敲减缺氧诱导因子-2α联合索拉非尼对肾癌细胞促凋亡作用的研究

Study on the Pro-apoptotic Effect of the HIF-2αKnockdown Combined with Sorafenib on Renal Cancer Cells

目的探讨敲减缺氧诱导因子-2α(HIF-2α)与索拉非尼联用的促肾癌凋亡作用。方法OS-RC-2细胞和786-O细胞在常氧或缺氧下,采用蛋白质印迹技术(Western blot)检测HIF-1α、HIF-2α蛋白表达;在缺氧下,将两种细胞分为对照组、索拉非尼组、Control shRNA组、索拉非尼+HIF-1αshRNA组、索拉非尼+HIF-2αshRNA组,用Western blot检测HIF-1α、HIF-2α、Caspase3蛋白表达,用流式细胞仪及TUNEL方法检测细胞凋亡率。结果HIF-1α和HIF-2α蛋白缺氧时均比常氧状态下升高。缺氧时,与对照组相比,索拉非尼可抑制两种细胞HIF-1α蛋白表达,促进HIF-2α蛋白表达。流式细胞凋亡率:与对照组比较,索拉非尼可促使两种细胞凋亡增加(P<0.05);与Control shRNA组比较,索拉非尼+HIF-2αshRNA组细胞凋亡增加,差异有统计学意义(P<0.05)。TUNEL凋亡率:与对照组比较,索拉非尼可促使两种细胞凋亡增加(P<0.05);与Control shRNA组比较,索拉非尼+HIF-2αshRNA组细胞凋亡增加,差异有统计学意义(P<0.05)。Pro-Caspase3表达下调,Cleaved Caspase3蛋白表达上调。结论缺氧环境下,敲减HIF-2α后与索拉非尼联用可通过Caspase3蛋白对肾癌细胞有协同抗凋亡作用。

Objective To investigate the pro-apoptotic effect of hypoxia-inducible factor-2α(HIF-2α)combined with sorafenib on renal cancer cells.Methods The protein expression of HIF-1αand HIF-2αwas detected by Western blot in the OS-RC-2 and 786-O cell lines under the normoxic or hypoxic condition.The OS-RC-2 and 786-O cell lines under the hypoxic condition were divided into the control group,sorafenib group,control shRNA group,sorafenib+HIF-1αshRNA group and sorafenib+HIF-2αshRNA group.The protein expression of HIF-1α,HIF-2αand Caspase 3 was detected by Western blot and the cell apoptosis was detected by flow cytometry and TUNEL assay.Results The protein expression of HIF-1αand HIF-2αwas higher under the hypoxic condition than under the normoxic condition.Sorafenib inhibited the protein expression of HIF-1αand promoted that of HIF-2αin the OS-RC-2 and 786-O cell lines when compared with the control group.As to the apoptosis rate of flow cytometry,sorafenib significantly increased the apoptosis of the two cell lines when compared with the control group(P<0.05)and the apoptosis was promoted more significantly in the sorafenib+HIF-2αshRNA group than in the control shRNA group(P<0.05).As to the apoptosis rate of TUNEL assay,sorafenib significantly increased the apoptosis of the two cells when compared with the control group(P<0.05)and the apoptosis was significantly elevated in the sorafenib+HIF-2αshRNA group when compared with the control shRNA group(P<0.05).The expression of pro-Caspase3 was down-regulated and that of cleaved Caspase3 was up-regulated.Conclusion The HIF-2αknockdown combined with sorafenib has a synergistic anti-apoptotic effect on renal cancer cells via the Caspase3 protein under the hypoxic condition.