**4. MiR/RISC-RNAm complex**

The recognition of the target RNAm takes place because of the complementarity of the sequence known as 'seed' (nucleotides 2–8) located in the 5′ extreme of the microRNA with the sequence of the target RNAm. Recognised sequences of target RNAm are in region 3′, non-translated (3′UTR) (60%), in codifying sequences (25%), in introns, in non-codifying RNA sequences and in 5′UTR. The degree of complementarity between microRNA and RNAm determines the silencing mechanism. When complementarity is 100%, targeted RNAm is downgraded, which mostly happens with plants. In animals, complementarity is 100% in the seed region, but not throughout the microRNA in such a way that a mechanism of inhibition of the target messenger takes place.

The effector complex headed by the Argonaut protein probably interacts with translational systems to inhibit the synthesis of proteins at the beginning or in the elongation step, depending, probably, on the nature of the miRNA and the target transcript [13, 14]. Most microRNAs described in the human body exert their inhibiting effect in the cytoplasm; nevertheless, there exist some microRNAs as miR-29b which has a terminal sequence of hexanucleotides which allows it to return to the nucleus where it possibly undertakes its functions.

The complexes formed by the microRNA/RISC-RNAm do not remain indefinitely in the cytoplasm, but they are rather transported towards structures of cytoplasmic processing, called P-bodies, where the downgrading of RNAm may take place due to deadenylation and decapping, or it is also stored and then separated from the repression complex and the P-body and returns to the translational machinery (**Figure 3**).

MicroRNAs participate in fundamental cell processes such as determining the cell lineage, apoptosis, proliferation, migration and regulation of the cell cycle, in which the translation of specific genes is highly precise and coordinated. MicroRNAs make up complex regulatory networks with its target genes, representing common mechanisms that have evolved

Drosha and DGCR8 (protein with a binding domain to double-strand RNA). This complex cuts the structure in a fork, becoming now a precursor miRNA (pre-miRNA) with a length of 60–80 nucleotides. The pre-miRNA is recognisable because of exportin 5 (nuclear exporting factor) and the nuclear protein Ran-GTP. Both transport pre-miRNA towards the cytoplasm. The Dicer and TRBP enzymes (proteins with binding domain to RNA) undertake a second cut in the base of the stem-handle and they generate an RNA molecule, double strand, of 18–24 nucleotides in size [12]. A great protein complex known as silencing complex induced by RNA (RISC) is associated with duplex RNA and separates both chains. RISC is a tetrameric complex made up of Dicer, TRBP (protein with binding domain to RNA), PACT (Activating Protein) and Ago2 (Protein of the Argonaut family). Ago2 identifies the target RNAm based on the complementarity with the associated single-strand microRNA. Recognised sequences of target RNAm are located mainly in region 3, non-translated (3′UTR). Generally speaking, only one strand is incorporated within RISC and the other one is downgraded. This miRNA

**Figure 2.** Biogenesis of the microRNA within the nucleus (a–c), maturing of the cytoplasm (d), formation of the microRNA-RISC complex which, depending on the sequence, shall return to the nucleus (e) or shall join its target RNAm to inhibit the translation (f) and finally the microRNA/RISC-RNAm complexes are stored in p-bodies where they are

guides RISC towards the target messenger inhibiting its translation (**Figure 2**) [13, 14].

The recognition of the target RNAm takes place because of the complementarity of the sequence known as 'seed' (nucleotides 2–8) located in the 5′ extreme of the microRNA with the sequence of the target RNAm. Recognised sequences of target RNAm are in region 3′,

**4. MiR/RISC-RNAm complex**

degraded or they return to the translation (g).

60 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**Figure 3.** Model which shows the effects of deregulation of miRNAs in the development of the glioma. Under normal physiological conditions, the expression of miRNAs is important for the induction of differentiation in the SNC and abrogation of the self-renewal of the stem cell. The loss of expression of miRNAs results in the creation of pre-malignant stem cells, which are hyperproliferative and non-differentiated, which may progress into a glioma of low to high grade. Additional oncogenic mutations may facilitate the malignant phenotype.

in mammals strengthening genetic regulation. At the same time, microRNAs are regulated by oncogenes, tumour-repressing genes, epigenetic mechanisms, genetic abnormalities and defects in the miRNA biogenesis machinery [15]. Each one of these mechanisms may contribute by themselves or, more likely, together, to alter the expression of miRNAs in cancer [15, 16].
