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Carcinogenesisvol.32no.5pp.713–722,2011doi:10.1093/carcin/bgr035

AdvanceAccesspublicationFebruary24,2011

Low-levelexpressionoflet-7aingastriccanceranditsinvolvementintumorigenesisbytargetingRAB40C

QiaoyuanYang1,2,y,ZhigangJie3,y,HongCao4,AnneR.Greenlee5,ChengfengYang6,FeiZou1andYiguoJiang1,2,?SchoolofPublicHealthandTropicalMedicine,SouthernMedical

University,18318NorthGuangzhouAvenue,Guangzhou510515,People’sRepublicofChina,2InstituteforChemicalCarcinogenesis,StateKeyLaboratoryofRespiratoryDisease,GuangzhouMedicalUniversity,195DongfengxiRoad,Guangzhou510182,People’sRepublicofChina,3DepartmentofGeneralSurgery,FirstAf?liatedHospital,Nanchang

University,Nanchang330006,People’sRepublicofChina,4DepartmentofGeneralSurgery,People’sHospitalofJiangxiProvince,Nanchang330006,People’sRepublicofChina,5TheJacksonLaboratory,BarHarbor,ME04609,USAand6DepartmentofPhysiologyandCenterforIntegrativeToxicology,MichiganStateUniversity,EastLansing,MI48824,USA

Towhomcorrespondenceshouldbeaddressed.Tel:t862081340186;Fax:t862081340724;

Email:jiangyiguo@yahoo.com

CorrespondencemayalsobeaddressedtoFeiZou.Tel:t862061648301;Fax:t862061648324;Email:zoufei26@163.com

?1Gastriccanceristhefourthmostcommoncancerandthesecondleadingcauseofcancermortalityworldwidebuttheunderlyingmolecularmechanismisnotentirelyclear.Theobjectiveofthisstudywastoexploretheroleoflet-7amicroRNA(miRNA)ingastrictumorigenesisandthepossiblecorrelationbetweenRAB40Candlet-7amiRNAingastriccancer.Wefoundthatex-pressionoflet-7aisreducedinhumangastriccancertissuesandcelllinesandtherewasasigni?cantcorrelationbetweentheleveloflet-7aexpressionandthestageofdifferentiation.Overexpres-sionoflet-7aresultedinadecreaseincellproliferationandG1arrest,signi?cantlysuppressedanchorage-dependentgrowthinvitroandthetumorigenicityofgastriccancercellsinanudemousexenograftmodel.Furthermore,wedemonstratedthatRAB40Cisregulateddirectlybylet-7aandplaysanessentialroleasamediatorofthebiologicaleffectsoflet-7aingastrictumori-genesis.Thisstudyrevealedthatlet-7aissigni?cantinsuppress-inggastriccancergrowthinvivoandinvitroandprovidedthe?rstevidencethatRAB40Cisnegativelyregulatedbylet-7aattheposttranscriptionallevelviabindingtothe3#-untranslatedregionofRAB40CmessengerRNAingastriccancer.Theresultsofthisstudysuggestthatlet-7aandRAB40Carepotentiallyusefultargetsforgastriccancerdiagnosisandtherapy.

Introduction

Gastriccanceristhefourthmostcommoncancerandthesecondleadingcauseofcancermortalityworldwidedespiteadecreasingincidenceinrecentdecades(1).Itremainsanimportantpublichealthburdenworldwide,especiallyindevelopingcountries.InChina,gastriccancerhasthehighestmortalityamongallcancersandtheoverallmortalityratehasincreasedsteadilyinthepast20years(2).However,themolecularmechanismsinvolvedingastriccancerarediverse,complexandnotfullyunderstood.

NewopportunitiesinthestudyofcancermolecularmechanismshavebeenprovidedbythediscoveryofmicroRNAs(miRNAs),

Abbreviations:CCK-8,cellcountingkit-8;DMEM,Dulbecco’smodi?edEagle’smedium;inhibitorNC,inhibitornon-speci?ccontrolmiRNA;mimicNC,mimicnon-speci?ccontrolmiRNA;mRNA,messengerRNA;miRNA,microRNA;PCR,polymerasechainreaction;RT,reversetranscription;siRNA,smallinterferingRNA;3#-UTR,3#-untranslatedregion.

yTheseauthorscontributedequallytothiswork.

aclassofshortnon-codingendogenousRNAsthatfunctionasnega-tiveregulatorsofgeneexpression(3).Asthemajorendogenoustrig-gersforposttranscriptionalsilencing,miRNAscannegativelyregulatetheexpressionofaprotein-codinggenebybindingwiththe3#-untranslatedregions(3#-UTRs)oftheirmessengerRNA(mRNA)targetsandthenrepressingexpressionofthetargetgenethroughmRNAdegradationortranslationalinhibition(4,5).miRNAsarepredictedtotargetmorethanone-thirdofhumangenesandeachmiRNAcancontrolhundredsoftargetgenes(6).Moreover,miRNAshavebeendemonstratedtobeevolutionarilyconservedandtoperformregulatoryfunctionsinnumerousbiologicalprocesses,includingdevelopmentaltiming,cellproliferation,apoptosis,metabolism,celldifferentiationandmorphogenesis(7–9).

