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OriginalArticleOpenAccessMicroRNA‐379suppressesosteosarcomaprogressionbytargetingPDK1ZhengLiDepartmentofOrthopaedicSurgery,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorJianxiongShenCorrespondingAuthor
E-mailaddress:shenjianxiong@medmail.com.cn
DepartmentofOrthopaedicSurgery,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
Correspondenceto:JianxiongSHEN.
E‐mail:shenjianxiong@medmail.com.cn
SearchformorepapersbythisauthorMatthewT.V.ChanDepartmentofAnaesthesiaandIntensiveCare,StateKeyLaboratoryofDigestiveDisease,LKSInstituteofHealthSciences,TheChineseUniversityofHongKong,HongKong,China
SearchformorepapersbythisauthorWilliamKaKeiWuDepartmentofAnaesthesiaandIntensiveCare,StateKeyLaboratoryofDigestiveDisease,LKSInstituteofHealthSciences,TheChineseUniversityofHongKong,HongKong,China
SearchformorepapersbythisauthorDepartmentofOrthopaedicSurgery,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorJianxiongShenCorrespondingAuthor
E-mailaddress:shenjianxiong@medmail.com.cn
DepartmentofOrthopaedicSurgery,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
Correspondenceto:JianxiongSHEN.
E‐mail:shenjianxiong@medmail.com.cn
SearchformorepapersbythisauthorMatthewT.V.ChanDepartmentofAnaesthesiaandIntensiveCare,StateKeyLaboratoryofDigestiveDisease,LKSInstituteofHealthSciences,TheChineseUniversityofHongKong,HongKong,China
SearchformorepapersbythisauthorWilliamKaKeiWuDepartmentofAnaesthesiaandIntensiveCare,StateKeyLaboratoryofDigestiveDisease,LKSInstituteofHealthSciences,TheChineseUniversityofHongKong,HongKong,China
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ShareonEmailFacebookTwitterLinkedInRedditWechatAbstractOsteosarcomaisthemostcommonprimarybonetumour.IncreasingevidencehasdemonstratedthepathogenicroleofmicroRNA(miRNAs)dysregulationintumourdevelopment.miR‐379waspreviouslyreportedtofunctionasanoncogenicortumour‐suppressingmiRNAinatissue‐dependentmanner.However,itsfunctioninosteosarcomaremainsunknown.Inthisstudy,wefoundthattheexpressionofmiR‐379wasdownregulatedinosteosarcomatissuesandcelllines.FurtherfunctionalcharacterizationrevealedthatmiR‐379suppressedosteosarcomacellproliferationandinvasioninvitroandretardedthegrowthofosteosarcomaxenograftsinvivo.Mechanistically,PDK1wasidentifiedasthedirecttargetofmiR‐379inosteosarcoma,inwhichPDK1expressionwasup‐regulatedandshowedinversecorrelationwithmiR‐379.EnforcedexpressionofPDK1promotedosteosarcomacellproliferationandrescuedtheanti‐proliferativeeffectofmiR‐379.ThesedatasuggestthatmiR‐379couldfunctionasatumour‐suppressingmiRNAviatargetingPDK1inosteosarcoma.
IntroductionOsteosarcomaisthemostcommonprimarytumourofbonewithanincidenceof4–5casespermillioninyoungadultsandadolescents1-4.Becauseoftheadvancementofitstreatment,includingsurgeryandmulti‐agentchemotherapy,the5‐yearsurvivalrateinpatientswithprimaryosteosarcomahasimprovedoverthepastseveraldecades5-8.However,thesurvivalrateforthistumourremainslow9-12.Therefore,itisnecessarytobetterunderstandthepathogenesisofosteosarcomaandidentifynovelMarkerstoimproveitstreatmentstrategies.
