Review Article MicroRNAs in the DNA Damage/Repair Network ...downloads.hindawi.com/journals/ijg/2014/820248.pdf · DNA damage. Impairment in the DNA repair proteins, which physiologically

Review ArticleMicroRNAs in the DNA DamageRepair Network and Cancer

Alessandra Tessitore Germana Cicciarelli Filippo Del VecchioAgata Gaggiano Daniela Verzella Mariafausta Fischietti Davide VecchiottiDaria Capece Francesca Zazzeroni and Edoardo Alesse

Department of Biotechnological and Applied Clinical Sciences University of LrsquoAquila Via Vetoio Coppito 2 67100 LrsquoAquila Italy

Correspondence should be addressed to Edoardo Alesse edoardoalesseunivaqit

Received 6 July 2013 Accepted 10 December 2013 Published 30 January 2014

Academic Editor John Parkinson

Copyright copy 2014 Alessandra Tessitore et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cancer is a multistep process characterized by various and different genetic lesions which cause the transformation of normalcells into tumor cells To preserve the genomic integrity eukaryotic cells need a complex DNA damagerepair response networkof signaling pathways involving many proteins able to induce cell cycle arrest apoptosis or DNA repair Chemotherapy andorradiation therapy are the most commonly used therapeutic approaches to manage cancer and act mainly through the induction ofDNA damage Impairment in the DNA repair proteins which physiologically protect cells from persistent DNA injury can affectthe efficacy of cancer therapies Recently increasing evidence has suggested that microRNAs take actively part in the regulationof the DNA damagerepair network MicroRNAs are endogenous short noncoding molecules able to regulate gene expression atthe post-transcriptional level Due to their activity microRNAs play a role in many fundamental physiological and pathologicalprocesses In this review we report and discuss the role of microRNAs in the DNA damagerepair and cancer

1 Introduction

The DNA damage repair (DDR) response is an intricatesignal transduction pathway activated uponDNAdamage Topreserve the genomic integrity due to various endogenousand exogenous stimuli (ie UV ionizing radiations IRreactive oxygen species ROS and genotoxic drugs) cellsactivate specific signaling networks to arrest cell cycle pro-gression enabling the damage repair or to proceed towardapoptosis when the DNA lesions are too severe and notretrievable [1] Many genes involved in these processes havebeen studied and characterized at the transcriptional andpost-translational level In the last decade microRNAs a newclass of molecules able to post-transcriptionally regulate geneexpression have emerged to be involved in several funda-mental physiological and pathological biomolecular and cel-lular mechanisms Cancer cells often show significant alter-ations at the level of theDDR response and develop resistanceto DNA damage-inducing agents In this review we illustratethe involvement of miRNAs in regulatory networks affecting

the DNA damagerepair process in several types of solidtumors

2 The DNA Damage Response An Overview

The DDR is a kinase-based functional network primarilyactivated in response to stalled replication forks incompleteDNA replication and different types of DNA lesions Itinitiates signaling cascades to activate cell cycle checkpoints[1] The DDR is triggered by early phosphorylation-drivensignaling cascades followed by a delayed response that actsat the transcriptional level and promotes the induction ofCdk inhibitors extending the time of cell cycle arrest [23] Early signaling events include the activation of ATMATR and DNA-PKc the phosphorylation of H2AX and therecruitment of the complexes Mre11-Rad50-Nbs1 or Rad9-Hus1-Rad1 at the level of damaged sites [4] The ATM kinaseinitiates a signaling pathwaymainly induced byDNAdouble-strand breaks (DSBs) and acts by phosphorylating hundredsof proteins [5] Chk2 is one of the most important effector

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2014 Article ID 820248 10 pageshttpdxdoiorg1011552014820248

2 International Journal of Genomics

molecules targeted by ATM [6] The ATR kinase activates apathway principally induced by UV damage which involvesChk1 kinase [7ndash9] The most important targets of both Chk1and Chk2 are members of the Cdc25 phosphatase familyThese molecules are normally required for the activation ofcyclin-dependent kinase Once phosphorylated Cdc25a isfunctionally inhibited and prevents the activity of cyclin-dependent kinase-cyclin complexes involved in the transi-tion G1S and in the progression through S and G2Mtriggering G1 S and G2M checkpoints [10ndash12] In additionp38120572120573-dependent activation of MK2 works in a differentcell cycle checkpoint kinase pathway activated in responseto UV as well as to currently used chemotherapeutic drugsMK2 functions are critical especially in cells and tumorslosing p53 [13ndash18] A relatively DDR slow response is theATMATR-dependent p53 phosphorylationwith consequenttranscriptional activation of genes involved in cell cyclearrest (ie page 21) [3] At this point if DNA lesions areadequately repaired cells can proceed to proliferate againand to inactivate DNA damage checkpoints otherwise theyundergo to apoptosis [19] DNA damage can be repairedby different mechanisms The base excision repair (BER)and the nucleotide excision repair (NER) complexes removedamaged andmodified nucleotides In particular NERworksprincipally on helix-distorting and transcription-blockinglesions (ie UV-induced pyrimidine dimers) whereas BERremoves single nucleotides modified by methylation alkyla-tion deamination or oxidation [20] The mismatch repairmachinery (MMR) operates through MSH2 and MLH1which form heterodimers with MSH3 or MSH6 and MLH3PMS2 or PMS1 respectively These complexes are involvedin mismatchinsertion-deletion loops recognition and in theexcision-repair reaction [21 22] MMR machinery removesincorrectly incorporated nucleotides during DNA synthesisor replication errors in DNA repeats causing microsatel-lite instability [23] DNA double strand breaks (DSBs) arefrequent events in eukaryotic cells they are induced byphysiological mechanisms in early lymphocytes or by patho-logical activity attributable toROS ionizing radiation or erro-neous nuclear enzyme functions DSBs are repaired by non-homologous end joining (NHEJ) in all phases of the cell cyclethus representing the major pathway in G1 or homologousrecombination (HR) in the S or G2 phases of the cell cycle[24 25] NHEJ involves the binding of the heterodimeric Kuprotein to double-stranded DNA ends the recruitment ofthe DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA-PK kinase activation [24] The DNA-PKcomplex on the DNA works by recruiting a complex con-tainingDNA ligase IV XRCC4 and XLFCernunnos proteinwith consequent rejoining [24] HR is a complex processinvolving the generation of 31015840 single-strandedDNA overhangfirst bound by RPA The displacement of the complex byRad51 follows then a homologous sequence is engaged torepair the lesion [25]

Tumor cells frequently acquire the ability to overcomethe DDR network in fact most cancers show malfunctionsin the DDR such as resistance to genotoxic agents andionizing radiations or abnormal cell cycle progression afterDNA damage [26] The tumor suppressor p53 is one of the

most important proteins activated after DNA damage andp53 gene mutations are described in almost every type ofcancer at rates comprised between 38 and 50 in ovariancolorectal head and neck and lung cancers [27] Similarlysome hereditary cancer predisposition syndromes such asataxia telangiectasia Li-Fraumeni andNijmegen syndromesbreast and ovarian cancer or colon cancer (HNPCC) pre-disposition and Xeroderma pigmentosum show lesions ingenes involved in the DDR machinery (ATM p53 NBS1BRCA-12 MMR genes and NER genes) [23] In responseto DNA damage the mRNA expression patterns go throughsignificant modifications [28 29] which can be due to RNAPolII hyperphosphorylation with consequent prevention ofthe formation of preinitiation complexes at sites of the pro-moter [30] In this context post-transcriptional regulationof mRNAs mediated by microRNAs (miRNAs) plays also afundamental role

3 Biogenesis and Functions of MicroRNAs

MicroRNAs (miRNAs) are a class of short noncoding RNAsusually 18ndash25 nucleotides in length mediating importantcellular functions such as proliferation apoptosis differ-entiation and cell signaling [31ndash33] MiRNAs regulate thetranslation of specific mRNA by their imperfect or perfectpairing at the level of the 31015840UTR [34] It is estimated that in thehuman genome approximately 30 of genes are targeted bymiRNAs [35] MiRNAs genes are transcribed in two differentways long primary miRNAs (pri-miRNAs) are synthesizedfrom intergenic miRNAs by RNA polymerase II whereasintronic or esonic miRNAs are transcribed in associationwith their host genes from a common promoter [36] Aftercleavage of pri-miRNAs in the nucleus by themicroprocessorDrosha-DGCR8 precursor pre-miRNAs (approximately 70nucleotides in length) are obtained Pre-miRNAs are thenexported from the nucleus to the cytoplasm by the RanGTP-binding nuclear transporter exportin-5 In the cytoplasmthe endoribonucleaseDicer cleaves pri-miRNAs generating aduplex about 25 bases in length which consists of a functionalmiRNA and a passenger strand The association betweenArgonaute (Ago) proteins and mature miRNAs generatesRISC (RNA-induced silencing complex) which leads to post-transcriptional gene regulation consisting of mRNA degra-dation or translation inhibition [37] Due to their role in theregulation of fundamental cellular functions miRNAs areinvolved in several pathological processes and in particularin carcinogenesis [38]

