ExpressionofliviningastriccancerandinductionofapoptosisinSGC-7901cellsbyshRNA-mediatedsilencingoflivingene

Reportperson

：王曉磊Studentid

：2014050448介導的shRNA能抑制肺癌細胞中l(wèi)ivin沉默基因的表達從而促進SGC-7901細胞凋亡

BackgroundBecauseofincreasedresistancetoapoptosisintumorcells,inhibitionofspecificantiapoptoticfactorsmayprovidearationalapproachforthedevelopmentofnoveltherapeuticstrategies.Livin,anovelinhibitorofapoptosisproteinfamily,hasbeenfoundtobeexpressedinvariousmalignanciesandissuggestedtohavepoorlyprognosticsignificance.However,nodataareavailableconcerningthesignificanceofliviningastriccancer.Inthisstudy,wedetectedtheexpressionoflivininhumangastriccarcinomaandinvestigatedtheapoptoticsusceptibilityofSGC-7901cellbyshRNAmediatedsilencingofthelivingene.背景由于腫瘤細胞抑制凋亡增殖,特定凋亡的抑制因素會對于發(fā)展新的治療策略提供一個合理途徑。Livin是一種凋亡抑制蛋白家族成員，在多種惡性腫瘤的表達中具有意義。但是,在有關胃癌方面沒有可利用的數(shù)據(jù)。在本研究中,我們發(fā)現(xiàn)livin基因在人類胃癌中的表達并調查了介導的shRNA能抑制肺癌細胞中l(wèi)ivin沉默基因的表達，從而促進SGC-7901細胞凋亡。1.Introduction

Gastriccancerisoneofthemostcommonmalignanciesintheworld.Mostpatientswiththisdiseasearediagnosedinadvancedstages,andlosethechanceofsurgicaleradication.Despitemuchprogressinchemotherapy,theoverallsurvivalofthepatientswithgastriccancerinadvancedstageisstillpoor.Resistanceofcancercellstochemoagentsmaycontributetofailureofthetreatment.Amongthereasonsofdrugresistance,inhibitedprocessofcellapoptosismayplayanimportantrole.1．介紹

胃癌是世界上最常見的惡性腫瘤之一。大多數(shù)患者被診斷為這個疾病的階段,在最佳時間的機會錯失了手術治愈。盡管有很大改善,但處于晚期胃癌的化療患者的總體存活率仍然很低。癌癥細胞化療耐抗性可能導致手術失敗。在耐藥的原因中,抑制細胞凋亡的過程會起重要作用。癌細胞常有抗凋亡增長的特征[1],介導其增加的阻力不同來刺激的細胞凋亡,如DNA損傷、缺氧、營養(yǎng)損失[2、3]。此外,在臨床實踐中細胞凋亡抵抗被認為是腫瘤手術失敗的主要原因,因此許多化療藥物和/或放射線療法都是通過誘導凋亡腫瘤死亡實現(xiàn)的[4]

Inthepresentstudy,weinvestigatedtheexpressionofliviningastriccancinomasandtheiradjacenttissues.Therelationshipbetweenlivinexpressionandclinicalpathologicparameterswasanalyzed.Furthermore,weexploredthefeasibilityofshRNAininhibitinglivingeneexpressionandtheapoptoticsusceptibilityofgastriccancercellbyshRNA-mediatedsilencingofthelivingene.在目前的研究中,我們調查了livin的表達在胃cancinomas及其鄰近組織。livin的表達和臨床病理參數(shù)之間的關系進行了分析。此外,我們探索了在抑制livin基因表達的shRNA可行性和胃癌易感性的凋亡細胞由shRNA介導的livin沉默基因。2.Patientsandmethods2.1.PatientsandtumorsamplesFortysamplesofgastriccarcinomaand13samplesofparacanceroustissueswerecollectedfromthepatientswhoreceivedgastrectomy(ageofpatientsrangingfrom29-77years).Thirteensamplesofbenigngastriclesion(chronicsuperficialgastritis)weregainedfromthepatientsundergoinggastricendoscopicexamination(ageofpatientsrangingfrom33-77years).ThesesampleswerecollectedfrompatientsadmittedtotheFirstAffiliatedHospitalofNanjingMedicalUniversity.ThepatientswithgastriccancerwerediagnosedasbeinginstageItoIVbasedonTNMclassification(UICC,2002).Tumorspecimenswereimmediatelyfrozeninliquidnitrogenaftersurgeryandstoredat-80℃untiluse.Informedconsentwasobtainedfromallpatients.2患者和方法

