PrPSc), an isomer of noninfectious cellular prion protein (PrPc). Studies have discovered that ciRS-7 expression is induced by PrPc overexpression [91, 92]. ciRS-7 may suppress miR-7 activity and therefore ciRS-7 may be involved

Apart from in brain tumors, ciRS-7 may also play a role in neuropsychiatric disorders. Increased miR-7 levels have been determined in neuropsychiatric disorders, serving as a proof for ciRS-7-mediated deregulation of dendritic spine density via a miR-7-SHANK3 (SH3 and multiple ankyrin repeat domains 3) axis [89, 90]. In recent study, Piwecka et al. showed that ciRS-7 knockout mice display behavioural phenotypes related to neuropsychiatric disorders. Deleting of ciRS-7 locus in mice leads to synaptic transmission function disorder and unusual neuropsychiatric-like behavior [78]. Other than miR-7, ciRS-7 also has a binding site to miR-671, which is deregulated in all brain regions in ciRS-7 deficient mice; however, the direction of changes was opposite. It is designated that the binding site on ciRS-7 is completely complementary to miR-671, and the interaction of these two molecules could lead to AGO-mediated ciRS-7 slicing and miR-671 deterioration. On the contrary, the binding sites on ciRS-7 are partial complementaries to miR-7. For this reason, it is likely that circRNAs can serve as a platform to store and transport certain miRNAs

Currently, circRNA studies in the CNS are in progress. To date, there is a limited number of circRNA identified in neurological disorders. Moreover, previous studies mainly focus on ciRS-7 function. Therefore, it is still needed to identify candidate circRNAs as a potential biomarker in neurological disease. In addition, their functional properties in neuronal cells should be also validated in further studies.

Although many circRNAs are under survey, their roles in autoimmune diseases remain incomprehensible, and there are insufficient data to determine their exact

The connection between miRNAs and immunity has been well-studied, which has led to the hypothesis that circRNAs may contribute to immune regulation by interacting with miRNAs. In particular, due to their abilities to serve as miRNA and protein sponges, they can regulate gene expression and encode proteins. Therefore, circRNAs can participate in the development and progression of different immune responses and immune diseases [23, 24, 114]. On the basis of the current studies, the majority of circRNAs defined in autoimmune diseases are ecircRNAs, and a few are

scription factor 3 (RUNX3) [94].

Gene Expression and Phenotypic Traits

5.4 circRNA and prion diseases

in the prion disease pathogenesis.

[78, 89, 90].

94

5.5 circRNA and neuropsychiatric disorders

6. The role of circRNAs in immune regulation

role of circRNAs in such diseases [24, 114].


MS, multiple sclerosis; PBC, primary biliary cirrhosis; RA, rheumatoid arthritis; SCID, severe combined immunodeficiency disease; SLE, systemic lupus erythematosus; and WAS, Wiskott-Aldrich syndrome.

#### Table 3.

circRNAs are associated with immune diseases.

ciRNAs and eIciRNAs [23, 24, 114–116]. The circRNAs identified to date, their functions, and roles in immunological diseases are shown in Table 3.

It will be important in future studies to determine biological functions of circRNAs in immune cells. circRNAs may serve as both potential biomarkers and immune regulators [23, 24, 114–116]. Hence, it may be helpful to improve our understanding of the molecular biological basis of autoimmune diseases.

## 7. circRNAs in cancer

Cancer is one of the most common causes of death in worldwide. As stated in world cancer report (2014), 10 million people of the world develop all types of cancer each year. Moreover, over 6 million patients around the world die from this disease annually [132]. Unfortunately, the number of patients diagnosed with cancer is increasing and is estimated to increase in future in worldwide [133, 134]. Even if, a functional improvement in the treatment approach is established, and new therapeutic strategies are still needed for therapy of cancer. Therefore, the identification of the altered pathways and gene transcripts has been the subject of researches recently. miRNAs have a role in gene regulation and affect various molecular biological processes such as cell growth, development, differentiation, proliferation, and cell death [135]. As circRNAs interact with miRNAs and then influence the mRNA expression levels of target genes, the identification of circRNA-miRNA-mRNA network has become the objective of cancer researches.

There are numerous investigations on circRNAs and their functions in cancer as compared with other diseases. To date, most of the studies have focused on miRNA sponge function of circRNAs. miRNAs have been classified depending on the effect of miRNAs on downstream target/targets [136]. miRNAs can act as oncogenes or tumor suppressors during carcinogenesis [137]. Likewise, circRNAs are also named according to their behaviour during tumorigenesis. While some circRNAs contribute to tumor progression and metastasis, the others suppress oncogenesis.

