**4. miRNA regulation of signal transduction for DNA damage**

miRNAs regulate multiple aspects of the DNA damage response pathway, including regulation of signal transduction of DNA damage, changing expression level of master regulatory proteins such as P53, modulating key protein expression in different types of DNA repair such as MMR, NER, NHEJ and HRR. Figure 2 and Table 1 summarize recently reported miRNAs associated with DNA damage and repair.

Roles of MicroRNA in DNA Damage and Repair 345

the miRNA-processing pathway (Dicer and Ago2) were knocked down. miRNA mediated gene regulation operates earlier than most other transcriptional responses following

H2AX, a histone variant, is an initial sensor protein for the DNA damage response. The function of H2AX is associated with DNA double strand break repair. miR-24 expression is up-regulated during hematopoietic cell differentiation into multiple lineages. miR-24 regulates H2AX expression through binding to its 3'-UTR. Both H2AX mRNA and protein levels are dramatically reduced by high levels of miR-24 in terminal differentiated human blood cells. miR-24 mediated suppression of H2AX in terminally differentiated blood cells renders them hypersensitive to gamma-irradiation, deficient in DSB repair, and susceptible

Wild-type p53-induced phosphatase 1 (Wip1) is an oncogene with critical function in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 reverses DNA damage–induced cell cycle checkpoints by dephosphorylating several key DNA damage responsive proteins. Recently, miRNAs are found to play an important role in suppressing Wip1 activity. Knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, and tumor formation in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16 expression results in marked regression of prostate tumor xenografts. The function of miR-15a and miR-16 is considered through their regulation of Wip1 expression. miR-16 can down-regulate the expression level of Wip1

Fig. 2. miRNAs directly regulate DNA repair

to chromosomal instability (Lal et al., 2009).

genotoxic stress (Pothof et al., 2009).

Fig. 1. MicroRNA biogenesis. A microRNA gene is transcribed by RNA polymerase II (RNAPII) to produce a pri-miRNA. The pri-miRNA is formed by RNase III family Drosha, cooperating in a complex with dsRNA-binding proteins DGCR8. The Drosha–DGCR8 complex processes the pri-miRNA into an ~70-nucleotide pre-miRNA, which is exported to the cytoplasm by expotin 5. The cytoplasm pre-miRNA is cleaved by Dicer, assisted by TRBP and AGO2, and yields an ~20-bp miRNA/miRNA\* duplex. One strand of the miRNA/miRNA\* duplex is preferentially incorporated into a miRNA-induced silencing complex (RISC), whereas the other strand is degraded (not shown). RISC recognizes target mRNAs and lets the miRNA binds to its target mRNA to suppress gene expression, either by mRNA cleavage or translational repression.

DNA damage activates the signal transduction process that leads to cell cycle arrest, which can lead to apoptosis or DNA repair. This DNA-damage response is mainly regulated at the transcriptional and posttranslational levels. Recent evidence suggests that miRNAs offer another degree of regulation at the posttranscriptional level in response to DNA damage. The DNA damage response to UV light was severely attenuated after the key components of

Fig. 1. MicroRNA biogenesis. A microRNA gene is transcribed by RNA polymerase II (RNAPII) to produce a pri-miRNA. The pri-miRNA is formed by RNase III family Drosha, cooperating in a complex with dsRNA-binding proteins DGCR8. The Drosha–DGCR8 complex processes the pri-miRNA into an ~70-nucleotide pre-miRNA, which is exported to the cytoplasm by expotin 5. The cytoplasm pre-miRNA is cleaved by Dicer, assisted by TRBP and AGO2, and yields an ~20-bp miRNA/miRNA\* duplex. One strand of the miRNA/miRNA\* duplex is preferentially incorporated into a miRNA-induced silencing complex (RISC), whereas the other strand is degraded (not shown). RISC recognizes target mRNAs and lets the miRNA binds to its target mRNA to suppress gene expression, either

DNA damage activates the signal transduction process that leads to cell cycle arrest, which can lead to apoptosis or DNA repair. This DNA-damage response is mainly regulated at the transcriptional and posttranslational levels. Recent evidence suggests that miRNAs offer another degree of regulation at the posttranscriptional level in response to DNA damage. The DNA damage response to UV light was severely attenuated after the key components of

by mRNA cleavage or translational repression.

Fig. 2. miRNAs directly regulate DNA repair

the miRNA-processing pathway (Dicer and Ago2) were knocked down. miRNA mediated gene regulation operates earlier than most other transcriptional responses following genotoxic stress (Pothof et al., 2009).

