MicroRNAs and DNA damage response

Abstract

The DNA damage response (DDR) pathways play critical roles in protecting the genome from DNA damage. Abrogation of DDR often results in elevated genomic instability and cellular sensitivity to DNA damaging agents. Many proteins involved in DDR are subjected to precise regulation at multiple levels, such as transcriptional control and posttranslational modifications, in response to DNA damage. MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. The expression levels of some miRNAs change in response to DNA damage. Some miRNAs, such as miR-24, 138, 96 and 182, have been implicated in DDR and/or DNA repair and affect cellular sensitivity to DNA damaging agents. In this review, we summarize recent findings related to the emerging roles of miRNAs in regulating DDR and DNA repair and discuss their potential in cancer therapy.

Keywords: :

• microRNA
• DNA damage response
• DNA repair
• chemotherapy
• radiotherapy
• DNA damaging agents

MicroRNAs (miRNAs) are a class of noncoding, endogenous, short (usually 21–25 nt) and single-stranded RNA molecules. MiRNA genes are located in both intragenic and intergenic regions throughout the genome, where they are transcribed independently or together with their host genes.¹ MiRNAs represent a new class of post-transcriptional regulators of gene expression and play important roles in many biological processes and disease phenotypes.² Particularly, the expression of miRNAs is responsive to various endogenous and exogenous stimuli, such as treatment with anticancer DNA damaging agents. Cells with defects in miRNA biogenesis machinery have abnormal cell cycle checkpoints and DNA repair.^3,4 Some miRNAs, such as miR-16, miR-24, miR-138 and the miR-183-96-182 cluster, have been implicated in DNA damage response (DDR) and DNA repair.^3,5-8 These findings suggest an important role for miRNAs in DDR and DNA repair pathways. Understanding the connection between miRNAs and DDR/DNA repair will advance our knowledge of cancer development and exploitation of miRNAs for cancer treatment. There are several excellent reviews about miRNAs and DDR/DNA repair.^9-11 Here, we provide up-to-date summaries of miRNA-DDR interactions, propose our models for the roles of miRNAs in DDR and discuss the therapeutic potential of the miRNAs that regulate DDR/DNA repair.

DNA Damage Response and DNA Repair

To preserve genomic stability, cells have developed elaborate response pathways to handle DNA lesions that are continuously caused by genotoxic agents arising from various sources.¹² This DDR is a signaling network initiated by lesion recognition and amplified by multiple mediator signaling proteins, which eventually activate downstream effectors to modulate cell fate by manipulating gene expression, cell cycle control, DNA repair, apoptosis and senescence.¹² Activation of cell cycle checkpoints at either G₁/S, intra-S or G₂/M gives cells time to employ DNA repair machineries to resolve DNA lesions. Multiple DNA repair pathways have evolved to resolve various DNA lesions. These pathways include single-strand break repair, homologous recombination repair, non-homologous end-joining repair, nucleotide excision repair, mismatch repair, translesion DNA synthesis, the Fanconi anemia pathway and others. DNA damage levels that exceed the repair capacity of cells lead to the activation of apoptosis or senescence.¹²

Three of the phosphoinositide 3-kinases (PI3K)-like protein kinases, ATM, ATR and DNA-PKcs, play a central role in controlling the cellular response to DNA damage.¹³ ATM, activated after detection of DSBs, phosphorylates numerous substrates, including Chk2, p53 and BRCA1, and plays central roles in regulating cell cycle checkpoint activation and DNA repair.¹⁴ ATR, which is activated upon detection of single-stranded DNA molecules present at DNA lesions, including DSBs and stalled replication forks, phosphorylates Chk1 and other substrates to control cell cycle checkpoint and genomic stability.^15,16 DNA-PKcs, activated upon induction of DSBs, phosphorylates itself and other substrates and plays an important role in DSB repair through non-homologous end joining.¹⁴

Tumor cells with defects in DDR or DNA repair are hypersensitive to certain types of DNA damaging and chemotherapeutic agents. For example, ATM-deficient cells are hypersensitive to ionizing radiation (IR).¹⁷ ATR-deficient cells are hypersensitive to multiple DNA damaging agents, including topoisomerase inhibitors, UV radiation and DNA crosslinking agents.¹⁸ Tumor cells with defective homologous recombination, such as breast and ovarian cancer cells with BRCA1 or BRCA2 deficiency, are hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors,¹⁹ which promote DSB formation during replication due to unrepaired single-strand DNA breaks.²⁰ Homologous recombination-deficient tumor cells are also sensitive to interstrand DNA crosslinking agents, such as cisplatin and carboplatin.²¹ Therefore, targeting DDR or DNA repair pathways is an important strategy for developing novel anticancer therapeutic agents and for chemosensitizing cancer cells to conventional chemotherapy.²²

