肺癌转移相关蛋白1lcmr1与细胞凋亡关系的研究-study on the relationship between lung cancer metastasis related protein 1 lcm r1 and apoptosis.docxVIP

（C端）功能区无相互作用。（4）从mRNA水平和蛋白水平验证了设计的LCMR1小干扰RNA对LCMR1有明显的敲减效果。（5）LCMR1与细胞凋亡关系研究发现，LCMR1下调导致：①凋亡细胞增加并出现明显凋亡峰；②活化的Caspase-3蛋白表达增加、活性增强。（6）LCMR1的凋亡机制研究发现，LCMR1下调导致：①活化的Caspase-8蛋白及活化的Caspase-9蛋白表达增多、活性增强；②肺腺癌A549（p53+/+）细胞凋亡增加，肺腺癌H1299（p53-/-）细胞凋亡无明显增加；③促凋亡因子Bax及Bid表达升高，抑凋亡因子Mcl-1表达下降。结论:（1）LCMR1与DEK（N端）功能区有相互作用，根据相互作用蛋白DEK功能区的线索，推测LCMR1与细胞凋亡相关。（2）成功设计并选取LCMR1的小干扰RNA，并验证其对LCMR1有明显的敲减效果。（3）LCMR1下降可引起细胞凋亡增加，LCMR1与细胞凋亡负相关。（4）LCMR1同时参与内源性及外源性凋亡通路。（5）LCMR1依赖P53基因通路影响细胞凋亡。（6）LCMR1通过Bcl-2家族基因调控细胞凋亡。关键词:肺癌；肺癌转移相关蛋白1；小干扰RNA；细胞凋亡TheRoleofLungCancerMetastasisRelatedProtein1(LCMR1)intheGenerationofApoptosisAbstractObjective:Lungcancermetastasisrelatedprotein1(LCMR1)isanovelgeneclonedfromPLA-801,apoorlydifferentiatedhumanlunglargecellcarcinomacellline,usingadifferentialdisplaypolymerasechainreactiontechniqueinourlaboratoryin2002.TheinformationofthisgenehasbeensubmittedtoNationalCenterforBiotechnologyInformation(NCBI),andaGenbankaccessionnumber(AY148462)hasbeenassigned.Inthepresentstudy,weemployedRNAinterference(RNAi)techniquetoknockdownLCMR1expressionandinvestigatedtheregulatoryroleofLCMR1inthegenerationoflungcancercellapoptosis.Methods:(1)TheN-terminalfunctiondomainandC-terminalfunctiondomainofDEKgene,aftersequenced,wereclonedintopcDNA3.0-flagplasmid,respectivelyandtheproteinswereacquiredbyusingtranscriptionandtranslationsysteminvitro.(2)TheinducingconditionofthepGEX-5T/LCMR1prokaryoticexpressionvectorwasoptimizedtoacquirestableexpressionofGST-LCMR1inE.colicells.(3)ThebindingdomainofinteractingproteinsLCMR1andDEKwasanalysedbyGSTpull-down.TheresultprovidedessentialcluesfortheinvestigationofLCMR1function.(4)RNAinterference(RNAi)approachwasusedtoknockdownLCMR1inlungcancercells.Quantitativereal-timeRT-PCRandWesternblotanalysiswereusedtoconfirmifLCMR1wassuccessfullyknockeddownbyRNAiatboththemRNAandproteinlevels.(5)TheimpactsofLCMR1knockdownontheapoptosisoflungcancercellswereassessedusingflowcytometry,quantitativereal-timeRT-PCRandWesternblotting.Results:(1)TheN-terminalfunctiondomainandC