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Q.Yangetal.

Fig.3.Thechangeoflet-7aexpressionandinhibitionofanchorage-dependentgrowthbyoverexpressionoflet-7a.(AandB)Thechangeoflet-7aexpressionafter1,2,3,4,7,10,14and21daysfromlet-7amimicandlet-7amimicNCtransfectedoruntransfectedAGS(A)orBGC-823(B)cells.(CandD)Inhibitionofanchorage-dependentgrowthbythelet-7amimicinAGS(C)andBGC-823(D)cells.(E)Representativeresultsofcolonyformationofuntransfected,NC-transfectedandmimic-transfectedAGSandBGC-823cells.Thedatain(A–D)areshownasmean±SDfromthreeindependentexperiments.?P,0.05;??P,0.01,comparedwiththelet-7amimicNC-transfectedgroup.Pvalueswereobtainedbyone-wayanalysisofvarianceorthenon-parametricKruskal–WallisHtestformultiplecomparisons.

clinicopathologicfactorsorprognosisincancerpatients(22,23,30).Inthepresentstudy,the75%let-7aexpressionofgastriccarcinomasampleswassigni?cantlylowerthanthatofmatchednormaltissues,suggestingthatreducedlet-7aexpressionisafrequenteventingastriccancer.Furthermore,wefoundthattheexpressionleveloflet-7awasassociatedwiththedifferentiationstageinpatientsandthesameresultswereobservedinthecells.Ouranalyticalresultsshowedthattumortissueofpatientsandcelllineswithlowerlevelsoflet-7atendedtohavepoordifferentiation.Thisobservationwasconsistentwithanearlierreportthatidenti?edthelossoflet-7expressionasamarkerforlessdifferentiatedcancer(31).Theseresultsindicatedthatlet-7aexpressionmightbeanimportantin-dicatorforgastriccancerdiagnosisandclinicalstage.

miRNAshavebeenshowntobeimportantinthedevelopmentandmaintenanceofnormalcellularfunction,andalterationinexpressionofmiRNAscanresultinhumancancerinitiationandtumorprogres-sion.Somestudieshavereportedthatlet-7miRNAwasamasterregulatorofcellproliferationandcellcyclepathwaysinlung,co-lorectalandhepaticcells(22,23,32).However,noinformationisavailableontheimpactofalteredlet-7aexpressionongastriccancercellproliferationpathways.Here,weshowthatoverexpressionoflet-7aresultedin40–50Tcreaseincellproliferationrateandtheeffect

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Fig.4.Suppressionoftumorgrowthbyoverexpressionoflet-7a.(A)Thetumorlatencywasthenumberofdaystotheonsetofpalpabletumorandthevaluesaregivenasmean±SD(n54pergroup).?P,0.05,comparedwithlet-7amimicNC-transfectedoruntransfectedcells-injectedgroup.(B)TumorgrowthcurvesweremeasuredafterinjectionofuntransfectedBGC-823cells,let-7amimicNCandlet-7amimic-transfectedBGC-823cells.ThetumorvolumeV(incubiccentimeter)wascalculatedusingtheformulavolumeV50.5LW2,whereListhelengthofthetumor(incentimeter),Wisthewidthofthetumor(incentimeter)andVisthemeantumorvolume(n54pergroup).(C)Tumorweight;thevaluesaregivenasmean±SD(n54pergroup).?P,0.05,comparedwithlet-7amimicNC-transfectedcells-injectedgroup.(D)Effectoflet-7aontumorigenesisinvivo.Thephotographshowsrepresentativefeaturesoftumorxenografts40daysafterinoculation.(E)RepresentativeimmunohistochemicalassayofKi-67intumorxenograftsofmiceinjectedwithuntransfectedcells,mimicNC-transfectedcellsorlet-7amimic-transfectedcells.Pvalueswereobtainedbyone-wayanalysisofvarianceformultiplecomparisons.