Biogenesis and Function of microRNAs

Maturation of miRNAs is a multistep process (Fig. 1). After being transcribed by RNA Pol II or Pol III in the nucleus,^1,23 the “stem-loop”-like primary-miRNAs (pri-miRNAs) are recognized and processed by the microprocessor complex that contains the RNase III enzyme, Drosha and its dsRNA-binding partner, DGCR8/Pasha.¹ The ~70 nucleotide excised hairpins (pre-miRNAs) are subsequently delivered to the cytosol by the exportin-5 (XPO5)-containing nuclear export protein complex.²⁴ Further processing of pre-miRNAs by the cytosolic RNase III enzyme, Dicer, with its dsRNA-binding partner TARBP2 (TRBP), generates miRNA duplexes.^25,26 Each strand of the duplex can be subsequently loaded into Argonaute (Ago) proteins to assemble the RNA-induced silencing complex (RISC),¹ depending on the thermodynamic stability at the 5′ or 3′ ends of miRNA duplexes.^27,28 The miRNA serves as a specificity factor for RISC by promoting the association of RISC with target mRNAs through base-paring interactions with complementary sites, mainly in the 3′-UTR of target mRNAs and sometimes in coding regions or 5′-UTR.^29,30 This leads to translational repression of the transcript, mRNA degradation or both.³¹ Interestingly, let-7 can bind to the 3′ end of its own primary transcript and provide a feedback loop, suggesting that miRNAs can also regulate the expression of noncoding RNAs.³² In addition to the canonical miRNA biogenesis pathway, minor pathways that bypass the requirement of the enzymatic activities of either Drosha^33,34 or Dicer^35-37 have also been identified.

Figure 1. Canonical miRNA biogenesis and its regulation by interacting partners. MiRNAs genes are transcribed by RNA polymerases II or III (Pol II/III) into pri-miRNAs from intergenic or intronic regions. The pri-miRNAs are recognized and processed by the DROSHA-DGCR8 complex to generate pre-miRNAs, which are subsequently released into cytosol by the XPO5-containing nuclear export receptor complex. The cytosolic pre-miRNAs will be further digested by the Dicer-TRBP complex to produce miRNA duplexes, which are unwinded and incorporated into the RISC complex for target recognition and silencing. Single-stranded miRNA can be decayed by the 5′-3′ exoribonuclease XRN2 or 3′-5′ exoribonuclease human polynucleotide phosphrylase (hPNPase). The unprocessed pre-miRNAs can also bind to MCPIP1, leading to degradation of pre-miRNAs. The biogenesis of miRNA is subjected to regulation by many proteins that bind to either pri-miRNAs or pre-miRNAs (refer to text for details).

MiRNA biogenesis is subjected to regulation by various mechanisms (Fig. 1). Some RNA binding proteins (such as DDX5, DDX17, SMADs, SNIP1, SF2 and TDP-43) bind to the stem regions of pri-miRNAs and mediate the interaction between the Drosha-DGCR8 complex and a subset of pri-miRNAs, leading to efficient processing.^38-43 The NF-90-NF-45 complex prevents access of the Drosha-DGCR8 complex to pri-miRNAs, causing a reduction of a subset of mature miRNAs.⁴⁴ Several RNA-binding proteins (such as TDP-43, Lin-28, KSRP) bind to the terminal loops of some pri-miRNAs and/or pre-miRNAs and promote or limit their processing.⁴² Binding of other proteins (such as p53, ERα) to these RNA binding proteins can modulate their regulatory roles in miRNA processing.^45,46 MCPIP1 can antagonize Dicer function to prevent the maturation of pre-miRNAs.⁴⁷ Furthermore, miRNA biogenesis is also associated with RNA-editing pathways.⁴⁸ For example, adenosine deaminases cause A-to-I editing of about 16% of human pri-miRNAs,^49-51 which can block the maturation of pri- or pre-miRNAs and/or change target specificity.^51,52 Deep sequencing of miRNA precursors also revealed extensive 3′ end uridylation and other modifications of pre-miRNAs.⁵³ In particular, Lin28 interacts with some miRNA precursors, including those of let-7, miR-107, miR-143 and miR-200c, to facilitate their terminal uridylation by TUT4 uridylyl transferase and subsequent degradation in stem cells.^54,55