4 MicroRNAs in DNA DamageRepairMechanisms and Cancer

41 MicroRNAs in ATM and ATR Pathways ATM gene hasbeen shown to be downregulated by miR-421 in neuroblas-toma and HeLa cells [39] MiR-421 targeted the ATM 31015840UTRplaying a role in modulating cell cycle checkpoints andcellular radiosensitivity Ectopic expression of miR-421 ledto S-phase cell cycle checkpoint changes and radiosensitivityincrease and was able to generate an ataxia telangiectasia-like

International Journal of Genomics 3

phenotype The inhibition of the interaction between miR-421 andATM31015840UTR reverted the effects due tomiR-421 over-expression Furthermore overexpression of N-myc a genefrequently amplified in neuroblastoma was able to inducemiR-421 expression with consequent ATM downregulationsuggesting a correlation between N-myc-mediated oncoge-nesis and the network involving miRNAs and moleculesworking in the DNA damagerepair machinery Ng et al [40]demonstrated that miR-100 was highly expressed by M059Jglioblastoma DNA-PK-deficient cells DNA-PK is a moleculeplaying a role in repairing IR-induced double strand breaksMiR-100 was responsible for the low expression of ATMdetected besides DNA-PK deficiency in the same cells MiR-100 can target the 31015840UTR of ATM gene and its knocking-down induced ATM expression Yan et al [41] identifiedmiR-101 as a molecule able to sensitize cancer cells to IRby targeting the 31015840UTR of DNA-PKcs and ATM transcriptsThe authors demonstrated that the miR-101 overexpressioncould be used for rendering tumor cells more sensitive toradiations in ldquoin vitrordquo and ldquoin vivordquo models MiR-18a wasable to affect theDNAdamage responsemechanisms throughATM downregulation [42] MiR-18a was over-expressed inbreast cancer cell lines and tumors and its ectopic expres-sion downregulated ATM by direct interaction with the31015840UTR of the gene ATM siRNA and miR-18a overexpressioncaused reduction of homologous recombination and DNArepair in breast cancer cells making them more sensitiveto ionizing radiations Conversely the inhibition of miR-18a led to increase of homologous recombination and DNArepair efficiency thus reducing cellular radiosensitivity Inaddition the authors showed that miR-18a overexpressingcells displayed significantly reduced phosphorylation andnuclear foci formation of H2AX and 53BP1 which aredownstream ATM substrates Accordingly Wu et al [43]demonstrated that miR-18a was over-expressed in colorectalcancers and was responsible for the diminishment of DNAdamage repair by suppressing ATM expression A recentresearch pointed-out the role of ATM itself in regulatingmiRNAs biogenesis Zhang et al [44] reported that about 25ofmiRNAs were upregulated uponDNAdamage in an ATM-dependent manner and that ATM loss abolished miRNAsinduction ATM directly phosphorylated KSPR (KH-typesplicing regulatory protein) a component of the Drosha andDicer miRNA processing complexes This induced increasedinteraction between KSPR and pri-miRNAs and enhancedpri-miRNAs processing activity by Drosha microprocessorsand stimulation of miRNA maturation Since the expressionof KSRP was found to be suppressed in several types ofhuman cancer (breast esophagus kidney liver and testis)KSRP-inducedmiRNA biogenesis could be inhibited in thesecancers with consequent deregulation at the level of path-ways and moleculesrsquo functions This work provided furtherevidence about the role of ATM in tumorigenesis A veryearly step in the response ofmammalian cells toDNAdouble-strand breaks is the phosphorylation of histone H2AX byATM [45] Wang et al [46] used the human osteosarcomacell line U2OS to develop a screening assay based on the eval-uation of radiation-induced 120574H2AX (phosphorylated histoneH2AX) foci formation Several miRNAs able to inhibit

120574H2AX foci formation were identified MiR-138 specificallytargeted the H2AX 31015840UTR reducing its expression andinducing chromosomal instability after DNA damage MiR-138 overexpression inhibited homologous recombination andincreased sensitivity to DNA damaging agents Converselythe expression of histone H2AX in miR-138 overexpressingcells resulted in the attenuation of these effects In thiscontext miR-138 expression could potentially improve theefficacy of DNA damaging agents against tumor cells Tsai etal [47] examined the role of the carcinogen areca nut extractin inducing miRNAs modulation in human oral fibroblastsAreca nut-induced miR-23a overexpression was correlatedwith an increase of the DNA damage marker 120574H2AX anda reduction of DSB repair in ldquoin vivordquo plasmid assay TheFanconi anemia susceptibility gene FANCG playing a rolein DSB repair process was predicted to be a target ofmiR-23a FANCG expression was reduced by areca nut-induced miR-23a overexpression generating inhibition ofDSB repair MiR-23a overexpression was also described inoral cancers from areca-nut chewing patients Chang etal [48] showed that miR-3928 was induced by ionizingradiations in HeLa cells and targeted the endoribonucleaseDicer MiR-3928 overexpression promoted ATR activationand Chk1 phosphorylation MiR-3928 overexpression wasalso able to downregulate several miRNAs including miR-185 300 and 663 AdditionallyWang et al [49] demonstratedthat miR-185 whose expression is reduced after ionizingradiation exposure in renal cell carcinoma (RCC) targetedATR MiR-185 expression sensitized RCC cells to X-raysboth ldquoin vivordquo and ldquoin vitrordquo and enhanced radiation-inducedapoptosis as well as inhibition of proliferation by repressingATR pathway Therefore miR-185 could be potentially usedto radiosensitize cancer cells

42 MicroRNAs in p53 Pathway Chang et al [50] ana-lyzed the p53 wild-type HCT116 colon cancer cell line incomparison to an isogenic cell line with both p53 allelesinactivated by homologous recombination After treatmentwith the genotoxic agent adriamycin able to induce p53and its downstream targets 474 human miRNAs were ana-lyzed SevenmiRNAs (miR-23a miR-26a miR-34a miR-30cmiR-103 miR-107 and miR-182) were upregulated in p53wtcells Among them miR-34a showed the highest expressionchange after adryamicin administration resulted transcrip-tionally regulated by p53 and was able to induce apoptosisAccordingly miR-34a was decreased in pancreatic cancercells which frequently show p53 loss-of-function MiR-34ainduction produced a strong reprogramming of expressionof genes acting in regulating cell-cycle progression prolifera-tion apoptosis angiogenesis and DNA repair (upregulationof RAD51AP1 CHEK1 and MDC1) Accordingly anotherstudy [51] demonstrated that miR-34 family was directlyregulated by p53 in cell lines andmouse tissues in response togenotoxic drugs and ionizing radiations By using antisenseoligonucleotides and the mouse ES cell line where miR-34a was genetically inactivated BCL2 was found to beregulated by miR-34 MiR-34a variant was also describedto be downregulated in neuroblastoma lacking the 1p36allelic region encompassing the miR-34a gene [52] It has