2.1?；颊吆湍[瘤樣本胃癌中四十個病例及接受胃切除手術的患者收集到的胃癌組織中13個病例(患者年齡從29~77歲)。其中良性胃潰瘍的13個病例(慢性淺表胃炎)患者在接受了胃內視鏡檢查(患者年齡從33~77歲)。這些病例均來自南京醫(yī)科大學第一附屬醫(yī)院。胃癌患者被診斷為TNM級的1~4階段(UICC，2002)。手術之后腫瘤標本就立即被凍結在液態(tài)氮中,儲存在-80℃直到使用為止。這是在所有的病人知情同意的情況下獲得的。2.2.RT-PCRprocedureTotalRNA(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.2.2逆轉錄聚合酶鏈反應技術程序總共RNA(2毫克)提取冷凍組織反轉錄進行，最后的體積2微升是用100pmolofoligo(dT)15和200UM-MLV與逆轉錄酶(promega、美國),根據(jù)制造商的說明。Aliquots對應的250微升cDNA被放大在PCR緩沖容器中，在最終的50微升中含25pmol/ml處理劑和1U聚合酶。每一個放大了35周期,一個周期的變性曲線在30s內到達94℃。熱處理在30s內59℃（livinandb-actin）擴大到30s內72℃。沒有RNA的病例作為陰性對照物包含在RT–PCR中。一系列常用的的livin和β-actin處理劑如下：livinα/βupstream，5＇-TCCACAGTGTGCAGGAGACT-3＇;livinα/βdownstream;5＇-TCCACAGTGTGCAGGAGACT-3＇;β-actinupstream,5＇-ACGGCACAAAGACGATGGAC-3＇β-actindownstream,5＇-AGCGCAAGTACTCCGTGTG-3＇。產(chǎn)品的尺寸分別為livinα/β是312/258bp，β-actin是501bp。2.3.WesternBlotAnalysisTissueswerehomogenizedwithlysisbuffer[50mMTris-HCl(pH7.5),250mMNaCl,0.1%NP40,and5mMEGTAcontaining50mMsodiumfluoride,60mMb-glycerol-phosphate,0.5mMsodiumvanadate,0.1mMphenylmethylsulfonylfluoride,10mg/mlaprotinin,and10mg/mlleupeptin.ThetotalproteinconcentrationwasdeterminedusingCoomassieBrilliantBlue.Proteinsampleswereelectrophoresedina10%denaturingSDSgelandtransferredtoPVDFmembrane(Roche,USA).Themembraneswereincubatedwithspecificprimaryantibodies,reactedwithaperoxidase-conjugatedsecondaryantibody(Cellsignalingtechnology,USA),andfinallyvisualizedbyenhancedchemiluminescence(Cellsignalingtechnology,USA).Monoclonalantibodiesrecognizinglivin(1:250)andactin(1:400)werepurchasedfromAlexesisInc.(USA)andSantaCruzBiotechnology(USA).2.3西方吸干技術分析病變同質性與沖力緩沖50mMTris-HCl(pH7.5),250mMNaCl,0.1%NP40和5mMEGTA包含50mM氟化鈉，60mMβ-丙三醇-磷酸鹽,0.5mM釩酸鈉,0.1mM苯甲基磺醯化氟10μg/ml亮抑蛋白酶肽。用考馬斯亮藍微盤比色法測定蛋白質含量。蛋白質樣品電泳10%變性SDS凝膠并轉移到PVDF膜(Roche、美國)。膜是培養(yǎng)特定的主要的抗體,與過氧化物酶繼發(fā)性抗體反應(細胞信號技術、美國),最后通過增強化學熒光達到可視化(細胞信號技術、美國)。Alexesis(美國)和散塔克魯茲生物技術(美國)購買的單克隆抗體livin(1:250)andactin(1:400)2.4.CelllinesandcellcultureWeselectedahumangastricadenocarcinomacelllinesforthisstudy.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.2.4細胞系和細胞培養(yǎng)我們選了一個人類胃腺癌細胞系在這一研究中。SGC-7901(上海細胞研究所,中國上海,)是一種附著中度分化胃腺的人類細胞株。線是胃癌細胞上皮細胞,并成長為附著細胞RPMI1640(HycloneInc,USA)含10%FCS(LifeTechnologies,Inc.),每毫升100個單位的青霉素和毫升100微克的鏈霉素(BioWhittaker)。在含有5%CO2空氣的條件下的37℃濕潤培養(yǎng)器中保存SGC-7901細胞。溶解順鉑和氟尿嘧啶(齊魯制藥廠、中國)在DMSO并且4℃儲存。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.2.5ShRNA的合成和PGPU/GFP/Neo/livin質粒的制造通過siRNASequence-Selector軟件設計并合成了Livin的ShRNA序列(上海生物技術有限責任公司。集團公司、中國)。序列如下(表1),然后被插入pGPU/GFP/Neo(上海GenePharma股份有限公司。中國)BbsIandBamH地址產(chǎn)生pGPU/GFP/Neo/livinandpGPU/GFP/Neo/Control質子。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)for24hbeforetransfectionThecellsweretransfectedwith4mg/wellofemptypGPU/GFP/Neo/vector,pGPU/GFP/Neo/livinorpGPU/GFP/Neo/ControlplasmidusingLi-pofectAMINE2000(LifeTechnologies,Inc.,GrandIsland,NY)accordingtothemanufacturer?