MicroRNAs(miRNAs)arehighlyconserved,endogenoussmallnoncodingRNAs(18–25nucleotidesinlength)thatrepressgeneexpressionbybindingtothe3′‐untranslatedregion(3′UTR)oftheirtargetmRNAs,inducingtheirdegradationortranslationalrepression13-16.SeveralstudieshavedemonstratedabnormalexpressionofmiRNAsindifferenttypesofcancer,includinggastriccancer,hepatocellularcarcinoma,glioblastoma,ovariancarcinoma,breastcancerandlaryngealcancer17-22.Moreover,increasingevidenceshowedthatmiRNAsplayimportantrolesinmanybiologicalprocesses,includingcellproliferation,apoptosis,metabolism,differentiationandinvasion23-26.SomestudieshavealsoidentifiedmiRNAsasnoveldiagnosticandprognosticbiomarkersforcancers27-30.
vPreviousstudiesshowedthataberrantexpressionofmiR‐379contributestocancerdevelopment31-33.Forinstance,Khanetal.demonstratedthattheexpressionofmiR‐379wassignificantlyreducedinbreastcancer,inwhichthismiRNAsignificantlyinhibitedcellproliferationbyrepressingcyclinB1expression34.Haenischetal.alsoshowedthatmiR‐379couldbeinducedbyrifampicin,therebyimpedingtheproteinoverexpressionofABCC2(multidrugresistanceassociatedprotein2)aftertreatmentwiththispregnaneXreceptorligandinHepG2cells35.However,theoncogenicroleofmiR‐379hasalsobeenreported.IthasbeendemonstratedthatmiR‐379expressionwaselevatedinbone‐metastaticprostatecancercelllinesandtissues.TheexpressionofmiR‐379wasalsocorrelatedwithshortenedprogression‐freesurvivalofpatientswithprostatecancer32.Inaddition,treatmentwithmiR‐379inhibitordecreasedbonemetastasisandincreasedsurvivalofmicetransplantedwithbonemetastaticARCaPMprostatecancercells32.Inosteosarcoma,theexpressionandfunctionofmiR‐379remainunknown.
Inthepresentstudy,wefoundthatmiR‐379expressionwasdownregulatedinosteosarcomatissuesandcelllines.FurtherinvestigationdemonstratedthatmiR‐379repressedtheinvitroandinvivomalignantphenotypesofosteosarcomacells.Importantly,weidentifiedPDK1asthedirecttargetofmiR‐379inosteosarcoma.
Atotalof30pairsofosteosarcomaandadjacentnon‐cancerbonetissues(located3cmfromthetumour)wereobtainedbetween2012and2014(seeTableS1fordemographicandclinicopathologicalinformation).Allsampleswereimmediatelyplacedinliquidnitrogenuntiluse.AllpatientshadprovidedconsentsandthisstudywasapprovedbytheinstitutionalethicalboardofPekingUnionMedicalCollegeHospitalandcompliedwiththeDeclarationofHelsinki.FourhumanosteosarcomacelllinesMG‐63(14yearsold,male),U2OS(15yearsold,female),SOSP‐9607(17yearsold,male),andSAOS‐2(11yearsold,female)andanon‐cancerosteoblasticcellline(hFOB)werepurchasedfromtheCellResourceCenterofChineseAcademyofMedicalSciences(Beijing,China).CellswereculturedinDMEM(Invitrogen,Carlsbad,CA,USA)andsupplementedwith10%foetalbovineserum(Gibco,GrandIsland,NY,USA).miR‐379mimicsandscrambledcontrol(GenePharma,Shanghai,China)weretransfectedintocellsat40–60%confluenceusingLipofectamine2000(Invitrogen,Carlsbad,CA,USA)accordingtothemanufacturer"srecommendations.ForPDK1overexpression,approximate4×105cellswereseededintoeachwellofa6‐wellplatethenightbeforefollowedbytransfectionwith2μgpcDNA3.1‐PDK1oremptyplasmidsDNAat60–80%confluenceusingLipofectamine2000.
TotalRNAwasisolatedfromcellsortissuesbyTrizolreagent(Invitrogen).TheexpressionofmiRNAandmRNAwasquantifiedbyquantitativereversetranscription(qRT)‐PCRontheiQ5Real‐TimePCRSystem(Bio‐Rad,Hercules,CA,USA).U6wasusedformiRNAnormalization36andGAPDHwasusedasacontrolformRNA(seeTableS2forprimersequences).Relativeexpressionwascalculatedusingthe2−ΔΔCtmethod.
