**11. Regulator mechanisms of miRNAs**

The regulation of miRNAs in cancer is undertaken by multiple mechanisms such as transcriptional regulation, epigenetic alterations, mutations, abnormalities, in the number of copies in DNA and defects in the biogenesis machinery for miRNAs. Each one of these mechanisms may contribute by themselves, or more probably to alter the expression of miRNAs in cancer [11, 15, 65]. Up next, each one of these regulation mechanisms is detailed.

The *transcriptional regulation* contributes to the alteration of expression patterns in miRNAs, an important example is that of miR-34b, a tumour suppressor which is regulated by the transcriptional factor p53. The inactivation of p53 in gliomas reduces the expression of miR-34, which makes it inhibit the cell proliferation, the progression of the cell cycle of G1/s, cell survival, 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].

profile of expression of miRNAs present in low-grade (G I, II) astrocytomas and in high-grade

One of the goals within cancer study is to develop non-invasive tests for the diagnosis and follow-up of patients; because of this, there is a great interest in the detection of nucleic acids that are circulating in serum and plasma. Serum and plasma contain a great number of stable miRNAs, despite the high content of ribonucleases in the plasma. This stability may be given by finding itself within the exosomes (organelles derived from endosomes), by chemical modifications or by associating with protein complexes such as RISC [60, 61]. Lawrie et al. [4] reported their first study regarding miRNAs, associated with tumours, in lymphoma patients' serums, and they found that the levels of miR-155, miR-210 and miR-21 were higher than those found in control serums of healthy patients. In this study, they related the high expression of miR-21 with a better prognosis. These results were consistent with previous results in biopsy material from lymphoma patients, in which high levels of miR-21 were associated with a better prognosis [4]. Chen et al. detected and sequenced 100 miRNAs in healthy patients' serums and in patients with lung and colorectal cancers, reporting specific expression patterns of tumour type. In this same study, they distinguished the miRNAs in the serums of other species of small nucleotides such as tRNA or downgraded RNA fragments, concluding that miRNAs are the main fraction present in serum [62, 63]. One of the first undertaken studies in astrocytoma patient serums was the one by Skog et al. in which they report that tumour cells on glioblastomas release microvesicles that contain microRNA, RNAm and angiogenic proteins [64]. These results indicate that patients with cancer present elevated levels of exosomes in plasma, derived from the tumour, in comparison with controls. Although normal cells may contribute to the population of exosomes in the peripheral circulation, the main source of circulating exosomes in cancer patients is originated in the tumour. Nevertheless, little is known about the mechanism by which miRNAs are generated in plasma and the biological impact of these molecules in distant sites of the body [61]. The discovery of miRNAs in serum opens the possibility of using them as biomarkers in different illnesses.

The regulation of miRNAs in cancer is undertaken by multiple mechanisms such as transcriptional regulation, epigenetic alterations, mutations, abnormalities, in the number of copies in DNA and defects in the biogenesis machinery for miRNAs. Each one of these mechanisms may contribute by themselves, or more probably to alter the expression of miRNAs in cancer

The *transcriptional regulation* contributes to the alteration of expression patterns in miRNAs, an important example is that of miR-34b, a tumour suppressor which is regulated by the transcriptional factor p53. The inactivation of p53 in gliomas reduces the expression of miR-34, which makes it inhibit the cell proliferation, the progression of the cell cycle of G1/s, cell survival,

[11, 15, 65]. Up next, each one of these regulation mechanisms is detailed.

astrocytomas (G III, IV) in paediatric population.

70 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**10. Expression of microRNAs in serum**

**11. Regulator mechanisms of miRNAs**

*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**).

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

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

neuroectodermic tumours, an altered expression of these proteins has also been observed.