ˉsinstructions.Forty-eighthoursaftertransfection,thecellswerepassagedat1:15(v/v)andculturedinmediumsupplementedwithGeneticin(G418)at1000g/mlfor4weeks.Stablytransfectedcloneswerepickedandmaintainedinmediumcontaining400g/mlG418foradditionalstudies.2.6SGC-7901的建立穩(wěn)定表達pGPU/GFP/Neo/livinandpGPU/GFP/Neo/Control轉染實驗,SGC-7901細胞被鍍成6孔板(3×105孔密度),,96孔板(1×104孔密度)和12孔板(1.5×105孔密度)轉染之前培養(yǎng)24小時。按照制造商的說明這些細胞被調控子用Li-pofectAMINE2000轉染4毫克/孔空的pGPU/GFP/Neo/vector，pGPU/GFP/Neo/livin或pGPU/GFP/Neo/Control質粒(生命技術公司、大島嶼,NY)。轉染48小時后,這些細胞被轉移在1:15(v/v)并用Geneticin(G418)1000克/毫升培養(yǎng)4周。穩(wěn)定轉染的克隆體取出并保存在媒介容器400g/mlG418用作另外的研究。2.7.Assayofanchorage-dependentcellgrowthParentcellsandcellsstablyexpressingemptypGPU/GFP/Neovector,pGPU/GFP/Neo/livinorpGPU/GFP/Neo/Controlwereseededinto6-wellplates.Cellsfromtriplicatewellswerecollectedeveryotherday.CellnumbersweredeterminedusingaCoultercounter(CoulterElectronics,Miami,FL).ThenumberofcellsperwellisreportedastheaverageSDattheindicatednumberofdaysafterplating.2．7依賴貼壁細胞細胞生長的測定親本細胞和細胞穩(wěn)定表達pGPU/GFP/Neo/vector,pGPU/GFP/Neo/livinorpGPU/GFP/Neo/Control被種到6孔盤子中。每隔一天收集三孔的細胞。使用計數(shù)器確定細胞數(shù)目(CoulterElectronics,Miami,FL).。種植一些天數(shù)后用平均SD記錄每孔細胞的數(shù)量。2.8.MTTassayCytotoxicitywasmeasuredbyMTTassay.Cellsgrowingexponentiallywereplatedonto96-wellplatesatadensityof10000cells/wellfor24h.Thecellswerethentreatedwithdifferentconcentrationsofdrugsfor48h.OnehundredmicrolitersofMTTstocksolution(1mg/ml)wereaddedtoeachwell,andthecellswerefurtherincubatedat37℃for4h.Thesupernatantwasreplacedwithisopropylalcoholtodissolveformazanproduction.Theabsorbanceatwavelength595nmwasmeasuredwithamicro-ELISAreader(ClinBio-128,SLT,Austria).Theratiooftheabsorbanceoftreatedcellsrelativetothatofthecontrolcellswascalculatedandexpressedasapercentageofcelldeath.2.8MTT測定通過MTT對細胞毒性進行了測量。呈幾何數(shù)增長的細胞被鍍在密度為10000細胞/孔的96孔板上作用24小時。接下來這些細胞被以不同濃度的藥物治療48小時。每孔加入100微升MTT溶液(1毫克/毫升),并且這些細胞放在37℃下培養(yǎng)四小時。上層清液用異丙醇代替溶解有色甲品。用micro-ELISA測量到吸光率的波長為595nm(ClinBio-128SLT,,奧地利)。治療細胞的吸光率相當于計算控制細胞的吸光率并用細胞死亡百分率顯示出來。2.9.FlowcytometryCellswerecollectedandfixedwithice-cold70%ethanolinPBSandstoredat-4℃untiluse.Afterresuspension,cellswereincubatedwith100mlofRNaseI(1mg/ml)and100mlofPI(400mg/ml)at37℃andanalyzedbyflowcytometry(BD,USA).2.9流式細胞術細胞被收集并加入冰冷的70%乙醇于PBS緩沖液中儲存在-4攝℃暖直到使用。懸浮后后，100ml核糖核酸酶I(1mg/ml)and100ml的聚酰亞胺(400μg/ml)37℃下培養(yǎng)細胞并用流式細胞術(BD,美國)進行分析。2.10.StatisticalanalysisDatawereexpressedasthemeansofatleastthreedifferentexperimentsSD.TheresultswereanalyzedbyStudent’st-test,andP<0.05wasconsideredstatisticallysignificant.2.10統(tǒng)計分析數(shù)據(jù)的展現(xiàn)要用至少三個不同實驗±SD的方法。實驗結果用學生的t檢驗來分析和當P

