shRNA沉默YAP基因对人肾癌786-O细胞增殖和凋亡的影响  

Influence of YAP gene silencing by shRNA on the proliferation and apoptosis of human renal cancer cell 786-O

作　　者：曹建佳 何江[2] 李兴森[3] 陈柯宏 盛夏[1] 李文宾[1] 李美材 黄飚[3] 王德林[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科,重庆400016 [2]重庆医科大学附属大学城医院消化神经中心,重庆401331 [3]重庆市綦江区人民医院泌尿外科.重庆401420

出　　处：《重庆医科大学学报》2014年第6期753-758,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30972999);重庆市自然科学基金资助项目(编号:cqpc2012jja1698);重庆市卫生局基金资助项目(编号:2013-2-082)

摘　　要：目的:探讨慢病毒干扰载体沉默Yes-相关蛋白(Yes-associated protein,YAP)对人肾癌786-O细胞增殖和凋亡的影响。方法:用细胞免疫荧光法检测肾癌786-O细胞中YAP蛋白表达情况;构建针对YAP基因的shRNA慢病毒干扰载体转染786-O细胞。RT-PCR和Western blot分别检测干扰786-O细胞前后YAP mRNA及蛋白的表达情况;CCK-8(cell counting kit-8)法检测沉默YAP后细胞增殖的改变;流式细胞仪(flow cytometry,FCM)检测细胞凋亡和周期的变化。结果:786-O细胞中YAP蛋白表达于细胞浆和细胞核;shRNA-YAP慢病毒干扰载体转染4 d后,可明显下调786-O细胞YAP mRNA及蛋白表达水平(P=0.000),并且明显抑制细胞增殖和促进细胞凋亡(P=0.000);细胞周期紊乱,G1期细胞明显增加,S期细胞明显降低(P=0.000)。结论:YAP-shRNA慢病毒干扰载体能有效抑制YAP基因在786-O细胞中表达,进而抑制细胞增殖并促进细胞凋亡。Objective：To investigate the effect of Yes-associated protein（YAP）gene silencing by shRNA on the proliferation and apoptosis of human renal cancer cells 786-O. Methods：Expression of YAP protein in 786-O cells was detected by immunofluorescence assay. Lentiviral interference vectors of shRNA-YAP were constructed successfully and were transfected into 786-O cells.Meanwhile blank control group and negative control group were divided. Expression of YAP mRNA and protein in 786-O cells was detected by RT-PCR and Western blot,respectively. CCK-8（cell counting kit-8）assay was applied to examine the effect of YAP silencing on the proliferation in 786-O cells. Apoptosis and cell cycle of 786-O cells were examined by flow cytometry. Results：YAP protein was expressed in cytoplasm and nucleus of 786-O cells. On 4 d after shRNA-YAP lentiviral interference vectors being infected,expression of YAP mRNA and protein in 786-O cells was significantly reduced（P=0.000）,cell proliferation was inhibited,and apoptosis was increased significantly（P=0.000）. YAP silencing resulted in cell cycle distribution disorder in 786-O cells,which the number of G1 phase cells was significantly increased while the number of S phase cells was significantly decreased compared with that of blank control group and negative control group（P=0.000）. Conclusion：shRNA-YAP lentiviral interference vectors can effectively decrease YAP gene expression in 786-O cells,inhibit cell proliferation and promote cell apoptosis.

关 键 词：肾细胞癌 Yes-相关蛋白 RNA干扰 慢病毒 

分 类 号：R737.11[医药卫生—肿瘤]