RecentlyacquiredevidencedemonstratesthatmiRNAscanberegulatorsincarcinogenesis.Calinetal.(10)showedthat.50%oftheknownmaturehumanmiRNAgenesarelocatedincancer-associatedgenomicregionsorinfragilesites,suggestingthatmiRNAsmighthaveanimportantroleinthepathogenesisofhumancancers.Moreover,differentcancertypeshavedistinctmiRNAex-pressionpro?les,andanincreasingnumberofmiRNAshavebeensuggestedtohaveimportantrolesintumorprogressionorintumorsuppression(11–13).IncreasedexpressionsofsomemiRNAs,suchasmiR-21andmiR-27a,havebeenfoundtoplaycrucialrolesingastrictumors(14,15).Inaddition,themiR-106b-25cluster,whichisupregulatedinhumangastrictumors,isinvolvedintheposttran-scriptionalregulationoftranscriptionfactorE2F1(16)andmiR-15bandmiR-16modulatemulti-drugresistancebytargetingB-celllymphoma/leukmia-2(BCL2)inhumangastriccancercells(17).Incontrast,miR-9,miR-141,miR-143,miR-145,miR-433andmiR-451aredownregulatedingastriccancerandthesemiRNAsactasanti-oncogenicmiRNAswithasigni?cantgrowthinhibitoryeffectongastriccancer(18–21).

Amongallhumancancer-relatedmiRNAs,thelet-7familyhasattractedthemostinterestbecauseitsfamilymembershavebeennotedtoexpressaberrantlyinhumancancers(22,23).ThefamilywasdiscoveredinitiallyinCaenorhabditiselegansandiscurrentlyoneofthemostimportantmembersofthemiRNAfamily.Thelet-7familyconsistsof11verycloselyrelatedgenesandmanyhumanlet-7genesmaptoregionsthatarealteredordeletedinhumantumors,indicatingthatthesegenesmightfunctionastumorsuppressors(22).Moreover,whenoverexpressedincoloncancercells,let-7miRNAleadstogrowthproliferationassociatedwithareducedlevelofRASprotein(24).let-7aisdownregulatedinBurkitt’slymphomaandithasbeenshowntobeananticancermiRNAthatrepressedC-MYCexpressionatthetranslationallevel(25).Recently,theimplicationoflet-7incarcinogenesishasbeenextendedtotherepressionofhigh-mobilitygroupA2,thuspreventingoncogenictransformationinmanytumors(26,27).These?ndingssuggestthatlet-7miRNAsparticipateactivelyintumorigenicprocessesandthetargetsin-volvedintheregulationoflet-7miRNAshavebeenassociatedwithvarioustumorigenicprocessesinadditiontothemiRNAsthem-selves.However,thedatafortherelationshipbetweengastriccarcinogenesisandtheexpressionoflet-7amiRNAareverylimited.Evidencecollectedtodateshowslet-7awaslinkedtothemodula-tionofdifferenttargetgenes,themostwell-knownbeingtheRASfamily.TheRASproteinsfunctionasthecriticalmolecularswitchforvarioussignalingpathwayscontrollingthediversebiologicalprocesses.RAB40CisamemberoftheRASfamily,whichplaysimportantrolesintumorigenesis.Withthehelpofabioinformaticanalysis,wefoundRAB40Ccontainedthelet-7abindingsiteandwasevolutionarilyconservedacross10species.Toourknowledge,thereisnoreportofworkinvestigatingtheroleoflet-7aorapossiblecorrelationbetweenRAB40Candlet-7amiRNAingastriccancers.

óTheAuthor2011.PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleaseemail:journals.permissions@oup.com

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Q.Yangetal.

Inthisstudy,weusedthequantitativereal-timepolymerasechainreaction(PCR)toexaminetheexpressionoflet-7amaturemiRNAin27matchedpairsofnormalandgastriccancertissuesfrompatients.Incomparisonwithnormaltissues,wefoundthattheexpressionoflet-7awassigni?cantlyloweringastrictumorspecimens.Importantly,in-creasedexpressionoflet-7asuppressedcellgrowthinvitroandtumorgrowthinvivoandwasassociatedwithdecreasedratesofcellpro-liferationandthecellcycle.Thisisthe?rstreportthatlet-7aisinvolvedintumorigenesisofgastriccancerbothinvitroandinvivo.Further-more,itiscon?rmedforthe?rsttimethatlet-7atargetsRAB40Cdi-rectly.BytargetingRAB40C,let-7asuppressesproliferationandanchorage-independentgrowthofhumangastriccancercells.Our?nd-ingssuggestthatlet-7aandRAB40Cmightbevaluabletoolsforde-velopinginterventionsaimedattreatinganddiagnosinggastriccancer.Materialsandmethods

Patientsandtissuesamples

In2008–09,27pairsofgastriccancertissuesandmatchednormalgastrictissueswereobtainedwithinformedconsentfrompatientsintheFirstAf?liatedHospitalofNanchangUniversity(Nanchang,China).Alltissuesampleswerecon?rmedhistopathologicallyandsnap-frozeninliquidnitrogen.Thenon-cancerousgastrictissuesweretaken3cmawayfromthetumor.Clinicopatho-logicinformationwasavailableforallsamplesandthestudywasapprovedbytheMedicalEthicsCommitteeofNanchangUniversityandtheMedicalEthicsCommitteeofGuangzhouMedicalUniversity.