oflet-7awasattenuatedbytheinhibitionoflet-7a.Moreimportantly,decreasedlevelsofproteinKi-67,whichhasbeenusedasamarkerforcellproliferation(33),weredetectedinthexenograftsofthelet-7amimic-transfectedcells.Itfurthercon?rmedthatcellproliferationwasaffectedbylet-7amiRNAingastriccancer.Flowcytometry-basedcellcycleanalysisrevealedatrendtowardanaccumulationofcellsinG1phaseinlet-7amimic-transfectedgastriccancercells.Conversely,let-7ainhibitorcausedasigni?cantincreaseinthefrac-tionofcellsinSphase.However,inthepresentstudy,wedidnotobserveanyapparentincreaseofthesub-G1populationoranyapo-ptosis-relatedmorphologicalchanges,suchasnuclearblebbingorcondensation,underthephasecontrastmicroscope(datanotshown).Thissuggeststhatgrowthsuppressioninducedbylet-7atransfectionwascausedbyinductionofG1arrestratherthanapoptosis.Thisstudycon?rmedtheresultsofthatearlierworkandextendedourknowledgeoftheinhibitoryeffectoflet-7aoncellproliferationandcellcyclecontrolingastriccancercells.Alltheresultsdemonstratedthattheconsistentlyreducedlet-7aexpressioningastriccancershouldbeafactorcontributingtothedevelopmentofthetumorratherthanbeingaffectedasaconsequenceofthedisease.

Gastrictumorxenograftmodelswereestablishedtoinvestigatetheantitumoreffectoflet-7ainvivorelevanttoour?ndingsinvitro.MiceinjectedwithBGC-823cellstransfectedwiththelet-7amimicshowedasigni?cantinhibitionoftumorxenografts,implyingthatoverexpressionoflet-7asuppressedtumorgrowthofgastriccancersigni?cantlyinnudemice.Therefore,therapeuticstrategiestointro-ducelet-7aintocancercellsmightbeusefulforretardingtheprocessoftumorigenesis.Experimentswiththexenograftcarcinomamodelindicatedthatonetransienttransfectionwithlet-7amimicissuf?cienttocausesubstantialinhibitionoftumorgrowth,whichraisesthepos-sibilitythatlet-7amimicmighthavepotentialtherapeuticvalue,con-sistentwiththeearlierstudies(34).

ItisgenerallyacceptedthatmiRNAsexerttheirfunctionthroughregulatingtheexpressionoftheirdownstreamtargetgenes(35).Thus,putativelet-7atargetswerepredictedusingTargetScanprograms.Computationalpredictionssuggestthatlet-7acantarget819

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Fig.5.let-7anegativelyregulatesRAB40CbybindingtotheRAB40C3#-UTR.(A)let-7amimicreducedRAB40CproteininAGSandBGC-823cellsandlet-7ainhibitorincreasedRAB40CproteinlevelsinGES-1cells.(B–C)AnalysisoftheproteinexpressionlevelsofRAB40Candcorrelationwithlet-7aexpressionin?vegastriccancercelllinesandtheGES-1cellline.Proteinexpressionwasdeterminedbywesternblotting(B).Theexpressionoflet-7aisplottedagainstproteinexpression(C).Coef?cientofdeterminationfromlogarithmicregressionmodel(R2)andPvaluesaregiven(R250.813;P50.014).(D–E)AnalysisoftheproteinexpressionlevelsofRAB40Candcorrelationwithlet-7aexpressionin27pairsofgastriccancertissuesandmatchednormalgastrictissues.Proteinexpressionwasdeterminedbywesternblotting(D)andtheexpressionoflet-7aisplottedagainstproteinexpression(E).Coef?cientofdeterminationfromlogarithmicregressionmodel(R2)andPvaluesaregiven(R250.451;P50.020).Thevalueoneachlanein(BandD)indicatestherelativeexpressionlevelofRAB40C,whichisrepresentedbytheintensityratiobetweenRAB40Candglyceraldehyde-3-phosphatedehydrogenasefragmentsineachlane.(F–G)Analysisofluciferaseactivity.let-7amimic(F)orinhibitor(G)inhibitedorenhancedwildtypebutnotmutantRAB40C3#-UTRreporteractivity.??P,0.01,comparedwiththemimicorinhibitorNCgroup.Pvalueswereobtainedbytwo-tailedStudent’st-test.Eachreporterplasmidwastransfectedatleasttwice(ondifferentdays),andeachsamplewasassayedintriplicate.

transcripts,withatotalof905conservedbindingsitesand125poorlyconservedbindingsites.RAB40CisamemberoftheRASfamily,whichplaysimportantrolesintheregulationofimmuneresponses,embryoandcelllineagedevelopment,cellcycleprogression,in?am-mationandoncogenesisandisinvolvedintheexecutionofimportantstepsintumorigenesis(36,37).Moreover,thedysregulationofRASfamilymembersisfrequentlyobservedinhumancancers(38–40).Inthisstudy,RAB40Cwassigni?cantlyoverexpressedingastrictumorsamplescomparedwiththematchednormaltissue.Basedonthebioinformaticanalysis,let-7abindstothe3#-UTRofRAB40CmRNAwithlowerfreeenergythanN-RasmRNA.Itmightstabilizeafavor-ableconformationthatfacilitatesthebindingbetweenlet-7aand3#-UTRsofRAB40C(41,42),whichpromptedustoexplorewhetherRAB40Cisafunctionaltargetoflet-7a.