CellproliferationwasdeterminedusingCCK‐8assay(Dojindo,Kumamoto,Japan)followingthemanufacturer"sinstruction.Theproliferationratewasmeasuredat0,24,48and72hrspost‐transfection(1×105cells).Forinvasionanalysis,cellswereseededontoaMatrigel‐coatedmembrane.Foetalbovineserumwasappendedandnon‐invadingcellswereremoved.CellsonthelowersurfaceoftheMatrigel‐coatedchamberwerestainedwith0.1%crystalviolet(Sigma‐Aldrich,St.Louis,MO,USA).Cellsontheentiremembranewerecounted.
Totaltissueorcellularproteinwasseparatedusing10%SDS‐PAGEandtransferredtopolyvinylidenedifluoridemembranes(Amersham,Buckinghamshire,UK).Themembraneswereblockedwith5%non‐fatmilkandthenincubatedwithprimaryantibodies(againstPDK1,GAPDHorKi‐67)for2hrs.AfterwashingwithTris‐bufferedsalinewithTween20,themembraneswereincubatedwithhorseradishperoxidase‐conjugatedsecondaryantibodyanddevelopedwithachemiluminescencedetectionkit(Millipore,Boston,MA,USA).
Themutant(MUT)orwild‐type(WT)3′UTRofPDK1wasclonedintopGL3luciferasepromotervector(Promega,Madison,WI,USA).CellswerecotransfectedwithmiR‐379mimicsorscrambledcontrolandthevectorscarryingPDK1MUTorWT3′UTRusingLipofectamine2000(Invitrogen).ActivitiesoffireflyandRenillaluciferasesweremeasuredusingadual‐luciferaseassaysystem(Promega).
MG‐63cells(5×106cellspermouse)wereinoculatedsubcutaneouslyintothedorsalflanksof6‐week‐oldfemalenudemice.Onday10,whentumoursreached~50mm3,miR‐379mimicsorscrambledcontrol(100nmolpermousein100μltotalvolumecontaining90μlphosphate‐bufferedsalineand10μlLipofectamine2000;5micepergroup)wasinjecteddirectlyintothetumourswith26‐gaugeneedlesevery3days.Thetumourwidth,lengthandweightweremeasuredevery3days.Micewerekilledbycervicaldislocationonthe25thdaysaftercancercellinoculation.Allexperimentalprocedureswereconductedinconformitywithinstitutionalguidelinesforthecareanduseoflaboratoryanimals,andprotocolswereapprovedbytheinstitutionalanimalethicscommitteeofPekingUnionMedicalCollege.
Cellswerefixedwith3.5%formaldehyde,permeabilizedwith0.1%TritonX‐100,blockedwith3%bovineserumalbuminand0.05%Tween20inPBSandthenincubatedwithprimaryantibody(dilutions1:1000,Ki‐67;Sigma‐Aldrich)overnightat4°C.Fluorescencewasmeasuredbyaconfocalmicroscope(TCSSP2;Leica,Mannheim,Germany).
Alldatawereshownasmean±S.D.DifferencesbetweentwogroupsandamongthreeormoregroupsweremeasuredbyStudent"st‐testandone‐wayANOVA,respectively.P0.05wasconsideredstatisticallysignificant.
TheexpressionofmiR‐379wasdownregulatedinosteosarcomatissuescomparedwiththeiradjacentnon‐canceroustissues(Fig.1A).Amongthem,24cases(80%)showedsignificantreductioninmiR‐379inosteosarcomatissues,whereassixcases(20%)showedup‐regulation.TheoverallexpressionofmiR‐379inosteosarcomatissueswassignificantlylowerthanthatincancer‐adjacenttissues(Fig.1B).Moreover,miR‐379expressionin4osteosarcomacelllines(MG‐63,U2OS,SOSP‐9607andSAOS‐2)waslowerascomparedwiththenon‐cancerosteoblasticcelllinehFOB(Fig.1C).Consistentwithourfindings,datafromthepublicdatabaseS‐MED(SarcomamicroRNAExpressionDatabase;http://www.oncomir.umn.edu/)indicatedthatthelevelsofmiR‐379inosteosarcomatissuesweremuchlowerthanthatinnormalbonetissues(Fig.1D).
