慢病毒介导shRNA沉默YAP基因对人前列腺癌LNCaP细胞增殖、凋亡及其雄激素受体的影响  

Effects of lentivirus-mediated shRNA silencing YAP gene on proliferation,apoptosis,and androgen receptor of human prostate cancer cell line LNCaP cells

作　　者：李美材 何江[2] 吴文强[3,4] 黄亮亮[1] 曾强锋 曹建佳 陈柯宏 盛夏[1] 李文宾[1] 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]重庆医科大学附属第一医院 [4]綦江区人民医院泌尿外科,重庆401420

出　　处：《重庆医科大学学报》2015年第4期576-582,共7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);重庆市卫生和计划生育委员会基金资助项目(编号:20143061)

摘　　要：目的:探讨慢病毒介导sh RNA沉默Yes-相关蛋白(Yes-associated protein,YAP)基因对人激素依赖性前列腺癌细胞株LNCa P细胞增殖、凋亡及其雄激素受体(androgen receptor,AR)的影响。方法:3条针对YAP基因的sh RNA慢病毒干扰载体感染LNCa P细胞,经嘌呤霉素筛选后建立稳定感染细胞系,real-time PCR、Western blot分别检测感染后各组细胞YAP、AR m RNA和蛋白水平;选取沉默效果最佳sh RNA慢病毒干扰载体组,以细胞增殖/毒性检测(cell counting kit-8,CCK-8)、平板克隆形成实验(colony formation)检测LNCa P细胞增殖能力;流式细胞仪(flow cytometry,FCM)检测LNCa P细胞凋亡改变。结果:重组慢病毒成功感染前列腺癌LNCa P细胞,经嘌呤霉素筛选后感染效率达95%以上,获得稳定沉默YAP基因LNCa P细胞系。与空白对照组和阴性对照组比较,感染sh RNA慢病毒干扰载体各组中YAP、AR m RNA及蛋白表达水平明显下调(P=0.000),细胞增殖受到明显抑制(P=0.000),细胞凋亡明显增加(P=0.000)。结论:慢病毒介导sh RNA沉默YAP基因能有效下调YAP在LNCa P细胞中表达,并抑制细胞增殖、诱导细胞凋亡;YAP可能作为AR配体参与AR信号通路的调节。Objective：To explore the effect of lentivirus-mediated sh RNA silencing Yes-associated protein（YAP）gene on proliferation,apoptosis,and androgen receptor of hormone dependent human prostate cancer LNCap cells. Methods：Three sh RNA for YAP gene lentiviral interference vectors were infected into LNCa P cells,then the LNCa P cells stably knockdown YAP gene expression were screened by puromycin. Real-time quantitative PCR（real-time PCR）and Western blot were used to detect m RNA and protein expression of YAP and androgen receptor（AR）after lentiviral interference vectors being infected respectively. sh RNA lentiviral vector with the best silence efficiency was acted as experiment group.CCK-8（cell counting kit-8） assay and colony formation were applied to evaluate the cell proliferation in LNCa P cells. Apop tosis of LNCa P cells was examined by flow cytometry（FCM）.Results：YAP-sh RNA lentiviral interference vectors were successfully infected into LNCa P cells. The infection efficiency of lentiviral interference vectors was more than 95% after puro-mycin selection and the stable YAP knockdown in LNCa P cells was established. Compared with those in control group and negative control group,expressions of YAP m RNA and protein were significantly reduced after YAP-sh RNA lentiviral interference vectors being infected（P=0.00）,meanwhile expressions of AR m RNA and protein were also significantly down-regulated（P=0.000）;in YAP-sh RNA lentiviral interference vectors group,cell proliferation was inhibited,and apoptosis was increased significantly（P=0.000）. Conclusions：lentivirus-mediated sh RNA silencing YAP gene can down-regulate the expression of YAP gene,inhibit the proliferation and promote apoptosis in LNCa P. YAP may act as a ligand of AR and regulate androgen receptor signaling pathway.

关 键 词：前列腺癌 雄激素受体 Yes-相关蛋白 慢病毒 

分 类 号：R737.25[医药卫生—肿瘤]