Celllinesandreagents

ThehumangastricepithelialcelllineGES-1andhumangastriccancercelllinesSGC-7901,BGC-823andHGC-27werepurchasedfromtheCellResourceCenterofXiangYaCentralLaboratory(Changsha,China).HumangastriccancercelllineMKN-28wasprovidedbytheLaboratoryAnimalCenteroftheFourthMilitaryMedicalUniversity(Xian,China).HumangastriccancercelllineAGSwasdonatedbytheSunYat-SenUniversityCancerCenter(Guangzhou,China).Allcells,exceptAGScells,weregrowninDulbecco’smodi?edEagle’smedium(DMEM)(Gibco,Carlsbad,CA)supplementedwith10%(vol/vol)fetalbovineserum,50U/mlpenicillinand50lg/mlstreptomycin.TheAGScellsweregrowninF12kmedium(Sigma,Louis,MO)supplementedwith10ttalbovineserum,with50U/mlpenicillinand50lg/mlstreptomycin.Allcelllineswereincubatedat37°Cinahumidi?ed5%(vol/vol)CO2atmosphere.RNAextractionandquantitativereal-time–PCR-baseddetectionoflet-7aandRAB40CmRNA

TotalRNAfromtissuesamplesandcelllineswasobtainedwiththeTRIzolòisolationreagent(Invitrogen,Carlsbad,CA)followingthemanufacturer’sin-structions.About100mgoftissuewashomogenizedin1mlofTRIzolreagentandthenenteredintothesamestepoftheanalysisascellsamples.Theconcentration,purityandamountoftotalRNAweredeterminedbyultravioletspectrometry(ND-1000spectrophotometer;NanoDropTechnologies,Wilmington,ThereverseDE).

transcription(RT)–PCRwasusedtodetecttheexpressionoflet-7aandtheRAB40Cgeneatthetranscriptlevelasdescribed(28).Brie?y,thismethodusestwo-stepRT–PCR.IntheRTstep,complementaryDNAwasre-versetranscribedfromtotalRNAsamplesusingtheReverTraAceqPCRRTkit(TOYOBO,Tokyo,Japan).Thequantitativereal-time–PCRfordetectingtheexpressionoflet-7ausingtheTaqManMicroRNAassay(AppliedBiosystems,FosterCity,CA)togetherwiththeTaqManUniversalPCRMasterMix(AppliedBiosystems)weredonewithanAppliedBiosystems7500real-timePCRsystem(AppliedBiosystems).Therelativequanti?cationoflet-7awascalculatedusingthe2àDDCtmethodnormalizedwithrespecttoRNU6Bastheinternalcontrolandrelativetoacalibratorsampleastheexternalcontrol.TheSYBRPremixExTaqTMKit(TaKaRa,Dalian,China)wasusedfordetectingtheexpressionofRAB40CmRNAfollowingthemanufacturer’sinstructions.Thedatawerealsocalculatedusingthe2–DDCtmethodnormalizedtotheindividualb-actinlevel.AllprimersweresynthesizedbyTaKaRa.Theprimerswereb-actinforward:CCCAGATCATGTTTGAGACCTandreverse:GAGTCCATCACGATGC-CAGTandRAB40Cforward:TCATCGACAAGCTTCCACTGandreverse:TTGGACCTCTTGAGGCTGTT.

miRNAandsmallinterferingRNAtransfections

Thelet-7amimicwasanRNAduplexwiththesequence:5#-UGAGGUA-GUAGGUUGUAUAGUU-3#and5#-CUAUACAACCUACUACCUCAUU-3#.Themimicnon-speci?ccontrolmiRNAsduplex(namedmimicNC)withasequenceof:5#-UUCUCCGAACGUGUCACGUTT-3#and5#-ACGUGA-CACGUUCGGAGAATT-3#wasnothomologoustoanyhumangenomesequence.Fortheinvivotumorigenicityassay,allpyrimidinenucleotidesin

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thelet-7amimicormimicNCweresubstitutedbytheir2#-O-methylanalogstoimproveRNAstability.Theanti-let-7a:5#-AACUAUACAACCUACUAC-CUCA-3#wasa2#-O-methyl-modi?edoligoribonucleotidedesignedaslet-7ainhibitor.Theinhibitornon-speci?ccontrolmiRNAs(namedinhibitorNC),withthesequence:5#-CAGUACUUUUGUGUAGUACAA-3#wasusedasanegativecontrolforanti-let-7aintheantagonismexperiment.AlltheRNAoligoribonucleotideswerepurchasedfromGenePharma(Shanghai,China).Ablankcontroltreatedwithonlythetransfectionreagentwasusedineverytransfectionexperiment.SmallinterferingRNAs(siRNAs)forhumanRAB40Csequencesense:5#-GGGACAUUGACCACUCAAATT-3#andantisense:5#-UUUGAGUGGUCAAUGUCCCTT-3#andscrambledsiRNAwerede-signedandsynthesizedbyGenePharma.