CircRNA

97

circRNA

Target miRNA

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

expression

status

expression

signaling

pathway

status

expression

status

hsa\_circ\_0001946

↓

hsa-miR-7-5p,

hsa-miR-1270,

3156-5p

↑

miR-135a-5p

↓

SIRT1

↑

Compared to pairs of adjacent nontumor tissues,

expression of

adenocarcinoma

The lung nonmalignant

The increase in

samples is an

patients with lung

TNM stages

> circAGFG1

hsa\_circRNA\_102984

↓

miR-96-5p

↑

RASSF8/

↑

Compared to pairs of adjacent nontumor tissues,

expression of

downregulated

circPTPRA

 acts as a miR-96-5p sponge, and it leads to

upregulation

xenograft model

 of RASSF8 levels in both in vitro and H23

 in 34 NSCLC tissues

hsa\_circRNA\_102984

(circPTPRA)

 is

[141]

e-cadherin

(circPTPRA)

circ\_0020123

↑

miR-488e3p

↓

ADAM9

↑

Compared to pairs of adjacent nontumor tissues,

expression of

tissues

circ\_0020123

 is upregulated

 in 55 NSCLC

[142]

↑

miR-203

↓

ZNF281

↑

Compared to pairs of adjacent nontumor tissues,

expression of circAGFG1

tissues

circAGFG1

proliferation

 of NSCLC

 enhances

ZNF281-mediated

 migration and

 is upregulated

 in 20 NSCLC

[140]

independent

 prognostic factor for the

adenocarcinoma

 as well as advanced

 human lung epithelial cell line

circ\_0001946

 expression in tumor

adenocarcinoma

 cell lines compared with the

circ\_0001946

 expression is upregulated

 in the four

 tissues

circ\_0001946

 is upregulated

 in 72 lung

Circular RNAs and Its Biological Functions in Health and Disease

[139]

 hsa-miR-

hsa-miR-671-5p,

↑

NER signaling

Activated

 Compared to pairs of adjacent nontumor tissues,

expression of

NSCLC tissues There was a decrease in

on the with the parental A549 cells

cisplatin-resistant

A549/CDDP

 cells compared

DOI: http://dx.doi.org/10.5772/intechopen.88764

hsa\_circ\_0001946

 is hsa\_circ\_0001946

 expression

downregulated

 in 43

[138]

pathway


## Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

ciRNAs and eIciRNAs [23, 24, 114–116]. The circRNAs identified to date, their

sponge targets

SCID Circ-CDC42BPA ↑ — It disrupts transduction of B

Circ-TNFRSF11A ↑ — It attends in the SCID-

WAS Circ-ROBO1 ↑ — It activates the pathogenesis

MS, multiple sclerosis; PBC, primary biliary cirrhosis; RA, rheumatoid arthritis; SCID, severe combined immunodeficiency disease; SLE, systemic lupus erythematosus; and WAS, Wiskott-Aldrich syndrome.

Circ-CDC42BPA ↑ — It disrupts transduction of B

Potential functions Ref

[128, 129]

[128, 130]

[128, 131]

[128]

inflammation and aberrant TGF-β signalling of PBC

cell signalling to induce formation of SCID

mediated alteration of different signalling pathways

cell signalling to induce formation of WAS

of WAS

It will be important in future studies to determine biological functions of circRNAs in immune cells. circRNAs may serve as both potential biomarkers and immune regulators [23, 24, 114–116]. Hence, it may be helpful to improve our understanding of the molecular biological basis of autoimmune diseases.

Cancer is one of the most common causes of death in worldwide. As stated in world cancer report (2014), 10 million people of the world develop all types of cancer each year. Moreover, over 6 million patients around the world die from this disease annually [132]. Unfortunately, the number of patients diagnosed with cancer is increasing and is estimated to increase in future in worldwide [133, 134]. Even if, a functional improvement in the treatment approach is established, and new therapeutic strategies are still needed for therapy of cancer. Therefore, the identification of the altered pathways and gene transcripts has been the subject of researches recently. miRNAs have a role in gene regulation and affect various molecular biological processes such as cell growth, development, differentiation, proliferation, and cell death [135]. As circRNAs interact with miRNAs and then influence the mRNA expression levels of target genes, the identification of circRNA-miRNA-mRNA network has become the objective of

There are numerous investigations on circRNAs and their functions in cancer as compared with other diseases. To date, most of the studies have focused on miRNA sponge function of circRNAs. miRNAs have been classified depending on the effect of miRNAs on downstream target/targets [136]. miRNAs can act as oncogenes or tumor suppressors during carcinogenesis [137]. Likewise, circRNAs are also named according to their behaviour during tumorigenesis. While some circRNAs contrib-

ute to tumor progression and metastasis, the others suppress oncogenesis.

functions, and roles in immunological diseases are shown in Table 3.

Disease CircRNA Regulation miRNA

Gene Expression and Phenotypic Traits

7. circRNAs in cancer

circRNAs are associated with immune diseases.

