敲降缺氧诱导因子的表达量对缺氧条件下肾癌细胞迁移能力的影响

目的探讨抑制缺氧诱导因子(hypoxia inducible factor,HIF)的表达对缺氧条件下的肾癌细胞活力、凋亡、迁移能力的影响及机制。方法实验分为5组:对照组(常氧条件)、缺氧组(未转染缺氧条件下细胞)、转染对照组(转染不含目的蛋白的siRNA)、siRNA-HIF1α组、siRNA-HIF2α组。应用MTT法观察细胞的活力,流式细胞术检测细胞的凋亡率,Transwell小室法分析细胞的迁移、侵袭能力变化,Western blot检测细胞中HIF1α/HIF2α、Cleaved Caspase-3、E-cadherin、Snail蛋白的表达量。结果缺氧显著降低细胞的凋亡率和Cleaved Caspase-3蛋白水平(P <0. 05),显著增加迁移、侵袭细胞数(P <0. 05)。与缺氧组相比,siRNA-HIF1α组细胞的凋亡率以及迁移能力的差异无统计学意义;但siRNA-HIF2α组细胞的凋亡率、Cleaved Caspase-3蛋白水平显著增加(P <0. 05),其迁移、侵袭细胞数显著降低(P <0. 05)。与对照组相比,抑制HIF1α和HIF2α的表达显著降低E-cadherin的表达(P <0. 05)、升高Snail的表达(P <0. 05);与缺氧组相比,siRNA-HIF1α组细胞中的E-cadherin、Snail的表达水平差异无统计学意义,siRNA-HIF2α组显著增加E-cadherin的表达水平(P <0. 05)、显著降低Snail的表达水平(P <0. 05)。结论敲降HIF2α的表达量抑制缺氧环境下肾癌细胞的活力、诱导肾癌细胞凋亡、降低肾癌细胞迁移能力,其作用机制可能与E-cadherin、Snail的表达量变化有关。

Effect of knockdown on the expression of hypoxia inducible factor on migration ability of renal cells under hypoxic condition

Purpose To investigate the effect of inhibiting hypoxia inducible factor 1α/2α(HIF1α/HIF2α) expression on cell activity,apoptosis and migration ability of renal carcinoma cells under hypoxia and its mechanism. Methods The cells were divided into 5 groups :control group (normoxic condition),hypoxia group (hypoxia cells without transfection),transfected control group (transfection of siRNA without target protein),siRNA-HIF1α group,siRNA-HIF2α group. The cell activity was measured by MTT assay. The cell apoptotic rate was analyzed by flow cytometry. The migration and invasion abilities of the cells were detected by Transwell chamber assay. The protein expression levels of HIF1α/HIF2α,Cleaved Caspase-3,E-cadherin and Snail in the cells were determined by Western blot. Results Hypoxia significantly decreased cell apoptosis rate and Cleaved Caspase-3 protein level ( P <0.05),increased the number of migration and invasion cells ( P <0.05). Compared with hypoxia group,the apoptotic rate and migration ability in siRNA-HIF1α group were not significantly different,however,the apoptosis rate and Cleaved Caspase-3 of siRNA-HIF2α group increased significantly ( P <0.05),and the cell number of migration and invasion decreased significantly ( P <0.05). The expression of E-cadherin was significantly lower than that in control group ( P <0.05) by inhibited the expression of HIF1α and HIF2α,and the expression of Snail was significantly higher ( P <0.05). Compared with hypoxia group,there was no significant difference in siRNA-HIF1α group with the expression of E-cadherin and Snail in cells,but the level of E-cadherin increased significantly and Snail was significantly decreased in siRNA-HIF2α group ( P <0.05). Conclusion Knockdown of HIF2α expression inhibits the cell activity in renal cancer cells under hypoxia,induces apoptosis and decreases the migration ability. The mechanism may be related with regulating the E-cadherin and Snail expression.