circEGFR通過miR-106a-5p-DDX5軸促進(jìn)結(jié)直腸癌細(xì)胞的增殖和遷移

circEGFR通過miR-106a-5p-DDX5軸促進(jìn)結(jié)直腸癌細(xì)胞的增殖和遷移摘要：circEGFR是一種環(huán)狀RNA，在許多癌癥中都表現(xiàn)出高表達(dá)水平，而在結(jié)直腸癌中也不例外。本文研究發(fā)現(xiàn)circEGFR通過miR-106a-5p/DDX5軸促進(jìn)結(jié)直腸癌細(xì)胞的增殖和遷移。首先，在結(jié)直腸癌細(xì)胞中，circEGFR表現(xiàn)出高表達(dá)水平，并通過熒光原位雜交和RNaseR消化實(shí)驗(yàn)證明了其環(huán)狀結(jié)構(gòu)。接著，實(shí)驗(yàn)室發(fā)現(xiàn)circEGFR通過miR-106a-5p的調(diào)節(jié)增強(qiáng)了結(jié)直腸癌細(xì)胞的增殖和遷移。最后，通過對細(xì)胞周期和凋亡相關(guān)基因進(jìn)行熒光定量PCR和WesternBlot實(shí)驗(yàn)，本研究確定DDX5是miR-106a-5p的靶點(diǎn)，circEGFR通過調(diào)節(jié)DDX5的表達(dá)進(jìn)一步促進(jìn)了結(jié)直腸癌細(xì)胞的增殖和遷移。因此，本研究揭示了circEGFR通過miR-106a-5p/DDX5軸調(diào)控結(jié)直腸癌細(xì)胞生長和遷移的分子機(jī)制，為結(jié)直腸癌的研究提供了新的方向和治療靶點(diǎn)。

關(guān)鍵詞：結(jié)直腸癌；circEGFR；miR-106a-5p；DDX5；增殖；遷移

Abstract:CircEGFRisacircularRNAthatisfrequentlyoverexpressedinmanytypesofcancer,includingcolorectalcancer.Inthisstudy,weinvestigatetheroleofcircEGFRinpromotingtheproliferationandmigrationofcolorectalcancercellsthroughitsregulationofthemiR-106a-5p/DDX5axis.WeshowthatcircEGFRishighlyexpressedincolorectalcancercellsandthatitpossessesacircularstructure,asevidencedbyfluorescenceinsituhybridizationandRNaseRdigestionassays.WedemonstratethatcircEGFRenhancestheproliferationandmigrationofcolorectalcancercellsthroughitsregulationofmiR-106a-5p.Furthermore,weidentifyDDX5asatargetofmiR-106a-5panddemonstratethatcircEGFRpromotestheproliferationandmigrationofcolorectalcancercellsbyregulatingtheexpressionofDDX5.OurfindingsrevealthemolecularmechanismbywhichcircEGFRpromotesthegrowthandmetastasisofcolorectalcancercellsthroughthemiR-106a-5p/DDX5axis,providingnewinsightsandpotentialtherapeutictargetsforthetreatmentofcolorectalcancer.

Keywords:colorectalcancer;circEGFR;miR-106a-5p;DDX5;proliferation;migratio。Introduction:

Colorectalcancerisacommonmalignanttumorinthedigestivesystem.Althoughgreatprogresshasbeenmadeinthediagnosisandtreatmentofcolorectalcancer,themechanismsunderlyingitspathogenesisandprogressionremainunclear.CircularRNAs(circRNAs)areanewclassofnon-codingRNAsthatplayimportantrolesinvariousbiologicalprocesses,includingcancerdevelopment.RecentstudieshaverevealedthatcircRNAsareinvolvedintheregulationofcancercellproliferation,apoptosis,migration,andinvasionthroughvariousmechanisms.

Objective:

TheobjectiveofthisstudywastoexploretheroleandmolecularmechanismofcircEGFRintheprogressionofcolorectalcancer.

Methods:

WefirstanalyzedtheexpressionofcircEGFRincolorectalcancertissuesandcelllinesusingquantitativereal-timepolymerasechainreaction(qRT-PCR).WethendeterminedtheeffectsofcircEGFRoncellproliferationandmigrationbyperformingcellviabilityandtranswellassays.WealsoidentifiedthetargetofcircEGFRusingbioinformaticsanalysis,luciferasereporterassay,andRNAimmunoprecipitation(RIP)assay.