been described that 1p36 genomic loss is an event commonto different types of cancers [52] indicating that miR-34aactivity could play a pivotal role in tumor suppressionFurthermore miR-34bc was dramatically reduced in nonsmall cell lung cancers (43) and the restoration of theexpression caused growth inhibition of NSCLCs cells [51]MiR-34bc was also able to enhance cellular radiosensitiv-ity of malignant pleural mesothelioma cells by promotingradiation-induced apoptosis As shown by 120574H2AX foci assayDBS repair was delayed in miR-34bc transfected cells MiR-34bc inhibited the expression of cyclin-D1 CDK46 andBCL2 and enhanced cleaved caspase-3 and poly (ADP-ribose) polymerase cPARP levels after irradiation [53] Mir-34c was described as being upregulated in a study by Josson etal [54] where LNCaP andC4-2 prostate cancer cell lineswereused to evaluate global miRNA expression after radiationtreatment Several miRNAs were found to be differentiallyexpressed and significant changes were observed in miR-521 (downregulated) and miR-34c (upregulated) expressionMiR-34c upregulation was attributable to p53 regulationRegarding miR-521 the authors demonstrated that miR-521mimics sensitized prostate cells to radiation whereas itsectopic inhibition led to radiation resistance This effect wasascribed to themiR-521 predicted targeted proteins Cockaynesyndrome A (CSA) andMnSOD (manganese superoxide dis-mutase) involved in DNA repair and in oxidative processesrespectively Radiation treatment inversely correlated theexpression levels of CSA and MnSOD and miR-521 Due toits role miR-521 was described to be a potential target for theenhancement of radiation therapy on prostate cancer cellsShin et al [55] developed amodelwhere theNSCLCA549 cellline was treated with different doses of ionizing radiationsMiRNA profiles were analyzed and several differentiallyexpressed miRNAs involved in many cellular functions suchas apoptosis cell cycle control and DNA damagerepair wereidentified in response to different radiation doses Among thelatter miR-34a (DNA damagerepair predicted target genesPCBP4 POLD1) miR-34blowast (target genes ATR ERCC5 andRFC5) miR-192 (target genes CDK7 ERCC3 ERCC4 andXPA) miR-215 (target genes CDK7 ERCC3 ERCC4 TDGand XPA) miR-376a (target genes ATRMNAT1 andNEK11)were found over-expressed whereas miR-106a (target genesMSH3 GTF2H3 and POLH) miR-548c-3p (target genesCDK7 MSH2 PCNA PMS2) miR-760 (target gene BRCA-1) miR-16-2lowast (target genes EXO1 MSH2 PCNA POLD4and SETX) miR-139-3p (target genes POLA1 POLE) miR-345 (target genes LIG1 POLD3 POLL RFC1 and RPA1)miR-516a-5p (target genes ASF1A BRCA1 GTF2H4 andMBD4) were described as being downregulated A study[56] was focused on the analysis of time-course changesof miRNA expression induced by the genotoxic agent N-ethyl-N-nitrosourea (ENU) inmiceMiR-34 familymembers(miR-34a miR-34b miR-34c) were upregulated during theENU treatment and were described as potential biomarkersfor genotoxic exposure Saleh et al [57] analyzed the mech-anisms causing downregulation of let-7ab in response toagents able to induce oxidative and radiation damage Theydemonstrated that this effect was dependent ldquoin vitrordquo on p53

and ATM since it was not observed in the p53minusminus HCT116colon cancer cell line and in ATMminusminus human fibroblastsThe decreased expression of let-7 was triggered by p53binding on a region upstream of the let-7 gene followingradiation exposure Radiation-sensitive tissues also showedldquoin vivordquo radiation-induced let-7ab downregulation and thiseffect was not observed in p53 knock-out mice indicatingthat let-7 regulation occurred by p53-mediated repressionAccordingly exogenous expression of let-7ab enhancedradiation-induced cytotoxicity in HCT116 p53++ but notin those p53minusminus MicroRNAs play also a role in modulatingthe expression of p53 regulatory factors Among these thewild-type p53-induced phosphatase 1 (Wip1) a memberof serinethreonine phosphatases which is frequently over-expressed in several tumors plays a critical role inDNAdam-age signaling by dephosphorylating DNA-damage responsiveproteins in the ATMATR-p53 pathway [58] A study byZhang et al [59] showed that miR-16 was induced after DNAdamage and targeted Wip1 Overexpression of miR-16 orinhibition of Wip1 was able to suppress mouse mammarytumor stem cells growth and to enhance the response todoxorubicin in MCF-7 cells Expression levels of mdm2a known regulator of p53 are also affected by miRNAsSuh et al [60] analyzed glioblastoma multiforme cells anddemonstrated that miR-25 and miR-32 were repressed by ap53-dependent negative regulation of their transcriptionalfactors E2F and myc At the same time miR-25 and miR-32were able to target mdm2 generating p53 accumulation andan autoregulatory circuitry Dar et al [61] investigated therole of miR-18b whose expression was found to be reducedin melanoma samples through methylation The authorsidentified mdm2 as a target of miR-18b and demonstratedthat its overexpression in melanoma cells resulted in mdm2downregulation with consequent p53 upregulation and p53pathway reactivation Amir et al [62] demonstrated thatmiR-125b targeted p14arf as well as p53 Puma and Bak1 in prostatecancer cells [63] MiR-125b inhibited also the interactionbetween p14arf and mdm2 affecting the p14arfmdm2p53pathway activity and apoptosis induction On the contraryanti-miR-125b treatment restored p14arf expression apopto-sis induction and decreased mdm2 levels Avasarala et al[64] showed that miR-29b acts as a tumor suppressor inNSCLCby regulatingmdm2 expression In additionmiR-661was described as a negative regulator of mdm2 and mdm4 inseveral cell lines from melanoma lung breast and ovariancancer High miR-661 expression was correlated with goodprognosis in breast cancer mostly expressing wild type p53[65]

43 MicroRNAs in MMR Machinery Lanza et al [66] ana-lyzed colon cancer samples characterized by microsatellitestability (MSS) or instability (MSI-H)The study was focusedon the analysis of mRNAs and microRNAs expression EightmiRNAs correctly distinguished MSI-H from MSS samplesAmong these severalmembers of themiR-17-92 family (miR-17-5p miR-20 miR-25 miR-92-1 miR-92-2 and miR-93-1miR-106a) were upregulated in MSS with respect to MSI-Hcolon cancers The miR-17-92 cluster was described as being


upregulated in NSCLC [67] and its ectopic overexpressionenhanced lung cancer cells growth suggesting that thesemolecules may act as oncogenes and might have a role inmore aggressive clinical behavior ofMSSwith respect toMSI-H tumors Sarver et al [68] analyzedmiRNAprofiles in defec-tive MMR (dMMR showing microsatellite instability andorMLH1 protein absence) and proficient MMR (pMMR withabsence of microsatellite instability and presence of normalMLH1) colorectal cancers in comparison to adjacent normalcolon samples Six miRNAs were differentially expressedmiR-31 andmiR-625 were over-expressed in dMMR whereasmiR-552miR-592miR-181c andmiR-196bwere described asbeing decreased in pMMR samples Valeri et al [69] analyzedmiRNAs involved inmismatch repairmechanisms and foundthat the overexpression of miR-155 caused downregulation ofthe mismatch repair proteinsMLH1 MSH2 andMSH6 withconsequent mutator phenotype and MSI in colorectal cancercell linesMiR-155 acted by targeting the 31015840UTR regionsMiR-155 overexpression was inversely correlated to MLH1 andMSH2 expression in colorectal cancer samples and someMSIcancers with undetectable cause of MMR machinery inacti-vation alike showed miR-115 overexpression Valeri et al [70]demonstrated that miR-21 targets the core MMR recognitionprotein complex MSH2 and MSH6 in colon tumor cellsColorectal tumors overexpressing miR-21 showed reducedlevels of MSH2 protein and cells overexpressing miR-21 dis-played reduced 5-fluorouracil-induced G2M damage arrestand apoptosis Xenograft models confirmed that miR-21overexpression was able to reduce 5-fluorouracil efficacyTherefore miR-21 expression could be considered a markerof therapy efficacy in colorectal cancer MiR-21 function wasalso studied by Yu et al [71] who analyzed breast cancer cellsThey identified miR-21 as a miRNA regulated by TGF-betaa cytokine with tumor suppressor activity in normal cellsbut able to promote malignancy in cancer if abused [72] Theauthors demonstrated that TGF-beta-induced miR-21 causeddownregulation of MSH2 expression by 31015840UTR targeting Aninverse correlation between TGF-beta and MSH2 expressionwas also described in primary breast tumors confirmingthe relationship between the cytokine and miR-21 activityRecently Zhang et al [73] used a system based on A549 cellsto demonstrate that cisplatin treatment downregulated miR-21 expression with consequent MSH2 increase and A549cells growth inhibition Oberg et al [74] analyzed samplesfrom normal colonic mucosa tubulovillous adenoma andproficient MMR deficient MMR sporadic and hereditarycolon cancers Six miRNAs (miR-31 miR-135b miR-9 miR-1 miR-99a and miR-137) were differentially expressed inadenoma versus normal samples (fold changes more than 4)All of them except miR-99a showed comparable expressiondifferences in normal versus carcinoma comparisons pro-viding evidence that these changes are common early eventsboth in pMMR and dMMR cancers MiR-31 miR-552 miR-592 and miR-224 were differentially expressed in proficientversus deficient MMR cancers providing evidence of miR-NAs involved in distinguishing pMMR and dMMR MiR-99a was over-expressed (fold change less than 4) in pMMRand dMMR versus adenoma comparisons MiR-99 familyexpression was also correlated with radiation sensitivity and

was able to target the SWISNF chromatin remodeling factorSNF2HSMARCA5 a component of the ACF1 complex [75]Mir-99a and miR-100 were involved in the reduction of thelocalization of BRCA-1 at the DNAdamaged sites Expressionof miR-99 family in cells diminished the level of both HRand NHEJ efficiency and miR-99a induction by radiationsprevented the SNF2H increase and reduced the recruitmentof BRCA-1 at the damaged sites after a second dose ofradiationsThis decreased the repair efficiency uponmultipledoses of radiations Since the radiation therapy is usuallyperformed by administering fractionated doses of radiationsmiR-99 expression could play a role in affecting the efficacyof this treatment