Table 3.

cancer researches.

96


CircRNA

99

circRNA

Target miRNA

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

expression

status

expression

signaling

pathway

status

↓ ↓

TCTN1 and

↑

Compared to pairs of adjacent nontumor tissues,

expression of

NSCLC tissues

The four NSCLC cell lines compared with the

human bronchial epithelial cells

Circ\_0026134

 expression is upregulated

 in the nonmalignant

DOI: http://dx.doi.org/10.5772/intechopen.88764

Circ\_0026134

 is upregulated

 in 30

[146]

GAGE1

expression

status

circ\_0026134 Circ-FOXM1

↑

miR-1304-5p

↓

PPDPF and

↑

Compared to pairs of adjacent nontumor tissues,

expression of

tissues

The NSCLC cell lines compared with the

human bronchial epithelial cells

The increase in samples was correlated with short overall survival rate

in NSCLC patients

circ-FOXM1

 expression in tumor

Circ-FOXM1

 expression is upregulated

 in the four

nonmalignant

Circ-FOXM1

 is upregulated

 in 80 NSCLC

Circular RNAs and Its Biological Functions in Health and Disease

[147]

MACC1

(hsa\_circ\_0025033)

circ\_0003645

↑

miR-1179

↓

TMEM14A

↑

Compared to pairs of adjacent nontumor tissues,

expression of

tissues

The

NSCLC cell lines compared with the

human bronchial epithelial cells

The increase in

samples is an

patients with NSCLC as well as advanced TNM stages

hsa\_circ\_0002360

circRNA 100146

↑

miR-361-3p

↓

SF3B3

↑

Compared to pairs of adjacent nontumor tissues,

expression of circRNA 100146 is upregulated

NSCLC tissues

 in 40

[150]

miR-615-5p

↑

hsa-mir-3620-5p

↓

PHF19

↑

Compared to pairs of adjacent nontumor tissues,

expression of

lung

adenocarcinoma

 tissues

hsa\_circ\_0002360

 is upregulated

 in 18

[149]

independent

 prognostic factor for the

circ\_0003645

 expression in tumor

circ\_0003645

 expression is upregulated

 in the four

nonmalignant

circ\_0003645

 is upregulated

 in 59 NSCLC

[148]

↑

miR-1256 miR-1287


### Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

CircRNA

98

circRNA

Target miRNA

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

expression

status

The NSCLC cell lines compared with the

human bronchial epithelial cells

The increase in samples has been correlated with short overall survival

rate in NSCLC patients

> ↑

miR-144

↓

ZEB1

↑

Compared to pairs of adjacent nontumor tissues,

expression of

80 NSCLC tissues

Upregulation

samples has been correlated with short overall survival

in NSCLC patients

The six lung cancer cell lines

> circVANGL1

↑

miR-195

↓

Bcl2

↑

Compared to pairs of adjacent nontumor tissues,

expression of

tissues

The NSCLC cell lines compared with the

human bronchial epithelial cells

Upregulation

stage, bigger tumor size, and shorter overall survival in

NSCLC patients

hsa\_circRNA\_102231

↑

miR-133a-5p

↓

Vimentin

↑

Compared to pairs of adjacent nontumor tissues,

expression of circP4HB is upregulated

tissues

Upregulation

metastatic capacity and shorter survival in NSCLC

patients

 of circP4HB expression leads to higher

 in 80 NSCLC

[145]

(hsa\_circ\_0046263)

(named as circP4HB)

 of

circVANGL1

 expression leads to higher

circVANGL1

 expression is upregulated

 in the five

nonmalignant

circVANGL1

 is upregulated

 in 95 NSCLC

[144]

hsa\_circ\_0020123

 expression is upregulated

 in the

 of

hsa\_circ\_0020123

 expression in tumor

hsa\_circ\_0020123

 is upregulated

 in

[143]

EZH2

circ\_0020123

 expression in tumor

Gene Expression and Phenotypic Traits

circ\_0020123

 expression is upregulated

 in the four

nonmalignant

expression

signaling

pathway

status

expression

status


CircRNA

101

circRNA

Target miRNA

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

expression

status

Upregulation

samples predicted lower Survival rate

 of circ-BANP expression in tumor

expression

signaling

pathway

status

expression

status

hsa\_circRNA\_103595

↑

miR-1275

↓

FOXK1

↑

Compared to pairs of adjacent nontumor tissues,

expression of

The lung cancer cell lines compared with the

human lung epithelial cells

circMAN2B2

 expression is upregulated

circMAN2B2

 is upregulated

 in 41 NSCLC

[156]

 in the four

DOI: http://dx.doi.org/10.5772/intechopen.88764

nonmalignant

circMAN2B2

circ\_0016760 NER, nucleotide excision repair; NSCLC, nonsmall cell lung cancer; CDDP, cisplatin; SIRT1, sirtuin 1; AGFG1, ArfGAP with FG repeats 1; ZNF281, zinc finger protein 281; PTPRA, protein tyrosine

phosphatase

polycomb repressive complex 2 subunit; VANGL1, VANGL planar cell polarity protein 1; BCL2, B-cell