The activity of the components of miRNA biogenesis machinery can be stimulated by post-translational modifications. For example, TRBP is phosphorylated by MAPK/ERK pathway upon growth factor stimulation, and this phosphorylation specifically regulates the expression of a subset of miRNAs.⁵⁶ Also, phosphorylation of Drosha is required for its nuclear localization.⁵⁷ These post-translational regulations of miRNA biogenesis proteins, together with the transcriptional control of pri-miRNAs, provide precise control over miRNA production under different physiological and pathological conditions, thereby relaying important roles in biological process and disease development. Furthermore, cellular miRNA levels also reflect the balance of biogenesis and turnover. Active miRNA degradation can be mediated by the 5′-3′ exoribonuclease XRN2⁵⁸ or the 3′-5′ exoribonuclease, human polynucleotide phosphorylase (hPNPase).^59,60

Modulation of microRNA Expression in DNA Damage Response

Expression of miRNAs is altered in response to DNA damage

Multiple normal cell models, such as human endothelial cells,⁶¹ human lymphoblast IM9 cells,^62,63 human thyroid cells⁶⁴ and mouse embryonic fibroblasts,⁶⁵ have been used to study IR-induced miRNA expression changes in various conditions. However, the lack of consistency among these cell lines indicates that the regulation of some miRNAs after DNA damage is cell-type specific. This is further supported by multiple studies in tumor cells. For example, Shin et al. identified a subset of miRNAs that were responsive to IR in A549 non-small cell lung cancer cells, but not in HCT116 colon carcinoma cells.^66,67 Niemoeller et al. found that only a few miRNAs are commonly upregulated in all six glioma and head and neck squamous cell carcinoma cell lines examined upon IR treatment.⁶⁸ Induction of miR-138 after IR treatment was observed in multiples cell lines, such as a HPV E6/E7-transformed lung epithelial cell line CRL-2741⁶⁹ and some glioma and head neck squamous cell carcinoma cell lines,⁶⁸ but not in IM9 B-cell lyphoblastoma cells,^63,66 A549 non-small cell lung cancer cells^63,66 or U2OS osteosarcoma cells.⁶

In addition to IR, treatment with other DNA damaging agents also modulates miRNA expression. For example, UV radiation alters the expression of miRNAs in both HeLa cells and normal human fibroblasts (NHFs).³ Hydrogen peroxide (H₂O₂) treatment induces a subset of miRNAs, such as the miR-16 family, the miR-106b family and the miR-183 cluster, in NHFs.⁷⁰ In another study, exposure to IR, H₂O₂ or etoposide causes differential miRNA expression in NHFs. In this study, some miRNAs can be modulated by two or all three types of DNA damage, while exposure to H₂O₂ and etoposide can modulate the expression of a unique set of miRNAs, respectively.⁷¹ Furthermore, cisplatin or 5-fluorouracil treatment, also elicits a unique miRNA response in two esophageal carcinoma cell lines (one adenocarcinoma and one squamous cell carcinoma).⁷² Thus, different DNA damaging agents can trigger different miRNA responses.

Mechanisms underlying DNA damage-induced miRNA response

Regulation of miRNA expression can occur at both transcriptional and post-transcriptional levels in response to DNA damage.⁷³ ATM, a key kinase that regulates DNA damage response, also plays a central role in miRNA expression after DNA damage, as loss of ATM prevents the alteration of a large set of miRNAs after DNA damage (Fig. 2).⁶⁵

Figure 2. A proposed model for the roles of miRNAs in DDR. DNA damage triggers the activation of PI3K-like kinases, such as ATM. ATM phosphorylates a subset of substrates (i.e., p53, KSRP, BRCA1 and ΔNp63α), which activate the transcription of some miRNA genes or enhance their processing through regulating the interaction between miRNA processing complex and pri- or pre-miRNAs. Activation of PI3K-like kinases may also suppress the transcription or processing of some miRNAs through unknown mechanisms. Activation of PI3K-like kinase-independent pathways may also contribute to miRNA response upon DNA damage. The differentially expressed miRNAs may modulate DDR through regulating the expression of DDR/DNA repair genes or their regulatory factors.