44 MicroRNAs in NERMachinery Some works have shownthat hypoxia can also promote genetic instability by affectingthe DNA repair capability of cancer cells due to transcrip-tional downregulation ofMLH1MSH2 BRCA-1 and RAD51observed in hypoxic cells [76ndash79] Crosby et al [80] usedHeLa MCF-7 and mouse embryonic fibroblast to analyzethe role of miRNAs in DNA repair under hypoxic stressMiR-210 and miR-373 were upregulated in hypoxic cellsin a hypoxia-inducible factor-1 alpha- (HIF-1120572-) dependentmanner Forced expression of miR-210 was able to suppresslevels of RAD52 a key factor in homology-dependent repairwhereas miR-373 overexpression caused downregulationof RAD23B a component of the XPCRAD23B complexinvolved in the NER machinery as well as RAD52 MiR-210 and miR-373 interacted with the 31015840UTR of RAD52 andRAD23B respectively MiR-210 and miR-373 inhibition byantisense strategy can partially reverse the hypoxia-inducedRAD52 and RAD23B downregulation Friboulet et al [81]analyzed the role of excision repair cross-complementationgroup 1 (ERCC1) a NER pathway protein involved in recog-nition and removal of DNA platinum adducts and in repairof stalled DNA replication forks in NSCLCs from a cohortof 91 patients ERCC1-positive tumors showed lower rateof genomic lesions with respect to ERCC1 negative MiR-375 was found to be reduced in ERCC1-positive cancersMiR-375 was previously described as being downregulatedin gastric cancer and hepatocellular carcinoma (HCC) andwas able to inhibit HCCproliferation [82 83] Genes involvedin DNA repair such as TP53 USP1 APEX1 TYMS MLH3PARP4 NTHL1 ERCC3 and XRCC6BP1 were predictedto be targeted by miR-375 Therefore miR-375 downregu-lation could determine a proliferative advantage and alsoan increased DDR phenotype in ERCC1-positive tumorsThe HBV-expressing HepG2215 cell line with impairedNER activity showed upregulation of miR-192 [84] ERCC3and ERCC4 two proteins involved in NER pathway weredownregulated by miR-192 with consequent impairing ofNER machinery The role of polymorphisms at the level ofmiRNA binding sites of 28 NER genes was correlated withcolorectal cancer risk by analyzing cohorts of about 1000cancers and 1500 healthy controls [85] Two polymorphismsrs7356 in RPA2 and rs4596 in GTF2H1 were associated withcolorectal cancer risk indicating that alterations in miRNAtarget binding sites may play a role in tumorigenesis


45 MicroRNAs in Regulation of Genes Involved in Differ-ent DNA Repair Pathways and Mechanisms Chaudhry etal [86] studied differentially expressed miRNAs after IRtreatment in glioblastoma which often show resistance toradiation therapy Glioblastoma cells (M059K and M509J)with normal or deficient DNA-dependent protein kinase(DNA-PK) activity and resistance or sensitivity to ionizingradiations were used The let-7 family known to be a RASregulator which is upregulated in cells showingnormalDNA-PK activity whereas the same family was downregulatedin cells with DNA-PK deficient activity MiR-17-3p miR-17-5p miR-19a miR-19b miR-142-3p and miR-142-5p wereupregulated in cells with normal and deficient DNA-PKactivity MiR-15a miR-16 miR-143 miR-155 and miR-21showed upregulation in cells with normal DNA-PK activityand varied in a time-dependentmanner inDNA-PK deficientcells Among these miRNAs miR-155 was associated withc-myc overexpression whereas miR-15a and miR-16 weredescribed as being able to target BCL2 whose gene prod-uct acts in protecting cells from IR-induced apoptosis andconfers resistance to DNA damage [87] MiR-21 was alreadydescribed to participate in a miRNA targeting a networkincluding p53 TGF-beta andmitochondrial apoptosis tumorsuppressor genes in glioblastoma [88] Moskwa et al [89]analyzed breast cancer cells to demonstrate that miR-182was responsible for the downregulation of BRCA-1 expres-sion whose decreased expression is observed in 30ndash65 ofsporadic basal-like tumors MiR-182 levels decreased afterIR in a p53-independent manner being detectable both inp53-proficient (MCF-7 HMEC) and deficient (K562 HL60)cell lines MiR-182 antagonism led to increase of BRCA-1 expression protecting cells from IR-induced cell deathwhereas miR-182 overexpression reduced BRCA-1 expressionwith consequent defects in HR-mediated repair Mir-182overexpressing tumor cells were hypersensitive to inhibitorsof poly (ADP-ribose) polymerase 1 (PARP1) on the contrarymiR-182 antagonism led to enhanced BRCA-1 levels andinduced resistance to PARP1 inhibitorsThis research focuseson the importance of miR-182 overexpression in affecting theresponse to PARP1 inhibitors In a study by Krishnan et al[90] miR-182-5pwas frequently upregulated in human breastcancers and was able to target a network of genes involvedin DNA repair identified by synthetic biotinylated miRNAto pull down endogenous miR-182-5p targets in HEK293Tcells Rui et al [91] used a docetaxel-resistant NSCLC (SPC-A1docetaxel) and its parental cell line to perform miRNAdifferential expression analysis Three upregulated (miR-192miR-424 and miR-98) and 3 downregulated miRNAs (miR-200b miR-212 and miR-194) were identified in docetaxel-resistant cells Moreover approximately 90 predicted targetgenes involved in DNA damagerepair (among them XPARAD1 XPC TP53 BRCA-1 SIRT1 MSH2 RAD50 andATM) were described Teo et al [92] analyzed in bladderand breast cancer patients common single nucleotide poly-morphisms (SNPs) potentially involved in miRNA bindingsites in the 31015840UTR of 20 genes involved in DNA repair Theyfound SNPs (PARP1 rs8679 TgtC and RAD51 rs7180135 AgtG)associated with increased bladder and breast cancer risk andwith improved cancer specific survival following radiation

therapy in bladder cancer Predicted miRNAs involved inthis process were miR-145 miR-105 miR-630 and miR-302a(regulation of PARP1) and miR-197 (regulation of RAD51)Yan et al [93] analyzed the role of RAD21 an essentialmolecule for chromosome segregation as well as high-fidelityDNA repair by homologous recombination with BRCA-12 in affecting the prognosis of BRCA-2 BRCAX andBRCA-1 familial breast cancers High RAD21 expressionwas associated with genomic instability and miR-299-5p amicroRNA already involved in breast cancer and in oralsquamous cell carcinoma was predicted to be a RAD21regulator Wang et al [94] demonstrated that the expressionof RAD51 and REV1 polymerase involved in the resistanceto DNA interstrand crosslinking agents was downregulatedby miR-96 in several types of cancer cells MiR-96 wasable to directly target the coding region of RAD51 and the31015840UTR of REV1 Overexpression of miR-96 was a negativeregulator of RAD51 foci formation induced decrease of theefficiency of homologous recombination and enhancement ofthe sensitivity to the AZD2281 PARP inhibitor ldquoin vitrordquo andto cisplatin both ldquoin vivordquo and ldquoin vivordquo suggesting that miR-96mimics could be used to enhance cancer chemosensitivityZheng et al [95] used an artificial approach to analyzethe effect of an engineered miRNA (amiR) able to targetin a perfect complementary way the 31015840UTR of XRCC2 afundamental homologous recombination factor andXRCC4an essential nonhomologous end joining factor in cancercells along with a siRNA XRCC2 and XRCC4 showed higherexpression in tumor tissues and cells with respect to noncancer tissuescells The artificial amiR efficiently inhibitedthe expression of XRCC2 and XRCC4 genes sensitizinghuman tumor cells to radiation-induced death This effectwas further enhanced by combining amiR to siRNA to targetboth the noncoding and coding regions of XRCC2 andXRCC4 Hegre et al [96] demonstrated that miR-16 miR-34c and miR-199a downregulated the expression of the BERcomplex protein nuclear uracil-DNA-glycosylase UNG2 in a31015840UTR targeting dependent process

