**3.1 How miRNAs work to silence genes**

To generate mature miRNAs, sequential steps are followed. First, RNA Polymerase II transcribes primary miRNAs (abbreviated as pri-miRNA). These molecules can be translated from both intergenic and intragenic regions [42]. Then in order to generate pre-miRNAs with hairpin precursors, pri-miRNAs are processed by Drosha, an RNase III enzyme and by Pasha, a dsRNA-binding protein [43]. After this step, pre-miRNAs get escorted out of the nucleus with the help of exportin 5 [44]. And the pre-miRNA gets further processed in cytoplasm by Dicer enzyme which is an RNase III endonuclease, this enzyme works to remove the hairpin loop and make a double stranded duplex miRNA [44]. Then this double stranded structure retains the active strand while passenger stand gets degraded. The active strand interacts with RNA-induced silencing complex (abbreviated as RISC) in order to function. RISC is a complex formed by multiple proteins with its key proteins being argonaute 2 (abbreviated as Ago2) and transactivation-responsive RNA-binding protein (abbreviated as TRBP), and it includes miRNA or siRNA in order to use them as a template [45]. With these RNA templates the complex is able to recognize their complementary mRNA. The characteristic features of the target gene for an effective binding can be; seed region, a target sequence that is conserved, 3′ untranslated region of miRNA available for binding but recent studies show the binding of a target may also happen in 5′ untranslated region, promoter regions or open reading frames [45]. The pairing of the miRNA template and its target mRNA differs between plant cells and animal cells. In the plant cells, the pairing is fully complementary between miRNA and mRNA. But in animal cells, this pairing is not fully complementary there are base mismatches even though this base pairing follows a pattern. But there is a small sequence with 2–8 nucleotides of length which is a perfect base pairing that is called seed region [46]. This region is a conserved heptametrical sequence that is always perfectly matched and it is mostly found towards the 5′ end of miRNA [47]. With the binding of mRNA and miRNA, downregulation is tried to be achieved and this can be achieved via enzymatic cleavage of the mRNA leading to its further destruction by the cell or blocking the translation by preventing ribosome subunit from binding to mRNA [48]. And the matching degree of target and miRNA plays a role in the decision of which mechanism will happen for downregulation to occur, if the target is fully complementary then cleavage of mRNA will happen but if it is not fully complementary stability alteration or repression of translation may occur [43].

#### **3.2 miRNAs in apoptosis**

miRNAs are known to have a regulatory effect on apoptosis via their regulation on both pro-apoptotic and anti-apoptotic genes. So, miRNAs can work to be both inhibitory and stimulatory depending on the miRNA and the cell context. Also, alteration of the expression of regulatory genes in the apoptotic process by miRNAs is not limited to one of the extrinsic and intrinsic pathways. And the effect of miRNAs can both be direct and indirect. For example, miR-21 is a miRNA that directly affects its target, inhibiting FasL in order to increase apoptosis but miR-130a is a miRNA that affects TRAIL resistance in order to effect other miRNAs that will eventually cause a change in apoptotic process [49].

For their indirect effects, miRNAs can be seen to function in both feedback and feedforward loops. Feedback loop effects can change depending on the cell context, miRNA and transcription factors as the regulators may have the same or opposite effects [50]. And, in feed-forward loops, transcription factors can be seen to regulate both the target gene and miRNA, which also regulates the transcription factor [50]. To regulate genes, miRNAs work together with transcription factors in a highly coordinated manner. Since they can show their effects on mRNAs after the transcription of said mRNA, they usually locate downstream to transcription factors [51].

#### *Recent Advancements in Apoptosis-Based Therapeutic Approaches for Cancer Targeting DOI: http://dx.doi.org/10.5772/intechopen.99202*

