介导的shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC7901细胞凋亡.docx
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介导的shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC7901细胞凋亡.docx
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介导的shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC7901细胞凋亡
ExpressionofliviningastriccancerandinductionofapoptosisinSGC-7901cellsbyshRNA-mediatedsilencingoflivingene
Background-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(P<0.05).FoursmallinterferingRNAeukaryoticexpression
vectorspecifictolivinwereconstructedbygenerecombination.Andoneofthemcanefficientlydecreasetheexpressionoflivin,theinhibitionofthegenewasnotlessthan70%(P<0.01).Therecombinatedplasmidswereextractedandtransfectedgastriccancercells.ThestablecloneswereobtainedbyG418screening,andwereamplifiedandcultured.Whenlivingenewassilenced,thereproductiveactivityofthegastriccancercellswassignificantlylowerthanthecontrolgroups(P<0.05).ThestudyalsoshowedthatIC50of5-FuandcisplatinongastriccancercellstreatedbyshRNAwasdecreasedandthecellsweremoresusceptibletoproapoptoticstimuli(5-Fuandcisplatin)(P<0.01).
Conclusions-CLivinisoverexpressedingastriccarcinomawitharelationshiptotumordifferentiationandlymphnodemetastases,whichissuggestedtobeoneofthemolecularprognosticfactorsforsomecasesofgastriccancer.ShRNAcaninhibitlivinexpressioninSGC-7901cellsandinducecellapoptosis.Livinmayserveasanewtargetforapoptosis-inducingtherapyofgastriccancer.
1.Introduction
Gastriccancerisoneofthemostcommonmalignanciesintheworld.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.Patientsandmethods
2.1.Patientsandtumorsamples
Fortysamplesofgastriccarcinomaand13samplesofparacanceroustissueswerecollectedfromthepatientswhoreceivedgastrectomy(ageofpatientsrangingfrom29-77years).Thirteensamplesofbenigngastriclesion(chronicsuperficialgastritis)weregainedfromthepatientsundergoinggastricendoscopicexamination(ageofpatientsrangingfrom33-77years).ThesesampleswerecollectedfrompatientsadmittedtotheFirstAffiliatedHospitalofNanjingMedicalUniversity.ThepatientswithgastriccancerwerediagnosedasbeinginstageItoIVbasedonTNMclassification(UICC,2002).Tumorspecimenswereimmediatelyfrozeninliquidnitrogenaftersurgeryandstoredat-80℃untiluse.Informedconsentwasobtainedfromallpatients.
2.2.RT-PCRprocedure
TotalRNA(2mg)extractedfromfrozentissueswasreversetranscribedinafinalvolumeof25mlwith100pmolofoligo(dT)15and200UM-MLVreversetranscriptase(promega,USA),accordingtothemanufacturer'sguidelines.
Aliquotscorrespondingto2.5mlcDNAwerethenamplifiedinPCRbuffercontaining25pmol/mleachprimerand1UTaqpolymeraseinafinalvolumeof50ml.Eachamplificationwasperformedfor35cycles,onecycleprofileconsistedofdenaturationat948Cfor30s,annealingat598C(livinandb-actin)for30sandextensionat728Cfor30s.AsamplewithoutRNAwasincludedineachRT¨CPCRasanegativecontrol.
Sequencesoflivinandβ-actinprimersusedareasfollows:
livina/bupstream,50-TCCACAGTGTGCAGGAGACT-30;livina/βdownstream,50-ACGGCACAAAGACGATGGAC-30;b-actinupstream,50-AGCGCAAGTACTCCGTGTG-30;β-actindownstream,50-AAGCAATGCTATCACCTCCC-30.Thesizeoftheamplifiedproductswere312/258bpforlivina/band501bpforb-actinrespectively.
2.3.WesternBlotAnalysis
Tissueswerehomogenizedwithlysisbuffer[50mMTris-HCl(pH7.5),250mMNaCl,0.1%NP40,and5mMEGTAcontaining50mMsodiumfluoride,60mMb-glycerol-phosphate,0.5mMsodiumvanadate,0.1mMphenylmethylsulfonylfluoride,10mg/mlaprotinin,and10mg/mlleupeptin.Thetotalproteinconcentration
wasdeterminedusingCoomassieBrilliantBlue.Proteinsampleswereelectrophoresedina10%denaturingSDSgelandtransferredtoPVDFmembrane(Roche,USA).Themembraneswereincubatedwithspecificprimaryantibodies,reactedwithaperoxidase-conjugatedsecondaryantibody(Cellsignalingtechnology,USA),andfinallyvisualizedbyenhancedchemiluminescence(Cellsignalingtechnology,USA).Monoclonalantibodiesrecognizinglivin(1:
250)andactin(1:
400)werepurchasedfromAlexesisInc.(USA)andSantaCruzBiotechnology(USA).
2.4.Celllinesandcellculture
Weselectedahumangastricadenocarcinomacelllinesforthisstudy.SGC-7901(ShanghaiInstituteofCellResearch,Shanghai,China)isanadherent,moderatelydifferentiated,humangastricadenocarcinomacellline.ThecelllinesaregastriccancerepithelialcellsandgrowasadherentcellsinRPMI1640(HycloneInc,USA)containing10%FCS(LifeTechnologies,Inc.),100units/mlpenicillin,and100mg/mlstreptomycin(BioWhittaker).SGC-7901cellsweremaintainedat378Cinahumidifiedincubatorwithanatmosphereof5%CO2.Cisplatinand5-fluorouracil(Qilupharmaceuticalfactory,China)weresolublizedinDMSOandstoredat48C.
2.5.ShRNAsynthesisandconstructionofPGPU/GFP/Neo/livinplasmids
ShRNAsequencesoflivinweredesignedbysoftwareofsiRNASequence-Selectorandsynthesized(ShanghaiBiotech,Ltd.Corp.,China).Thesequencesasfollowing(Table1)thenwereinsertedintoBbsIandBamHsitesofthepGPU/GFP/Neo(ShanghaiGenePharmaCo.LtdChina)togeneratepGPU/GFP/Neo/livinandpGPU/GFP/Neo/Controlplasmids,respectively.
2.6.EstablishmentofSGC-7901stabletransfectantsexpressingpGPU/GFP/Neo/livinandpGPU/GFP/Neo/Control
Fortransfectionexperiments,SGC-7901cellswereplatedinto6-wellplates(3?
á105cells/well),96-wellplates(1×104cells/well)and12-wellplates(1.5×105cells/well)for24hbeforetransfection
Thecellsweretransfectedwith4mg/wellofemptypGPU/GFP/Neo/vector,pGPU/GFP/Neo/livinorpGPU/GFP/Neo/ControlplasmidusingLi-pofectAMINE2000(LifeTechnologies,Inc.,GrandIsland,NY)accordingtothemanufacturer?
ˉsinstructions.Forty-eighthoursaftertransfection,thecellswerepassagedat1:
15(v/v)andcultured
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- shRNA 抑制 肺癌 细胞 livin 沉默 基因 表达 从而 促进 SGC7901