Figure1OpeninfigureviewerPowerPointmiR‐379expressionwasdownregulatedinosteosarcomatissuesandcelllines.(A)TheexpressionlevelsofmiR‐379weremeasuredin30osteosarcomasamplesbyqRT‐qPCR.(B)TheexpressionofmiR‐379inosteosarcomatissueswassignificantlylowerthanthatincancer‐adjacenttissues.(C)miR‐379expressionwasdeterminedinfourosteosarcomacelllines(MG‐63,U2OS,SOSP‐9607,andSAOS‐2)andanon‐cancerosteoblasticcellline(hFOB)usingqRT‐PCR.(D)ExpressiondataofmiR‐397innormalbonetissuesandosteosarcomatissueswereextractedfromthepublicdatabaseS‐MED.
miR‐379mimicsorscrambledcontrolwastransfectedintoMG‐63andU2OS.TheefficiencyofmiR‐379transfectionwasconfirmedbyqRT‐PCR(Fig.2A).RestoredexpressionofmiR‐379inhibitedMG‐63andU2OScellproliferation(Fig.2B).Inaddition,themRNAandproteinexpressionofKi‐67,aproliferationmarker,wasdecreasedinthesetwocelllinesupontransfectionwithmiR‐379mimics(Fig.2CandD).
Figure2OpeninfigureviewerPowerPointmiR‐379inhibitedosteosarcomacellproliferation.(A)TheexpressionofmiR‐379inMG‐63andU2OScellswasdeterminedbyqRT‐qPCR.(B)TransfectionofmiR‐379mimicsinhibitedMG‐63andU2OScellproliferation.(C)miR‐379suppressedthemRNAexpressionofKi‐67inMG‐63andU2OScells.(D)TheproteinexpressionofKi‐67inMG‐63andU2OScellswasdeterminedbyWesternblots.*P0.05;**P0.01and***P0.001.
CellinvasionassaywasperformedtomeasuretheeffectofmiR‐379onthecellinvasiveabilityinmiR‐379‐transfectedMG‐63andU2OScells.DatashowedthatrestoredexpressionofmiR‐379inhibitedbothMG‐63andU2OScellinvasion(Fig.3).
Figure3OpeninfigureviewerPowerPointmiR‐379inhibitedosteosarcomacellinvasion.Trans‐wellinvasionassayswereconductedusingMG‐63andU2OScellstransfectedwithmiR‐379mimicsorscrambledcontrol.Therelativeratioofinvadedcellsperfieldwasshown,***P0.001.
UsingTargetScan,weidentifiedPDK1asthetentativetargetofmiR‐379(Fig.4A).Toconfirmthispossibility,luciferasereporterassaywasperformed.TherelativeluciferaseactivityofthereportercontainingPDK1WT‐3′UTRwassignificantlydecreasedbyabout60%uponmiR‐379transfectionascomparedwiththereportercontainingPDK1MUT‐3′UTRinbothMG‐63andU2OScells(Fig.4B).RestoredexpressionofmiR‐379alsorepressedthemRNAandproteinexpressionofPDK1inbothMG‐63andU2OScells(Fig.4CandD).
Figure4OpeninfigureviewerPowerPointPDK1wasthedirecttargetofmiR‐379inosteosarcoma.(A)The3′UTRofPDK1mRNAcontainsthebindingsequenceofmiR‐379.(B)RelativeluciferaseactivityoftheindicatedPDK1reporterconstructinMG‐63cellsisshown.FireflyluciferasevalueswerenormalizedtoRenillaluciferaseactivityandplottedasrelativeluciferaseactivity.(C)RelativeluciferaseactivityoftheindicatedPDK1reporterconstructinU2OScellsisshown.(DandE)ThemRNAexpressionofPDK1in(D)MG‐63and(E)U2OScellswasmeasuredbyqRT‐PCR.(FandG)TheproteinexpressionofPDK1in(F)MG‐63and(G)U2OScellswasmeasuredbyWesternblots,***P0.001.