H2AX, a histone variant, is an initial sensor protein for the DNA damage response. The function of H2AX is associated with DNA double strand break repair. miR-24 expression is up-regulated during hematopoietic cell differentiation into multiple lineages. miR-24 regulates H2AX expression through binding to its 3'-UTR. Both H2AX mRNA and protein levels are dramatically reduced by high levels of miR-24 in terminal differentiated human blood cells. miR-24 mediated suppression of H2AX in terminally differentiated blood cells renders them hypersensitive to gamma-irradiation, deficient in DSB repair, and susceptible to chromosomal instability (Lal et al., 2009).

Wild-type p53-induced phosphatase 1 (Wip1) is an oncogene with critical function in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 reverses DNA damage–induced cell cycle checkpoints by dephosphorylating several key DNA damage responsive proteins. Recently, miRNAs are found to play an important role in suppressing Wip1 activity. Knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, and tumor formation in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16 expression results in marked regression of prostate tumor xenografts. The function of miR-15a and miR-16 is considered through their regulation of Wip1 expression. miR-16 can down-regulate the expression level of Wip1

Roles of MicroRNA in DNA Damage and Repair 347

miRNAs are involved in DNA repair by regulating critical components of the DNA repair pathways, such as P53. As a transcription factor, the tumor suppressor P53 is a powerful regulator of diverse cellular processes including cell-cycle arrest, DNA repair, apoptosis and cellular senescence. P53 and its signaling pathway, play a pivotal role in maintaining genomic stability and tumor suppression (Levine et al., 2004; Levine et al., 2006). Recently, P53 activity was found to be widely regulated by a number of miRNAs. These miRNAs either directly target the 3' UTR of P53 or indirectly regulate P53 activity by modulating proteins associated with P53 (Figure 3). Among these miRNAs, miR-504 negatively regulate p53 expression through binding to two DNA *cis* element located in the P53 3' UTR. Ectopic expression of miR-504 reduces the protein level of P53 and impairs P53-mediated apoptosis and cell cycle arrest (Hu et al., 2010b). miR-125b is another negative regulator of P53 in both zebrafish and humans (Le et al., 2009). Knocking down of miR-125b increased the expression level of P53; and overexpression of miR-125b suppressed the expression of P53. Interestingly, miR-125b was downregulated when the Zebrafish embryo was exposed to gamma irradiation, corresponding to

In addition to the direct binding to P53, several miRNA including miR-34a, miR-29 and miR-122 can indirectly modify P53 activity (Fornari et al., 2009; Park et al., 2009; Yamakuchi et al., 2008). miR-34a is a direct transcriptional target of P53 (Chang, et al., 2007; Corney, et al., 2007; Raver-Shapira, et al., 2007). P53 can up-regulate miR-34a expression by binding to a palindromic sequence located in miR-34a promoter region. miR-34a can positively regulate P53-dependent apoptosis through another intermediate protein, SIRT1 (Yamakuchi, et al., 2008). miR-34 inhibition of SIRT1 leads to an increase in acetylated P53. As a result, miR-34 suppression of SIRT1 ultimately leads to P53 mediated apoptosis in human colon cancer cells. miR-29 family members directly suppress P85a and CDC42, both of which negatively regulate P53. As a result, miR-29 positively up-regulates P53 level and induces apoptosis and DNA repair in a P53-dependent manner (Park, et al., 2009). miR-122 is a liver-specific miRNA accounting for 70% of the total miRNA population. miR-122 can down-regulate the expression of cyclin G1, which has the potential to inhibit P53 activity and promote cancer development. From a therapeutic perspective, miR-122 has potential to become a miRNA based therapy for

MMR corrects erroneous deletion, insertion, or mis-incorporation of bases resulting from DNA replication, DNA recombination, or DNA damage. Human mutS homolog 2 (hMSH2) and mutL homolog 1 (hMLH1) function as core proteins in MMR. They form heterodimers with protein homologs hMSH3 or hMSH6 (Fishel, 2001). The over-expression of miR-21 is linked to progression of human colorectal cancer (Link et al., 2010; Ng et al., 2009). It was reported recently that miR-21 directly targeted the 3′ UTRs of both the hMSH2 and hMSH6 mRNAs (Valeri et al., 2010a). Protein levels of hMSH2 and hMSH6 in the cells transfected with a locked nucleic acid (LNA) against miR-21 were significantly increased over the control cells. In addition, the over-expression of miR-21 was inversely correlated with the down regulation of hMSH2 in colorectal cancer tumors. Because the hMSH2-hMSH6 heterodimer is the key initiation component of MMR, the down regulation of hMSH2 is

miR-155 also plays a critical role in MMR. Over-expression of miR-155 reduced the levels of the human mismatch repair genes MLH1, MSH2 and MSH6 in a colorectal cancer cell line.

