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

摘要:

目的：探讨慢病毒介导shRNA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LNCaP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shRNA慢病毒干扰载体感染LNCaP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 mRNA和蛋白水平；选取沉默效果最佳shRNA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LNCaP细胞增殖能力；流式细胞仪（flow cytometry，FCM）检测LNCaP细胞凋亡改变。结果：重组慢病毒成功感染前列腺癌LNCaP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LNCaP细胞系。与空白对照组和阴性对照组比较，感染shRNA慢病毒干扰载体各组中YAP、AR mRNA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shRNA沉默YAP基因能有效下调YAP在LNCaP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。

Abstract:

Objective：To explore the effect of lentivirus-mediated shRNA silencing Yes-associated protein（YAP） gene on prolifera－tion，apoptosis，and androgen receptor of hormone dependent human prostate cancer LNCap cells. Methods：Three shRNA for YAP gene lentiviral interference vectors were infected into LNCaP cells，then the LNCaP cells stably knockdown YAP gene expression were screened by puromycin. Real-time quantitative PCR（real-time PCR） and Western blot were used to detect mRNA and protein ex－pression of YAP and androgen receptor（AR） after lentiviral interference vectors being infected respectively. shRNA 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 LNCaP cells. Apoptosis of LNCaP cells was examined by flow cytometry（FCM）. Results：YAP-shRNA lentiviral interference vectors were suc－cessfully infected into LNCaP cells. The infection efficiency of lentiviral interference vectors was more than 95% after puro-mycin selection and the stable YAP knockdown in LNCaP cells was established. Compared with those in control group and negative control group，expressions of YAP mRNA and protein were significantly reduced after YAP-shRNA lentiviral interference vectors being infected（P=0.00），meanwhile expressions of AR mRNA and protein were also significantly down-regulated（P=0.000）；in YAP-shRNA lentiviral interference vectors group，cell proliferation was inhibited，and apoptosis was increased significantly（P=0.000）. Conclusions：lentivirus-mediated shRNA silencing YAP gene can down-regulate the expression of YAP gene，inhibit the proliferation and promote apoptosis in LNCaP. YAP may act as a ligand of AR and regulate androgen receptor signaling pathway.