Here,wedemonstratethatRAB40Cisregulateddirectlybylet-7a.ThisisevidentattheleveloftheRAB40Cproteinisdysregulatedbythechangeoflet-7aexpressionwithoutanychangeintheamountofRAB40CmRNA.Inaddition,therewasasigni?cantinversecorrela-tionoflet-7aexpressionwiththelevelofRAB40Cproteiningastriccancercelllinesandgastrictumortissues.Tothebestofourknowl-edge,thisisthe?rstreportshowingacorrelationbetweenRAB40CmRNAandlet-7amiRNAinclinicalsamplesofhumancancer.More-over,mutationofthelet-7a-bindingsiteabolishedtheeffectoflet-7aontheregulationofRAB40C?uorescenceintensity.Importantly,de-pletionofRAB40CbysiRNAtransfectionpartlyrescuesthereducedcellularproliferationandtheplatingef?ciencyofcellsinsoftagarobserveduponlet-7aup-regulation,furtherdemonstratingthatRAB40Cisatargetoflet-7aandsuggestinganessentialroleforRAB40Casamediatorofthebiologicaleffectsoflet-7aingastrictumorigenesis.Thisisthe?rststudytoshowthatRAB40Cisnega-tivelyregulatedbylet-7aattheposttranscriptionallevelviabindingtothe3#-UTRofRAB40CmRNAingastriccancercells.

Insummary,wereportthatlet-7aexpressionwasdecreasedinhumangastriccancertissuesandcelllines.Ourstudyextendedthe

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Fig.6.TheeffectofRAB40CsiRNAandlet-7amimiconcellproliferationandanchorage-independentgrowth.(A)SuppressionofRAB40CmRNAexpressionlevelsbyRAB40CsiRNAtransfection.(B)SuppressionofRAB40CproteinexpressionlevelsbyRAB40CsiRNAtransfection.(C)TheeffectofRAB40CsiRNAandlet-7amimiconcellproliferation.Relativecellgrowthiscomparedwiththeuntransfectedgroup.(D)TheeffectofRAB40CsiRNAandlet-7amimiconanchorage-independentgrowthofcells.Dataaregivenasmean±SDfromthreeindependentexperiments.?P,0.05;??P,0.01,comparedwiththesiRNANCgroup.Pvalueswereobtainedbyone-wayanalysisofvarianceorthenon-parametricKruskal–WallisHtestformultiplecomparisons.

impactoflet-7atogastriccancerbyshowingforthe?rsttimethatlet-7adecreasedproliferationandcellcycleingastriccancercellsandinhibitedtumorgrowthinnudemice.Theseeffectsarepossiblyduetodown-regulationofRAB40Cbylet-7a.Therefore,miRNAs,inpar-ticularlet-7a,mightserveaspotentiallyusefultargetsforgastriccancerdiagnosisandtherapy.Futurestudieswillbeaimedatdevel-opingstrategiestouselet-7aandRAB40Casatherapeuticregimen.Supplementarymaterial

SupplementaryFiguresS1–S4canbefoundathttp://carcin.oxfordjournals.org/Funding

NationalNaturalScienceFoundationofChina(30771780,30972443toJ.Y.);NaturalScienceFoundationofGuangdongProvince(07117550,9251018201000004toJ.Y.);ScienceandTechnologyProgramofGuangzhouBureauofEducation(08A093toJ.Y.,08A092toY.Q.);ScienceandTechnologyPillarProgramofJiangxiProvince(2009zDS16000toC.H.).Acknowledgements

WethankXinZhouandWeiLi(DepartmentofGeneralSurgery,FirstAf?l-iatedHospitalofNanchangUniversity)forcollectingtheclinicopathologicinformationforthepatients.WethankRongLiu(InstituteofRespiratoryDiseases,FirstAf?liatedHospitalofGuangzhouMedicalUniversity)foras-sistancewiththenudemiceassays.

Con?ictofInterestStatement:Nonedeclared.

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