介导的shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC-7901细胞凋亡

ExpressionofliviningastriccancerandinductionofapoptosisinSGC-7901cellsbyshRNA-mediatedsilencingoflivingeneBackground-Becauseofincreasedresistancetoapoptosisintumorcells,inhibitionofspecificantiapoptoticfactorsmayprovidearationalapproachforthedevelopmentofnoveltherapeuticstrategies.Livin,anovelinhibitorofapoptosisproteinfamily,hasbeenfoundtobeexpressedinvariousmalignanciesandissuggestedtohavepoorlyprognosticsignificance.However,nodataareavailableconcerningthesignificanceofliviningastriccancer.Inthisstudy,wedetectedtheexpressionoflivininhumangastriccarcinomaandinvestigatedtheapoptoticsusceptibilityofSGC-7901cellbyshRNAmediatedsilencingofthelivingene.Methods-ThemRNAandproteinexpressionoflivinwereanalyzedbyRT-PCRandwesternblotassay.Therelationshipbetweenlivinexpressionandclinicalpathologicparameterswasinvestigated.ThesmallinterferingRNAeukaryoticexpressionvectorspecifictolivinwasconstructedbygenerecombination,andthenucleicacidwassequenced.ThenitwastransfectedintoSGC-7901cellsbyLipofectamin2000.RT-PCRandWesternblotassaywereusedtovalidategene-silencingefficiencyoflivininSGC-7901cells.StablecloneswereobtainedbyG418screening.Thecellapoptosiswasassessedbyflowcytometry(FCM).Cellgrowthstateand50%inhibitionconcentration(IC50)of5-FUandcisplatinwasdeterminedbyMTTmethod.Results-TheexpressionoflivinmRNAandproteinweredetectedin19of40gastriccarcinomacases(47.5%)andSGC-7901cells.Noexpressionoflivinwasdetectedintumoradjacenttissuesandbenigngastriclesion.Thepositivecorrelationwasfoundbetweenlivinexpressionandpoordifferentiationoftumorsaswellaslymphnodemetastases(P0.05).FoursmallinterferingRNAeukaryoticexpressionvectorspecifictolivinwereconstructedbygenerecombination.Andoneofthemcanefficientlydecreasetheexpressionoflivin,theinhibitionofthegenewasnotlessthan70%(P0.01).Therecombinatedplasmidswereextractedandtransfectedgastriccancercells.ThestablecloneswereobtainedbyG418screening,andwereamplifiedandcultured.Whenlivingenewassilenced,thereproductiveactivityofthegastriccancercellswassignificantlylowerthanthecontrolgroups(P0.05).ThestudyalsoshowedthatIC50of5-FuandcisplatinongastriccancercellstreatedbyshRNAwasdecreasedandthecellsweremoresusceptibletoproapoptoticstimuli(5-Fuandcisplatin)(P0.01).Conclusions-CLivinisoverexpressedingastriccarcinomawitharelationshiptotumordifferentiationandlymphnodemetastases,whichissuggestedtobeoneofthemolecularprognosticfactorsforsomecasesofgastriccancer.ShRNAcaninhibitlivinexpressioninSGC-7901cellsandinducecellapoptosis.Livinmayserveasanewtargetforapoptosis-inducingtherapyofgastriccancer.1.IntroductionGastriccancerisoneofthemostcommonmalignanciesintheworld.Mostpatientswiththisdiseasearediagnosedinadvancedstages,andlosethechanceofsurgicaleradication.Despitemuchprogressinchemotherapy,theoverallsurvivalofthepatientswithgastriccancerinadvancedstageisstillpoor.Resistanceofcancercellstochemoagentsmaycontributetofailureofthetreatment.Amongthereasonsofdrugresistance,inhibitedprocessofcellapoptosismayplayanimportantrole.Cancercellsareoftencharacterizedbyincreasedresistancetoapoptosis[1],whichmediatestheirincreasedresistancetovariousstimuliofcellapoptosis,suchasDNAdamage,hypoxia,nutrient-deprivation[2,3].Moreover,apoptosisresistanceisconsideredtobeamajorcauseoftherapeuticfailurefortumorsinclinicalpractice,sincemanychemo-and/orradiotherapeuticagentsfunctionthroughtheinductionofapoptotictumordeath[4].Inhibitorofapoptosisprotein(IAPs)isanovelfamilyofintracellularproteinswhichsuppressapoptosisinducedbyavarietyofstimuli[5,6],includingviralinfection,chemotherapeuticdrugs,staurosporin,growthfactorwithdrawal,andbycomponentsofthetumornecrosisfactor-a(TNF-a)/Fasapoptoticsignalingpathways[7¨C9].TheIAPsconsistsofagroupofstructurallyrelatedproteinswithantiapoptoticproperties[10],andmayplayasubstantialroleinpreventingtumorcellfromapoptosis,andhasbecomethefocusofresearchinrecentyears.AnovelmemberofthisfamilyisML-IAP/livin/KIAP/BIRC7(inthefollowingtermedlivin)whichhastwoisoforms,livinaandlivinb[11¨C14].Ithasbeenshownthatover-expressionofthelivincanblockapoptosisinducedbyavarietyofproapoptoticstimuli[12].Interestingly,livingenehasbeenfoundtoberestrictivelyexpressedintumorcells,butnot,ortolesseramountsinmostnormaladulttissues[11¨C15],andmaycontributetotumorigenesisbyallowingmalignantcelltoavoidapoptoticcelldeath.Soinhibitionoflivinexpressionmayrepresentaninterestingtherapeuticstrategy.Inthepresentstudy,weinvestigatedtheexpressionofliviningastriccancinomasandtheiradjacenttissues.Therelationshipbetweenlivinexpressionandclinicalpathologicparameterswasanalyzed.Furthermore,weexploredthefeasibilityofshRNAininhibitinglivingeneexpressionandtheapoptoticsusceptibilityofgastriccancercellbyshRNA-mediatedsilencingofthelivingene.2.Patientsandmethods2.1.PatientsandtumorsamplesFortysamplesofgastriccarcinomaand13samplesofparacanceroustissueswerecollectedfromthepatientswhoreceivedgastrectomy(ageofpatientsrangingfrom29-77years).Thirteensamplesofbenigngastriclesion(chronicsuperficialgastritis)weregainedfromthepatientsundergoinggastricendoscopicexam