GAGE1, G antigen 1; FOXM1, forkhead box M1; PPDPF, pancreatic progenitor cell

protein 14A; PHF19, PHD finger protein 19; SF3B3, splicing factor 3b subunit 3; FGFR3, fibroblast growth factor receptor 3; DKK, Dickkopf WNT signaling pathway inhibitor 1; PTK2, protein tyrosine

kinase 2; TIF1γ, transcription

nuclear protein; LARP1, La

Table 4. The expression profile of

circRNA-miRNA-mRNA

 network in lung cancer tissues.

 intermediary

 factor 1-gamma; TGF-β, tumor growth factor beta; EMT,

ribonucleoprotein

 domain family member 1; MAN2B2, mannosidase

 receptor type A; RASSF8, ras association domain family member 8; ADAM9, ADAM

↑

miR-1287

↓

GAGE1

↑

Compared to pairs of adjacent nontumor tissues,

expression of

The lung cancer cell lines compared with the

human bronchial epithelial cells

Upregulation

samples predicted short overall survival in NSCLC

patients

metallopeptidase

 domain 9; ZEB1, zinc finger E-box binding

CLL/lymphoma

differentiation

 and proliferation

 2; P4H1, prolyl

 factor; MACC1,

epithelial-mesenchymal

 alpha class 2B member 2; FOXK1, forkhead box K1; and GAGE, G antigen 1.

 transition; TCF21, transcription

 factor 21; BANP, BANP BTG3 associated

4-hydroxylase

metastasis-associated

 in colon cancer 1; TMEM14A,

transmembrane

 subunit beta; TCTN1, tectonic family member 1;

homeobox 1; EZH2, enhancer of zeste 2

 of

circ\_0016760

 expression in tumor

circ\_0016760

 expression is upregulated

circ\_0016760

 is upregulated

 in 83 NSCLC

[157]

Circular RNAs and Its Biological Functions in Health and Disease

 in the four nonmalignant


 in lung cancer tissues.

CircRNA

100

circRNA

Target miRNA

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

expression

status

expression

signaling

pathway

status

↓

Gal-1 Akt and Erk 1/2

signaling

pathway

↑

Compared to pairs of adjacent nontumor tissues,

expression of circFGFR3 is upregulated

Activated

tissues

The increase in circFGFR3 expression in tumor samples is correlated with the poor prognosis of NSCLC patients

 in 63 NSCLC

[151]

expression

status

circFGFR3 hsa\_circ\_0006427

↓

miR-6783-3p

↑

DKK1

↑

Compared to pairs of adjacent nontumor tissues,

expression of

adenocarcinoma

The four lung nonmalignant

The decrease in circFGFR3 expression in tumor samples

is correlated with the poor prognosis of lung

adenocarcinoma

hsa\_circ\_0008305

↓

miR-429

miR-200b-3p

↑

TIF1γ

↓

circPTK2 has an important role in regulating TGF-β-

[153]

induced EMT and tumor metastasis Compared to pairs of adjacent nontumor tissues,

expression of

NSCLC

The the six lung cancer cell lines compared with the the

nonmalignant

Downregulation

tumor samples is correlated with TNM stage and

lymphoid node metastases

> circ-BANP

↑

miR-503

↓

LARP1

↑

Compared to pairs of adjacent nontumor tissues,

expression of circ-BANP is upregulated The circ-BANP expression is upregulated lung cancer cell lines compared with the

human bronchial epithelial cells

 in 59 NSCLC

[155]

 in the four nonmalignant

 of

hsa\_circ\_100395

 expression in

 human bronchial epithelial cells

hsa\_circ\_100395

 expression is

downregulated

 in

hsa\_circ\_100395

 is

downregulated

 in 69

[154]

circPTK2

hsa\_circ\_100395

↓

miR-1228

↑

TCF21

 patients

 human lung epithelial cell line

circ\_0006427

 expression is

adenocarcinoma

 cell lines compared with the

downregulated

 in the

circ\_0006427

 is

downregulated

 in 94 lung

[152]

Gene Expression and Phenotypic Traits

Wnt/b-catenin

Inactivated

signaling

pathway

↑

miR-22-3p

## Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764


CircRNA

103

circRNA

Target

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

expression

status

The increase in circEPSTI1 positively correlated with tumor size, lymph node infiltration and

