**3. The role of miRNAs in cancer**

Croce's group established for the first time an association of mRNAs with cancer, indicating an alteration of miR-15a/16-1 cluster, in chronic lymphocytic leukemia [15]. Further functional analyses have demonstrated that miR-15 and miR-16 can target and suppress the expression of BCL2 oncogene, inducing the apoptosis. [16]. Through exploring the role of miRNAs, Croce's group has demonstrated that miRNA profiling could be taken into consideration for characterizing the malignant phenotype [17], opening a new perspective for identifying new cancer-specific miRNAs. Interestingly, for poorly differentiated tumors, tissue miRNA profiling has revealed better diagnosis than mRNA profiling, highlighting their role as tumor biomarkers [18]. An important feature of miRNAs, given by their high stability in formalin-fixed paraffin embedded (FFPE) tissues, blood including serum and plasma, as well as other biological fluids such as urine, tears, breast milk, saliva, and seminal fluids, makes them important candidates for the discovery of new minimally invasive biomarkers [19, 20]

Therefore, a myriad of studies describing the role of miRNAs in cancer development have been provided, with more than 21,565 papers that are published in PubMed today, when "miRNA, miR, microRNA, and cancer" are used as a string search.

Alteration of miRNA expression in cancer is due to genetic and epigenetic events. Genetic alterations include: chromosomal rearrangements or loss of heterozygosis (LOH) (e.g. miR-15a/16-1), gene amplification (e.g. miR-17-92 cluster, miR-155), deletions (e.g. let-7 family member), or mutations (e.g. miR-16) [15]. Moreover, genetic alteration may occur in the PCGs involved in the synthesis of the protein components of the Drosha, DGCR8, Exportin 5, Dicer, and AGO2, the main enzymes that process the biogenesis and activation of miRNAs. Pre- and posttranscriptional controls of not only miRNA biogenesis but also epigenetic events, including methylation and acetylation, were also related to aberrant expression of tumor miRNAs [21, 22]. Not lastly, the presence of the single-nucleotide polymorphism (SNP) mutations in the miRNA-coding genes may lead to the alterations of mature miRNA structure, reducing its specificity to the mRNA target [23].

Functionality studies have demonstrated that the expression of oncogenes and tumor-suppressor genes in cancer is closely controlled by miRNAs (**Figure 2**). Such as, miRNAs that target and modulate the oncogenic expression are defined as tumor-suppressor miRNAs (TS-miRNAs), while the miRNAs that modulate the expression of tumor-suppressor genes are known as oncomiRs [24]. Genetic and epigenetic alterations occurring in cancer lead to "gain of function" of oncomiRs and inactivation or "loss of function" of TS-miR (**Figure 2**), which translate into regulating the expression of their targets through downregulation of tumorsuppressor genes and upregulation of oncogenes, respectively [25]. miRNAs are involved in all hallmarks of cancer, including self-sufficiency in growth signals (let-7 family, miR-21), insensitivity to antigrowth signals (e.g. miR-17-92 cluster, miR-195), evasion from apoptosis (e.g. miR-34a, miR-185, miR-15/miR-16), limitless replicative potential (e.g. miR-372/373 cluster, miR221/222), angiogenesis (e.g. miR-210, miR-26, miR-15b, miR-155), invasion and metastases (e.g. miR-10b, miR-31, miR-200 family, miR-21, miR-15b), reprogramming energy metabolism

**35**

**Figure 2.**

*inactivation of tumor development.*

*MiRNA-Based Therapeutics in Oncology, Realities, and Challenges*

(e.g. miR-23a/b, miR-378, miR-143, miR-15b), evading immune destruction (e.g. miR-124, miR-155, miR-17-92), tumor-promoting inflammation (miR-23b, miR-155, let-7d), and genomic instability (miR-21, miR-155, miR15b) [22, 26, 27].

