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

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。

Abstract:

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 immunofluores－cence 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 si－lencing 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 in－fected，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.