ATAD2对人胶质瘤细胞系转移及上皮间质转换的影响

ATAD2对人胶质瘤细胞系转移及上皮间质转换的影响秦彦昌;贾栋;于庆伟2(ATAD2)对人胶质瘤细胞的影响.方法real-timePCRWesternblotATAD2U87MG、U251.U87MGU251(control)、脂质体对照组(mock)、ATAD2siRNA(转染特异性siRNA)和ATAD2overexpressionATAD2)4,MTTTranswellblotU87MGU251ATAD2,ATAD2siRNAU87MGU251overexpressionATAD2ToanalyzetheeffectofATAD2ongliomacelllines.MethodsATAD2levelinU87MG,U251andnormalhumanastrocyteswasdetectedbyRT-PCRandWesternblot.U87MGandU251cellsweredividedintofourgroups:control,mock(lipofectioncontrol),ATAD2siRNA(transfectedwithATAD2siRNA)andATAD2overexpression(transfectedwithATAD2).Cellproliferation,migrationandinvasionwererespectivelymeasuredbyMTTandTranswellassay.ThelevelofE-cadherin,N-cadherinandVimentinwasmeasuredbyWesternblot.ResultsATAD2washighlyexpressedinU87MGandU251cells.Comparedwithcontrol,cellproliferation,invasion,migration,N-cadherinandVimentinexpressionweredecreasedbyATAD2siRNAwhiletheywerepromotedbyATAD2overexpression(P<0.05).E-cadherinexpressionwasupregulatedbyATAD2siRNAanditwasinhibitedbyATAD2overexpression(P<0.05).ConclusionsATAD2participantsinhumangliomacellsmetastasisviaepithelial-mesenchymaltransition(EMT).【期刊名称】《基础医学与临床》【年(卷),期】2017(037)004【总页数】6页(P512-517)【关键词】三磷酸腺苷酶家族蛋白2;胶质瘤;侵袭;迁移;上皮间质转换【作者】秦彦昌;贾栋;于庆伟【作者单位】第四军医大学唐都医院神经外科,陕西西安710038;第四军医大学唐都医院神经外科,陕西西安710038;第四军医大学唐都医院神经外科,陕西西安710038【正文语种】中文R651神经胶质瘤是常见脑肿瘤，发病率近年不断上升[1]，其侵袭性较高，手术难以完59.8%[22(ATPasefamily，AAAdomaincontaining2，ATAD2)在胃癌[3]、肝癌[4]和结直肠癌[5]等多种恶性肿瘤中发挥重要作用，在胶质瘤中的作用却未见报道。本文旨在研究ATAD2对胶质瘤细胞的影响。主要试剂人恶性胶质瘤细胞系U87MG和U251(ATCC)；人正常星形胶质细胞Astrocyte(中科院上海细胞所)；ATAD2siRNA序列(上海吉玛公司合成)；MTT试剂盒(华美生物公司)；Transwell(MilliporeLipofetamineTM2000和反转录试剂盒(InvitrogenTrizolATAD2GAPDH兔抗人多克隆抗体和辣根过氧化酶(HRP)IgG(Abcam方法80nmol/LsiRNA-脂质体复合物及ATAD2-脂质体复合物，ATAD2siRNAATAD2siRNAATAD2过表达组(ATAD2overexpressionATAD2-脂质体复合物。细胞分为对照组(control)、脂质体对照组(mock)、ATAD2siRNAATAD2overexpression42×105/670%～80%汇合时换无血清培养基，LipofetamineTM2000ATAD2siRNAATAD2MTT法测细胞增殖：将细胞以3×103个/孔接种于96孔板，分别培养24、48、7296hMTT5g/L4h。弃去上清，加入二甲亚砜，振荡10min充分溶解紫色结晶物，酶标仪上测定490nm处各孔A值。每组实验重复3次。Transwell[6]测定细胞侵袭，10053TranswellMatrigel，其他步骤同侵袭。Real-timePCRATAD2mRNATrizolRNA，反转录cDNAcDNAATAD25′-GAGCTGGATCTCTCTCCGGT-3′，5′-TCTTAAAGCACGTGTCCGGT-3′；β-actin5′-CCGCACCACTGGCATTGTCAT-3′，5′-CAGCACCGTGTTGGCGTAGAGG-3′。条件：94℃2min；94℃，1min；55℃1min；72℃1min，共40个循环；72℃，10min。WesternblotATAD2(epithelial-mesenchymaltransition，EMT)标志蛋白的表达：提取细胞总蛋白测浓度，20μgSDS-PAGE1hATAD2、上皮钙黏素(E-cadherin)、神经钙黏素(N-cadherin)或波形蛋白(Vimentin42hECLGAPDH统计学分析数据以均数±标准差表示，用SPSS22.0软件统计分析，多组间比较用单因素方差分析，两两比较采用LSD和Dunnett’sT3检验。