ATM regulates miRNA expression through several different mechanisms. One mechanism is the ATM-dependent phosphorylation of KH-type splicing regulatory protein (KSRP).⁶⁵ KSRP is an interacting factor of both Drosha and Dicer and regulates the maturation of a subset of miRNAs by recognizing the GGG triplets in their terminal loop region.⁷⁴ ATM-mediated phosphorylation of KSRP at S132, S274 and S670, significantly strengthens the interaction between KSRP and pri-miRNAs and also increases KSRP activity in miRNA processing.⁶⁵ Whether phosphorylation of KSRP at those sites is important for DDR remains to be determined. Since ATR and ATM share many of the same substrates, it will be interesting to determine whether KSRP is also phosphorylated by ATR in response to UV or replication stress, and whether KSRP phosphorylation plays a role in UV (or replication stress)-induced miRNA alteration.

Interestingly, while all of the 29 KSRP-dependent miRNAs are induced in an ATM-dependent manner in response to neocarzinostain (a radiomimetic agent) treatment,⁷⁴ an additional 42 miRNAs are induced in an ATM-dependent but KSRP-independent manner.⁶⁵

One possible KSRP-independent mechanism of ATM-mediated miRNA regulation is through p53. In response to DNA damage, ATM activates p53 by multiple mechanisms, such as Chk2-dependent phosphorylation of p53.^75,76 p53 regulates miRNA expression both by transcriptional regulation and post-transcriptional regulation. For example, p53 promotes the processing of a subset of pri-miRNAs through interaction with the DDX5 RNA helicase⁴⁵ and upregulates the transcription of pri-miR-34a-c after DNA damage.⁷⁷ Seven of the 10 miRNAs that were induced by doxorubicin in a p53-dependent manner are also induced by DNA damage in an ATM-dependent manner in MEFs,^45,65 suggesting that p53-mediated regulation is a mechanism by which ATM controls miRNA expression. However, only three of the seven miRNAs (miR-15a, miR-16 and miR-26b) are induced in a KSRP-dependent manner. This suggests that p53 and KSRP can regulate pri-miRNA processing differently.

Other mechanisms also contribute to ATM-dependent miRNA induction after DNA damage. ATM-dependent phosphorylation of ΔNp63α causes alteration of miRNA expression, both by transcriptionally modulating miRNA expression and by upregulating Dicer to stimulate miRNA maturation after cisplatin treatment.^78-80 BRCA1, activated by ATM upon DNA damage, facilitates the processing of some pri-miRNAs through direct interaction with Drosha, DDX5 and pri-miRNAs.⁸¹ However, these mechanisms cannot explain the ATM-dependent reduction of some miRNAs. Thus, there are likely additional ATM substrates that are relevant to ATM-dependent reduction of those miRNAs. Identification of such substrates is important for our understanding of the mechanisms of DNA damage-induced alteration of miRNA expression.

A subset of miRNAs responds to DNA damage in an ATM-independent, but DNA-PKcs or ATR-dependent, manner.^82,83 Although ATR and DNA-PKcs may share some substrates with ATM, they may differentially regulate other substrates and the expression of some miRNAs. Supporting this notion, Chaudhry et al. examined the expression of miRNAs in DNA-PKcs-deficient (M059J) and -proficient (M059K) glioma cells derived from the same patient.⁸² They found that miR-17-3p, miR-17-5p, miR-19a/b, miR-142-3p and miR-142-5p are upregulated in both cell lines, whereas miR-15a, miR-16, miR-143, miR-155 and miR-21 are upregulated only in DNA-PKcs-proficient cells, suggesting that their upregulation is dependent upon DNA-PKcs.⁸² In another study, miR-709 is induced in mouse testes possibly through ATR-dependent upregulation of Rfx-1, the host gene of miR-709.⁸³

Kinases other than ATM, ATR and DNA-PKcs, such as MAPKs, are also activated in response to DNA damage.⁸⁴ Their roles in DNA damage-induced miRNA response are far from being understood. Interestingly, Ago2 is phosphorylated at S387 by p38 MAPK, and this phosphorylation promotes the localization of the RISC complex to P-bodies.⁸⁵ Phosphorylation of Ago2 at site Y529 by an unidentified kinase may also prevent small RNA binding.⁸⁶ TRBP is phosphorylated by MAPK/ERK, and this phosphorylation modulates miRNA production by stabilizing the Dicer/TRBP complex.⁵⁶ Whether these phosphorylation events can be induced by UV or other types of DNA damage needs to be tested.