Astrocytomas and miRNAs: Are They Useful? http://dx.doi.org/10.5772/intechopen.77419 73

Currently, miRNAs are categorised as oncogenes and tumour suppressants in such a manner that a future therapeutic strategy must be headed to inhibiting or activating the altered miRNA, in this sense, in recent years, a therapy of re-expression of microRNAs. The main advantage of miRNA therapy is that its re-expression may influence the expression of hundreds of genes involved in several cell strands and routes. The main obstacle for an effective therapy is the insertion of miRNAs within the cell, because they are molecules that do not freely enter, they are unstable and therefore they may degrade after crossing the membrane of plasma. Another important part is controlling the levels of re-expression of miRNAs to avoid their expression beyond the physiological levels. Another challenge is achieving the antineoplastic agents to cross the haematoma-encephalic barrier. To overcome this inconvenience, different strategies are being developed, such as the intranasal application of oligonucleotides, which is a non-invasive method for the transport of therapeutic agents; unites nucleic acids to cationic lipids, introducing the therapeutic agent by a conjugation with membrane lipids. The *in vitro* studies done with cell strands, antagomiRs, are introduced to cells uniting to their region 5′ a cholesterol molecule; in this way, antagomiR crosses the cell membrane and inhibits the action of the miRNA, sequestering it and uniting by a complementarity of bases, avoiding the inhibition of the target RNAm. Nevertheless, cancer is a complex illness and patients with the same diagnosis may have different genetic and epigenetic alterations and polymorphic variations;

In the development of the brain, several microRNAs have been identified with a differential expression profile, for which the differentiation strategy in cancers represents a new approach. There are two focuses on this regard: on one side, there are miRs which favour the growth of the tumour through the inhibition of the cell differentiation, and the maintenance of a small population of tumour stem cells (cells which retain properties of stem cells). In this case, therapies must be directed to these cell under-populations, introducing molecules which block the functions of the miR (antagomiR) [49]. On the contrary, it is known that the overexpression of some miRs such as miR-451 stimulates the CD133+ cells of GBM to differentiate themselves and lose their character of stem cells [67]. MiR-21 regulates several oncogenic routes and strands, for which it participates in the development and progress of gliomas. This makes it a potential therapeutic target in order to treat these tumours. In the same manner, the therapy headed to restore the levels of miR-34a may achieve anti-tumour effects by inducing their differentiation [66]. MiR-124 and 137 inhibit the expression of the RNAm of CDK6, protein CDK6, and they phosphorylate RB in GBM cells, which demonstrate their potential value in treating this illness. Besides, miR-124 and miR-137 have a potent anti-proliferation effect and pro-differentiation effect in GBM CD133+ and CD133− cells [40] (**Figure 12**).

In the following figure, the re-expression of miR-124 is described as a differentiation therapy

The mechanisms, which alter the expression of miRNAs, are resumed in **Figure 11**.

therefore, the incorporation of customised medicine is necessary.

in GBM.

**12. miRNAs as therapeutic targets**

**Figure 10.** Epigenetic mechanisms regulate the transcription of miRNAs. (A) A CpG island regulates the transcription of an intergenic miRNA. (B) A CpG island regulates the transcription of a gene that harbours an miRNA. (C) An intronic miRNA has its own transcriptional starting point, which is regulated through CpGs. (D) A factor of transcription recruits DNA-modifying enzymes and histones so as to epigenetically regulate a gene that harbours an miRNA which is surrounded by CpGs.

**Figure 11.** Diverse mechanisms which alter the expression and functionality of miRNAs in human cancer.

*Defects in biogenesis* of miRNAs. Proteins which participate in the biogenesis of miRNAs may find themselves altered in cancer. In a study that spanned 67 lung cancer patients, a low expression on Dicer1 levels was determined, associated with a poor differentiation of tumour cells and short post-surgery survival [65]. The Argonaut proteins, components of the RISC complex, are in chromosome 1 and are deleted frequently in Wilms' tumours; in neuroectodermic tumours, an altered expression of these proteins has also been observed. The mechanisms, which alter the expression of miRNAs, are resumed in **Figure 11**.
