**9.2. MiR-128**

the survival of the cell by a coordinated competence between the entrance in phase S and signalization routes of the growth factor that stimulates the cell proliferation [55]. The high expression of miR-221 in high-grade astrocytomas and cell strands, and they strongly imply

**Figure 7.** MiR-124 and miR-137 has CDK6 as target, CDK6 which is a regulator of the cell cycle and differentiation.

**Figure 6.** The miR-221 oncogene promotes the progression of the cell cycle because it inhibits the translation of the tumour suppressor p27kip1, whose reduction causes the expression of CDK and, with it, the progress of the cell cycle [52].

There are three genes that codify for miR-124 and are located in different regions; thus, we have miR-124-1 located in chromosome 8p23.1, miR-124-2 located in 8q12.3 and miR-124-3 in

that it is a candidate to becoming a specific tumour marker (**Figure 7**) [59].

PTBP1 is also one of its targets and is related with alternative 'splicing'.

66 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**proliferation**

**9.1. MiR-124**

**9. Tumour-suppressing microRNA: neural differentiation and** 

MiR-128-1 is located in chromosome 2q21.3 and miR-128-2 in 3p22.3. It is an miRNA specific to the brain, where it finds itself enriched. On the other hand, in gliomas and glioma cell strands, its expression is lowered [25, 38, 42]. Zhang et al. studied the expression of miR-128 in astrocytomas GII, GIII and GIV and in cell strands, and they observed that it lowers itself progressively as the grade of the tumour increases. Its tumour-suppressing characteristics were evidenced when transfecting miR-128 in glioma cell strands, observing an inhibition in cell proliferation [25]. Godlewski et al. proved the low expression of miR-128 in gliomas and in cell strands and focused in finding a target that was related with cell differentiation and self-renewal. MiR-128 makes up for an important biological target against the 'tumour stem cells' which are characteristic and part of the origin of the glioma (**Figure 9**) [42].

**Figure 8.** MiR-128 has Bmi and E2F as its main targets.

is the most sensible way to predict the clinical diagnosis for patients with low-degree astrocytomas. These results suggest that miR-181 may maintain the state of differentiation in normal brain cells for which their diminution would induce the loss of differentiation in tumour cells. The identification of target may provide information regarding the cell differentiation (**Table 1**). The findings that are registered in the study up to now represent starting studies; nevertheless, it has been established that the deregulated expression of miRNAs participates in the tumourigenesis in several types of tumours such as GBM. Data are scarce regarding the differential expression of miRNAs in low- and high-grade astrocytomas in children. In children, low-degree astrocytomas are the most common; nevertheless, high-grade astrocytomas take place frequently and in advanced clinical studies. In paediatric population, the profile of expression of miRNAs in low- (GI and GII) and high-grade astrocytomas (GIII and GIV) is unknown. With methods of cell and molecular biology, it is possible to generate information regarding the biological behaviour of these molecules and to establish molecular markers which may be used to identify and differentiate the different grades of astrocytomas that have malignity characteristics, despite being low grade. The goal of this work is to determine the

> **Biological function** *Oncogene/tumour suppressant*

*Oncogene*

*Oncogene*

G0/G1) *Oncogene*

Low G III, IV Differentiation,

Proliferation and anti-apoptosis *Oncogene*

Proliferation: cell cycle

proliferation: cell cycle *Suppressor tumour*

Proliferation: cell cycle *Tumour suppressor*

Induces differentiation, inhibits proliferation *Tumour suppressor*

Induces apoptosis, inhibits invasion and

*Tumour suppressor*

**Table 1.** Expression of microRNAs in normal brain and in astrocytomas, their functions and validated targets.

growth

Regulates the progression of the cell cycle (arrest in

**Target RNAm**

**Experimentally validated**

p63, JMY, TOPORS, TP53BP2, TGFβR2/3, DAXX, HNRPK, PDCD4, RECK, TIMP3, LRRFIP1

CDKN1B/p27 CDKN1C/p57, BIRC1

PTBP1 (neural differentiation), CDK6

REST 683

Astrocytomas and miRNAs: Are They Useful? http://dx.doi.org/10.5772/intechopen.77419 69

CCNE (codifies cyclinE1) 968

E2F3a, BMI1 785

CDK6 468

Not reported 892

**Number of possible targets**

210

307

1299

**miRNA Normal brain Type of** 

MiR-21 Basal High G II,

miR-221 Basal High G II,

miR-15b Basal High (cell

MiR-137 Abundant Degrees III

miR-181a Abundant G II, III, IV,

MiR-124 Abundant, specific

miR-128 Abundant, specific

**glioma and expression**

MiR-9 Abundant High GIV Differentiation

III, IV

III, IV

strand glioma U118)