Cellswereseededontosix-wellplates(3?105cellsperwell)thedaybeforetransfectionswereperformed.Cells($60%con?uent)weretransfectedwithlet-7amimic(50nmol/L),mimicNC(50nmol/L),inhibitor(100nmol/L)orinhibitorNC(100nmol/L)usingLipofectamineTM2000(Invitrogen)andtransfectionef?ciency(.90%)wascon?rmedwiththeuseoftheSilencer5-carboxy?uorescein-labeledNegativeControl(GenePharma).ForthemiRNAandsiRNAcombinationexperiments,cellsweretransfectedwithlet-7amimic(50nmol/L)for24h.ThesecellswerethencotreatedwithRAB40CsiRNA(100nmol/L)orsiRNANC(100nmol/L)foranother24h.TotalRNAandproteinwereprepared1or2daysaftertransfectionandwereusedforRT–PCRorwesternblotanalysistovalidatetheknockdownef?ciency.

Cellproliferationassay

Thecellproliferationassaywasdonewithacellcountingkit-8(CCK-8;Dojindo,Tokyo,Japan)at24haftertransfection.Brie?y,5000transfectedGES-1,AGSorBGC-823cellswereplatedperwellin96-wellplatesat24haftertransfectionandculturedin100llofcellculturemediumperwellfor24hinnormalconditions.Afterincubationfor24h,20llofCCK-8reagentwasaddedtoeachwellandincubatedat37°Cfor1h.Theabsorbanceat450nm(A450)andat650nm(A650)weremeasuredwithaSynergy2micro-platereader(BioTek,Winooski,VT).The?nalabsorbancewascalculatedasA650àA450,andcellviabilitywasnormalizedas:

eFinalabsorbancetreated=finalabsorbancecontrolT?100%:

Colonyformationassay

TransfectedGES-1,AGSandBGC-823cellsweretrypsinized,countedandseededatadensityof1000cellsper60mmculturedishinnormalculturemediumforcolonyformationassayandincubatedfor10daysat37°Cinahumidi?ed5%CO2atmosphere.Duringcolonygrowth,theculturemediumwasreplacedevery3daysandacolonywascountedonlyifitcontained.50cells.Colonyformationandgrowthwerevisualizedbystainingwithcrystalvioletandthecolonyformationratewascalculatedas:

eNumberofcolonies=numberofseededcellsT?100%:

Cellcycleanalysis

Cellswereharvestedat24haftertransfectionand?xedin70%ice-coldethanol,treatedwithRNAseA,stainedwith50mg/mlpropidiumiodideand0.1mg/mlRNaseAforDNAcontentanalysisby?owcytometrywithaFACSCalibursystem(BectonDickinson,Franklin,NJ).Thepercentageofcellpop-ulationineachphasewascalculatedwithFlowJoFACSanalysissoftware(TreeStar,Ashland,OR).

Softagarassay

Softagarplateswerepreparedintriplicatein60mmdisheswithabottomlayerof0.6%(wt/vol)Nobleagar(Sigma)inserum-freeDMEM.Thereafter,trans-fectedcellsweretrypsinizedand1000cellsperdishwereseededontothebottomagarlayeraftermixingwith0.3%NobleagarinDMEMsupplementedwith10ttalbovineserumandallowedtoharden.Toassesscellviabilitybeforeplatinginsoftagar,thenumberofcellswasdeterminedbystainingwithtrypanblue.Thedisheswereincubatedfor3weeksat37°Cinahumidi?ed5%CO2atmosphereandscoredforclones.Theresultisexpressedasthenumberofcoloniescontainingatleast50cellsperwell.

Tumorigenicityassaysinnudemice

Five-week-oldBalb/cnudemicewereprovidedbyGuangzhouUniversityofTraditionalChineseMedicine(Guangzhou,China).Allexperimentalproce-duresinvolvinganimalswereinaccordancewiththeGuidefortheCareandUseofLaboratoryAnimalsandwereinaccordwiththeinstitutionalethicalguidelinesforexperimentswithanimals.TransfectedandcontrolBGC-823cellsweretrypsinized,collectedbycentrifugationandsuspendedinDMEM.

A0.2mlsampleofculturemediumcontaining5?106cellswasinjectedsubcutaneouslyintotheright-handsideoftheposterior?ankofeachmouse.Themicewerehousedinapathogen-freeenvironmentandmonitoredevery5daysfortumorgrowth.Themicewerekilledafter40daysandtheweightandvolumeofeachtumorweredetermined.Thetumorxenograftswereexcisedandweighed,thenusedfortheextractionoftotalRNAandimmunohistochem-icalassays.Thetumorlatencytimewasdeterminedasthetimetoappearanceofpalpabletumorandthevolume(V)wascalculatedasdescribed(29).Immunohistochemistry

Ki-67proteinexpressioninthecancertissuesofnudemicewasdetectedusingthestreptavidin–peroxidasecomplexmethodwithaHistostain-pluskit(ZhongshanGoldenBridgeBiotechnology,Beijing,China).Rabbitanti-humanKi-67antibodies(Boster,Wuhan,China)wereusedatadilutionof1:400asprimaryantibodies.Colordevelopmentwasachievedwith3#,3#-diaminobenzidine,whichstainedpositivecellsbrown.Normalrabbitserumandsecondaryantibodyalonewereusedasnegativecontrols.