*The role of miRNAs in cancer and their use of miRNA-based therapy.* **(A)** *miRNAs that function as oncomiRs and TS-miRs. Tumors are characterized by aberrant upregulation of oncomiRs that lead to downregulation of tumor-suppressor genes and inactivation of TS-miRs reflected in overexpression of oncogenes. All of these contribute to tumor development, invasion, and metastasis as well as decrease cell death.* **(B)** *The use of anti-miRNA therapies leads to block the oncomiR activities, resulting in the upregulation of tumor-suppressor genes, while substituting TS-miR therapies increases the cellular level of TS-miRs, leading to the inactivation of oncogenes. The effects of miRNA-based therapy indicate an increase in cell death concomitantly with* 

The choice to use mRNA-based therapies is based on the fact that the expression of mRNAs in tumor cells is altered, and tumor phenotype can be changed by the

**4. Strategies used for miRNA-based therapies**

**4.1 miRNA inhibition therapies for oncomiRs**

modulation of the miRNA expression [28].

*DOI: http://dx.doi.org/10.5772/intechopen.81847*

*MiRNA-Based Therapeutics in Oncology, Realities, and Challenges DOI: http://dx.doi.org/10.5772/intechopen.81847*

#### **Figure 2.**

*Antisense Therapy*

biomarkers [19, 20]

**3. The role of miRNAs in cancer**

At the moment, 48,885 mature miRNA products from 271 species, including 2654 mature human miRNAs, have been reported in the latest available miRNA database (miRBase release 22; http://www.mirbase.org/) [14]. In normal phenotype, by their modulatory effects, miRNAs maintain the cell physiology, while by their aberrant expression, miRNAs lead to the validation of many diseases including cancer.

Croce's group established for the first time an association of mRNAs with cancer, indicating an alteration of miR-15a/16-1 cluster, in chronic lymphocytic leukemia [15]. Further functional analyses have demonstrated that miR-15 and miR-16 can target and suppress the expression of BCL2 oncogene, inducing the apoptosis. [16]. Through exploring the role of miRNAs, Croce's group has demonstrated that miRNA profiling could be taken into consideration for characterizing the malignant phenotype [17], opening a new perspective for identifying new cancer-specific miRNAs. Interestingly, for poorly differentiated tumors, tissue miRNA profiling has revealed better diagnosis than mRNA profiling, highlighting their role as tumor biomarkers [18]. An important feature of miRNAs, given by their high stability in formalin-fixed paraffin embedded (FFPE) tissues, blood including serum and plasma, as well as other biological fluids such as urine, tears, breast milk, saliva, and seminal fluids, makes them important candidates for the discovery of new minimally invasive

Therefore, a myriad of studies describing the role of miRNAs in cancer development have been provided, with more than 21,565 papers that are published in PubMed today, when "miRNA, miR, microRNA, and cancer" are used as a string search. Alteration of miRNA expression in cancer is due to genetic and epigenetic events. Genetic alterations include: chromosomal rearrangements or loss of heterozygosis (LOH) (e.g. miR-15a/16-1), gene amplification (e.g. miR-17-92 cluster, miR-155), deletions (e.g. let-7 family member), or mutations (e.g. miR-16) [15]. Moreover, genetic alteration may occur in the PCGs involved in the synthesis of the protein components of the Drosha, DGCR8, Exportin 5, Dicer, and AGO2, the main enzymes that process the biogenesis and activation of miRNAs. Pre- and posttranscriptional controls of not only miRNA biogenesis but also epigenetic events, including methylation and acetylation, were also related to aberrant expression of tumor miRNAs [21, 22]. Not lastly, the presence of the single-nucleotide polymorphism (SNP) mutations in the miRNA-coding genes may lead to the alterations of

mature miRNA structure, reducing its specificity to the mRNA target [23].

Functionality studies have demonstrated that the expression of oncogenes and tumor-suppressor genes in cancer is closely controlled by miRNAs (**Figure 2**). Such as, miRNAs that target and modulate the oncogenic expression are defined as tumor-suppressor miRNAs (TS-miRNAs), while the miRNAs that modulate the expression of tumor-suppressor genes are known as oncomiRs [24]. Genetic and epigenetic alterations occurring in cancer lead to "gain of function" of oncomiRs and inactivation or "loss of function" of TS-miR (**Figure 2**), which translate into regulating the expression of their targets through downregulation of tumorsuppressor genes and upregulation of oncogenes, respectively [25]. miRNAs are involved in all hallmarks of cancer, including self-sufficiency in growth signals (let-7 family, miR-21), insensitivity to antigrowth signals (e.g. miR-17-92 cluster, miR-195), evasion from apoptosis (e.g. miR-34a, miR-185, miR-15/miR-16), limitless replicative potential (e.g. miR-372/373 cluster, miR221/222), angiogenesis (e.g. miR-210, miR-26, miR-15b, miR-155), invasion and metastases (e.g. miR-10b, miR-31, miR-200 family, miR-21, miR-15b), reprogramming energy metabolism