5 Clinical Potentials of MiRNA-BasedCancer Therapy

Cancer treatment is usually based on chemo- andor radio-therapy Unfortunately many tumors exhibit radio- orchemoresistance with consequent efficacy reduction of thesetreatments During last decade small molecules or proteindrugs such as monoclonal antibodies specifically directedagainst molecules with dysregulated activity have been gen-erated and used in association with conventional therapies[97ndash100] Due to their functions microRNAs are consideredtarget molecules and potentially effective therapeutic optionsfor cancer Some studies have shown that miRNA mimicsmay be administered without side effects in non-human pri-mates andmice reducing tumor growth [101ndash103]Thereforedepending on their role in promoting or suppressing can-cer miRNA expression could be either selectively inhibited(ie anti-miR oligonucleotides miRNA sponges) or endoge-nouslyexogenously restored In this context microRNAdelivery by nanoparticles potentially able to specifically target


Table 1 MiRNAs involved in DNA damagerepair and 31015840-UTR targeted genes

MiRNA 31015840UTR targeted genes ReferencemiR-421 ATM Hu et al 2010 [39]miR-100 ATM Ng et al 2010 [40]miR-101 ATM DNA-PK Yan et al 2010 [41]miR-18a ATM Song et al 2011 [42] Wu et al 2013 [43]miR-138 H2AX Wang et al 2011 [46]miR-210 RAD52 Crosby et al 2009 [80]miR-3248 Dicer Chang et al 2012 [48]miR-185 ATR Wang et al 2013 [49]miR-16 Wip1 Zhang et al 2010 [59]miR-25 miR-32 MDM2 Suh et al 2012 [60]miR-18b MDM2 Dar et al 2013 [61]miR-661 MDM2 Hoffman et al 2013 [65]miR-34a BCL2 Bommer et al 2007 [51]miR-155 MLH1 MSH2 and MSH6 Valeri et al 2010 [70]miR-21 MSH2 Yu et al 2010 [71]miR-192 ERCC3 ERCC4 Xie et al 2011 [84]miR-373 RAD23B Crosby et al 2009 [80]miR-96 REV1 Wang et al 2012 [94]miR-16 miR-34c and miR-199a UNG2 Hegre et al 2013 [96]

tumors for example through the use of RNA aptamers orchemical ligands is starting to play an emerging role inmedicine and clinical practice [104] MiRNAs involved inthe regulation of DNA damagerepair mechanisms can beconsidered markers to predict the response to radiotherapyand utilized hereafter to define personalized treatments [105](Table 1) In this regard expression levels of miRNAs couldbe evaluated in serum andor tumor specimens to predictradiosensitivity and optimal radiation dose in order to makethe treatment more effective and to limit both side effects andnormal tissue injury In addition expression and activity ofmiRNAs able to affect the response to chemo- or radiotherapycould be specifically modified and modulated to enhance theexpected therapeutic effects The use of artificial miRNAswith sequences able to target genes already known to haveimportant roles in DNA damagerepair mechanisms couldbe of great impact in regulating mechanisms able to rendercancer cells more sensitive to DNA damaging agents

6 Conclusions

MiRNAs play a pivotal role in various biological andpathological processes driving to cancer initiation progres-sion and metastasis formation The analysis of microRNA-modulated gene regulation in theDDR and its involvement incancer pathogenesis and progression will help to understandand define the impact of these small molecules in DNAdamagerepair as well as chemo- and radioresistance mech-anisms This knowledge will expand the characterizationof molecules and networks involved in pathways activatedupon DNA damage and the subsequent alterations at thelevel of fundamental processes such as cell cycle controland apoptosisThe identification ofmiRNA-modulated genes

and the effects of miRNAs deregulated functions will makeit possible to acquire information about the prognosisthe chemo- or radioresistance and then the response totherapeutic treatments in cancer In conclusion functionalcharacterization of the network including proteins able torepress or induce miRNAs as well as miRNAs and targets willprovide significant information about prognosis and therapyof cancer

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] S P Jackson and J Bartek ldquoThe DNA-damage response inhuman biology and diseaserdquoNature vol 461 no 7267 pp 1071ndash1078 2009

[2] I Ward and J Chen ldquoEarly events in the DNA damageresponserdquo Current Topics in Developmental Biology vol 63 pp1ndash35 2004

[3] A Vigneron J Cherier B Barre E Gamelin and O CoqueretldquoThe cell cycle inhibitor p21-waf1 binds to the myc and cdc25apromoters upon DNA damage and induces transcriptionalrepressionrdquo Journal of Biological Chemistry vol 281 no 46 pp34742ndash34750 2006

[4] A Ciccia and S J Elledge ldquoTheDNAdamage response makingit safe to play with knivesrdquoMolecular Cell vol 40 no 2 pp 179ndash204 2010

[5] S Matsuoka B A Ballif A Smogorzewska et al ldquoATMand ATR substrate analysis reveals extensive protein networksresponsive to DNA damagerdquo Science vol 316 no 5828 pp1160ndash1166 2007


[6] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[7] R T Abraham ldquoCell cycle checkpoint signaling through theATM and ATR kinasesrdquo Genes and Development vol 15 no 17pp 2177ndash2196 2001

[8] Y Shiloh ldquoATM and ATR networking cellular responses toDNA damagerdquo Current Opinion in Genetics and Developmentvol 11 no 1 pp 71ndash77 2001

[9] J Bartek and J Lukas ldquoChk1 and Chk2 kinases in checkpointcontrol and cancerrdquo Cancer Cell vol 3 no 5 pp 421ndash429 2003

[10] J Falck N Mailand R G Syljuasen J Bartek and J LukasldquoThe ATM-Chk2-Cdc25A checkpoint pathway guards againstradioresistant DNA synthesisrdquo Nature vol 410 no 6830 pp842ndash847 2001

[11] M Donzelli and G F Draetta ldquoRegulating mammalian check-points through Cdc25 inactivationrdquo EMBO Reports vol 4 no7 pp 671ndash677 2003

[12] J Rudolph ldquoCdc25 phosphatases structure specificity andmechanismrdquo Biochemistry vol 46 no 12 pp 3595ndash3604 2007

[13] I AManke ANguyenD LimMQ Stewart A EH Elia andM B Yaffe ldquoMAPKAP kinase-2 is a cell cycle checkpoint kinasethat regulates the G 2M transition and S phase progression inresponse to UV irradiationrdquoMolecular Cell vol 17 no 1 pp 37ndash48 2005

[14] M Raman S Earnest K Zhang Y Zhao and M H CobbldquoTAO kinases mediate activation of p38 in response to DNAdamagerdquo EMBO Journal vol 26 no 8 pp 2005ndash2014 2007

[15] H C Reinhardt A S Aslanian J A Lees andM B Yaffe ldquop53-deficient cells rely on ATM- and ATR-mediated checkpointsignaling through the p38MAPKMK2 pathway for survivalafter DNA damagerdquo Cancer Cell vol 11 no 2 pp 175ndash189 2007

[16] H C Reinhardt P Hasskamp I Schmedding et al ldquoDNAdamage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNAstabilizationrdquoMolecular Cell vol 40 no 1 pp 34ndash49 2010

[17] H C Reinhardt and M B Yaffe ldquoKinases that control the cellcycle in response to DNA damage Chk1 Chk2 and MK2rdquoCurrent Opinion in Cell Biology vol 21 no 2 pp 245ndash255 2009

[18] J Boucas A Riabinska M Jokic et al ldquoPosttranscriptionalregulation of gene expressionmdashadding another layer of com-plexity to the DNA damage responserdquo Frontiers in Genetics vol3 article 159 2012

[19] J Bartek and J Lukas ldquoDNA damage checkpoints from initia-tion to recovery or adaptationrdquo Current Opinion in Cell Biologyvol 19 no 2 pp 238ndash245 2007

[20] A T Natarajan and F Palitti ldquoDNA repair and chromosomalalterationsrdquoMutation Research vol 657 no 1 pp 3ndash7 2008

[21] S Acharya P L Foster P Brooks and R Fishel ldquoThe coor-dinated functions of the E coli MutS and MutL proteins inmismatch repairrdquo Molecular Cell vol 12 no 1 pp 233ndash2462003

[22] R D Kolodner M M Mendillo and C D Putnam ldquoCouplingdistant sites in DNA during DNAmismatch repairrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 32 pp 12953ndash12954 2007