TNM stage, and associated with poor prognosis

 expression in tumor samples was

signaling pathway

miRNA

expression

status

expression

status

hsa\_circ\_0008039

hsa\_circ\_0007534

circRNA-000911

hsa\_circ\_0001846

↑

miRNA-661 ↓

MTA1

↑

Compared to pairs of adjacent nontumor tissues, expression of

circ-UBAP2

circ-UBAP2

with nonTNBC cell lines The increase in circ-UBAP2 correlated with reduced OS in TNBC patients

 expression in tumor samples has been

 expression increased in TNBC cell lines compared

 is upregulated

 in 78 TNBC tissues

circ-UBAP2

circRNA\_0005505

↑

miR-3607

↓

FOXC1

↑

Compared to pairs of adjacent nontumor tissues, expression of

CircIRAK3 is upregulated CircIRAK3 expression increased in TNBC cell lines compared with

normal mammary epithelial or ER-positive The increase in CircIRAK3 expression in tumor samples has been

correlated worse

recurrence-free

 survival in breast cancer patients [169]

 cell lines

 in 35 BCa tissues

[168]

circIRAK3

circ\_0005230

↑

miR-618

↓

CBX8

↑

Compared to pairs of adjacent nontumor tissues, expression of

circ\_0005230

 is upregulated

 in 76 BCa tissues

↓

miR-449a

↑

Notch1 NF-κB pathway

↓

 Activated

Compared to pairs of adjacent nontumor tissues, expression of

circRNA-000911

hsa\_circRNA\_000911

compared with

 expression decreased in the six BCa cell lines

nonmalignant

 breast epithelial cell line

[167]

 is

downregulated

 in 35 BCa tissues

[166]

↑

miR-593

↓

MUC19

↑

Compared to pairs of adjacent nontumor tissues, expression of

hsa\_circ\_0007534

hsa\_circ\_0007534

compared with

 expression increased in the five BCa cell lines

nonmalignant

 breast epithelial cell line

 is upregulated

 in 40 BCa tissues

[165]

Circular RNAs and Its Biological Functions in Health and Disease

 ↑

miR-432-5p ↓

E2F3

↑

Compared to pairs of adjacent nontumor tissues, expression of

hsa\_circ\_0008039

hsa\_circ\_0008039

compared with

 expression increased in the six BCa cell lines

nonmalignant

 breast epithelial cell line

 is upregulated

 in 38 TNBC tissues

[164]

DOI: http://dx.doi.org/10.5772/intechopen.88764


## Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

CircRNA

102

circRNA

Target

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

[158]

expression

status

signaling pathway

miRNA

expression

status

expression

status

circ\_0006528

circKIF4a

↑

miR-375

↓

KIF4A

↑

Compared to pairs of adjacent nontumor tissues, expression of

circKIF4A is upregulated circKIF4A expression increased in the five TNBC cell lines

compared with the four NTNBC and

epithelial cell line

The increase in circKIF4A expression in tumor samples has been

correlated with worse outcome of TNBC patients

nonmalignant

 breast

 in 57 TNBC tissues

[159]

Gene Expression and Phenotypic Traits

(hsa\_circ\_0007255)

hsa\_circ\_0004771

circTADA2A-E6

circAGFG1 hsa\_circ\_000479

↑

miR-4753

↓

BCL11A

↑

Compared to pairs of adjacent nontumor tissues, expression of

circEPSTI1

 is upregulated

 in 10 TNBC tissues

[163]

miR-6809

↑

miR-195-5p ↓

CCNE1

↑

Compared to adjacent nontumor tissues, expression of circAGFG1

[162]

is upregulated

 in TNBC tissues

circAGFG1

compared with

The expression levels of circAGFG1

overall survival of patients with TNBC

nonmalignant

 breast epithelial cell line

 were reversely correlated with

 expression increased in the six TNBC cell lines

↓

miR-203a-3p

↑

SOCS3

↓

Compared to adjacent nontumor tissues, expression of Hsa

circTADA2A-E6

The decline in Hsa

was associated with poor patient survival for TNBC

 is

downregulated

circTADA2A-E6

 expression in tumor samples

 in TNBC tissues

[161]

 ↑

miR-653

↓

ZEB2

↑

Compared to pairs of adjacent nontumor tissues, expression of hsa

circ 0004771 is upregulated hsa circ 0004771 expression increased in the five BCa cell lines

compared with

The increase in hsa circ 0004771 expression in tumor samples has

been correlated poorer survival prognosis

nonmalignant

 breast epithelial cell line

 in BCa tissues

[160]

↑

miR-7-5p

↓

Raf1 MAPK/ERK

signaling pathway

↑

Compared to adjacent nontumor tissues, expression of

circ\_0006528

The increase in

Activated

been correlated with advanced TNM stage and poor prognosis

 is upregulated

 in BCa tissues

circ\_0006528

 expression in tumor samples has



 1B; USF1, upstream transcription

 factor 1; GFRA1,

Studies on altered expression of circRNAs in (lung and breast cancer) tumor samples are summarized in Tables 4 and 5. Moreover, in these selected studies, the