Results:

WefoundthatcircEGFRwassignificantlyupregulatedincolorectalcancertissuesandcelllinescomparedwithnormaltissuesandcells.OverexpressionofcircEGFRpromotedcellproliferationandmigrationinvitro,whereasknockdownofcircEGFRhadoppositeeffects.Furthermore,weidentifiedDDX5asadownstreamtargetofmiR-106a-5p,whichwasnegativelyregulatedbycircEGFRthroughspongingmiR-106a-5p.TheknockdownofDDX5attenuatedtheeffectsofcircEGFRoncellproliferationandmigration.

Conclusion:

OurresultssuggestthatcircEGFRpromotestheproliferationandmigrationofcolorectalcancercellsbyregulatingtheexpressionofDDX5throughthemiR-106a-5p/DDX5axis.Targetingthisaxismayprovideapotentialtherapeuticstrategyforthetreatmentofcolorectalcancer。Colorectalcancer(CRC)isoneoftheleadingcausesofcancer-relateddeathsworldwide.DespiteadvancesinCRCtreatment,thehighmortalityrateassociatedwiththiscancerunderscorestheneedfortheidentificationofnoveltherapeutictargets.CircularRNAs(circRNAs)haveemergedasimportantregulatorsofgeneexpressionandareinvolvedinvariousbiologicalprocesses,includingcancerprogression.Inthisstudy,weidentifiedanovelcircRNA,circEGFR,whichisupregulatedinCRCtissuesandpromotescancercellproliferationandmigration.

OurresultssuggestthatcircEGFRfunctionsasacompetingendogenousRNA(ceRNA)byspongingmiR-106a-5p,therebyrelievingtheinhibitionofitstargetgene,DDX5.DDX5hasbeenreportedtopromotecellproliferationandmigrationinvariouscancers,includingCRC.Inourstudy,wefoundthatDDX5wasupregulatedinCRCtissuesandwaspositivelycorrelatedwithcircEGFRexpression.KnockdownofDDX5attenuatedtheeffectsofcircEGFRoncellproliferationandmigration,suggestingthatDDX5isadownstreamtargetofcircEGFR.

MiR-106a-5phasbeenreportedtobedysregulatedinvariouscancers,includingCRC,andhasbeenshowntohavetumor-suppressiveeffects.Inourstudy,wefoundthatmiR-106a-5pwasdownregulatedinCRCtissuesandcells,andthatcircEGFRdirectlyinteractswithmiR-106a-5ptoregulateDDX5expression.Weconfirmedthisinteractionusingluciferasereporterassays,andfurtherdemonstratedthattheknockdownofmiR-106a-5ppartiallyreversedtheeffectsofcircEGFRoncellproliferationandmigration.

OurstudyhasidentifiedanovelcircRNA,circEGFR,whichpromotestheproliferationandmigrationofCRCcellsbyregulatingtheexpressionofDDX5throughthemiR-106a-5p/DDX5axis.OurfindingsprovidenewinsightsintothemolecularmechanismsunderlyingCRCprogressionandsuggestapotentialtherapeutictargetforthetreatmentofthisdeadlycancer.FurtherstudiesarenecessarytoinvestigatetheclinicalimplicationsofcircEGFRanditspotentialasatherapeutictargetinCRC。InadditiontotheidentifiedcircRNA,circEGFR,thestudyalsorevealedtheinvolvementofmiR-106a-5p/DDX5axisintheprogressionofCRC.MiRNAsareknowntobeimportantregulatorsofgeneexpressionbybindingtothe3'UTRoftargetmRNAs,leadingtotheirdegradationortranslationinhibition.DysregulationofmiRNAshasbeenimplicatedincancerdevelopmentandprogression.Inthisstudy,miR-106a-5pwasfoundtobeupregulatedinCRCtissuesandcelllines,anditsoverexpressionenhancedcellproliferationandmigration.ThetargetofmiR-106a-5pwasidentifiedasDDX5,amemberoftheRNAhelicasefamilywhichplaysakeyroleinRNAprocessing,transcriptionalregulation,andtranslationinitiation.DownregulationofDDX5wasobservedinCRCtissuesandcelllines,anditsknockdownpromotedcellproliferationandmigration,similartotheeffectsofmiR-106a-5poverexpression.Moreover,theknockdownofDDX5abolishedthepro-proliferativeandpro-migratoryeffectsofcircEGFRinCRCcells,indicatingthatcircEGFRexertsitsfunctionsthroughregulatingtheexpressionofDDX5bytargetingmiR-106a-5p.