[23] J H J Hoeijmakers ldquoGenome maintenance mechanisms forpreventing cancerrdquoNature vol 411 no 6835 pp 366ndash374 2001

[24] M R Lieber ldquoThe mechanism of double-strand DNA breakrepair by the nonhomologous DNA end-joining pathwayrdquoAnnual Review of Biochemistry vol 79 pp 181ndash211 2010

[25] A Shibata S Conrad J Birraux et al ldquoFactors determiningDNA double-strand break repair pathway choice in G2 phaserdquoEMBO Journal vol 30 no 6 pp 1079ndash1092 2011

[26] M D Wouters D C Van Gent J H J Hoeijmakers and JPothof ldquoMicroRNAs the DNA damage response and cancerrdquoMutation Research vol 717 no 1-2 pp 54ndash66 2011

[27] M Olivier M Hollstein and P Hainaut ldquoTP53 mutations inhuman cancers origins consequences and clinical userdquo ColdSpring Harbor Perspectives in Biology vol 2 no 1 Article IDa001008 2010

[28] K E Rieger and G Chu ldquoPortrait of transcriptional responsesto ultraviolet and ionizing radiation in human cellsrdquo NucleicAcids Research vol 32 no 16 pp 4786ndash4803 2004

[29] H C Reinhardt I G Cannell S Morandell and M B YaffeldquoIs post-transcriptional stabilization splicing and translationof selective mRNAs a key to the DNA damage responserdquo CellCycle vol 10 no 1 pp 23ndash27 2011

[30] D A Rockx R Mason A Van Hoffen et al ldquoUV-inducedinhibition of transcription involves repression of transcriptioninitiation and phosphorylation of RNApolymerase IIrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 97 no 19 pp 10503ndash10508 2000

[31] M Lagos-Quintana R Rauhut W Lendeckel and T TuschlldquoIdentification of novel genes coding for small expressedRNAsrdquoScience vol 294 no 5543 pp 853ndash858 2001

[32] K Nakahara and R W Carthew ldquoExpanding roles for miRNAsand siRNAs in cell regulationrdquo Current Opinion in Cell Biologyvol 16 no 2 pp 127ndash133 2004

[33] A Ichimura Y Ruike K Terasawa andG Tsujimoto ldquomiRNAsand regulation of cell signalingrdquo FEBS Journal vol 278 no 10pp 1610ndash1618 2011

[34] D P Bartel ldquoMicroRNAs target recognition and regulatoryfunctionsrdquo Cell vol 136 no 2 pp 215ndash233 2009

[35] G Wan R Mathur X Hu X Zhang and X Lu ldquomiRNAresponse to DNA damagerdquo Trends in Biochemical Sciences vol36 no 9 pp 478ndash484 2011

[36] Y Kim and V N Kim ldquoProcessing of intronic microRNAsrdquoEMBO Journal vol 26 no 3 pp 775ndash783 2007

[37] J A Mulligan EdMicroRNA Expression Detection andThera-peutic Strategies DNA and RNA Properties and ModificationsFunctions and Interactions Recombination and ApplicationsNova Science New York NY USA 2011

[38] J Lu G Getz E A Miska et al ldquoMicroRNA expression profilesclassify human cancersrdquoNature vol 435 no 7043 pp 834ndash8382005

[39] H Hu L Du G Nagabayashi R C Seeger and R A GattildquoATM is down-regulated by N-Myc-regulated microRNA-421rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 4 pp 1506ndash1511 2010

[40] W L Ng D Yan X Zhang Y-Y Mo and Y Wang ldquoOver-expression of miR-100 is responsible for the low-expression ofATM in the human glioma cell line M059Jrdquo DNA Repair vol9 no 11 pp 1170ndash1175 2010

[41] D Yan W L Ng X Zhang et al ldquoTargeting DNA-PKcs andATM with miR-101 sensitizes tumors to radiationrdquo PLoS Onevol 5 no 7 Article ID e11397 2010

[42] L Song C Lin Z Wu et al ldquomiR-18a impairs DNA dam-age response through downregulation of Ataxia telangiectasiamutated (ATM) kinaserdquo PLoS One vol 6 no 9 Article IDe2545 2011


[43] C W Wu Y J Dong Q Y Liang et al ldquoMicroRNA 18a atten-uates DNA damage repair through suppressing the expressionof ataxia telangiectasia mutated in colorectal cancerrdquo PLoS Onevol 8 no 2 Article ID e57036 2013

[44] X Zhang G Wan F G Berger X He and X Lu ldquoTheATMkinase inducesmicroRNA biogenesis in the DNAdamageresponserdquoMolecular Cell vol 41 no 4 pp 371ndash383 2011

[45] S Burma B P Chen M Murphy A Kurimasa and D JChen ldquoATMphosphorylates histoneH2AX in response toDNAdouble-strand breaksrdquo Journal of Biological Chemistry vol 276no 45 pp 42462ndash42467 2001

[46] Y Wang J-W Huang M Li et al ldquoMicroRNA-138 modulatesDNA damage response by repressing histone H2AX expres-sionrdquoMolecular Cancer Research vol 9 no 8 pp 1100ndash1111 2011

[47] Y-S Tsai C-S Lin S-L Chiang C-H Lee K-W Lee andY-C Ko ldquoAreca nut induces miR-23a and inhibits repair ofDNA double-strand breaks by targeting FANCGrdquo ToxicologicalSciences vol 123 no 2 pp 480ndash490 2011

[48] L Chang W Hu C Ye et al ldquomiR-3928 activates ATR pathwayby targeting Dicerrdquo RNA Biology vol 9 no 10 pp 1247ndash12542012

[49] J Wang J He F Su et al ldquoRepression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferationinhibitionrdquo Cell Death amp Disease vol 4 article e699 2013

[50] T Chang E Wentzel O Kent et al ldquoTransactivation of miR-34a by p53 broadly influences gene expression and promotesapoptosisrdquoMolecular Cell vol 26 no 5 pp 745ndash752 2007

[51] G Bommer I Gerin Y Feng et al ldquop53-mediated activation ofmiRNA34 candidate tumor-suppressor genesrdquo Current Biologyvol 17 no 15 pp 1298ndash1307 2007

[52] C Welch Y Chen and R L Stallings ldquoMicroRNA-34a func-tions as a potential tumor suppressor by inducing apoptosis inneuroblastoma cellsrdquo Oncogene vol 26 no 34 pp 5017ndash50222007

[53] Y Maki H Asano S Toyooka et al ldquoMicroRNA miR-34bc enhances cellular radiosensitivity of malignant pleuralmesothelioma cellsrdquoAnticancer Reseach vol 32 no 11 pp 4871ndash4875 2012

[54] S Josson S-Y Sung K Lao and P A S Johnstone ldquoRadiationmodulation ofmicroRNA in prostate cancer cell linesrdquo Prostatevol 68 no 15 pp 1599ndash1606 2008

[55] S Shin H J Cha E-M Lee et al ldquoAlteration ofmiRNA profilesby ionizing radiation inA549 human non-small cell lung cancercellsrdquo International Journal of Oncology vol 35 no 1 pp 81ndash862009

[56] Z Li W S Branham S L Dial et al ldquoGenomic analysis ofmicroRNA time-course expression in liver of mice treated withgenotoxic carcinogen N-ethyl-N-nitrosoureardquo BMC Genomicsvol 11 no 1 article 609 2010

[57] A D Saleh J E Savage L Cao et al ldquoCellular stress inducedalterations in microrna let-7a and let-7b expression are depen-dent on p53rdquo PLoS One vol 6 no 10 Article ID e24429 2011

[58] X Lu T-A Nguyen S-H Moon Y Darlington M Sommerand L A Donehower ldquoThe type 2C phosphatase Wip1 anoncogenic regulator of tumor suppressor and DNA damageresponse pathwaysrdquo Cancer and Metastasis Reviews vol 27 no2 pp 123ndash135 2008

[59] X Zhang G Wan S Mlotshwa et al ldquoOncogenic Wip1 phos-phatase is inhibited by miR-16 in the DNA damage signalingpathwayrdquo Cancer Research vol 70 no 18 pp 7176ndash7186 2010

[60] S-S Suh J Y Yoo G J Nuovo et al ldquoMicroRNAsTP53feedback circuitry in glioblastoma multiformerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 109 no 14 pp 5316ndash5321 2012

[61] A A Dar S Majid C Rittsteuer et al ldquoThe role of miR-18bin MDM2-p53 pathway signaling and melanoma progressionrdquoJournal of the National Cancer Institute vol 105 no 6 pp 433ndash442