**34**

*The role of miRNAs in cancer and their use of miRNA-based therapy.* **(A)** *miRNAs that function as oncomiRs and TS-miRs. Tumors are characterized by aberrant upregulation of oncomiRs that lead to downregulation of tumor-suppressor genes and inactivation of TS-miRs reflected in overexpression of oncogenes. All of these contribute to tumor development, invasion, and metastasis as well as decrease cell death.* **(B)** *The use of anti-miRNA therapies leads to block the oncomiR activities, resulting in the upregulation of tumor-suppressor genes, while substituting TS-miR therapies increases the cellular level of TS-miRs, leading to the inactivation of oncogenes. The effects of miRNA-based therapy indicate an increase in cell death concomitantly with inactivation of tumor development.*

(e.g. miR-23a/b, miR-378, miR-143, miR-15b), evading immune destruction (e.g. miR-124, miR-155, miR-17-92), tumor-promoting inflammation (miR-23b, miR-155, let-7d), and genomic instability (miR-21, miR-155, miR15b) [22, 26, 27].

## **4. Strategies used for miRNA-based therapies**

### **4.1 miRNA inhibition therapies for oncomiRs**

The choice to use mRNA-based therapies is based on the fact that the expression of mRNAs in tumor cells is altered, and tumor phenotype can be changed by the modulation of the miRNA expression [28].

MiRNA inhibition therapy is used to suppress the expression of oncomiRs that are frequently overexpressed in human cancers and reestablish the normal expression of tumor-suppressor genes that are targeting directly (**Figure 2**). The therapy for miRNA inhibition includes the following agents: antisense anti-miR oligonucleotides (AMOs), locked nucleic acid (LNA) anti-miRs, antagomiRs, miRNA sponges, and small molecule inhibitors of miRNAs (SMIRs) [27]. The principle of this therapy consists of an isolation of the endogenous miRNAs in an unrecognizable configuration, leading to inactivating and excluding the mature miRNAs from the RISC.

AMOs are single-stranded, chemically modified antisense oligonucleotides of about 17–22 nucleotides that are complementary to a miRNA of interest [28]. These antisense oligonucleotides anneal to the complementary mature miRNAs and inhibit their interaction with specific mRNA targets.

LNA anti-miRs represent an example of a modified antisense anti-miR oligonucleotide [29]. LNA-modified oligonucleotides present a higher thermal stability and affinity for their miRNA target molecules, as well as a higher aqueous solubility and increased metabolic stability for *in vivo* delivery [30].

The antagomiRs are single-stranded RNA molecules of about 23 nucleotides in length complementary to miRNA targets that are chemically modified to increase the stability of the RNA and protect it from degradation [31]. One of the most important aspects of using these agents is due to their lack of inducing any immune response.

miRNA sponges represent a class of RNAs that include multiple artificial binding sites similar to those found in the endogenous miRNA targets. The expression vectors represent the source of miRNA sponge transcription, thus reducing the miRNA's effects and increasing the expression of the miRNA's native targets [32].

SMIRs are small molecules that suppress the miRNA biogenesis or block the interaction between a miRNA and the target. The inhibition therapy using SMIRs is an encouraging one due to the reduced time of production, approval, and cost [33].

## *4.1.1 Discussion*

Krützfeldt et al. [31] demonstrated that intravenous administration of several antagomiRs toward miR-16, miR-122, miR-192, and miR-194 leads to a significant reduction in the corresponding endogenous miRNAs.

Moreover, an important positive effect observed in this study was that after the administration of antagomiR-122, the cholesterol levels in plasma have decreased. Due to the fact that, so far, the therapy using antagomiRs did not induce a significant immune response, it is worth into consideration the development of a promising antisense therapy based on antagomiRs.

One of the main advantages of using locked nucleic acid (LNA) anti-miRs is that they present a higher thermal stability, high-affinity Watson-Crick hybridization with their RNA target molecules, higher aqueous solubility, and increased metabolic stability for *in vivo* delivery. Overexpression of miR-21 is a common place in glioblastomas, and Griveau et al.'s [34] study was able to silence miR-21 in U87MG glioblastoma cell line, using a LNA conjugated to lipid nanocapsules (LNC). Another advantage of using LNA-LNC complexes in combination with external beam radiation is represented by the improvement of cell sensitivity to treatment.

