The Role Of Structure-Specific Endonuclease FEN1 In Cervical Cancer Treatment And Its Mechanism Study

Objectives:Cervicalcancerisoneofthemostcommoncausesofcancer associated mortality among affected women in the world.Atpresent,treatmentwithweeklycisplatinplusradiotherapyisthestandardregimenfor cervicalcancer,especially forlocaladvanced cervicalcancer.However,patientswhoinitiallyrespondedtocisplatintherapyoftendevelopresistance to the drug during subsequenttreatment.The potentialnephrotoxicity,ototoxicityandhighlyemeticeffectsofcisplatinalsolimititsusetospecial populations.DNAflapendonuclease-1（FEN1）isanucleasewhichpossesses flapendonuclease（FEN）activity,gapdependentendonuclease（GEN）activity andexonuclease（EXO）activity,andplaysessentialrolesin Okazakifragment maturationof DNAreplicationand DNArepairpathways.FEN1overexpression hasbeenfoundinmanyformsofcancer,and FEN1inhibitorhasbeenshownto sensitize DNAdamagerelatedchemotherapydrugs.However,whether FEN1deficiencyoractivityinhibitionishelpforradiotherapyforcervicalcanceris stillunknown.Since FEN1playsacentralrolein DNAmetabolism,FEN1knockoutmice modelisembryoniclethalat9.5days.Itishardtostudythefunctionsof FEN1duringcellproliferationandmetabolism processbyomicstechnology,which ispowerfulandeffectiveforthecomprehensivestudyof FEN1.Inthisstudy,weaimedtostudythepotentialfunctionof FEN1during cervicalcancercellproliferation,thefunctionof FEN1inhibitortosuppress cervicalcancercellgrowth,andthesensitizingeffectsof FEN1inhibitorto radiotherapyforkillingthecervicalcancer,whichwouldhelpfulforclinical application.Besides,we also detected and analyzed the gene expression profilesof FEN1knockdowncells,whichwouldlayafoundationanddefinethe researchdirectionsforcomprehensivestudyof FEN1functions.Methods:1.Analyzetheexpressionof FEN1andγH2AXinthe Cervical cancersamplesand normaltissuesbythe Cancer Genome Atlas（TCGA）database,and enrichtheγH2AX markersin FEN1^highsamplesand FEN1^Lowsamplesthrough GSEAgeneenrichmentanalysis.Anddetecttheexpression levelsof FEN1andγH2AX in Helacellswhichweretreatedwithionizing radiation（2hourslater）.2.Theeffectof FEN1inhibitor SC13onionizingradiationsensitivityof Helacellswasstudiedthroughtheanalysisof Helacellvitality,colonyforming abilityandcellapoptosis.3.The FEN1genein293Tcellswasknockoutby CRISPRtechnology,observed the abilityofcellcolonyformation and the effecton ionizing radiationsensitivityafterknockoutof FEN1gene.4.Establishnudemousetumormodeltostudytheeffectof FEN1inhibitor SC13synergisticionizingradiationonthetumorigenicabilityof Helacellsin mice.5.Using¹⁸F-FDG Micro-PET display technology to evaluate the sensitizingeffectsof FEN1inhibitorwithionizingradiationtreatmentonthe nudemousetumormodel.6.Construct FEN1geneknock-down293Tcelllinebytransfectedwith FEN1-si RNAandvalidatedbywesternblotassay.7.Theeffectof FEN1geneknock-downoncellproliferationwouldbe analyzedby CCK-8cellproliferationassay.8.Extractthetotal RNA from FEN1geneknock-downandnormalcell linesandreverselytranscribeintoc DNAfor RNA-seqanalysis.9.The RNA-seq results would be statistically analyzed by KEGG enrichmentanalysisand GO analysistoscreenoutdifferentiallyexpressed genes.10.Selectedgeneswhichshoweddifferentialexpressionlevelbetween normaland FEN1knock-downcellstrainswouldbedetectedtheexpression levelby Real-timequantitative PCRtechnology.Results:1.FEN1wasoverexpressedincervicalcancerandupregulated furtherbyionizingradiationinduction.2.FEN1inhibitor SC13canenhancetheionizingradiationsensitivityofthe Hela,whichwasmainlycausedbyinducingapoptosis.3.The FEN1genein293Tcellswasknockoutby CRISPRtechnology,and itwasfoundthatknockoutof FEN1expressioncanenhancetheionizing radiationsensitivityofthe293Tcells.4.The FEN1inhibitor SC13hadaradiationsensitizationeffectontumor formationinmicewithcervicalcancercells.5.Itwas found thatthe standardized uptake value（SUV）ofeach intervention group waslowerthan the controlgroup,¹⁸F-FDG Micro-PET displaytechnologywasgoodstrategyforthetherapeuticefficacyevaluation ofcervicalcancerxenograftmicemodel.6.FEN1playedanimportantroleincellgrowth,andknock-downof FEN1geneinhibitedtheproliferationof293Tcells.7.RNA-seqanalyseswereperformedon293Tnormaland FEN1gene knock-downcells.From theanalysis,5,730differentiallyexpressiongenes wereidentifiedbetweenthetwogroups,3,117（54%）geneswereupregulated and2,613（46%）genesweredownregulatedinthe FEN1geneknock-down group.8.The differentially expressed geneswere analyzed through DAVID databases,thedatarevealedthatnucleicacidsrelatedmetabolismsandcell cyclerelatedmetabolismsweresignificantlyinterruptedinthe FEN1gene knock-downgroup.Fortheupregulatedgeneclustersofthe FEN1gene knock-down group,most genes are related to morphogenesis or developmentofcellularorganinbiologicalprocesscategory,andprotein bindingactivityinmolecularfunctioncategory.4.KEGG enrichment analysis of the pathway revealed that FEN1down-regulationwasassociatedwith RNAtransport,ribosomalbiogenesisand RNAdegradation,aswellasviralinfectionandtumor-relatedpathways5.Sixupregulatedgenesandfourdownregulatedgeneswerechosenfor furtheranalysisbyq RT-PCRtoconfirm theexpressionprofilesof FEN1gene knock-downcells.Thedatashowedthatalltheq RT-PCRresultswereingood agreementwiththe RNA-seqanalysis.Conclusion:Inthisstudy,wedeterminedif FEN1inhibitor SC13could sensitizeradiotherapyofcervicalcancercell.Wedemonstratedthat FEN1is overexpressed in Hela celland upregulated furtherby ionizing radiation induction.Wealso showedthat FEN1inhibitorenhancesionizingradiation sensitivityofcervicalcancerinvitroandinvivo,andthisbeneficialeffectwas largelydueto theinductionofapoptosis.Furthermore,thepresentstudy demonstrated that FEN1deregulation willsuppresscellproliferation,and interruptnucleicacidsrelatedmetabolismsandcellcyclerelatedmetabolisms.Itwasalsofoundthat FEN1canbeinvolvedinnon-coding RNAprocessing,ribosomal RNAprocessing,transfer RNAprocessingandribosomalbiogenesis,which laid some foundationto understand the role of FEN1nucleasesin regulatingcellgrowth,DNArepairandcancertherapy.Italsoprovidesclues forunderstandingtheroleofother RAD2familynucleasesincellgrowthand metabolism.