Low G II, III, IV

and IV, low

low

**Figure 9.** Possible transport route of miRNAs in serum, and their final destination to receptor tissue cells.

### **9.3. MiR-137**

Located in the chromosome 1p21.3, Silber et al. studied the expression of several miRNAs during the differentiation of adult neural stem cells and observed an increase of miR-137 24-fold. This miRNA is considered a strong anti-proliferation factor and a cell pro-differentiator, with tumour-suppressing activity in gliomas, and may be of therapeutic relevance [42]. In high-grade astrocytomas, the expression of miR-137 is lowered. One of its validated targets through the reporting system of luciferase is CDK6, which regulates the progress of the cell cycle and differentiation, suggesting that miR-137 mediates the inhibition of CDK6, which can, in part, cause proliferation and differentiation of CBM cells (**Figure 8**) [54].

### **9.4. MiR-181**

The miR-181 family is made up of miR-181a located in 9q33.3, miR-181b in 1q32.1 and miR-181c located in 19p13.13; miR-181a and miR-181b are enriched in a normal brain. Ciafre et al. studied the expression profile in patients with glioblastoma, finding a low expression of miR-181a, miR-181b and miR-181c in 20–30% of cases. In cell strands, a low expression was also observed, being miR-181a the one with the lowest expression, followed by miR-181b. In this case, low expression was correlated with the lack of differentiation of tumour cells [38]. In the same manner, Shi et al. studied a small series of gliomas in grades II, III and IV and observed a low expression of miR-181a and miR-181b associated with the grade of tumour. They also transfected glioma cell strands with both miRs and they observed an inhibition of the growth, induction of apoptosis and inhibition of the invasion. These effects were more evident with miR-181b [55, 56]. Conti et al. studied the expression of miR-181 in different grades of astrocytoma from a diffuse astrocytoma, grade II up to GBM GIV and observed the low regulation of mir-181b in all grades; nevertheless, the expression levels of miR-181a and miR-181c were similar to those on a normal brain [24]. Zhi et al. studied a total of 124 astrocytomas ranging from GI to GIV and they found low levels of miR181b which were associated with low survival. The authors also mention that miR-181b is the most sensible way to predict the clinical diagnosis for patients with low-degree astrocytomas. These results suggest that miR-181 may maintain the state of differentiation in normal brain cells for which their diminution would induce the loss of differentiation in tumour cells. The identification of target may provide information regarding the cell differentiation (**Table 1**).

The findings that are registered in the study up to now represent starting studies; nevertheless, it has been established that the deregulated expression of miRNAs participates in the tumourigenesis in several types of tumours such as GBM. Data are scarce regarding the differential expression of miRNAs in low- and high-grade astrocytomas in children. In children, low-degree astrocytomas are the most common; nevertheless, high-grade astrocytomas take place frequently and in advanced clinical studies. In paediatric population, the profile of expression of miRNAs in low- (GI and GII) and high-grade astrocytomas (GIII and GIV) is unknown. With methods of cell and molecular biology, it is possible to generate information regarding the biological behaviour of these molecules and to establish molecular markers which may be used to identify and differentiate the different grades of astrocytomas that have malignity characteristics, despite being low grade. The goal of this work is to determine the

**9.3. MiR-137**

**9.4. MiR-181**

Located in the chromosome 1p21.3, Silber et al. studied the expression of several miRNAs during the differentiation of adult neural stem cells and observed an increase of miR-137 24-fold. This miRNA is considered a strong anti-proliferation factor and a cell pro-differentiator, with tumour-suppressing activity in gliomas, and may be of therapeutic relevance [42]. In high-grade astrocytomas, the expression of miR-137 is lowered. One of its validated targets through the reporting system of luciferase is CDK6, which regulates the progress of the cell cycle and differentiation, suggesting that miR-137 mediates the inhibition of CDK6, which

The miR-181 family is made up of miR-181a located in 9q33.3, miR-181b in 1q32.1 and miR-181c located in 19p13.13; miR-181a and miR-181b are enriched in a normal brain. Ciafre et al. studied the expression profile in patients with glioblastoma, finding a low expression of miR-181a, miR-181b and miR-181c in 20–30% of cases. In cell strands, a low expression was also observed, being miR-181a the one with the lowest expression, followed by miR-181b. In this case, low expression was correlated with the lack of differentiation of tumour cells [38]. In the same manner, Shi et al. studied a small series of gliomas in grades II, III and IV and observed a low expression of miR-181a and miR-181b associated with the grade of tumour. They also transfected glioma cell strands with both miRs and they observed an inhibition of the growth, induction of apoptosis and inhibition of the invasion. These effects were more evident with miR-181b [55, 56]. Conti et al. studied the expression of miR-181 in different grades of astrocytoma from a diffuse astrocytoma, grade II up to GBM GIV and observed the low regulation of mir-181b in all grades; nevertheless, the expression levels of miR-181a and miR-181c were similar to those on a normal brain [24]. Zhi et al. studied a total of 124 astrocytomas ranging from GI to GIV and they found low levels of miR181b which were associated with low survival. The authors also mention that miR-181b

can, in part, cause proliferation and differentiation of CBM cells (**Figure 8**) [54].