Westernblotting

Totalproteinswerepreparedbystandardproceduresandquanti?edbytheBCAmethod(Jiancheng,Nanjing,China).A20lgsampleofproteinwasmixedwith5?sodiumdodecylsulfate/polyacrylamidegelelectrophoresissamplebuffer(Weijia,Guangzhou,China)andboiledfor5minbeforesodiumdodecylsulfate–polyacrylamidegelelectrophoresis(15%polyacrylamidegel)andtransfertoapolyvinylidenedi?uoridemembrane(MilliporeCorp.,Bedford,MA).Themembranewasblockedwith5%non-fatdrymilkinphosphate-bufferedsalinefor45minat37°Cwithagitation.RAB40Cproteinandglyceraldehyde-3-phosphatedehydrogenaseonthesamemembranewerequanti?edbydividingthemembraneintotwopiecesaccordingtothemolec-ularmassofprestainedproteinstandards(Weijia).Thepieceofthemembranewiththegreatermolecularmassproteinswasincubatedwithaprimaryanti-bodyforratanti-humanRAB40C(Epitomics,Burlingame,CA)ataconcentra-tionof1:1000at37°Cfor1h.Theotherpieceofthemembranewasincubatedwithaprimaryantibodyformouseanti-humanglyceraldehyde-3-phosphatedehydrogenase(Epitomics)ataconcentrationof1:1500.SignalsweredetectedbysecondaryantibodieslabeledwithIRDye800(RocklandImmunochemi-cals,Gilbertsville,PA)andsignalintensitywasdeterminedwithanOdysseyInfraredImagingSystem(Li-CorBiosciences,Lincoln,NE).

Luciferasereporterassay

Thefull-length3#-UTRofRAB40Cgene(GenBankaccessionnumberNM_021168;length1650bp)wasampli?edfromGES-1cellscomplementaryDNAandclonedintotheNotlandXholsitesinapsiCHECK-2vector(Prom-ega,Madison,WI)downstreamofthereportergene.TheprimersforRAB40Cwereforward:5#-AGCTTTGTTTAAACCGGGATGGGCGCGGGGATG-3#andreverse:5#-AGCTTTGTTTAAACTGTGGGGACCACAGCTGAAT-TAC-3#.Tointroducemutationsintolet-7atargetsitesintheRAB40Ccodingregion,primersweredesignedforsite-directedmutagenesisbasedonwildpsiCHECK-2-RAB40C3#-UTRplasmidthatresultedinthedestructionofthelet-7atargetsitewithoutalteringtheaminoacidsequenceofRAB40C.Thesitesweremutatedasfollows:beforemutagenesis,CUACCUC;aftermutagenesis,AUCGAGC.TheprimersforRAB40Cmtwereforward:5#-CA-CAGCACTGGTGATCACCTATCGCTCCTGTCCTCAGGCCGTGCGGC-3#andreverse:5#-GCCGCACGGCCTGAGGACAGGATCGATAGGTGAG-CACCAGTGCTGTG-3#.ThesegmentoftheRAB40C3#-UTRcontainingthemutatedlet-7atargetsequence.wasalsoclonedintothepsiCHECK-2Luciferasevector(Promega).TheresultingwildandmutatedRAB40Cexpres-sionvectorswerecon?rmedbysequencing.TheconstructofreportervectorwasperformedbyLandbiology(Guangzhou,China).

Fortheluciferasereporterassay,GES-1cells(50%con?uentin24-wellplates)werecotransfectedwith0.5lgofpsiCHECK-2vectorconstructswithorwithout20lMlet-7amimicorinhibitorfor48hbyLipofectamine2000(Invitrogen).At48haftertransfection,theactivityofRenillaluciferaseand?re?yluciferasewasmeasuredwiththeDual-luciferaseReporterAssaySystem(Promega).Relativeluciferaseactivitywasnormalizedwith?re?yluciferaseactivityandthencomparedwiththepsiCHECK-2vectorcontrol.Statisticalanalysis

AllstatisticalanalysiswasdonewithSPSS13.0software.Valuesareexpressedasmean±SD.DifferencesbetweengroupswereanalyzedbyStudent’st-testorthenon-parametricMann–WhitneyU-testforcomparisonoftwogroupsandone-wayanalysisofvarianceorthenon-parametricKruskal–WallisHtestformultiplecomparisons.Nonlinearregressionanalysisbetweengroupswasusedwithlogarithmicregressionmodelbymethodofcurveestimation.Allexperi-mentsweredoneatleastintriplicateandthelevelofstatisticalsigni?cancewassetatP,0.05foralltests.