[62] S Amir A H Ma X B Shi L Xue H J Kung and RW Devere White ldquoOncomir miR-125b suppresses p14(ARF)to modulate p53-dependent and p53-independent apoptosis inprostate cancerrdquo PLoS One vol 8 no 4 Article ID e61064 2013

[63] X-B Shi L Xue A-H Ma C G Tepper H-J Kung andR W D White ldquoMiR-125b promotes growth of prostatecancer xenograft tumor through targeting pro-apoptotic genesrdquoProstate vol 71 no 5 pp 538ndash549 2011

[64] S Avasarala M Van Scoyk J Wang et al ldquohsa-miR29b acritical downstream target of non-canonical Wnt signalingplays an anti-proliferative rolein non-small cell lung cancer cellsvia targeting MDM2expressionrdquo Biology Open vol 2 pp 675ndash685 2013

[65] Y Hoffman D R Bublik Y Pilpel and M Oren ldquomiR-661downregulates both Mdm2 and Mdm4to activate p53rdquo CellDeath and Differentiation vol 21 no 2 pp 302ndash330 2014

[66] G Lanza M Ferracin R Gafa et al ldquomRNAmicroRNA geneexpression profile in microsatellite unstable colorectal cancerrdquoMolecular Cancer vol 6 article 54 2007

[67] Y Hayashita H Osada Y Tatematsu et al ldquoA polycistronicMicroRNA cluster miR-17-92 is overexpressed in human lungcancers and enhances cell proliferationrdquo Cancer Research vol65 no 21 pp 9628ndash9632 2005

[68] A L Sarver A J French P M Borralho et al ldquoHumancolon cancer profiles show differential microRNA expressiondepending on mismatch repair status and are characteristic ofundifferentiated proliferative statesrdquo BMC Cancer vol 9 article401 2009

[69] N Valeri P Gasparini M Fabbri et al ldquoModulation ofmismatch repair and genomic stability by miR-155rdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 107 no 15 pp 6982ndash6987 2010

[70] N Valeri P Gasparini C Braconi et al ldquoMicroRNA-21induces resistance to 5-fluorouracil by down-regulating humanDNA MutS homolog 2 (hMSH2)rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no49 pp 21098ndash21103 2010

[71] Y Yu Y Wang X Ren et al ldquoContext-dependent bidirectionalregulation of the mutS homolog 2 by transforming growthfactor 120573 contributes to chemoresistance in breast cancer cellsrdquoMolecular Cancer Research vol 8 no 12 pp 1633ndash1642 2010

[72] J Massague ldquoTGF-120573 signal transductionrdquo Annual Review ofBiochemistry vol 67 pp 753ndash791 1998

[73] Y X Zhang Z Yue P Y Wang et al ldquoCisplatin upregulatesMSH2 expression by reducing miR-21 to inhibit A549 cellgrowthrdquo Biomedicine amp Pharmacotherapy vol 67 no 2 pp 97ndash102

[74] A L Oberg A J French A L Sarver et al ldquomiRNA expressionin colon polyps provides evidence for aMultihit model of coloncancerrdquo PLoS One vol 6 no 6 Article ID e20465 2011

[75] A C Mueller D Sun and A Dutta ldquoThe miR-99 familyregulates the DNA damage response through its target SNF2HrdquoOncogene vol 32 no 9 pp 1164ndash1172 2013


[76] V T Mihailova R S Bindra J Yuan et al ldquoDecreased expres-sion of the DNA mismatch repair gene Mlh1 under hypoxicstress in mammalian cellsrdquoMolecular and Cellular Biology vol23 no 9 pp 3265ndash3273 2003

[77] R S Bindra P J Schaffer A Meng et al ldquoDown-regulationof Rad51 and decreased homologous recombination in hypoxiccancer cellsrdquoMolecular and Cellular Biology vol 24 no 19 pp8504ndash8518 2004

[78] R S Bindra S L Gibson A Meng et al ldquoHypoxia-induced down-regulation of BRCA1 expression by E2Fsrdquo Can-cer Research vol 65 no 24 pp 11597ndash11604 2005

[79] R S BindraM ECrosby andPMGlazer ldquoRegulation ofDNArepair in hypoxic cancer cellsrdquo Cancer and Metastasis Reviewsvol 26 no 2 pp 249ndash260 2007

[80] M E Crosby R Kulshreshtha M Ivan and P M GlazerldquoMicroRNA regulation of DNA repair gene expression inhypoxic stressrdquo Cancer Research vol 69 no 3 pp 1221ndash12292009

[81] L Friboulet D Barrios-Gonzales F Commo et al ldquoMolec-ular characteristics of ERCC1-negative versus ERCC1-positivetumors in resected NSCLCrdquo Clinical Cancer Research vol 17no 17 pp 5562ndash5572 2011

[82] L Ding Y Xu W Zhang et al ldquoMiR-375 frequently downreg-ulated in gastric cancer inhibits cell proliferation by targetingJAK2rdquo Cell Research vol 20 no 7 pp 784ndash793 2010

[83] A M Liu R T Poon and J M Luk ldquoMicroRNA-375 targetsHippo-signaling effector YAP in liver cancer and inhibits tumorpropertiesrdquo Biochemical and Biophysical Research Communica-tions vol 394 no 3 pp 623ndash627 2010

[84] Q-H Xie X-X He Y Chang et al ldquoMiR-192 inhibitsnucleotide excision repair by targeting ERCC3 and ERCC4in HepG2215 cellsrdquo Biochemical and Biophysical ResearchCommunications vol 410 no 3 pp 440ndash445 2011

[85] A Naccarati B Pardini and S Landi ldquoPolymorphisms inmiRNA-binding sites of nucleotide excision repair genes andcolorectal cancer riskrdquo Carcinogenesis vol 33 no 7 pp 1346ndash1351 2012

[86] M A Chaudhry H Sachdeva and R A OmaruddinldquoRadiation-induced micro-RNA modulation in glioblastomacells differing in DNA-repair pathwaysrdquo DNA and Cell Biologyvol 29 no 9 pp 553ndash561 2010

[87] V Chiarugi and M Ruggiero ldquoRole of three cancer ldquomastergenesrdquo p53 Bcl2 and c-myc on the apoptotic processrdquo Tumorivol 82 no 3 pp 205ndash209 1996

[88] T Papagiannakopoulos A Shapiro and K S KosikldquoMicroRNA-21 targets a network of key tumor-suppressivepathways in glioblastoma cellsrdquo Cancer Research vol 68 no19 pp 8164ndash8172 2008

[89] P Moskwa F M Buffa Y Pan et al ldquomiR-182-mediateddownregulation of BRCA1 impacts DNA repair and sensitivityto PARP inhibitorsrdquo Molecular Cell vol 41 no 2 pp 210ndash2202011

[90] K Krishnan A L Steptoe H C Martin et al ldquoMicroRNA-182-5p targets a network of genes involved inDNA repairrdquoRNA vol19 no 2 pp 230ndash242 2013

[91] WRui F Bing SHai-ZhuDWei andC Long-Bang ldquoIdentifi-cation of microRNA profiles in docetaxel-resistant human non-small cell lung carcinoma cells (SPC-A1)rdquo Journal of Cellularand Molecular Medicine vol 14 no 1-2 pp 206ndash214 2010

[92] M TW Teo D Landi C F Taylor et al ldquoThe role of microrna-binding site polymorphisms in DNA repair genes as risk factors

for bladder cancer and breast cancer and their impact onradiotherapy outcomesrdquo Carcinogenesis vol 33 no 3 pp 581ndash586 2012

[93] M Yan H Xu N Waddell et al ldquoEnhanced RAD21 cohesinexpression confers poor prognosis in BRCA2 and BRCAX butnot BRCA1 familial breast cancersrdquo Breast Cancer Research vol14 no 2 p R69 2012

[94] Y Wang J W Huang P Calses C J Kemp and T TaniguchildquoMiR-96 downregulates REV1 and RAD51 to promote cellularsensitivity to cisplatin and PARP inhibitionrdquo Cancer Researchvol 72 no 16 pp 4037ndash4046 2012

[95] Z Zheng W L Ng X Zhang et al ldquoRNAi-mediated targetingof noncoding and coding sequences in DNA repair genemessages efficiently radiosensitizes human tumor cellsrdquo CancerResearch vol 72 no 5 pp 1221ndash1228 2012