**Figure 9.** Possible transport route of miRNAs in serum, and their final destination to receptor tissue cells.

68 Glioma - Contemporary Diagnostic and Therapeutic Approaches


**Table 1.** Expression of microRNAs in normal brain and in astrocytomas, their functions and validated targets.

profile of expression of miRNAs present in low-grade (G I, II) astrocytomas and in high-grade astrocytomas (G III, IV) in paediatric population.

migration and cell invasion [66], and correction. Another example is miR-451; in this case, it is known that there are two transcription factors, SMAD3 and SMAD4, separated by 157 pb and whose binding sequence is in 1135 pb upstream from the miR-451 sequence. Both factors increase the transcription of miR-451 and induce the inhibition of growth and proliferation [67].

Astrocytomas and miRNAs: Are They Useful? http://dx.doi.org/10.5772/intechopen.77419 71

*Epigenetic mechanisms* may regulate up to a certain degree, the imbalance of miRNAs in tumour cells [68]. The methylation of DNA and modification of histones play a predominant role in the remodelling of chromatin and the general regulation of expression of genes that codify proteins. The hyper-methylation of CpG islands associated with specific miRNAs has been proposed as one of the mechanisms by which a low expression of miRNAs in tumour cells has been observed. The epigenetic silencing of miRNAs that act as tumour suppressors is emerging as an important alteration in cancers. Lujambio et al. studied the expression profile for several miRNAs in cells derived from a metastatic ganglion, and afterwards, the cells were treated with a de-methylating agent, observing that there was some re-expression of some miRNAs such as miR-148a, miR-34b/c and miR-9 [68]. The regulation of miR-124 is given, partly, due to epigenetic mechanisms, which was observed in a cell strand for colon cancer. No expression of miR-124 was observed here, but when cells were treated with a de-methylating agent, their expression was restored and, at the same time, correlated with the inhibition of one of its targets, CDK6. This result is due to miR-124 being located within a great CpG island, which, in a normal colon tissue, would be hypo-methylated, but in colon, tumour finds itself hyper-methylated [69]. In the same manner, the epigenetic silencing of miR-124 was evidenced when treating glioma cell strands with 5-aza-2′-deoxicitidine (a methylation inhibitor) and TSA (histone deacetylase inhibitors), increasing the expression of miR-124 [45]. In gliomas, miR-137 is partially regulated by epigenetic mechanisms, and its expression was increased 12-fold when astrocytoma cell strands were treated with de-methylating agents. This suggests that epigenetic modifications for regulating

sequences in CpG islands may contribute to silencing miR-137 in GBM [45] (**Figure 10**).

tions may be found in pri-, pre- and mature sequences of miRNAs [16].

eration through the proto-oncogene c-Myc [15].

Somatic mutations and/or in the germinal line, identified in miRNAs, are scarce. Some of the most recent findings have taken place in chronic lymphocytic leukaemia (CLL) [30]. In this illness, 42 genes which codify microRNAs were sequenced and five microRNAs with mutations were found. In the case of solid tumours, 15 miRNAs were evaluated in 91 epithelial-origin tumour cell strands and mutations were found in one case, a variation in the sequence of the precursor miRNA, and 15 variations in the sequence of primary miRNAs [15]. These muta-

The *abnormality in the number of DNA copies* is one of the mechanisms which modify the expression and functioning of genes. It is calculated that close to 50% of genes that codify human miRNAs and are registered are located in fragile areas, in regions with minimal loss of heterozygosity (LOH), minimal amplification regions and breaking regions. In chronic lymphocytic leukaemia, region 13q14 is deleted in over 50% of cases, and in this place, there is miR-16-1 and miR-15a. These two miRNAs have Bcl-2 as a target and work as tumour suppressors in this illness. The deletion of these miRNAs has also been identified in pituitary adenomas, ovary adenomas and breast cancer. In patients with lymphoma, the amplification of C13orf25 located in 13q31-32 has been described; in it, seven polycystronic miRNAs have been located. This group of miRNAs work as oncogenes, altering the balance between apoptosis and prolif-