Furthermore, DNA damage may modulate the expression of miRNA biogenesis genes. As mentioned above, cisplatin can stimulate the transcription of Dicer in a ΔNp63α-dependent manner.⁸⁰ It is also possible that the enzymatic activities of Drosha and Dicer may be altered upon DNA damage by post-translational modifications of themselves or their binding partners, such as DGCR8, TRBP, DDX5 and DDX17. Interestingly, DDX17 is phosphorylated at two SQ sites (ATM/ATR consensus sites),⁸⁷ though the functional relevance of this phosphorylation in DNA damage-induced miRNA response needs to be studied.

Functions of miRNAs in DNA Damage Response

Although expression of a lot of miRNAs is altered in response to DNA damage, the roles for these miRNAs in DDR are still poorly understood. The conceptual models of the function of DDR-regulated miRNAs are shown in Figure 3. In Model A, upregulated miRNAs in DDR promote DDR by suppressing the negative regulators of DDR. In Model B, downregulated miRNAs in DDR release their inhibitory roles on DDR by targeting positive regulators of DDR. In Model C, upregulated miRNAs in DDR target positive regulators of DDR, or downregulated miRNAs target negative regulators of DDR. This results in fine-tuning DDR, or shutting off DNA repair upon the resolution of DNA damage or in the case of extensive and irreparable DNA damage. In Model D, DNA damage changes the interaction between unaltered microRNAs and the positive or negative regulators of DDR/DNA repair. Recent studies have shed some light on DDR-regulated miRNAs’ functions by identifying their targets relevant to cell cycle checkpoints and DNA repair (Table 1), both of which cooperate to prevent propagation of damaged genetic materials.

Figure 3. Conceptual models of the functions of miRNAs in DDR. (A) Upregulated miRNAs in DDR may promote DDR by suppressing the negative regulators of DDR; (B) downregulated miRNAs in DDR may release their inhibitory roles on DDR by targeting positive regulators of DDR; (C) upregulated miRNAs in DDR may target positive regulators of DDR or downregulated miRNAs may target negative regulators of DDR to reach an optimal DDR or to shut off DNA repair once completed or in the case of extensive damage; (D) DNA damage may change the interaction between unaltered microRNAs and the positive or negative regulators of DDR/DNA repair. Examples for miRNAs and their targeting DDR regulators are shown in brackets.

Table 1. MicroRNAs that directly regulate the expression of DDR genes

DDR genes	Function in DDR	miRNAs	References
ATM	DNA repair	miR-421, miR-100, miR-101	^99 ^- ^101
H2AX	DNA repair	miR-24, miR-138	^6 ^, ^7
BRCA1	DNA repair	miR-182, miR-146a, miR-146b-5p	^8 ^, ^102
RAD23B	DNA repair	miR-373	^134
RAD51	DNA repair	miR-96	^5
RAD52	DNA repair	miR-210, miR-373	^134
REV1	DNA repair	miR-96	^5
REV3L	DNA repair	miR-25, miR-32	^135
MSH2	DNA repair	miR-21, miR-155	^103 ^, ^114
MLH1	DNA repair	miR-155	^103
FANCG	DNA repair	miR-23a	^136
p53	cell cycle checkpoint, apoptosis	miR-125b, miR-504, miR-25, miR-30d	^137 ^- ^139
Wee1	cell cycle checkpoint	miR-128a, miR-155, miR-516–3p, miR-195	^140 ^, ^141
Wip1	cell cycle checkpoint	miR-16	^88
Cdc25A	cell cycle checkpoint	miR-16, miR-21, miR-322, miR-424, miR-503, miR-449a/b	^3 ^, ^89 ^, ^142 ^, ^143
c-myc	cell cycle checkpoint	miR-145, let-7	^144 ^, ^145
p21	cell cycle	miR-17, miR-106a/b	^96 ^, ^146
p27	cell cycle	miR-221/222	^147
E2F	cell cycle	miR-17–92, miR-20a, miR-34a, let-7b	^92
CDK2	cell cycle	miR-302, miR-372, miR-885–5p	^148 ^- ^150