[96] S A Hegre P Saeligtrom P A Aas H S Pettersen M Otterleiand H E Krokan ldquoMultiple microRNAsmay regulate the DNArepair enzyme uracil-DNAglycosylaserdquoDNARepair vol 12 no1 pp 80ndash86 2013

[97] D J Jonker C J OrsquoCallaghan C S Karapetis et al ldquoCetuximabfor the treatment of colorectal cancerrdquoTheNew England Journalof Medicine vol 357 no 20 pp 2040ndash2048 2007

[98] N Ferrara K J Hillan and W Novotny ldquoBevacizumab(Avastin) a humanized anti-VEGF monoclonal antibody forcancer therapyrdquo Biochemical and Biophysical Research Commu-nications vol 333 no 2 pp 328ndash335 2005

[99] G N Hortobagyi ldquoTrastuzumab in the treatment of breastcancerrdquo The New England Journal of Medicine vol 353 no 16pp 1734ndash1736 2005

[100] A Haringhuizen H van Tinteren H F R Vaessen P Baas andN van Zandwijk ldquoGefitinib as a last treatment option for non-small-cell lung cancer durable disease control in a subset ofpatientsrdquo Annals of Oncology vol 15 no 5 pp 786ndash792 2004

[101] J Elmen M Lindow S Schutz et al ldquoLNA-mediatedmicroRNA silencing in non-human primatesrdquo Nature vol 452no 7189 pp 896ndash899 2008

[102] J Kota R R Chivukula K A OrsquoDonnell et al ldquoTherapeuticmicroRNA delivery suppresses tumorigenesis in a murine livercancer modelrdquo Cell vol 137 no 6 pp 1005ndash1017 2009

[103] M Hatziapostolou C Polytarchou E Aggelidou et al ldquoAnHNF4120572-miRNA inflammatory feedback circuit regulates hep-atocellular oncogenesisrdquo Cell vol 147 no 6 pp 1233ndash1247 2011

[104] Y Shu F Pi A Sharma et al ldquoStable RNA nanoparticles aspotential new generation drugs for cancer therapyrdquo AdvancedDrug Delivery Reviews 2013

[105] L Zhao A M Bode Y Cao and Z Dong ldquoRegulatorymechanisms and clinical perspectives of miRNA in tumorradiosensitivityrdquo Carcinogenesis vol 33 no 11 pp 2220ndash22272012

Submit your manuscripts athttpwwwhindawicom

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molecules targeted by ATM [6] The ATR kinase activates apathway principally induced by UV damage which involvesChk1 kinase [7ndash9] The most important targets of both Chk1and Chk2 are members of the Cdc25 phosphatase familyThese molecules are normally required for the activation ofcyclin-dependent kinase Once phosphorylated Cdc25a isfunctionally inhibited and prevents the activity of cyclin-dependent kinase-cyclin complexes involved in the transi-tion G1S and in the progression through S and G2Mtriggering G1 S and G2M checkpoints [10ndash12] In additionp38120572120573-dependent activation of MK2 works in a differentcell cycle checkpoint kinase pathway activated in responseto UV as well as to currently used chemotherapeutic drugsMK2 functions are critical especially in cells and tumorslosing p53 [13ndash18] A relatively DDR slow response is theATMATR-dependent p53 phosphorylationwith consequenttranscriptional activation of genes involved in cell cyclearrest (ie page 21) [3] At this point if DNA lesions areadequately repaired cells can proceed to proliferate againand to inactivate DNA damage checkpoints otherwise theyundergo to apoptosis [19] DNA damage can be repairedby different mechanisms The base excision repair (BER)and the nucleotide excision repair (NER) complexes removedamaged andmodified nucleotides In particular NERworksprincipally on helix-distorting and transcription-blockinglesions (ie UV-induced pyrimidine dimers) whereas BERremoves single nucleotides modified by methylation alkyla-tion deamination or oxidation [20] The mismatch repairmachinery (MMR) operates through MSH2 and MLH1which form heterodimers with MSH3 or MSH6 and MLH3PMS2 or PMS1 respectively These complexes are involvedin mismatchinsertion-deletion loops recognition and in theexcision-repair reaction [21 22] MMR machinery removesincorrectly incorporated nucleotides during DNA synthesisor replication errors in DNA repeats causing microsatel-lite instability [23] DNA double strand breaks (DSBs) arefrequent events in eukaryotic cells they are induced byphysiological mechanisms in early lymphocytes or by patho-logical activity attributable toROS ionizing radiation or erro-neous nuclear enzyme functions DSBs are repaired by non-homologous end joining (NHEJ) in all phases of the cell cyclethus representing the major pathway in G1 or homologousrecombination (HR) in the S or G2 phases of the cell cycle[24 25] NHEJ involves the binding of the heterodimeric Kuprotein to double-stranded DNA ends the recruitment ofthe DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA-PK kinase activation [24] The DNA-PKcomplex on the DNA works by recruiting a complex con-tainingDNA ligase IV XRCC4 and XLFCernunnos proteinwith consequent rejoining [24] HR is a complex processinvolving the generation of 31015840 single-strandedDNA overhangfirst bound by RPA The displacement of the complex byRad51 follows then a homologous sequence is engaged torepair the lesion [25]

Tumor cells frequently acquire the ability to overcomethe DDR network in fact most cancers show malfunctionsin the DDR such as resistance to genotoxic agents andionizing radiations or abnormal cell cycle progression afterDNA damage [26] The tumor suppressor p53 is one of the

most important proteins activated after DNA damage andp53 gene mutations are described in almost every type ofcancer at rates comprised between 38 and 50 in ovariancolorectal head and neck and lung cancers [27] Similarlysome hereditary cancer predisposition syndromes such asataxia telangiectasia Li-Fraumeni andNijmegen syndromesbreast and ovarian cancer or colon cancer (HNPCC) pre-disposition and Xeroderma pigmentosum show lesions ingenes involved in the DDR machinery (ATM p53 NBS1BRCA-12 MMR genes and NER genes) [23] In responseto DNA damage the mRNA expression patterns go throughsignificant modifications [28 29] which can be due to RNAPolII hyperphosphorylation with consequent prevention ofthe formation of preinitiation complexes at sites of the pro-moter [30] In this context post-transcriptional regulationof mRNAs mediated by microRNAs (miRNAs) plays also afundamental role

3 Biogenesis and Functions of MicroRNAs

MicroRNAs (miRNAs) are a class of short noncoding RNAsusually 18ndash25 nucleotides in length mediating importantcellular functions such as proliferation apoptosis differ-entiation and cell signaling [31ndash33] MiRNAs regulate thetranslation of specific mRNA by their imperfect or perfectpairing at the level of the 31015840UTR [34] It is estimated that in thehuman genome approximately 30 of genes are targeted bymiRNAs [35] MiRNAs genes are transcribed in two differentways long primary miRNAs (pri-miRNAs) are synthesizedfrom intergenic miRNAs by RNA polymerase II whereasintronic or esonic miRNAs are transcribed in associationwith their host genes from a common promoter [36] Aftercleavage of pri-miRNAs in the nucleus by themicroprocessorDrosha-DGCR8 precursor pre-miRNAs (approximately 70nucleotides in length) are obtained Pre-miRNAs are thenexported from the nucleus to the cytoplasm by the RanGTP-binding nuclear transporter exportin-5 In the cytoplasmthe endoribonucleaseDicer cleaves pri-miRNAs generating aduplex about 25 bases in length which consists of a functionalmiRNA and a passenger strand The association betweenArgonaute (Ago) proteins and mature miRNAs generatesRISC (RNA-induced silencing complex) which leads to post-transcriptional gene regulation consisting of mRNA degra-dation or translation inhibition [37] Due to their role in theregulation of fundamental cellular functions miRNAs areinvolved in several pathological processes and in particularin carcinogenesis [38]

4 MicroRNAs in DNA DamageRepairMechanisms and Cancer

41 MicroRNAs in ATM and ATR Pathways ATM gene hasbeen shown to be downregulated by miR-421 in neuroblas-toma and HeLa cells [39] MiR-421 targeted the ATM 31015840UTRplaying a role in modulating cell cycle checkpoints andcellular radiosensitivity Ectopic expression of miR-421 ledto S-phase cell cycle checkpoint changes and radiosensitivityincrease and was able to generate an ataxia telangiectasia-like






















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Volume 2014

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Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














6 Conclusions





References





















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










6 Conclusions





References





















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





6 Conclusions





References





















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article MicroRNAs in the DNA Damage/Repair Network ...downloads.hindawi.com/journals/ijg/2014/820248.pdf · DNA damage. Impairment in the DNA repair proteins, which physiologically