**5. miRNA regulation of core components of DNA damage response** 

the up-regulation of P53 protein induced by the irradiation exposure.

hepatocellular carcinoma (HCC) patients (Fornari, et al., 2009).

**6. Functions of miRNAs in mismatch repair (MMR)** 

likely to suppress MMR, and ultimately enhance tumor progression.


by targeting the 3' UTR of Wip1. As a result, the Wip1 protein level is significantly deceased, which prevents a premature inactivation of ATM/ATR signaling and allows a functional completion of the early DNA damage response (Zhang et al., 2010).

Table 1. miRNAs involved in DNA repair (notes: - means inhibite and + means stimulate)

ATM is a serine/threonine kinase that transfers the DNA damage signals to down-steam events, such as cell cycle arrest, apoptosis and DNA repair (Lavin, 2008; Shiloh, 2003). ATM plays a critical role in the maintenance of genomic stability by activating cell cycle checkpoints and promoting DNA double-strand breaks repair. M059J is a human malignant glioma cell line with high sensitivity to ionizing radiation due to low-expression of ATM. The low-expression of ATM is related to miR-100 (Ng et al., 2010). Both computational analysis and luciferase reporter gene assay indicate that miR-100 can target the 3'-UTR of ATM. miR-100 was found to be highly-expressed in M059J cells by RNase protection assay and qRT-PCR. Up-regulation of miR-100 in M059K cells reduces ATM expression and renders them hypersensitive to ionizing radiation, while Knock-down of miR-100 promotes ATM expression in M059J cells. These results indicate that the low-expression of ATM in M059J cells is mainly due to the high expression of miR-100.

Another miRNA miR-421 is also involved in ATM regulation. miR-421 suppresses the expression of ATM by targeting the 3' UTR of ATM. Ectopic expression of miR-421 lead to a deficient cell cycle checkpoint in S-phase and increased sensitivity to ionizing radiation (Hu et al., 2010a). Blocking the interaction between miR-421 and ATM with chemically synthesized oligonucleotides rescued the defective phenotype caused by miR-421 over expression, suggesting that ATM mediates the effect of miR-421 on cell-cycle checkpoints followed by radiation.

by targeting the 3' UTR of Wip1. As a result, the Wip1 protein level is significantly deceased, which prevents a premature inactivation of ATM/ATR signaling and allows a functional

miR-373 NER RAD23B - Crosby, Kulshreshtha. 2009 miR-21 MMR MSH2, MSH6 - Valeri, Gasparini. 2010) miR-155 MMR MLH1, MSH2 - Volinia, Calin. 2006 miR-182 HRR BRCA1 - Moskwa, Buffa. 2011 miR-210 HRR RAD52 - Crosby, Kulshreshtha. 2009 miR-373 HRR RAD52 - Crosby, Kulshreshtha. 2009

miR-34a P53 SIRT1 + Yamakuchi, Ferlito. 2008) miR122 P53 Cyclin G1 + Fornari, Gramantieri. 2009

Table 1. miRNAs involved in DNA repair (notes: - means inhibite and + means stimulate) ATM is a serine/threonine kinase that transfers the DNA damage signals to down-steam events, such as cell cycle arrest, apoptosis and DNA repair (Lavin, 2008; Shiloh, 2003). ATM plays a critical role in the maintenance of genomic stability by activating cell cycle checkpoints and promoting DNA double-strand breaks repair. M059J is a human malignant glioma cell line with high sensitivity to ionizing radiation due to low-expression of ATM. The low-expression of ATM is related to miR-100 (Ng et al., 2010). Both computational analysis and luciferase reporter gene assay indicate that miR-100 can target the 3'-UTR of ATM. miR-100 was found to be highly-expressed in M059J cells by RNase protection assay and qRT-PCR. Up-regulation of miR-100 in M059K cells reduces ATM expression and renders them hypersensitive to ionizing radiation, while Knock-down of miR-100 promotes ATM expression in M059J cells. These results indicate that the low-expression of ATM in

Another miRNA miR-421 is also involved in ATM regulation. miR-421 suppresses the expression of ATM by targeting the 3' UTR of ATM. Ectopic expression of miR-421 lead to a deficient cell cycle checkpoint in S-phase and increased sensitivity to ionizing radiation (Hu et al., 2010a). Blocking the interaction between miR-421 and ATM with chemically synthesized oligonucleotides rescued the defective phenotype caused by miR-421 over expression, suggesting that ATM mediates the effect of miR-421 on cell-cycle checkpoints

completion of the early DNA damage response (Zhang et al., 2010).