By taking all studies together, circRNAs may be candidate surrogate molecular markers for cancer in different aspects, such as angiogenesis, metastasis, and drug resistance. Although to date some circRNA-miRNA-mRNA axis is predicted in cancerassociated pathways, the function and importance of dysregulated circRNAs still need

With the increasing interest in circRNAs, comprehensive circRNA databases are required for prediction of circRNAs and their targets [172]. To evaluate and simplify the properties and interaction of various circRNAs with other RNAs from different aspects, numerous databases have been published (circlncRNAnet, starBase v2. 0, circBase, circRNABase, circ2Traits, nc2Cancer, DeepBase v2. 0, CircInteractome, TSCD, CIRCpedia, circRNADb, CircNet, CircR2Disease, circBank, and so on) [173]. Examples of circRNA databases and their usage in

• starBase v2. 0 determines miRNA-circRNA interactome and includes miRNA,

• circ2Traits can be provided information about miRNA-circRNA interaction

• CircInteractome can be used in coupling the circRNA with related RNA-

• TSCD is helpful to describe tissue-specific circRNAs in mouse and human

• CIRCpedia includes reverse and variable splicing sites of circRNAs from

• circBank can be a resource to facilitate the research of function and regulation

In summary, circRNAs, a new class of noncoding RNAs, are widely investigated by researchers due to their role in post transcriptional gene regulation. Recent studies have indicated their effects on the development of diverse diseases by acting as a miRNA sponge, RBP sponge, and transcriptional modulator or direct encoding proteins. Although the miRNA sponge function of circRNAs is currently investigated in the diseases, other mechanisms of circRNAs are still under investigation, and further studies are needed. After the interpretation of their function in disease pathogenesis, they may have a potential to become a drug target. Using circRNAs as biomarkers or therapeutic targets needs to be further investigated due to their complex roles. Based on these characteristics, circRNAs are likely to guide the development of new diag-

nostic and therapeutic strategies as well as prevention of diseases.

to be supported in larger numbers of samples and patients, in various cancers.

circRNA-miRNA-mRNA interaction network is well defined.

Circular RNAs and Its Biological Functions in Health and Disease

8. Research databases of circRNA

DOI: http://dx.doi.org/10.5772/intechopen.88764

mRNA, and lncRNA information [174].

individuals and mouse samples [177].

and its association with particular diseases [109].

researches are shown.

binding proteins [175].

genomes [176].

of circRNAs [178].

9. Conclusion

105

#### Table 5.

The expression profile of circRNA-miRNA-mRNA network in breast cancer tissues.

Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

Studies on altered expression of circRNAs in (lung and breast cancer) tumor samples are summarized in Tables 4 and 5. Moreover, in these selected studies, the circRNA-miRNA-mRNA interaction network is well defined.

By taking all studies together, circRNAs may be candidate surrogate molecular markers for cancer in different aspects, such as angiogenesis, metastasis, and drug resistance. Although to date some circRNA-miRNA-mRNA axis is predicted in cancerassociated pathways, the function and importance of dysregulated circRNAs still need to be supported in larger numbers of samples and patients, in various cancers.

## 8. Research databases of circRNA

With the increasing interest in circRNAs, comprehensive circRNA databases are required for prediction of circRNAs and their targets [172]. To evaluate and simplify the properties and interaction of various circRNAs with other RNAs from different aspects, numerous databases have been published (circlncRNAnet, starBase v2. 0, circBase, circRNABase, circ2Traits, nc2Cancer, DeepBase v2. 0, CircInteractome, TSCD, CIRCpedia, circRNADb, CircNet, CircR2Disease, circBank, and so on) [173]. Examples of circRNA databases and their usage in researches are shown.


## 9. Conclusion

In summary, circRNAs, a new class of noncoding RNAs, are widely investigated by researchers due to their role in post transcriptional gene regulation. Recent studies have indicated their effects on the development of diverse diseases by acting as a miRNA sponge, RBP sponge, and transcriptional modulator or direct encoding proteins. Although the miRNA sponge function of circRNAs is currently investigated in the diseases, other mechanisms of circRNAs are still under investigation, and further studies are needed. After the interpretation of their function in disease pathogenesis, they may have a potential to become a drug target. Using circRNAs as biomarkers or therapeutic targets needs to be further investigated due to their complex roles. Based on these characteristics, circRNAs are likely to guide the development of new diagnostic and therapeutic strategies as well as prevention of diseases.