<0.05時被認為是具有統(tǒng)計學顯著性。3.Results3.1.ExpressionofliviningastriccarcinomasInthepresentstudy,forthefirsttime,weevaluatedbyRT¨CPCRandwestenblotthepresenceoflivinexpressionin40gastriccancinomas,13para-canceroustissuesand13benignlesionsofgastricmucosa.Inpara-canceroustissuesandbenignlesionsofgastricmucosa,nodetectablelevelsofeithermRNAisoformswererevealed,whileamongtumortissues,19/40(47.5%)showedmRNAandproteinexpressionoflivinaandlivinb(Figs.1and2).Livinexpressioncorrelatedwithsomeoftheknownprognosticvariables,suchashistologicgradeandlymphnodemetastasis,butnotwithage,sexuality,stageandtumorinfiltrationextent(Table2).結果3.1livin在胃腸癌中的表達在目前的研究中,我們第一次驗證了逆轉錄聚合酶鏈反應技術和西方吸干技術的存在在40胃癌中,13癌組織和13良性病變胃粘膜損傷。在癌組織和良性病變胃粘膜損傷中,每個mRNA亞型不可見水平被發(fā)現(xiàn)后,在腫瘤組織中,19/40(47.5%)顯示出mRNA及l(fā)ivinaα和livinβ蛋白質表達(Figs.1and2).。livin表達與預后變量相關,如組織學惡性度和淋巴結轉移,但包括年齡、性別、階段和腫瘤細胞浸潤程度(表2)。3.2.CharacterizationofstabletransfectantsexpressingpGPU/GFP/Neo/livinandpGPU/GFP/Neo/ControlWeestablishedSGC-7901stabletransfectantswitheitherpGPU/GFP/Neo/livin,pGPU/GFP/Neo/Controlplasmid,oremptypGPU/GFP/Neo/vector(Fig.3).SomeclonesfromeachtransfectionwereselectedandanalyzedbyRT-PCRandWesternblottodeterminethelivinmRNAandproteinexpression,andotherswereselectedforexpansionandadditionalstudies.Asshownin(Figs.4and5),theleveloflivinmRNAandproteininSGC-7901pGPU/GFP/Neo/livin2transfectantswasreducedbymorethan90%.ThesuppressionoflivinexpressionwasnotobservedinpGPU/GFP/Neo/livin1transfectantsandnegativecontrol.SoSGC-7901pGPU/GFP/Neo/livin2transfectantswaschosenforsubsequentexperiment.3.2。穩(wěn)定轉染表達pGPU/GFP/Neo/livin與pGPU/GFP/Neo/Control的特征我們建立了SGC-7901與任一pGPU/GFP/Neo/livin穩(wěn)定轉染，pGPU/GFP/Neo/Control質粒，或空pGPU