Involved Protein Effect

miRNA Pathway Target Net Reference

miR-24 DDR H2AX - Lal, Pan. 2009 miR-16 DDR Wip1 + Zhang, Wan. 2010 miR-100 DDR ATM - Ng, WL. 2010 miR-101 DDR ATM - Yan, Ng. 2010 miR-421 DDR ATM - Hu, Du. 2010)

miR-101 NHEJ DNA-PKcs Yan, Ng. 2010 miR-29 P53 P85a, CDC42 + Park, Lee. 2009

miR-125b P53 P53 - Le, Teh. 2009 miR-504 P53 P53 - Hu, Chan. 2010

M059J cells is mainly due to the high expression of miR-100.

followed by radiation.

### **5. miRNA regulation of core components of DNA damage response**

miRNAs are involved in DNA repair by regulating critical components of the DNA repair pathways, such as P53. As a transcription factor, the tumor suppressor P53 is a powerful regulator of diverse cellular processes including cell-cycle arrest, DNA repair, apoptosis and cellular senescence. P53 and its signaling pathway, play a pivotal role in maintaining genomic stability and tumor suppression (Levine et al., 2004; Levine et al., 2006). Recently, P53 activity was found to be widely regulated by a number of miRNAs. These miRNAs either directly target the 3' UTR of P53 or indirectly regulate P53 activity by modulating proteins associated with P53 (Figure 3). Among these miRNAs, miR-504 negatively regulate p53 expression through binding to two DNA *cis* element located in the P53 3' UTR. Ectopic expression of miR-504 reduces the protein level of P53 and impairs P53-mediated apoptosis and cell cycle arrest (Hu et al., 2010b). miR-125b is another negative regulator of P53 in both zebrafish and humans (Le et al., 2009). Knocking down of miR-125b increased the expression level of P53; and overexpression of miR-125b suppressed the expression of P53. Interestingly, miR-125b was downregulated when the Zebrafish embryo was exposed to gamma irradiation, corresponding to the up-regulation of P53 protein induced by the irradiation exposure.

In addition to the direct binding to P53, several miRNA including miR-34a, miR-29 and miR-122 can indirectly modify P53 activity (Fornari et al., 2009; Park et al., 2009; Yamakuchi et al., 2008). miR-34a is a direct transcriptional target of P53 (Chang, et al., 2007; Corney, et al., 2007; Raver-Shapira, et al., 2007). P53 can up-regulate miR-34a expression by binding to a palindromic sequence located in miR-34a promoter region. miR-34a can positively regulate P53-dependent apoptosis through another intermediate protein, SIRT1 (Yamakuchi, et al., 2008). miR-34 inhibition of SIRT1 leads to an increase in acetylated P53. As a result, miR-34 suppression of SIRT1 ultimately leads to P53 mediated apoptosis in human colon cancer cells. miR-29 family members directly suppress P85a and CDC42, both of which negatively regulate P53. As a result, miR-29 positively up-regulates P53 level and induces apoptosis and DNA repair in a P53-dependent manner (Park, et al., 2009). miR-122 is a liver-specific miRNA accounting for 70% of the total miRNA population. miR-122 can down-regulate the expression of cyclin G1, which has the potential to inhibit P53 activity and promote cancer development. From a therapeutic perspective, miR-122 has potential to become a miRNA based therapy for hepatocellular carcinoma (HCC) patients (Fornari, et al., 2009).

#### **6. Functions of miRNAs in mismatch repair (MMR)**

MMR corrects erroneous deletion, insertion, or mis-incorporation of bases resulting from DNA replication, DNA recombination, or DNA damage. Human mutS homolog 2 (hMSH2) and mutL homolog 1 (hMLH1) function as core proteins in MMR. They form heterodimers with protein homologs hMSH3 or hMSH6 (Fishel, 2001). The over-expression of miR-21 is linked to progression of human colorectal cancer (Link et al., 2010; Ng et al., 2009). It was reported recently that miR-21 directly targeted the 3′ UTRs of both the hMSH2 and hMSH6 mRNAs (Valeri et al., 2010a). Protein levels of hMSH2 and hMSH6 in the cells transfected with a locked nucleic acid (LNA) against miR-21 were significantly increased over the control cells. In addition, the over-expression of miR-21 was inversely correlated with the down regulation of hMSH2 in colorectal cancer tumors. Because the hMSH2-hMSH6 heterodimer is the key initiation component of MMR, the down regulation of hMSH2 is likely to suppress MMR, and ultimately enhance tumor progression.

miR-155 also plays a critical role in MMR. Over-expression of miR-155 reduced the levels of the human mismatch repair genes MLH1, MSH2 and MSH6 in a colorectal cancer cell line.