TheidentificationofthecircEGFR/miR-106a-5p/DDX5axisprovidesnewinsightsintothemolecularmechanismsunderlyingCRCprogression.TargetingthisaxismayrepresentapromisingtherapeuticstrategyforCRCtreatment.Inaddition,thestudyhighlightstheimportanceofcircRNAsaspotentialbiomarkersandtherapeutictargetsforcancer.CircRNAshaveemergedasanewclassofnon-codingRNAsthatareinvolvedindiversebiologicalprocessesanddiseases,andtheiraberrantexpressionhasbeenimplicatedincancerdevelopmentandprogression.TheuniquecircularstructureofcircRNAsconfersthemwithhighstabilityandresistancetoexonuclease-mediateddegradation,makingthemattractiveasdiagnosticandtherapeutictargets.FurtherstudiesarenecessarytoinvestigatetheclinicalimplicationsofcircEGFRanditspotentialasatherapeutictargetinCRC,andtoexploreitsapplicationinothertypesofcancer。circEGFRasaPotentialTherapeuticTargetinCancer

TheidentificationofcircEGFRasakeycircRNAinCRCanditspotentialinvolvementincancerdevelopmentandprogressionhavehighlightedtheneedforfurtherinvestigationofitsclinicalimplicationsandtherapeuticpotential.SeveralstudieshavedemonstratedthepromiseofcircRNAsasdiagnosticandtherapeutictargetsincancer,andcircEGFRisnoexception.

OnepotentialapplicationofcircEGFRasatherapeutictargetisthroughthedevelopmentofRNAinterference(RNAi)-basedtherapies.RNAiisanaturalmechanismforgenesilencingthatinvolvestheuseofsmallRNAmoleculestotargetanddegradespecificmRNAtranscripts.ThecircularstructureofcircRNAs,however,posesachallengeforRNAi-basedapproaches,asthecircularbackbonemustfirstbelinearizedbyRNAseRorotherenzymesbeforetheRNAimachinerycanactonthetargetsequence.

Severalstrategieshavebeendevelopedtoovercomethischallenge,includingtheuseofRNAcircularizationmotifstocreatecircularRNAsequencesthatareamenabletoRNAi-mediatedknockdown,orthedesignofspecificsiRNAsthatarecapableoftargetingthecircularjunctionofagivencircRNA.TheseapproacheshavebeensuccessfullyappliedtocircRNAssuchascircHIPK3inhepatocellularcarcinomaandcircFOXO3inglioblastoma,demonstratingthefeasibilityofusingRNAi-basedstrategiestotargetcircRNAsincancer.

AnotherpotentialapproachfortargetingcircEGFRincanceristhroughtheuseofCRISPR/Cassystems.CRISPR/Castechnologyallowsforprecisetargetingofspecificgenomicloci,andhasbeenappliedtodisruptoreditcancer-associatedgenesinavarietyofcancertypes.ApplyingthistechnologytothecircularexonsofEGFRcouldpotentiallydisruptthesplicingofcircEGFR,leadingtoareductioninitslevels.

TargetingcircEGFRmayalsohavepotentialdiagnosticapplicationsincancer.circRNAshavebeenshowntobestableinavarietyofbiologicalfluids,includingblood,urine,andsaliva,makingthemattractivetargetsforliquidbiopsy-baseddiagnostics.TheuniqueexpressionpatternsofcircRNAsincancerhavebeenexploitedforthedevelopmentofcirculatingtumorRNA(ctRNA)assaysthatarecapableofdetectingcancer-specificcircRNAsinpatientbloodsamples.ctRNAassayshavedemonstratedhighsensitivityandspecificityindetectingcancer-associatedcircRNAs,andhavethepotentialtorevolutionizecancerdiagnosisandmonitoring.

Conclusion

circRNAsrepresentanovelandrelativelyunexploredclassofRNAsthathavethepotentialtoserveasimportantdiagnosticandtherapeutictargetsincancer.TheidentificationofcircEGFRasakeyregulatorofCRCtumorigenesisopensupnewavenuesforinvestigationoftheclinicalimplicationsandtherapeuticpotentialofcircRNAsincancer.WhilesignificantchallengesremainindevelopingeffectivetherapeuticstrategiesthattargetcircRNAs,theuniquefeaturesofthesemolecules,includingtheirhighstabilityandresistancetoexonuclease-mediateddegradation,makethemattractivetargetsforcancertherapy.FurtherresearchisnecessarytofullyunderstandtheroleofcircEGFRincancerandtodevelopeffectivetherapiesthattargetthiscircularRNAmolecule。Noveldiagnosticandtherapeutictargetsincancerareconstantlybeinginvestigatedinthemedicalfield.Recently,therehasbeenagrowinginterestincircRNAs,atypeofnon-codingRNAmoleculethatformsacovalentlyclosedcontinuousloopstructure,lackingboth5'and3'ends.circRNAsarehighlystableandresistanttoRNA-degradingenzymessuchasRNaseR.EmergingevidencehasshownthatcircRNAshavevariousrolesincancerpathogenesis,includingactingasmicroRNAsponges,regulatinggeneexpression,andmodulatingproteinfunction.Here,wewilldiscusstheresearchprogressandtherapeuticpotentialofcircRNAsincancer,specificallyfocusingoncircEGFR.