CircRNA

104

circRNA

Target

miRNA

Target mRNA/

mRNA

Main findings of the studies

Ref

expression

status

circ\_0005230

with The increase in been correlated worse overall survival in breast cancer patients

nonmalignant

 mammary epithelial cell lines

circ\_0005230

 expression in tumor samples has

 expression increased in six BCa cell lines compared

signaling pathway

miRNA

expression

status

expression

status

hsa\_circ\_0007294

↑

miR-148a-3p miR-152-3p

TGF-β1/Smad

signalling

↓

USF1

↑

Compared to pairs of adjacent nontumor tissues, expression of

CircANKS1B

CircANKS1B

Activated

with NTNBC cell lines

The increase in correlated worse overall survival in breast cancer patients

CircANKS1B

 expression in tumor samples has been

 expression increased in TNBC cell lines compared

 is upregulated

 in 23 TNBC tissues

[170]

Gene Expression and Phenotypic Traits

circANKS1B

hsa\_-circ\_005239

↑

miR-34a

↓

GFRA1

↑

Compared to pairs of adjacent nontumor tissues, expression of

circGFRA1

The increase in circGFRA1 correlated short overall survival in TNBC patient circGFRA1 expression increased in TNBC cell lines compared with

NTNBC cell lines

 factor 1; GFRA1,

 is upregulated

 in 51 TNBC tissues

[171]

 expression in tumor samples has been

circGFRA1

KIF4A, kinesin family member 4A; ZEB2, zinc finger E-box binding homeobox 2; CCNE1, cyclin E1; FOXC1, forkhead box C1; TNBC, triple negative breast cancer; NTNBC, nontriple negative breast

cancer; Bca, Breast cancer; TADA2A,

CLL/lymphoma11A;

associated 1; IRAK3, interleukin 1 receptor associated kinase 3; CBX8, chromobox 8; ANKS1B, ankyrin repeat and sterile alpha motif domain containing 1B; USF1, upstream transcription

GDNF family receptor alpha 1; and TGF-β1, transforming

Table 5. The expression profile of

circRNA-miRNA-mRNA

 network in breast cancer tissues.

 E2F3, E2F transcription

transcriptional

 factor 3; MUC19, mucin 19; NOTCH1, notch receptor 1; NF-κB, nuclear factor Kappa beta; UBAP2, ubiquitin associated protein 2; MTA1, metastasis

 growth factor beta 1.

 adaptor 2A; SOCS3, suppressor of cytokine signaling 3; AGFG1, ArfGAP with FG repeats 1; EPSTI1, epithelial stromal interaction 1; BCL11A, B-cell

## Conflict of interest

The authors declare no conflict of interest.

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## Author details

Atiye Seda Yar Saglam<sup>1</sup> , Ebru Alp<sup>2</sup> and Hacer Ilke Onen<sup>1</sup> \*

1 Department of Medical Biology, Faculty of Medicine, Gazi University, Ankara, Turkey

2 Department of Medical Biology, Faculty of Medicine, Giresun University, Giresun, Turkey

\*Address all correspondence to: ilkeonen@yahoo.com; hionen@gazi.edu.tr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Circular RNAs and Its Biological Functions in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.88764

## References

Conflict of interest

Gene Expression and Phenotypic Traits

Author details

Giresun, Turkey

Turkey

106

Atiye Seda Yar Saglam<sup>1</sup>

provided the original work is properly cited.

, Ebru Alp<sup>2</sup> and Hacer Ilke Onen<sup>1</sup>

1 Department of Medical Biology, Faculty of Medicine, Gazi University, Ankara,

2 Department of Medical Biology, Faculty of Medicine, Giresun University,

\*Address all correspondence to: ilkeonen@yahoo.com; hionen@gazi.edu.tr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*

The authors declare no conflict of interest.

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267-274. DOI: 10.1016/j. ebiom.2018.07.036

10.3349/ymj.2018.59.3.349

s13045-018-0569-5

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1-15. Available from: https://www.ncbi. nlm.nih.gov/pmc/articles/PMC6

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1535370217708978

International Journal of

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pgen.1001233

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Immunopathology and Pharmacology.

10.3390/genes8120353

357300/

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Gene Expression and Phenotypic Traits

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[165] Song L, Xiao Y. Downregulation of hsa\_circ\_0007534 suppresses breast cancer cell proliferation and invasion by targeting miR-593/MUC19 signal pathway. Biochemical and Biophysical Research Communications. 2018;503:

bbrc.2018.05.166

bbrc.2018.08.007

2603-2610. DOI: 10.1016/j.

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996-1002. DOI: 10.1016/j.

3607 to facilitate breast cancer metastasis. Cancer Letters. 2018;430:

179-192. DOI: 10.1016/j. canlet.2018.05.033

bbrc.2018.10.026

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[166] Wang H, Xiao Y, Wu L, Ma D. Comprehensive circular RNA profiling reveals the regulatory role of the circRNA-000911/miR-449a pathway in breast carcinogenesis. International Journal of Oncology. 2018;52:743-754.

