Livin靶向RNA干扰对MDA-MB-231细胞增殖和凋亡的影响要点解析.docVIP

Livin靶向RNA干扰对MDA-MB-231细胞增殖和凋亡的影响 　　摘　要:目的　研究凋亡抑制蛋白Livin靶向RNA干扰MDA-MB-231对细胞增殖和凋亡的影响,探讨其可能的机制。方法　构建3个靶向Livin的siRNA真核表达载体,分别转染乳腺癌细胞MDA-MB-231后,通过RT-PCR、Western blot技术检测干扰前后MDA-MB-231细胞Livin、Caspase-3和Smac/DIABLO在mRNA、蛋白水平的表达;采用MTT法检测对细胞增殖的抑制作用,流式细胞法测细胞周期,TUNEL法检测细胞凋亡以及观察细胞形态变化。结果　成功构建了针对Livin基因的3个特异性siRNA表达载体。它们不同程度地抑制了MDA-MB-231细胞Livin基因的表达,以si-Livin2抑制效率最高,它对Livin的mRNA和蛋白的抑制率分别达76. 4%, 70. 2%。si-Livin2转染48 h后,与未转染组相比,细胞增殖活力受到抑制,转染48 h抑制率达36. 1%。细胞周期重新分布, G0/G1期上升为(71. 75±1. 31)%, S期下降为(18. 06±1·46)%;细胞凋亡率上升为(13·28±1·65)%;电镜下可见典型的凋亡细胞特征。细胞内Caspase-3, Smac/DIABLO的mRNA、蛋白表达有所上升。结论　Livin靶向RNA干扰通过上调Caspase-3, Smac/DIABLO表达有效地抑制MDA-MB-231细胞增殖,促进凋亡。 　　关键词:乳腺癌;Livin; RNA干扰;增殖;凋亡;MDA-MB-231细胞株 　　中图法分类号:R329. 25;R394. 3;R737. 9文献标识码:A Effects ofRNAi targeting Livin on proliferation and apoptosis ofMDA-MB-231 cellsLIFan,WANG Xiao-yi,CHEN Li,TIAN Tian,LI Jia,REN Guo-sheng(DepartmentofGeneralSurgery,The FirstAffiliatedHospital ofChongqingMedicalUniversity,Chongqing 400016,China)　　Abstract:Objective　To study themechanisms bywhich theRNA targetingLivin regulates the prolifera-tion and apoptosis ofMDA-MB-231 cells.Methods　Three siRNA eukaryotic expression vectors targetingLivinwere constructed and transfected into breast cancerMDA-MB-231 cells. The changes ofLivin,Caspase-3 andSmac/DIABLO expressions inMDA-MB-231 cellswith orwithoutRNAi transfectionwere detected by quantita-tiveReal-Time PCR andWestern blotting. The proliferation ofMDA-MB-231 cellswas detected byMTT assay,their cell cycles by flow cytometry and their apoptosis by TUNEL assay and theirmorphological changes ob-served by opticalmicroscope and electron microscope.Results　Three specific siRNA eukaryotic expressionvectors targetingLivinwere successfully constructed. They all could inhibit the expressions ofLivinmRNA andprotein to different degree,ofwhich si-Livin2 showed the highest inhibition rate. Forty-eight hours after si-Livin2 transfection,the proliferation ofMDA-MB-231 cellswas strongly inhibited,with the inhibit