ATAD2U87MGU251U87MGU251ATAD2mRNA1)。ATAD2和对照组比较，ATAD2siRNA组ATAD2表达降低，ATAD2overexpression组升高(P<0.05)(图2,3)。ATAD2U87MGU251连续观察96h，发现在第48hATAD2siRNA对细胞增殖出现明显抑制作用，ATAD2过表达显示出促进细胞增殖(P<0.05)(图4)。ATAD2U87MGU251与对照组比，ATAD2siRNA组穿膜和迁移细胞数均减少，ATAD2overexpression组穿膜和迁移细胞数均增多(P<0.05)(图5,6)。ATAD2U87MGU251EMT和对照组相比，ATAD2siRNA组细胞E-cadherin表达升高，N-cadherin和vimentin表达降低；ATAD2overexpression组则相反(P<0.05)(图7)。向，一些分子显示出良好的抑制胶质瘤作用[78]，对与胶质瘤生物学特性相关分子进行研究意义重大。ATAD2CmycαAAAC-MycU87MGU251ATAD2ATAD2U87MGU251细胞侵袭和转移是恶性肿瘤主要致死原因，EMT[12EMTE-cadherinN-cadherinVimentinATAD2EMTATAD2siRNAE-cadherinVimentinATAD2ATAD2EMT综上所述，本研究首次发现ATAD2在胶质瘤细胞中高表达，ATAD2影响胶质瘤细胞增殖、侵袭和迁移，可能与ATAD2调节EMT有关。【相关文献】MeteF,OzkayaE,ArasS,etal.AssociationbetweengenepolymorphismsinTIM1,TSLP,IL18R1andchildhoodasthmainTurkishpopulation[J].IntJClinExpMed,2014,7:1071-1077.StuppR,HegiME,MasonWP,etal.EffectsofradiotherapywithconcomitantandadjuvanttemozolomideversusradiotherapyaloneonsurvivalinglioblastomainarandomisedphaseⅢstudy:5-yearanalysisoftheEORTC-NCICtrial[J].LancetOncol,2009,10:459-466.ZhangMJ,ZhangCZ,DuWJ,etal.ATAD2isoverexpressedingastriccancerandservesasanindependentpoorprognosticbiomarker[J].ClinTranslOncol,2015,18:776-781.HwangHW,HaSY,BangH,etal.ATAD2asaPoorPrognosticMarkerforHepatocellularCarcinomaafterCurativeResection[J].CancerResTreat,2015,47:853-861.LuoY,YeGY,QinSL,etal.ATAD2OverexpressionIdentifiesColorectalCancerPatientswithPoorPrognosisandDrivesProliferationofCancerCells[J].GastroenterolResPract,2015,2015:936564.PanW,WangH,JianweiR,etal.MicroRNA-27apromotesproliferation,migrationandinvasionbytargetingMAP2K4inhumanosteosarcomacells[J].CellPhysiolBiochem,2014,33:402-412.HuangSK,QianJX,YuanBQ,etal.SiRNA-mediatedknockdownagainstNUF2suppressestumorgrowthandinducescellapoptosisinhumangliomacells[J].CellMolBiol(Noisy-le-grand),2014,60:30-36.WangX,TongX,GaoH,etal.SilencingHIWIsuppressesthegrowth,invasionandmigrationofgliomacells[J].IntJOncol,2014,45:2385-2392.CiroM,ProsperiniE,QuartoM,etal.ATAD2isanovelcofactorforMYC,overexpressedandamplifiedinaggressivetumors[J].CancerRes,2009,69:8491-8498.CaronC,LestratC,MarsalS,etal.FunctionalcharacterizationofATAD2asanewcancer/testisfactorandapredictorofpoorprognosisinbreastandlungcancers[J].Oncogene,2010,29:5171-5181.FouretR,LaffaireJ,HofmanP,etal.AcomparativeandintegrativeapproachidentifiesATPasefamily,AAAdomaincontaining2asalikelydriverofcellproliferationinlungadenocarcinoma[J].ClinCancerRes,2012,18