let-7aingastriccancer

Results

Expressionoflet-7aisreducedinhumangastriccancertissuesandcelllines

Theclinicopathologicdatafor27patientsaregiveninTableI.BasedontheresultsofTaqManreal-timePCR,wefoundtheexpressionoflet-7ain21cancertissueswaslowerthanthatinthematchednormaltissues(Figure1A).Theexpressionoflet-7awassigni?cantlyloweringastriccancertissuesthanthatinnormaltissues,withamedianchangeof0.38-fold(Figure1B).Next,weexaminedthecorrelationofthetumortissue(T)/normaltissue(N)ratiosforlet-7aexpressionwiththeclinicopath-ologicfactorsgiveninTable1.TheT/Nratiosinpatientswithpoordifferentiationweresigni?cantlylowerthanthosewithmoderateorgooddifferentiation(TableI;Figure1C).However,therewasnosig-ni?cantdifferencebetweenthegroupsdividedbyanyotherclinico-pathologicfeature,includinggender,tumorsize,histologiccelltype,lymphnodemetastasisorvenousinvasion.Tocon?rmtheassociationbetweentheexpressionoflet-7aandgastriccancer,?vecelllinesde-rivedfromgastriccancerswithvariousdegreesofdifferentiationwereselectedtodetectlet-7aexpression:MKN-28(welldifferentiated),AGS(welldifferentiated),SGC-7901(moderatelydifferentiated),BGC-823(poorlydifferentiatedorundifferentiated)andHGC-27(un-differentiated).Thedatashowedthattheexpressionoflet-7awassig-ni?cantlydownregulatedinMKN-28(0.63-fold),AGS(0.33-fold),SGC-7901(0.28-fold),BGC-823(0.23-fold)andHGC-27(0.13-fold)cellscomparedwithnormalhumangastricepithelialcelllineGES-1(Figure1D).

let-7aregulatescellproliferationandthecellcycle

Weevaluatedthetransfectionef?ciencybydeterminingthepercentageofcellscontainingthe5-carboxy?uorescein-labelednon-speci?cmiRNAcontrolandtheresultsindicatedsuccessfultransfectionof5-carboxy?uorescein-labelednon-speci?cmiRNAcontrolinto.90%ofcellsofallthecelllinesusedinthisstudy.TaqManreal-timePCRrevealedthetransfectedlet-7amimicsigni?cantlyincreasedthelevelsoflet-7a(Figure2A),whereasthetransfectedlet-7ainhibitoreffec-tivelyinhibitedtheexpressionoflet-7a(Figure2B)comparedwithmimicNCorinhibitorNCcellsorwithuntransfectedcellsinallsixcelllines,whichdemonstratedthatthetransfectedlet-7amimicandinhibitorwerefunctional.

TableI.TheClinicopathologicfactorsof27gastriccancerpatientsandcomparisonoflet-7aexpressionindifferentgroupsClinicopathologicfactor

No.ofRelativeP

patients

let-7aexpressionMin–max(median)Age(years).60190.04–8.97(0.47)0.313 6080.08–2.00(0.25)GenderMale170.04–8.97(0.47)0.335Female

100.04–2.63(0.25)Tumorsize(cm).370.10–2.72(0.82)0.580 3

200.04–8.97(0.31)HistologiccelltypeWelldifferentiated

60.10–8.97(2.06)0.018

Moderatelydifferentiated80.16–2.63(0.45)Poorlydifferentiated130.04–2.00(0.19)LymphnodemetastasisAbsent120.04–2.72(0.51)0.727Present

150.04–8.97(0.35)VenousinvasionAbsent90.04–8.97(0.20)0.862

Present

18

0.04–2.72(0.49)

715

Q.Yangetal.

Fig.1.Levelofexpressionofmaturelet-7aingastriccancertissue

specimensandcelllines.Maturelet-7aexpressionwasanalyzedbyTaqManreal-timePCRandnormalizedtoU6Bexpression.(A)Thecomparisonoflet-7aexpressionbetweenmatchednormalgastricandgastriccancertissuesin27patients.T,gastriccancertumortissue;N,pair-matchedadjacentnon-tumortissue.(B)Thedataforlet-7aexpressionwereanalyzedusingtheMann–WhitneyU-test.(C)Theratiosoftumortonormaltissueforlet-7aexpressionarepresentedastherelativetumortissue/normaltissueratio(T/Nratio)oflet-7aexpression.TheT/Nratioswereanalyzedstatisticallyinpatientsofdifferentpathologicalgrade(Kruskal–WallisHtest,P50.018).Horizontallinesshowthemedianvalueofeachgroup.W,welldifferentiated;M,moderatelydifferentiated;P,poorlydifferentiated.(D)Theexpressionoflet-7ain?vegastriccancercelllinesrelativetothenormalgastriccelllineGES-1(Kruskal–WallisHtest,P50.016).N,normalgastricepithelialcellline;W,welldifferentiatedcancercellline;M,moderatelydifferentiatedcancercellline;P,poorlydifferentiatedcancercellline;U,undifferentiatedcancercellline.Thedataareshownasmedianwithinterquartilerangeforthreeindependentexperiments.

WeusedtheCCK-8assaytomeasurecellproliferationinvitro.TheAGSandBGC-823celllines,whichhadthehighestleveloflet-7aexpressionandthesmalleststandarddeviation(Figure2A),wereusedinanoverexpressionstudy.AGSandBGC-823cellstransfectedwiththelet-7amimicshowedasigni?cantreductioninthenumberofmetabolicallyactivecellscomparedwiththosetransfectedwithmimicNCortheuntransfectedcontrol(Figure2C).TheGES-1cellline,whichhadthelowestleveloflet-7aexpression(Figure2B),wasusedforaninhibitionstudy.Anincreaseof58.8%incellgrowthwasobservedinGES-1cellsaftertransfectionwithalet-7ainhibitorcomparedwithinhibitorNC(Figure2C).

