慢病毒介导的YAP基因沉默对肾癌细胞增殖和凋亡的影响

发布时间：2019-05-13 12:18

【摘要】：目的:探讨慢病毒干扰载体沉默Yes-相关蛋白（Yes-associatedprotein, YAP）对人肾癌786-0细胞增殖和凋亡的影响。 方法:体外培养肾癌786-0细胞，构建针对YAP基因的shRNA慢病毒干扰载体并转染786-0细胞。RT-PCR和Western blot分别检测干扰786-0细胞前后YAP及其下游转录因子TEAD1mRNA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情况。 结果:构建了3条针对YAP基因的YAP-shRNA慢病毒干扰载体和1条阴性对照慢病毒载体并成功转染786-0细胞；YAP-shRNA慢病毒干扰载体转染96小时后，可显著下调786-0细胞YAP mRNA及蛋白表达水平(P=0.000)，并且TEAD1mRNA及蛋白表达水平也明显下降(P=0.000)；与空白对照组和阴性对照组细胞相比，转染组细胞增殖明显抑制，细胞凋亡显著增加(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786-0细胞中表达，进而抑制细胞增殖并促进细胞凋亡。
[Abstract]:Aim: to investigate the effect of lentivirus interference vector silencing Yes- associated protein (Yes-associatedprotein, YAP) on proliferation and apoptosis of human renal cell carcinoma 786 cells. Methods: 786 cells of renal cell carcinoma were cultured in vitro. The shRNA lentivirus interference vector targeting YAP gene was constructed and transfected into 786 / 0 cells. The expression of YAP and its downstream transcription factor TEAD1mRNA and protein were detected by RT-PCR and Western blot before and after interfering with 786 / 0 cells, respectively. the expression of YAP and its downstream transcription factor TEAD1mRNA and protein were detected before and after interference. CCK-8 (cell counting kit-8) was used to detect the changes of cell proliferation after silencing YAP, and flow cytometry (flow cytometry, FCM) was used to detect the changes of apoptosis and cycle. Results: three YAP-shRNA lentivirus interference vectors targeting YAP gene and one negative control lentivirus vector were constructed and successfully transformed into 786 cells. After 96 hours of transfection with YAP-shRNA lentivirus interference vector, the expression of YAP mRNA and protein in 786 / 0 cells was significantly down-regulated (P 鈮

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