In the intrinsic pathway, p53 and BCL-2 families play an important role. MiRNAs can alter their expression to regulate the intrinsic pathway. As miRNAs regulate the levels of p53, this tumor suppressor actually has an effect on the miRNAs as well by functioning to regulate miRNA expression and maturation [52]. For an example of p53 regulating miRNAs and how its mutation can cause a change, we can look at miR-16 and miR-143. Their processing is dependent on the interaction between p53 and Drosha complex so if there is a mutation in the DNA binding domain of p53, their processing cannot be achieved and cell proliferation will be suppressed [45]. Activation of p53 is found to be increasing the expression of 30 or more miRNAs including miRNAs like let7a, miR-34a and miR-15a/16 which are tumor suppressors [53]. BCL-2 is an anti-apoptotic protein that is generally overexpressed in tumors. Three pro-apoptotic miRNAs, miR-24, miR-195 and miR-365, work to down-regulate BCL2 expression via their binding to BCL-2 gene's 3′ untranslated region [53]. With this interaction, pro-apoptotic miRNAs lead to apoptosis. Extrinsic pathway is also regulated by miRNAs. Some miRNAs were found to regulate TRAIL-induced apoptosis directly and indirectly [43]. miR-221 and miR-222 can be an example of this regulation since they are found to have altering expressions between TRAIL resistant and sensitive cells, resistant cells being the ones with up-regulation of these miRNAs [43]. Another example can be miR-200c since it directly targets FAP-1, a phosphatase that works to inhibit apoptosis [43].

An example to miRNAs with effects not limited to one site is miR-21. We can observe its effects on both non-small cell lung carcinoma (NSCLC) and diffuse large B-cell lymphoma (DLBCL). In NSCL, miR-21 effects apoptosis via its inhibition on PI3K/Akt/NF-kB pathway and also it is found that miR-21 targets apoptosis-stimulating protein of p53 (ASPP2) which is a protein that functions in tumorigenesis [54]. And it was found that in early-stage samples of NSCL cells, miR-21 expression was increased when compared to the control [55]. The experiments revealed that in NSCL cells, miR-21 down-regulation led to the repression of EMT signaling pathway, cell migration and invasion, and miR-21 inhibition led to triggering of apoptosis [54]. Both *in vitro* and *in vivo*, miR-21 inhibited PI3K/Akt/ NF-κB signaling pathway and promoted caspase-dependent pathway of apoptosis. MiR-21 also is known to have high expression levels in B-cell lymphoma. In DLBCL, its effect on apoptosis can be seen via regulation of phosphatase and tensin homolog (PTEN). The expression level of miR-21 in patient samples was found to be more than the healthy samples, and these levels were also negatively correlated with expression level of PTEN [14]. Other miRNAs that have an effect on PTEN are miR-130 family. This family of miRNAs which corresponds to miR-130b, miR-301a and miR-301b, are found to have high expression levels in bladder cancer samples compared to normal ones. Via their regulation upon PTEN they also regulate focal adhesion kinase (FAK) and Akt phosphorylation, and lead to cell migration and invasion increase in bladder cancer. Experiments showed that the inhibition of this family causes down-regulation of FAK and Akt phosphorylation and this effects cell migration and invasion negatively, so it can be said that they have an important role in the progression of bladder cancer [56].

As it can be seen from the examples, the effects of miRNAs are diversifiable depending on the gene they are affecting or their expression level. The alteration of their expression leads to interchangeable role of miRNAs as oncogenes or tumor suppressors. Generally, the miRNAs that are down-regulated in the cancer tissues are considered to be tumor suppressors, as pro-apoptotic miRNAs they work for apoptosis to happen. miR-7, miR335 and miR-608 are examples of this type of miRNAs since they target BCL-2 family, and miR-203 and miR-143 can be other examples as they target PKC family [57]. On the other hand, other miRNA examples can be seen as upregulated in the cancer tissues, as antiapoptotic miRNAs they induce apoptosis to allow uncontrollable proliferation. miR-197, miR-21 and miR-212 can be the examples of these kind of miRNAs [57].

Our current research based on developing target specific drug candidates over breast cancer cell lines. The array studies indicated that non-coding RNA hsa-miR-215 greatly enhance the inhibitor compound efficiency on MCF-7 and MDA-MB-231 breast cancer cell lines. The expression of hsa-miR-215 decreases in breast cancer cell lines compared to non-cancerous breast MCF10-A cell line. Over expressing this miRNA by transfecting into the cancerous cell lines drive the cells to apoptosis. Therefore, synergetic effect of the inhibitor compound along with hsamiR-215 mimic augment anticancer treatment. A nanocarrier is being developed with hsa-miR-215 and inhibitor compound (patent pending). This formulation is designed for apoptosis-based therapeutic approach for breast cancer treatment. The utilization of carrier system along with miRNAs and inhibitor compounds introduced in this study for therapeutic purposes has the potential of clinical applications.