In line with Model A, a number of studies find that some DNA damage-inducible miRNAs target the negative regulators of cell cycle checkpoint. The miR-16 family miRNAs, such as miR-16 and miR-15a/b, are induced by multiple DNA damage agents. Cdc25A and Wip1 are critical phosphatases that regulate DNA damage-induced cell cycle checkpoint. Induction of miR-16 leads to cell cycle checkpoint activation by suppressing Cdc25A and Wip1.^3,88 MiR-21, induced upon IR irradiation, represses Cdc25A and modulates cell cycle checkpoint activation.⁸⁹ The miR-34 family miRNAs (miR-34a, 34b and 34c) are induced by DNA damage in a p53-dependent manner. Induction of miR-34a can regulate the G₁/S checkpoint by modulating multiple genes (E2F, cyclinE2, CDK4 and CDK6),^77,90 whereas miR-34c regulates the S-phase checkpoint by targeting c-myc.⁹¹ The let-7 family miRNAs are also induced by DNA damage. Overexpression of let-7a induces G₁/S cell cycle arrest by targeting E2F2 and cyclin D2 in prostate cancer cells.⁹² Overexpression of let-7b arrests primary fibroblasts at G₂/M by direct repression of Cdc34 (a subunit of SCF E3 ligase complex), which leads to stabilization of Wee1 kinase.⁹³ Overexpression of let-7b also downregulates the expression of cyclins D1/D3/A and Cdk4 in melanoma cells.⁹⁴ Furthermore, miR-24 can activate G₁/S cell cycle checkpoint mainly by targeting E2F2.⁹⁵

In agreement with Model B, downregulation of some miRNAs is required for efficient cell cycle checkpoint activation. For example, miR-106b is downregulated in IR-irradiated LNCaP prostate cancer cells, and this downregulation is required for p21-mediated G₂/M cell cycle arrest.⁹⁶ Some of the miR-17 family miRNAs (three paralog clusters: miR-17–92, miR-106a-363 and miR-106b-25) are involved in cell cycle control. Inhibition of miR-17 or miR-20a leads to an E2F1-associated DDR and G₁ checkpoint activation.⁹⁷ MiR-17-5p acts specifically at the G₁/S boundary by targeting more than 20 genes involved in the G₁/S transition.⁹⁸ Furthermore, downregulation of some miRNAs allows rapid accumulation of DNA repair proteins following DNA damage. Among these miRNAs, miR-421, miR-101 and miR-100 suppress the expression of ATM.^99-101 MiR-101 also targets DNA-PKcs,¹⁰¹ and miR-138 and miR-24 both target histone H2AX.^6,7 The miR-183-96-182 polycistronic miRNA cluster, which is rapidly downregulated in response to IR, may affect DNA repair through miR-182-mediated suppression of BRCA1⁸ or miR-96-mediated suppression of RAD51 and REV1.⁵

There are also some miRNAs that are upregulated in response to DNA damage and target DNA repair or DDR genes. These miRNAs may impose a threshold on the activation of DNA repair or DDR, thereby buffering the cellular system for optimal activation of DNA repair and DDR (Fig. 3, Model C). For example, miR-146a and miR-146b-5p, which target BRCA1,¹⁰² are induced in response to DSBs.⁶⁵ MiR-155, induced upon DNA damage, targets MLH1 and MSH2 and negatively regulates mismatch repair.¹⁰³ In addition, the MLH1-PMS2 heterodimer facilitates the processing of mir-422a, which, in turn, suppresses MLH1 through base pairing with the MLH1 3′-untranslated region.¹⁰⁴ These miRNAs may provide an opportunity for cells to choose DNA repair pathways in certain cell types or provide a way for cells to shut down DNA repair machinery after extensive damage. Moreover, the induction of Dicer expression is in concert with the induction of let-7 and miR-103/107 miRNAs after DNA damage, both of which can target Dicer.^105-108 This suggests that a feedback control by let-7 and miR-103/107 may limit the extent of Dicer activity to reach an optimal activation of DDR.

Some DNA damage non-responsive miRNAs may also regulate DDR (Fig. 3, model D). For example, although miR-19 level was not changed after UV irradiation, UV prevents the binding of miR-19 to its target site in the 3′UTR of RhoB by dissociating the AU-rich element binding protein HuR from an adjacent binding site, thereby protecting the cell from apoptosis.¹⁰⁹ This observation suggests that DNA damage may alter the function of miRNA in DDR through modulating the environment for efficient miRNA-target interaction. Further work is needed to evaluate the contribution of this type of regulation in DDR.

