诱导/封闭自噬相关基因7对肾癌786-0细胞自噬的调控作用研究

Effect of overexpression/knockdown Atg7 on the autophagy of 786-0 renal clear carcinoma cell lines

目的探讨诱导/封闭Atg7对肾癌786-0细胞自噬的调控作用。方法构建上调Atg7的慢病毒载体plenti6.3-Atg7,下调Atg7的慢病毒载体sh-Atg-1lv、sh-Atg-2lv,空载对照慢病毒载体sh-scramb-con lv、plenti6.3-GFP,分别转染肾癌786-0细胞系,未转染的786-0细胞系为空白对照。RT-PCR和Western blot检测各组Atg7的表达,研究plenti6.3-Atg7激活Atg7效率和sh-Atg-1lv、sh-Atg-2lv抑制Atg7效率。Western blot检测上述各组细胞LCII/LC3I蛋白比值,研究上调及下调Atg7对肾癌786-0细胞自噬的激活及抑制作用。结果经过筛选,成功构建稳定上调Atg7的786-0/plenti6.3-Atg7xbx细胞系、稳定下调Atg7的786-0/sh-Atg-2lv细胞系、空载对照786-0/sh-scramb-con lv、786-0/plenti6.3-GFP细胞系。与空白对照组比较,Atg7上调组786-0细胞LCII/LC3I比值显著升高(3.31±0.12 vs.2.23±0.10 P<0.01),具有稳定的自噬激活作用;Atg7下调组的786-0细胞LCII/LC3I比值显著降低(1.45±0.11 vs.2.23±0.10 P<0.01),具有稳定自噬抑制作用。结论上调Atg7对肾癌786-0细胞系具有自噬激活作用,下调Atg7对肾癌786-0细胞系具有自噬抑制作用。

Objective To explore the regulative effect of autophagy-related gene Atg7 on the autophagy of 786-0 cell line in renal cell carcinoma （RCC） .Methods Atg7-overexpression plasmid plenti6 .3-Atg7 and Atg7 specific microRNA-express-ing plasmid sh-Atg-2 lv were established and identified .Plenti6 .3-Atg7 and sh-Atg-2 lv were transfected into 786-0 cells ,West-ern blot was used to detect LCII/LC3I respectively .Results 786-0 cells with Atg7-overexpression and Atg7-knockdown were obtained .Cells with Atg7-overexpression showed increased LC3II/LC3I compared with controls（3 .31 ± 0 .12 vs .2 .23 ± 0.10 , P〈0 .01） ,while cells with Atg7-knockdown showed reduced LC3II/LC3I compared with controls（1 .45 ± 0 .11 vs .2.23 ± 0.10 ,P〈0 .01） .Conclusions 786-0 cells with Atg7-overexpression promote autophagy while 786-0 cells with Atg7-knock-down inhibit autophagy .