Roles of MicroRNA in DNA Damage and Repair 349

is a core component of mammalian NHEJ and involves a catalytic subunit (DNA-PKcs) that can act as a regulatory element. DNA-PKcs is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. Therefore, DNA-PKcs is an essential factor for NHEJ. Yan et al. found that miR-101 could efficiently target DNA-PKcs and ATM via binding to their 3'- UTRs. Up-regulating miR-101 efficiently reduced the protein levels of DNA-PKcs and ATM in tumor cells, and most importantly, sensitized the tumor cells to radiation in vitro and in vivo (Yan et al., 2010). Radiotherapy kills tumor-cells by inducing DNA double strand breaks (DSBs). However, the efficient repair of double strand breaks in tumors frequently prevents successful treatment. Therefore, miR-101 could be used to target DNA DSB repair genes, in order to sensitize

**9. Functions of miRNAs in homologous recombination repair (HRR)** 

HRR is the most widely used repair mechanism which can accurately repair DNA double strand breaks. HRR reconstitutes the genetic information using the sister chromatid as a template. Several proteins are involved in the HRR process. Rad 52 protein recognizes double-strand breaks and adheres to the free ends of the break while the Rad51 protein, together with tumor-suppressor protein BRCA1, searches the undamaged sister chromatid

Both miR-210 and miR-373 were up-regulated in hypoxic cells. Up-regulation of miR-210 significantly suppressed the expression level of RAD51, while up-regulation of miR-373 inhibited the expression of RAD52. The modulation of miR-210 to RAD51 and miR-373 to RAD52 were verified by microarray analysis and luciferase reporter gene assay. Both of the miRNAs can bind to the binding sites in the 3' UTRs of their respective target mRNAs (Crosby, et al., 2009). Thus, hypoxia-inducible miR-210 and miR-373 regulate HRR via

BRCA1 is a constituent of several different protein complexes and is a key protein for HRR. Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients (Mueller and Roskelley, 2003). It was recently reported that miR-182 down-regulated BRCA1 expression. As a result, the HRR efficiency for DNA double strand break repair was impaired (Moskwa et al., 2011; Yao and Ventura, 2011). Antagonizing miR-182 enhanced BRCA1 protein level, which, in turn, protected cells from irradiation exposure. Over-expressing of miR-182 reduced BRCA1 protein level, which impaired HRR efficiency and rendered cells hypersensitive to irradiation. The impaired HRR phenotype due to miR-182 over-expression was able to be fully rescued by overexpressing of BRCA1. Thus, these data demonstrate miR-182-mediated down-regulation of

miRNAs appear to be involved in DNA damage and repair in many ways. miRNA biogenesis, including miRNA gene transcription and miRNA maturation processes, is readily altered in response to DNA damage. miRNAs regulate the ATM and P53 that are the regulators of the global induction of miRNA biogenesis upon DNA damage. miRNAs are also involved in signal transduction processes that leads to cell cycle arrest, apoptosis or DNA repair upon DNA damage. miR-100 and miR-421 can regulate expression of ATM, a

tumors to radiation and improve tumor radiotherapy.

for homologous pairing (Haber, 2000; Orelli and Bishop, 2001).

targeting RAD51 and RAD52.

BRCA1 suppresses HRR.

**10. Conclusion** 

In addition, high expression of miR-155 was inversely correlated with the low expression of MLH1 and MSH2 protein in human colorectal cancer. More importantly, human tumors with unexplained MMR inactivation showed miR-155 over expression (Valeri et al., 2010b; Volinia et al., 2006). These results indicate that increased expression of miR-155 downregulates MMR genes and results in an increase in genomic instability.

Fig. 3. miRNA indirectly regulates DNA repair through P53

miR-504 and miR-125b directly bind to the P53 3'-UTR and down-regulate P53 activity. miR-34a positively up-regulates P53 through SIRT1 inhibition, a negative regulator of P53. miR-29 down-regulates the P85a regulatory subunit of PI3K, which enhances P53 activity through the negative feedback loop between PI3K-AKT-MDM2 and P53.