Thus, many DDR and DNA repair genes are subjected to precise control at the post-transcriptional level by miRNAs. Some of these genes are targeted by multiple miRNAs, while some miRNAs can also target multiple DDR/DNA repair genes. Many miRNAs are altered mildly in DNA damage, and the small change in expression of individual miRNA may not produce a significant effect on DDR. However, the collective effects of multiple miRNAs that target the same gene or a common pathway (such as cell cycle checkpoint or homologous recombination repair) may be important for an effective cellular response to DNA damage.

Interestingly, a new class of small non-coding RNAs, other than miRNAs, is also important for DDR. Wei et al. identified that a new class of Dicer-processed small non-coding RNAs, called DSB-induced small RNAs (diRNAs), can be generated at DNA damage sites in both Arabidopsis and human cells.⁴ These diRNAs are important for the activation of homologous recombination repair, possibly by directing certain chromatin modifications.⁴ Francia et al. further demonstrated that site-specific DICER and DROSHA RNA products, but not miRNAs, are required for efficient foci formation of several DDR proteins, including phospho-ATM, 53BP1, MDC1 and ATM/ATR substrates (recognized by phospho S/TQ antibody).¹¹⁰ However, this does not rule out the possibility that some specific miRNAs are required for efficient recruitment and accumulation of certain DDR proteins in the presence of substantial DNA damage.

MiRNAs Serve as Therapeutic Agents or Targets for Improving Cancer Treatment with DNA Damaging Agents

Because of the key role of DDR and DNA repair in cancer treatment with DNA damaging agents, targeting DDR and DNA repair is becoming a very attractive strategy to overcome chemo-/radio-resistance.²² In particular, miRNAs are promising agents for improving the efficacy of conventional cancer therapy with DNA damaging agents, attributing to their active involvement in DDR and their ability to target DDR components to control cellular response to DNA damaging agents.

For example, inhibition of ATM by miR-101, miR-100 and miR-421 or inhibition of DNA-PKcs by miR-101 causes increased cellular sensitivity to IR.^99-101 Inhibition of H2AX expression by either miR-24 or miR-138 promotes cellular sensitivity to IR and/or genotoxic agents.^6,7 Several other IR-responsive miRNAs, such as miR-521, miR-127, let-7g, miR-125a and miR-189, can also modulate radiosensitivity by targeting DDR genes,^61,111 although the mechanisms are less clear. Furthermore, miR-34a negatively regulates cellular resistance to 5-FU by regulating Sirt1 and E2F3 in DLD-1 colon cancer cells,¹¹² while miR-21 promotes resistance to 5-FU by suppressing a mismatch repair gene, MSH2, in colon cancer cells.^113,114 MiR-143 and miR-145, whose expression is induced by DNA damage through p53, target MDM2 and, thus, provide positive feedback to strengthen the function of p53 in mediating DNA damage-induced cell death.¹¹⁵

To identify miRNAs that modulate chemosensitivity by inhibiting homologous recombination DNA repair, we have developed a cell-based high-throughput screen assay. We transfected individual miRNA mimics into a human osteosarcoma cell line U2OS, irradiated cells with IR and monitored RAD51 foci formation using automated fluorescence microscopy (Huang et al., manuscript in preparation). By screening the human miRNA mimic library, we identified a set of miRNAs that inhibit IR-induced RAD51 foci formation, including miR-96.⁵ MiR-96 targets both RAD51 and REV1, an error-prone Y-family DNA polymerase required for translesion synthesis across the unhooked interstrand crosslinks.^116,117 Overexpression of miR-96 promotes cellular hypersensitivity to both cisplatin and a PARP inhibitor, AZD2281.⁵ Interestingly, Moskwa et al. reported that overexpression of miR-182, another member of the miR-183-96-182 cluster, inhibits BRCA1 and induces hypersensitivity to PARP inhibitors in vitro and in a xenograft model.⁸ However, we did not observe significant effects of miR-182 or miR-183 on chemosensitivity to cisplatin or AZD2281,⁵ suggesting a potential cell type-specific miRNA-target interaction.

