RESEARCHOpenAccessCircularRNAcircRHOT1promoteshepatocellularcarcinomaprogressionbyinitiationofNR2F6expressionLiyanWang,HaiyanLong,QinghuaZheng,XiaotongBo,XuhuaXiaoandBinLi*AbstractBackground:Increasingevidencehasrevealedacloserelationshipbetweennon-codingRNAsandcancerprogression.CircularRNAs(circRNAs),arecentlyidentifiednewmemberofnon-codingRNAs,aredemonstratedtoparticipateindiversebiologicalprocesses,suchasdevelopment,homeostaticmaintenanceandpathologicalresponses.ThefunctionsofcircRNAsincancerhavedrawnwideattentionrecently.Untilnow,theexpressionpatternsandrolesofcircRNAsinhepatocellularcarcinoma(HCC)haveremainedlargelyunknown.Methods:BioinformaticsmethodwasusedtoscreendifferentiallyexpressednovelcircRNAsinHCC.Northernblotting,qRT-PCR,insituhybridization(ISH)andRNA-FISHwereutilizedtoanalyzedtheexpressionofcircRHOT1inHCCtisues.CCK8,colonyformation,EdUassayswereusedtoanalyzeproliferationofHCCcells.TranswellassaywasutilizedtoanalyzeHCCcellmigrationandinvasion.FACSwasusedforapoptosisanalysis.Xenograftexperimentswereusedtoanalyzetumorgrowthinvivo.Massspectrum,RNApulldown,RIPandEMSAwasutilizedtotesttheinteractionbetweencircRHOT1andTIP60.RNA-sequencingmethodwasusedtoanalyzethedownstreamtargetgeneofcircRHOT1.Results:WeidentifiedcircRHOT1(hsa_circRNA_102034)asaconservedanddramaticallyupregulatedcircRNAinHCCtissues.HCCpatientsdisplayinghighcircRHOT1levelpossessedpoorprognosis.Throughinvitroandinvivoexperiments,wedemonstratedcircRHOT1significantlypromotedHCCgrowthandmetastasis.Regardingthemechanism,weconductedaRNApulldownwithabiotin-labeledcircRHOT1-specificprobeandfoundthatcircRHOT1recruitedTIP60totheNR2F6promoterandinitiatedNR2F6transcription.Moreover,NR2F6knockoutinhibitedgrowth,migrationandinvasion,whereasrescuingNR2F6incircRHOT1-knockoutHCCcellsrescuedtheproliferationandmetastasisabilitiesofHCCcells.Conclusion:Takentogether,circRHOT1inhibitsHCCdevelopmentandprogressionviarecruitingTIP60toinitiateNR2F6expression,indicatingthatcircRHOT1andNR2F6maybepotentialbiomarkersfo...