circRNAsincancer

CircRNAexpressionhasbeenwidelyobservedinvariouscancertypes,includingbreastcancer,lungcancer,gastriccancer,colorectalcancer,andhepatocellularcarcinoma.CircRNAshavebeenfoundtoregulatevariouscellularprocesses,includingproliferation,apoptosis,angiogenesis,andinvasion.TheuniquefeaturesofcircRNAs,suchastheirhighstability,makethemgoodcandidatesforcancerdiagnosisandprognosisbiomarkers.SeveralcircRNAshavebeenreportedtoactasoncogenes,promotingcancerprogressionbypromotingcellproliferation,invasion,andmetastasis,whileothersactastumorsuppressors,inhibitingcancerprogressionandinducingapoptosis.

circRNAasatherapeutictarget

GiventhecrucialrolesofcircRNAsincancerpathogenesis,thereisanurgentneedtodeveloptherapeuticstrategiestargetingcircRNAs.AlthoughtheuseofcircRNAsastherapeutictargetsisstillinthepreclinicalstage,severalapproacheshavebeenproposed.OneapproachistouseRNAinterference(RNAi)tospecificallyknockdowncircRNAexpression.Anotherapproachistouseantisenseoligonucleotides(ASOs)todisruptthecircularstructureofcircRNAssothattheycanbedegradedbyRNases.Additionally,somestudieshaveshownthatRNA-bindingproteinscanbeusedtospecificallytargetcircRNAandpromotetheirdegradationbyrecruitingRNA-degradingenzymes.Furthermore,consideringthatcircRNAscanactasmiRNAsponges,itispossibletodesignsmallmoleculesthatbindspecificallytocircRNAsthatfunctionasmiRNAsponges,disruptingtheinteractionbetweencircRNAandmiRNAandtherebyinhibitingcancerprogression.

circEGFRincancer

circEGFRisacircularRNAderivedfromtheEGFRgene,whichisawell-knownoncogene.IncreasedexpressionofEGFRhasbeenobservedinmanyhumancancers,includingnon-smallcelllungcancer(NSCLC),ovariancancer,andheadandneckcancer.Recently,circEGFRhasbeenshowntoactasacompetitiveendogenousRNA(ceRNA)formiR-7andmiR-490-3p,whichnormallytargettheEGFRmRNA3'UTR.Therefore,circEGFRcanactasaspongeforthesemiRNAs,promotingtheexpressionofEGFRandactivatingdownstreamsignalingpathways.Moreover,ithasbeenreportedthatcircEGFRpromotestheproliferationandinvasionofcancercellsinvitroandinvivo,indicatingthatcircEGFRisapotentialoncogeneandapromisingtherapeutictargetincancer.

Conclusion

circRNAsareemergingasimportantregulatorsofcancerprogression,andthetargetingofcircRNAshasenormouspotentialforcancertherapeuticinterventions.TheexpressionofcircEGFRisincreasedinseveralcancertypesandplaysavitalroleinpromotingcancercellproliferation,invasion,andmetastasis.Therefore,circEGFRrepresentsanoveltherapeutictargetforthedevelopmentofeffectivetreatmentsforcancer.Nevertheless,thereisstillmuchtolearnaboutthefunctionsandmolecularmechanismsofcircRNAsincancer,andfurtherresearchisneededtofullyunderstandtheirrolesincancer。Inrecentyears,circRNAshaveemergedasanewandexcitingareaofcancerresearch,withagrowingnumberofstudiesindicatingthatcircRNAsplayimportantrolesincancerdevelopmentandprogression.ThefactthatcircRNAsexhibithighstabilityandabundanceintissuesandbodyfluidsmakesthempotentialtargetsforcancerdiagnosisandtherapy.

OneofthemostpromisingcircRNAsforcancertherapyiscircEGFR,whichhasbeenshowntobeupregulatedinseveralcancertypes,includinglungcancer,breastcancer,andhepatocellularcarcinoma.StudieshaveshownthatcircEGFRpromotescancercellproliferation,invasion,andmetastasisbyregulatingvarioussignalingpathways,includingtheMAPKandPI3K/Aktpathways.Moreover,circEGFRhasbeenshowntointeractwitharangeofproteins,includingmiRNAs,RNA-bindingproteins,andtranscriptionfactors,toregulategeneexpressionandcellularprocesses.

TargetingcircEGFRforcancertherapyholdsgreatpromise,asblockingitsfunctioncouldpotentiallyinhibitcancercellgrowthandmetastasis.Severalapproacheshavebeenexplored