Given the fact that some miRNAs can target multiple genes involved in DDR, modulating endogenous miRNA expression may be a promising way to overcome chemoresistance in cancer treatment. Overexpression of miRNA can be achieved by ectopic expression of mature miRNAs or their precursors. Suppression of endogenous miRNAs can be reached with anti-miR oligonucleotides or miRNA sponges, which express a transgene-containing multiple tandem binding sites for endogenous miRNA.¹¹⁸ MiRNA-masking antisense oligonucleotides can also be used to protect particular targets of miRNAs.¹¹⁹

Multiple technologies have been developed to achieve systemic delivery of miRNA mimics or anti-miRs. They include application of chemically modified oligonucleotides, adenoviral or lentiviral-based delivery and nanoparticle-based delivery.^120-124 Chemical modification of oligonucleotides, such as adding sugar and phosphate modifications into the active and passenger strands of duplex miRNA mimics or anti-miRs, improves their activities and stability.^125,126 Recent studies with modified nanoparticles also provide some potential selective targeting tools for delivering small RNAs in vivo.^127,128 Substantial studies provide solid evidence for the use of miRNAs as therapeutic agents or tools in cancer therapy, but the side effects of miRNA therapy also need much attention. This is important since each miRNA may target numerous transcripts and high expression of miRNA mimics may interfere with the endogenous miRNAs or siRNAs by saturating the RISC complex. Therefore, the safety of miRNA formulations needs to be extensively evaluated in disease models.

Conclusions and Future Directions

In summary, miRNA expression is modulated in response to DNA damage at either the transcriptional or post-transcriptional level. Some of these miRNAs can regulate the expression of a wide range of DDR/DNA repair genes and modulate cellular sensitivity to DNA damaging agents. This level of regulation occurs at intervals between rapidly induced post-translational modifications and late-initiating transcriptional control,¹²⁹ thus having fundamental roles on the integrity of DDR. However, many questions remain to be addressed for a more complete understanding of the importance of miRNA response in DDR.

First, we need to evaluate whether DNA damage triggers post-translational modifications (i.e., phosphorylation) of canonical miRNA biogenesis proteins to regulate miRNA maturation. For example, phosphorylation of Drosha at S300 or S302 was shown to be necessary for its nuclear retention and pri-miRNA processing,⁵⁷ but analyses of other proteins involved in the miRNA biogenesis are incomplete. It is possible that these phosphorylation events are mediated by ATM, other PI3K-like kinases, their downstream kinases or completely different kinases.

Second, it is important to elucidate a mechanism by which some miRNAs are downregulated in DDR, since downregulation of certain miRNAs may release their inhibitory roles on cell cycle checkpoint control or DNA repair, allowing efficient DDR.

Third, it should be tested whether miRNAs can be edited to modulate their stability and/or activities in DDR.

Lastly, it will be interesting to investigate whether DNA damage triggers secretion of certain miRNAs that are taken up by surrounding cells to modulate the activation of DDR and cellular sensitivity to DNA damaging agents. Recent studies found that miRNAs can be secreted into serum by different mechanisms and taken up by adjacent cells.¹³⁰ A significant number of miRNAs were found to be rapidly upregulated in the blood of radiotherapy-treated mice or patients.^131-133 Whether these secreted miRNAs play roles in DDR needs to be studied.

Fully addressing these questions will help to understand the functions of miRNAs in DDR and provide solid evidence for designing novel strategies for cancer treatment.

Abbreviations:
miRNA	=	microRNA
pri-miRNA	=	primary miRNA
pre-miRNA	=	precursor miRNA
DDR	=	DNA damage response
DSBs	=	double-strand breaks
PARP	=	poly(ADP-ribose) polymerase
IR	=	ionizing radiation
NHFs	=	normal human fibroblasts

Acknowledgments

We appreciate all the members of Taniguchi lab for helpful discussion. We thank Dr. Muneesh Tewari, Dr. Kiranjit Dhillon, Jen-wei Huang and Philamer Calses for critical reading of the manuscript. This work was supported by Howard Hughes Medical Institute, the NIH/ NHLBI (R21 HL092978 to T.T.), the NIH/ NCI (R01 CA125636 to T.T.), Fanconi Anemia Research Fund (to T.T.) and the NIH (P30 DK56465 to Y.W. and T.T.). Y.W. is a research fellow supported by Canadian Institute of Health Research.