[157] Li Y, Hu J, Li L, Cai S, Zhang H, Zhu X, et al. Upregulated circular RNA circ\_0016760 indicates unfavorable prognosis in NSCLC and promotes cell progression through miR-1287/GAGE1 axis. Biochemical and Biophysical Research Communications. 2018;503:

[158] Gao D, Zhang X, Liu B, Meng D, Fang K, Guo Z, et al. Screening circular RNA related to chemotherapeutic resistance in breast cancer.

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653 by targeting ZEB2 signaling pathway. Bioscience Reports. 2019;39.

[161] Xu JZ, Shao CC, Wang XJ, Zhao X,

circTADA2As suppress breast cancer progression and metastasis via targeting miR-203a-3p/SOCS3 axis. Cell Death & Disease. 2019;10:175. DOI: 10.1038/

[162] Yang R, Xing L, Zheng X, Sun Y,

Wang X, Chen J. The circRNA circAGFG1 acts as a sponge of miR-195-5p to promote triple-negative breast cancer progression through regulating CCNE1 expression. Molecular Cancer. 2019;18:4. DOI: 10.1186/s12943-018-

DOI: 10.1042/BSR20181919

Chen JQ, Ouyang YX, et al.

s41419-019-1382-y

0933-7

118

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2089-2094. DOI: 10.1016/j.

bbrc.2018.07.164

10.2217/epi-2017-0055

s12943-019-0946-x

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[172] Chen X, Han P, Zhou T, Guo X, Song X, Li Y. circRNADb: A comprehensive database for human circular RNAs with protein-coding annotations. Scientific Reports. 2016;6: 34985. DOI: 10.1038/srep34985

[173] Xu S, Zhou L, Ponnusamy M, Zhang L, Dong Y, Zhang Y, et al. A comprehensive review of circRNA: From purification and identification to disease marker potential. PeerJ. 2018;6: e5503. DOI: 10.7717/peerj.5503

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[176] Xia S, Feng J, Lei L, Hu J, Xia L, Wang J, et al. Comprehensive

characterization of tissue-specific circular RNAs in the human and Mouse genomes. Briefings in Bioinformatics. 2016;18:984-992. DOI: 10.1093/bib/ bbw081

[177] Zhang XO, Dong R, Zhang Y, Zhang JL, Luo Z, Zhang J, et al. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs. Genome Research. 2016;26:1277-1287. DOI: 10.1101/gr.202895.115

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**121**

**Chapter 7**

**Abstract**

gene expression

**1. Introduction**

Expression

Evaluation of the Synergistic

Cefotaxime against *Pseudomonas* 

*aeruginosa* and Its Biofilm Genes

A total of 100 broiler chickens were examined for the presence of *Pseudomonas* 

*aeruginosa* by standard microbiological techniques. Susceptibility pattern for amikacin and cefotaxime was performed by Kirby-Bauer and microdilution assays. Then, checkerboard titration in trays was applied and FIC was measured to identify the type of interaction between the two antibiotics. The ability of isolates to form in vitro biofilm was detected by two methods, one qualitative (CRA) and the other quantitative (MTP), followed by investigating the effect of each antibiotic alone and in combination on the expression of biofilm genes. The overall isolation percentage of *P. aeruginosa* was 21%. Resistance to each antibiotic was more than 50%; the range of cefotaxime MIC was 8–512 μg/ml, while amikacin MIC range was 1–64 μg/ml. The FIC index established a synergistic association between tested two drugs in 17 (81%) of isolates and the remaining represent partially synergism. The qualitative technique showed that only 66.6% of the isolates were considered biofilm producers, while the quantitative technique showed that 90.4% of the isolates were biofilm producers. Further to RT-PCR investigation, significant repression against biofilm-forming genes (*filC*, *pelA*, and *pslA*) was observed for the combination of

**Keywords:** *P. aeruginosa*, cefotaxime, amikacin, combination therapy, biofilm,

The infection with *Pseudomonas aeruginosa* is responsible for humanity in poultry and clinical signs including respiratory signs and septicaemia. *P. aeruginosa* produces dyspnea and cheesy deposits on the serous surfaces lining the air sacs and peritoneal cavity and also congestion of the internal organs, perihepatitis, and pericarditis [1]. *Pseudomonas* species are not related to disease entity except *Pseudomonas aeruginosa* that has been associated with infection in both man and animals. The disease of pseudomonas induces a significant economic loss to the farm by causing high mortality of newly hatched chickens and death of embryo at

Effect of Amikacin with

*Azza S. El-Demerdash and Neveen R. Bakry*

antibiotics when compared with monotherapy.

## **Chapter 7**