TheexpressionofPDK1wasup‐regulatedinosteosarcomatissuescomparedwithcorrespondingnon‐cancerousadjacenttissues(Fig.5A).Amongthem,26cases(87%)showedsignificantlyhigherlevelsofPDK1inosteosarcomasamples,whereasfourcases(13%)exhibiteddownregulation.TheoverallexpressionofPDK1inosteosarcomatissueswassignificanthigherthanthatincancer‐adjacenttissues(Fig.5B).Moreover,thePDK1levelswereinverselycorrelatedwithmiR‐379expression(r2=−0.347;P0.001;Fig.5C).PDK1expressionwasalsoincreasedinthefourosteosarcomacelllines(MG‐63,U2OS,SOSP‐9607,andSAOS‐2)ascomparedwithhFOB(Fig.5D).
Figure5OpeninfigureviewerPowerPointPDK1expressionwasup‐regulatedinosteosarcomaandwasinverselycorrelatedwithmiR‐379expression.(A)TheexpressionlevelsofPDK1weremeasuredin30osteosarcomasamplesbyqRT‐qPCR.(B)PDK1expressionwasup‐regulatedinosteosarcomatissuescomparedwithcancer‐adjacenttissues.(C)PDK1levelswereinverselycorrelatedwithmiR‐379expression(r2=−0.347;P0.001).(D)PDK1expressionwasmeasuredin4osteosarcomacelllines(MG‐63,U2OS,SOSP‐9607,andSAOS‐2)andanon‐cancerousosteoblasticcellline(hFOB)usingqRT‐PCR.
ThePDK1expressionvectorwasconstructedtorestorePDK1expression.TheproteinlevelofPDK1wasup‐regulatedinMG‐63cellsupontransfectionwithPDK1vector(Fig.6A).EnforcedexpressionofPDK1persepromotedMG‐63andU2OScellproliferation(Fig.6BandC).Importantly,enforcedexpressionofPDK1rescuedmiR‐379‐mediatedinhibitionofcellproliferationinbothcelllines(Fig.6DandE).EctopicexpressionofPDK1promotedtheU2OScellinvasion(Fig.6F).
Figure6OpeninfigureviewerPowerPointPDK1downregulationwasinvolvedinthetumour‐suppressingfunctionofmiR‐379.(A)TheproteinexpressionofPDK1wasmeasuredbyWesternblots.(BandC)EnforcedexpressionofPDK1promoted(B)MG‐63and(C)U2OScellproliferation.(DandE)EnforcedexpressionofPDK1rescuedthemiR‐379‐mediatedinhibitionofcellproliferationin(D)MG‐63and(E)U2OScells.(F)EctopicexpressionofPDK1promotedtheU2OScellinvasion.Therelativeinvasioncellwasshown.*P0.05;**P0.01and***P0.001.
MG‐63cellswereinoculatedsubcutaneouslyinposteriorflanksofnudemicetostudythetherapeuticeffectofmiR‐379invivo.Whentumoursreached50mm3,miR‐379mimicsorscrambledcontrolwasinjecteddirectlyintothetumours.After4weeks,wefoundthatinjectionwithmiR‐379mimicsincreasedtheintratumourallevelsofmiR‐379(Fig.7A)andrepressedthegrowthofMG‐63xenograftscomparedwithscrambledoligonucleotides‐injectedtumours(Fig.7B).Consistentwiththetumourgrowthcurve,theweightoftumoursinjectedwithmiR‐379wassignificantlylowerthanscrambledcontrol‐injectedtumours(Fig.7C).TheproteinexpressionofPDK1wasalsolowerinmiR‐379‐injectedtumours(Fig.7D).Moreover,themRNAexpressionofKi‐67wasalsoreducedbymiR‐379injection(Fig.7E),accompaniedbylessKi‐67‐positivecells(Fig.7F).