AsshowninFigure2D,DNAcontentanalysisby?owcytometryrevealedlet-7arestorationinducedanaccumulationofAGScellsinthepercentageofcellsattheG1phasefrom55.38to64.87%andareductionofcellsinSphasefrom37.94to26.55%comparedwiththemimicNCgroupandthiseffectoncellcyclewasobservedalsoforBGC-823cells(Figure2E).Incontrast,thepercentageofGES-1cellstransfectedwithlet-7ainhibitorintheG1phasewasdecreasedfrom

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75.01to60.35%,whereasthepercentageofcellsintheSphasewasincreasedfrom20.61to35.76%comparedwiththeinhibitorNCgroup(Figure2F).These?ndings,togetherwiththecellproliferationresults(Figure1C),illustratedthatlet-7acouldbeatumorsuppressorgeneingastriccancer.

let-7asuppressesanchorage-dependentgrowthinvitroandtumorgrowthinvivo

Thesigni?cantreductionoflet-7aoncellgrowthinvitropromptedustoexplorethepossiblebiologicalsigni?canceoflet-7aintumorigen-esis.AGSandBGC-823cellswereusedintheseassays.Totesthowlongthepromotionoflet-7abythelet-7amimiccanbesustained,wemeasuredthelet-7alevelsafter1,2,3,4,7,10,14and21daysfromtransfectionbyTaqManreal-timePCR.Wefoundthatthepromotioneffectlasteduptoatleast10daysinlet-7amimic-transfectedAGScells(Figure3A)and14daysinlet-7amimic-transfectedBGC-823cells(Figure3B)comparedwithmimicNC-transfectedcells.Thecol-onynumbersofAGSandBGC-823cellsthatformedinsoftagarmediumweredecreasedat3weeksaftertransfectionwiththelet-7amimic(Figure3CandD).Furthermore,AGSandBGC-823cellstrans-fectedwiththelet-7amimicdisplayedfewerandsmallercoloniescomparedwiththemimicNC-transfectedanduntransfectedcells(Fig-ure3E).Takentogether,theseresultssuggestthattheinitialpromotionoflet-7amightbesuf?cienttoinhibittumorgrowthandpromptedustoinvestigatetheroleoflet-7aontumorgrowthinvivo.

PoorlydifferentiatedBGC-823cellswereincludedinthissteptofurthercon?rmthepromotioneffectontumorgrowthinvivoinducedbytransfectionofthelet-7amimic.Theemergingrateoftumorandtumorlatencytimeshowedasigni?cantdifferencebetweenthemiceinjectedwithlet-7amimic-transfectedcellsandthemimicNCgroup(Figure4A).Itisofconsiderableinterestthattumorsderivedfromcellstransfectedwiththelet-7amimicgrewsubstantiallymoreslowlythanthemimicNCgroupthroughouttumorgrowth(Figure4B).Theaverageweightoftumorsderivedfromcellstransfectedwiththelet-7amimicwasonly63.2%ofthatderivedfromthecellstransfectedwiththemimicNC(Figure4CandD).Therewasnosigni?cantdifferenceoftumorvolume,tumoraverageweightorotherindicesbetweenthemimicNC-transfectedcellgroupandtheuntransfectedcellgroup.Additionally,theexpressionoftheproliferationmarkerKi-67wassigni?cantlylowerintumorxenograftsofthelet-7amimicgroupcomparedwiththemimicNCanduntransfectedgroups(Figure4E).Theseresultsindicatethatthereducedtumorgrowthisprobablytobeduetodecreasedproliferationinducedbylet-7a.

RAB40Cisadirecttargetoflet-7a

Abioinformaticanalysisidenti?edRAB40Casahypotheticaltargetgeneoflet-7a,asidenti?edbyTargetScanalghorithms(http://www.targetscan.org/).Thelet-7atargetingsitesinRAB40C3#-UTRareconservedamongmammalsandthecorrespondingsequencesoftheRAB40Cmutated3#-UTRsareshowninsupplementaryFigureS1,availableatCarcinogenesisOnline.TheputativesecondaryRNAhy-brid(http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/)forhumanlet-7aandRAB40CorN-RasmRNAwithminimalfreeenergyisshowninsupplementaryFiguresS2andS3,availableatCarcinogenesisOnline.Ouranalysisindicatesthatlet-7ahasamorestablesecondarystructurewithlowerfreeenergywithRAB40CcomparedwiththevalidatedtargetN-Ras.