#### **4.2 miRNA replacement therapies for tumor-suppressor miRNAs**

Also defined as miRNA restoration therapy, the replacement therapy with miRNAs includes the following agents: small molecules, synthetic miRNA mimics, and DNA plasmids encoding a miRNA gene that epigenetically alters endogenous expression of miRNAs [35].

**37**

*MiRNA-Based Therapeutics in Oncology, Realities, and Challenges*

Small molecules in miRNA replacement therapy are represented by hypomethylating agents (Decitabine or 5-azacytidine) and enoxacin, exerting a role in the

miRNA mimics are double-stranded synthetic RNAs, which are aimed to compensate the lack of tumor-suppressor miRNAs by replacing the lost miRNAs. These chemically structures are loaded into RISC to provide the downstream inhibition of

One of the main challenges of miRNA replacement therapy is represented by finding the most suitable, efficient, and specific delivery system. The efficacy of this therapy is significantly decreased by an unsuitable size of the vector or by gene expression. Since miRNAs can be introduced into cells using a similar technique to small interference RNAs [36], it is recommended to improve those techniques based on the insertion of synthetic miRNA mimics, DNA plasmids, and small molecules,

An important aspect that is worth considering in miRNA cancer therapy aims to use miRNA delivery systems. One of these delivery systems including microvesicles and exosomes aim to block miRNA-entrapped exosomes released by tumors. It is already demonstrated that miRNA-entrapped exosomes secreted by tumor cells can regulate gene expression in the receiving cells by binding to their target mRNAs [37]. The use of some agents that block specific miRNAs (such as LNA anti-miR-21 and LNA anti-miR-29a) in tumor cells could lead to the reduction in miRNAentrapped exosomes, released by cancer cells [38]. However, an ideal delivery system meets the following criteria: protects the miRNAs from early degradation in the bloodstream, efficient distribution to the target cells, facilitates cellular uptake, does not induce an immune response, and made of biocompatible and biodegrad-

The most commonly used vectors for miRNA delivery include viral and nonviral vectors. Previous data demonstrated that viral vectors mainly caused an immune response; therefore, the focus of the actual studies is on developing efficient nonviral vectors. Nonviral vectors are classified into three main groups, including polymeric vectors (polyethyleneimines, atelocollagen, polylactic-coglycolic acid, polyamidoamine dendrimers), lipid-based carriers (positively, negatively or neutral charged), and inorganic materials (gold, diamond, silica, and

The delivery system based on viral vectors transfers the pri-miRNA or mature miRNAs, usually a TS-miR, into a plasmid, which contains a viral promoter, an antibiotic resistance gene, and a restriction enzyme gene, to the tumor cells. After nuclear integration of the miRNA and further transcription, the mature miRNA represses the translation and/or induces the degradation of the target mRNA [41].

One of the most studied classes of polymeric vectors was represented by polyethylenimines (PEIs) but was removed from clinical studies due to their high toxicity, given by an excessive positive charge, low biological degradation, and inactivation in serum caused by a nonspecific protein. Ibrahim et al. [42] have demonstrated that by using low molecular weight, PEIs as system delivery for miR-145 and miR-33a would

as well as to improve the quality of molecules used for this type of therapy.

*DOI: http://dx.doi.org/10.5772/intechopen.81847*

nonspecific miRNA expression.

the target mRNAs [27].

**4.3 miRNA delivery systems**

able materials [39].

ferric oxide) [40].

*4.3.1 Discussion*

*4.2.1 Discussion*

#### *MiRNA-Based Therapeutics in Oncology, Realities, and Challenges DOI: http://dx.doi.org/10.5772/intechopen.81847*

Small molecules in miRNA replacement therapy are represented by hypomethylating agents (Decitabine or 5-azacytidine) and enoxacin, exerting a role in the nonspecific miRNA expression.

miRNA mimics are double-stranded synthetic RNAs, which are aimed to compensate the lack of tumor-suppressor miRNAs by replacing the lost miRNAs. These chemically structures are loaded into RISC to provide the downstream inhibition of the target mRNAs [27].