Figure7OpeninfigureviewerPowerPointmiR‐379repressedthegrowthofMG‐63xenograftsinnudemice.(A)InjectionwithmiR‐379mimicsincreasedthelevelsofmiR‐379inMG‐63xenograftsasdeterminedbyquantitativeRT‐PCR.(B)Graphrepresentingtumourvolumesattheindicateddaysduringtheexperimentforthetwogroups:scrambledcontrolandmiR‐379mimics(n=5pergroup).(C)AveragetumourweightofscrambledcontrolandmiR‐379mimics‐injectedmiceattheendoftheexperiment(25dayspost‐miRNAinjection).(D)PDK1proteinexpressionwasmeasuredbyWesternblots.(E)ThemRNAexpressionofKi‐67wasmeasuredbyqRT‐PCR.(F)Ki‐67‐positivecellswerecountedinthetwogroups:scrambledcontrolandmiR‐379mimics.*P0.05and***P0.001.
Inthisstudy,weinvestigatedtheexpressionandfunctionofmiR‐379inosteosarcoma.WefoundthatthemiR‐379expressionwasdownregulatedinosteosarcomatissuesandcelllines.FurtherinvestigationdemonstratedthatmiR‐379repressedcellproliferationandinvasionintwoosteosarcomacellslines(MG‐63andU2OS)andinhibitedinvivotumourigenicity.Mechanistically,ourdataindicatedthatPDK1wasthedirecttargetofmiR‐379inosteosarcoma,inwhichPDK1wasup‐regulatedandshowedinversecorrelationwithmiR‐379.Inaddition,wedemonstratedthatenforcedexpressionofPDK1promotedosteosarcomacellproliferationandPDK1downregulationwasinvolvedinthetumour‐suppressingfunctionofmiR‐379.ThesedatasuggestthatmiR‐379repressedthedevelopmentofosteosarcoma.
Inthepresentstudy,weidentifiedPDK1asthedirecttargetofmiR‐379inosteosarcoma.WefirstusedtheTargetScantoidentifyPDK1followedbyvalidatingthephysicalinteractionofits3′UTRwithmiR‐379usingtheluciferaseassay.Mutationofthepredictedbindingsequenceabrogatedsuchinteractions.ThedownregulationofPDK1atmRNAandproteinlevelsuponmiR‐379transfectionwasalsoconfirmedinMG‐63andU2OS.Importantly,theinversecorrelationbetweenPDK1andmiR‐379inclinicalsampleswasdemonstrated.PDK1isacriticalcomponentoftheoncogenicphosphoinositide3‐kinasesignallinganditsoverexpressionhasbeendocumentedinbreastcancer37,acutemyeloidleukaemia38andmultiplemyeloma39.Activationoflargenumberofproteins,includingAkt,someproteinkinaseCisoforms,S6KandSGK,byPDK1hasalsobeenreported.Inbreastcancer,targetingPDK1inhibitsmigrationandexperimentalmetastasis40.Herein,wedemonstratedforthefirsttimetheup‐regulationofPDK1anditsmitogenicactioninosteosarcoma.OurdataalsosuggestedthatderepressionofPDK1bymiR‐379downregulationmaybeanimportantoncogeniccascadeinosteosarcoma.
OneofthemajorlimitationsofthepresentstudyistheuseoftransienttransfectionofmiR‐379insteadofstableexpressioninosteosarcomacelllines.Furthermore,knockdownmiR‐379inothernon‐cancerousosteoblasticcelllinesshouldfurthersubstantiateitstumour‐suppressingroleinosteosarcoma.Inconclusion,wefoundthattheexpressionofmiR‐379wasdownregulatedinosteosarcomatissuesandcelllines,andoverexpressionofmiR‐379suppressedtheosteosarcomacellproliferationandinvasionandtumourgrowth.ThefunctionofmiR‐379wasmediatedbythedownregulationofPDK1.ThesedatasuggestedthatmiR‐379downregulationmayplaycrucialrolesinosteosarcomadevelopmentandthatmiR‐379maybeapotentialtherapeutictargetforthetreatmentofosteosarcoma.
ThisworkwassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina(NSFC)(grantnumbers:81401847,81272053and81330044).
Pleasenote:Thepublisherisnotresponsibleforthecontentorfunctionalityofanysupportinginformationsuppliedbytheauthors.Anyqueries(otherthanmissingcontent)shouldbedirectedtothecorrespondingauthorforthearticle.
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