**3.5. Antiapoptosis and microRNAs**

Among the tumor-associated miRNAs, miR-21 is frequently overexpressed in various types of tumors, including GBMs (7, 9), and plays a critical role in cell death and apoptosis. Knock‐ down of miR-21 in cultured GBM cells triggered activation of caspases and led to increased apoptotic cell death (8). miR-21 knockdown disrupts glioma growth *in vivo* and displays synergistic cytotoxicity when combined with the agent tumor necrosis factor-related apoptosis (s-TRAIL), leading to an increase in caspase activity (95). In particular, it was reported that the downregulation of miR-21 in GBM-derived cell lines resulted in an increased expression of a specific target, PDCD4 (programmed cell death 4), a known tumor suppressor gene, with a consequent decrease in proliferation and increase in apoptosis (96). Furthermore, miR-21 regulated a network of p53, TGF-b, and mitochondrial apoptosis tumor suppressor genes in GBM cells, targeting HNRPK (heterogeneous nuclear ribonucleoprotein K), TP53BP2 (tumor protein p53-binding protein 2), and p63, a member of the p53 family of genes (97).

miR-92a and miR-92b, upregulated in glioma, target BCL2L11 (98) and NLK (99), respectively. Their inhibition promotes tumor-suppressive phenotypes through the induction of apoptosis. In addition, miR-93 was found to be upregulated in glioma specimens and resulted in an enhancement in cell survival, promoting sphere formation, and augmenting tumor growth by suppressing the expression of ITGB8 target (61).

miR-221 and 222 were extensively investigated in glioma. These miRNAs were found to be upregulated in this cancer, and both were reported to target the cell growth-suppressive cyclindependent kinase inhibitors p27 and p57 (100).

## **3.6. Genome instability and microRNAs**

Genomic instability is defined as a high frequency of mutations within the genome, including changes in nucleic acid sequences, chromosomal rearrangements, or aneuploidy. The stability of the human genome is maintained by multiple mechanisms such as the cell cycle checkpoint, DNA damage response, and mitotic separation machinery. Defects in the DNA damage response could cause genomic DNA mutations, deletions, insertions, or gross chromosomal gains and losses upon cell division and subsequently lead to cancer. Genomic instability is present in GBM and affects the prognosis of patients.

Several members of the miR-106b-25 cluster and its paralog miR-17-92 cluster were associated with DNA replication and mitosis. Overexpression of miR-106b-5p in glioma tumor cells significantly promoted cell proliferation, suggesting a role of this miRNA in cell cycle regula‐ tion (101). A mechanistic study revealed that two target genes, retinoblastoma-like 1 (RBL1) and RBL2, were involved in miR-106b-5p's regulation of cell proliferation (101). RBL proteins were involved in genomic instability, coinciding with decreased DNA methylation and increased acetylation of histone H3.

p27, one of the cyclin-dependent kinase inhibitor, inhibits cell cycle progression at the G1 phase by blocking the activation of cyclin-CDK complexes (102). The downregulation of p27 expression by miRNAs could abrogate the cell cycle checkpoints and could increase DNA damages in GBM cells. The regulatory effect of miR-221/222 on p27 and the subsequent effect on cell proliferation were also demonstrated in GBM cells (103). Moreover, p27 inhibition abrogated the growth advantage of cells with miR221/miR-222 downregulation (103).

DNA mismatch repair (MMR) is a system for recognizing and correcting insertions, deletions, and misincorporated bases at DNA replication and recombination. Deficiency of MMR system can be a cause for the development and progression of GBMs. There are two essential members of the DNA MMR genes, MutS homologs and MutL homologs. Computational algorithms predicted, and subsequently *in vitro* studies confirmed, several essential MMR genes, includ‐ ing MLH1, MSH2, and MSH6, as potential binding sites for miR-155. miR-155 is known to elevate its expression levels in primary and secondary GBMs (94).

As an important sensor in the DNA damage response, p53 functions to block the cell cycle to repair damaged DNA. Several miRNAs affect p53 or p53-regulated genes via different mechanisms. For instance, miR-125b, which has been shown to play a potential role in the development of glioma stem cells (104), directly targets p53 by binding to 3′-UTR of p53 and negatively regulates p53 expression (105). In addition, miR-125b negatively regulates MXD1 expression, an adaptor protein for MAD2L2 in the spindle assembly checkpoint. Moreover, miR-29, commonly downregulated in GBMs, indirectly upregulates p53 by targeting PIK3R1 and CDC42 (106). Previous studies have shown that miR-34 is directly regulated by p53 and represses various oncogenes, such as CCND1, CCNE2, CDK4, MET, MYC, SNAI1, and SIRT1. However, these miR-34-targeting oncogenes are upregulated in GBM, because miR-34 is usually downregulated (107). Several studies have shown that miR-34 is indispensable for the DNA damage response, and miR-34a-regulated genes are strongly related with DNA repair and apoptosis (108).

Ataxia telangiectasia mutated (ATM) is an important mediator in connecting DNA damage signals to downstream events, including damage repair. miR-101, a miRNA downregulated in GBM, could directly target ATM via the canonical action mechanism (109). In addition, miR-101 also targets PRKDC (protein kinase, DNA activated, catalytic polypeptide) to regulate nonhomologous end joining of DNA double-strand breaks (109).
