HIF-2α基因与人肺腺癌A549细胞株对顺铂耐药性的关系

Relationship between HIF-2α Gene and Chemotherapy Resistance of Lung Adenocarcinoma A549 Cells to Cisplatin

目的:探讨小干扰RNA(small interfering RNA,siRNA)沉默缺氧诱导因子2α(hypoxia inducible factor-2 alpha,HIF-2α)基因表达后,人肺腺癌A549细胞株对顺铂耐药性的变化。方法:构建靶向HIF-2α基因的siRNA真核表达载体并转染A549细胞,采用Real-time PCR检测HIF-2αmRNA的表达,采用Western blotting检测HIF-2α及多药耐药基因1(multidrug resistance-1,Mdr-1)蛋白的表达,采用MTT法检测顺铂处理后细胞的存活率。结果:在HIF-2αsiRNA转染A549细胞后24、48 h后,HIF-2α在mRNA和蛋白水平的表达与空白对照组相比均显著下调(均P<0.01),Mdr-1蛋白的表达水平也明显降低(均P<0.05)。与空白对照组相比,HIF-2αsiRNA组细胞对顺铂的敏感性明显增加,各时间点的IC50值均显著降低,差异有统计学意义(均P<0.01)。结论:靶向HIF-2α的siRNA表达载体能够有效抑制HIF-2α基因的表达,从而逆转A549细胞对顺铂的化疗耐药性。

Objective：To investigate the effect of silencing hypoxia inducible factor-2 alpha （HIF-2α） gene expression by small interfering RNA （siRNA） on chemotherapy resistance of A549 cells to cisplatin.Methods：The siRNA eukaryotic expression vectors targeting HIF-2α gene were constructed and transfected into A549 cells.The expression of HIF-2α mRNA was detected by real-time PCR.The expression of HIF-2α and multidrug resistance-1 （Mdr-1） proteins were detected by Western blotting.MTT assay was used to explore the survival rate of A549 cells under the treatment of cisplatin.Results：After transfection of HIF-2α siRNA into A549 cells for 24 and 48 h,the expression levels of HIF-2α mRNA and protein were both down-regulated significantly compared with those in the blank group （all P＜0.01）.Meanwhile,the protein expression level of Mdr-1 also decreased significantly （all P＜0.05）.The sensitivity of A549 cells to cisplatin was significantly increased in HIF-2α siRNA transfection group compared with that in the blank group.The IC50 value at each time point was significantly decreased,and the difference was statistically significant （all P＜0.01）.Conclusions：The siRNA expression vector targeting HIF 2α constructed in this study can effectively inhibit the expression of HIF-2α gene,and thus reversing the chemotherapy resistance of A549 cells to cisplatin.