Next,wefoundoverexpressionoflet-7ainAGSandBGC-823cellseffectivelydecreasedtheleveloftheRAB40Cprotein.Ablockingstrategywasfurtheradaptedbyintroducingthelet-7ainhibitorintoGES-1cells,whichincreasedtheleveloftheRAB40Cprotein(Figure5A).Inbothcases,neitherthelet-7amimicnortheinhibitoraffectedthemRNAofRAB40C(supplementaryFigureS4isavailableatCarcinogenesisOnline),suggestingposttranscriptionalregulationofRAB40Cbylet-7aingastriccancercelllines.Afurtherhintaboutthepotentialroleoflet-7aintheregulationofRAB40Cexpressioncamefromtheanalysisof?vedifferentgastriccancercelllines(MKN-28,AGS,SGC-7901,BGC-823andHGC-27)thatshowed

let-7aingastriccancer

Fig.2.Expressionoflet-7aaftertransfectionwiththelet-7amimicorinhibitorinsixgastriccelllinesandoverexpressionorknockdownoflet-7aingastriccellsaffectedcellgrowthinvitro.TotalRNAwasisolated24haftertransfectionforthedetectionoflet-7aandmaturelet-7aexpressionwasanalyzedbyTaqManreal-timePCRandnormalizedtoU6Bexpression.(A)Expressionoflet-7ainlet-7amimic-transfected,mimicNC-transfectedanduntransfectedgastriccells.(B)Expressionoflet-7ainlet-7ainhibitor-transfected,inhibitorNC-transfectedanduntransfectedgastriccells.(C)Overexpressionorknockdownofsigni?cantlet-7a-affectedcellproliferationinAGS,BGC-823orGES-1cellsasmeasuredbytheCCK-8assay.(DandE)Overexpressionoflet-7aresultedinG1arrestinAGS(D)andBGC-823(E)cells.(F)Knockdownoflet-7aenhancedSphasecellsinGES-1cells.(A–F)Thedataareshownasmean±SDforthreeindependentexperiments,eachdoneintriplicate.?P,0.05,??P,0.01,comparedwiththelet-7amimicNC-orinhibitorNC-transfectedgrouporwithuntransfectedgroup.Pvalueswereobtainedbyone-wayanalysisofvarianceorthenon-parametricKruskal–WallisHtestformultiplecomparisons.

asigni?cantreversednonlinearcorrelationbetweenlet-7alevelsandtheRAB40Cprotein(Figures5BandC).Toassesstheclinicalrele-vanceofthese?ndings,weexaminedthecorrelationbetweentheleveloftheRAB40Cproteinwithlet-7aexpressioninthe27matchednormalandcancertissues(Figure5D).Therewasareversednonlinearcorre-lationbetweentheleveloftheRAB40Cproteinandlet-7aexpression(Figure5E).Theseresultssupportthepremisethatdown-regulationoflet-7aincreasesthelevelofRAB40Cgeneingastriccancer.

Tocon?rmthedirectinteractionbetweenlet-7aanditsbindingsitewithinRAB40CmRNA,ahumanRAB40C3#-UTRfragmentcontain-ingawildtypeormutantlet-7a-bindingsequencewascloneddown-streamoftheluciferasereportergene.ThepsiCHECK-2vectorwascotransfectedinGES-1cellsinassociationwithlet-7amimicorin-hibitor.GES-1cellstransfectedwithpsiCHECK-2vectorshowed$50Tcreaseor30%increaseoftherelativeluciferaseactivitywhencotransfectedwithlet-7amimicorinhibitor(Figure5FandG).Thelet-7aseedsequencewasmutagenizedintheclonedRAB40C3#-UTRregiontogeneratethepsiCHECK-2-RAB40Cmtvector.Nosigni?cantchangeoftherelativeluciferaseactivitywasobservedfollowingthecotransfectionofthismutagenizedvectorwithlet-7amimicorinhibitor(Figure5FandG).These?ndingsshowedadirectinteractionbetweenlet-7aandRAB40CmRNAandindicatedthatlet-7amightsuppressgeneexpressionthroughthelet-7abindingse-quenceatthe3#-UTRofRAB40C.

RAB40Cmediatesthelet-7aeffectoncellproliferationandanchorage-independentgrowth

Tocon?rmthatthelet-7aeffectoncellproliferationandanchorage-independentgrowthisassociatedwithitsmodulationofRAB40C,GES-1cellsweretransfectedwithsiRNAtargetingRAB40CorcontrolsiRNA.TransfectionofAGScellswithsiRNAforRAB40CeffectivelysuppressedRAB40CmRNAexpression(Figure6A)andRAB40Cpro-tein(Figure6B).AsimilareffectwasobservedinBGC-823cells(Figure6AandB).RAB40Cdepletionsigni?cantlyalleviatedtheanti-proliferativeeffectoflet-7aupregulationinAGScellsandBGC-823cellsasdeterminedbytheCCK-8assay(Figure6C).Furthermore,RAB40CsiRNAattenuatedtheplatingef?ciencyofcellsinsoftagar,whichwasdecreaseduponthetransfectionoflet-7amimicinAGScellsandBGC-823cells(Figure6D).ThesedataprovidedfurtherevidencethatRAB40Cwastargetedbylet-7aandthereforesuggestedthatRAB40Cmediatesthelet-7aeffectoncellproliferationandan-chorage-independentgrowthingastriccancercells.Discussion

miRNAsarebecomingincreasinglyrecognizedasregulatorymole-culesinhumancancers,whichhavebeendemonstratedtofunctionasoncogenesortumorsuppressors.let-7ahasbeensuggestedtofunctionasatumorsuppressorandhaveastrongcorrelationwith

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