HIF-2α对自噬介导的肝癌细胞顺铂耐药的影响及机制

Effect of HIF-2αon cisplatin resistance in human hepatocellular carcinoma cells and its mechanism

目的探究低氧诱导因子-2α(HIF-2α)对自噬介导的人肝癌阿霉素耐药细胞株(HepG2/ADM)顺铂(cisplatin)耐药的影响及机制,以期为疾病的临床治疗提供新思路。方法将人肝癌细胞HepG2和HepG2/ADM分为7组并加入相应试剂:(1)HIF-2α-siRNA+cisplatin组;(2)HIF-2α-siRNA组;(3)NC-siRNA组;(4)3-甲基腺嘌呤(3-MA)+cisplatin组;(5)cisplatin组;(6)3-MA组;(7)对照组。采用Western blot法检测微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、Beclin 1、HIF-2α蛋白表达水平,流式细胞仪检测细胞凋亡率,CCK-8试剂盒检测细胞增殖抑制率。结果cisplatin组HepG2、HepG2/ADM细胞中LC3-Ⅱ、Beclin 1、HIF-2α蛋白表达水平及细胞凋亡率较对照组均显著升高(均P<0.05)。3-MA组HepG2/ADM细胞中LC3-Ⅱ、Beclin 1蛋白表达水平较对照组均显著下降(均P<0.05)。3-MA+cisplatin组HepG2/ADM细胞凋亡率较cisplatin组、3-MA组和对照组均显著升高(均P<0.05);cisplatin组HepG2/ADM细胞凋亡率较3-MA组和对照组均显著升高(均P<0.05)。HIF-2α-siRNA组HepG2/ADM细胞中HIF-2α、LC3-Ⅱ、Beclin 1蛋白表达水平较NC-siRNA组均显著下降(均P<0.05)。HIF-2α-siRNA+cisplatin组HepG2/ADM细胞凋亡率较HIF-2α-siRNA组、NC-siRNA组和cisplatin组均显著升高(均P<0.05);HIF-2α-siRNA组和NC-siRNA组HepG2/ADM细胞凋亡率较cisplatin组均显著降低(均P<0.05)。沉默HIF-2α表达后,随cisplatin处理浓度增加,HepG2/ADM细胞增殖抑制率升高。结论HIF-2α可能通过调节自噬影响肝癌细胞HepG2/ADM的cisplatin耐药性,因此临床上可通过降低HIF-2α表达水平,提高cisplatin对肝癌细胞HepG2/ADM的杀伤作用,为晚期肝癌治疗提供新思路。

Objective To explore the effect and mechanism of hypoxia inducible factor-2α(HIF-2α)on cisplatin resistance in hepatocellular carcinoma HepG2/ADM cells.Methods Human hepatocellular carcinoma HepG2/ADM cells were divided into(1)HIF-2α-siRNA+cisplatin group.(2)HIF-2α-siRNA group.(3)NC-siRNA group.(4)3-MA+cisplatin group.(5)cisplatin group.(6)autophagy inhibitor 3-methyladenine(3-MA)group.(7)control group,and treated accordingly.The protein expression levels of microtubule associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),Beclin 1 and hypoxia inducible factor-2α(HIF-2α)were detected by Western blotting(WB).The apoptosis rate was detected by flow cytometry,and the inhibition rate of cell proliferation was detected by CCK-8 kit.Results The apoptotic rate of HepG2 and HepG2/ADM cells and the protein expression levels of LC3Ⅱ,Beclin 1 and HIF-2αin cisplatin group were significantly higher than those in control group(all P<0.05).The protein expression levels of LC3Ⅱand Beclin 1 in HepG2/ADM cells in 3-mA group were significantly lower than those in control group(all P<0.05).The apoptosis rate of HepG2/ADM cells in 3-mA+cisplatin group was significantly higher than that in cisplatin group,3-mA group and control group(all P<0.05),the apoptosis rate of HepG2/ADM cells in cisplatin group was significantly higher than that in 3-mA group and control group(all P<0.05).The expression levels of HIF-2α,LC3Ⅱand Beclin 1 in HepG2/ADM cells in HIF-2α-siRNA group were significantly lower than those in NC-siRNA group(all P<0.05).The apoptosis rate of HepG2/ADM cells in HIF-2α-siRNA+cisplatin group was significantly higher than that in HIF-2α-siRNA group,NC-siRNA group and cisplatin group(all P<0.05),the apoptosis rate of HepG2/ADM cells in HIF-2α-siRNA group and NC-siRNA group was significantly lower than that in cisplatin group(all P<0.05).The proliferation inhibition rate of HepG2/ADM cells increased with the increase of cisplatin concentration after silencing HIF-2αexpression.Conclusion HIF-2α may affect the cisplatin resistance of hepatocellular carcinoma HepG2/ADM cells through regulating autophagy.