ERCC1和BRCA2基因共沉默有效逆转耐药肺癌细胞对顺铂的耐药性

Co-silencing of ERCC1 and BRCA2 genes effectively reverse resistance of cisplatin resistant lung cancer cells to cisplatin

目的:探讨沉默ERCC1和BRCA2基因后逆转顺铂耐药肺癌细胞株(A549/DDP)对顺铂耐药性的效应及其分子机制。方法:合成针对ERCC1和BRCA2基因的siRNAs(ERCC1-siRNA和BRCA2-siRNA),用脂质体转染试剂将其分别或共同转染于A549/DDP细胞。蛋白质印迹法测定转染前后A549/DDP细胞ERCC1和BRCA2蛋白的表达以确定转染成功,并检测转染前后A549/DDP细胞经顺铂诱导的FANCD2单泛素化水平及BRCA1和RAD51蛋白表达水平;CCK-8法测定转染前后A549/DDP细胞对顺铂的敏感性;Annexin V/PI流式细胞术测定转染前后A549/DDP细胞凋亡率;免疫荧光法测定转染前后A549/DDP细胞核内FANCD2核聚小体和RAD51核聚小体的表达。结果:A549/DDP细胞转染ERCC1-siRNA和BRCA2-siRNA后ERCC1和BRCA2蛋白表达均明显降低(P<0.05),表明转染有效。转染ERCC1-siRNA后顺铂诱导的FANCD2蛋白单泛素化水平和核聚小体表达均明显降低。转染BRCA2-siRNA后顺铂诱导的BRCA1和RAD51蛋白表达及RAD51核聚小体表达明显降低。与此同时,转染ERCC1-siRNA或BRCA2-siRNA均可增加A549/DDP细胞对顺铂的敏感性,以及增强顺铂诱导的细胞凋亡作用,共转染ERCC1-siRNA和BRCA2-siRNA则进一步增强顺铂对A549/DDP的细胞毒性作用。结论:沉默ERCC1和BRCA2基因可通过抑制A549/DDP细胞株范可尼贫血通路和同源重组通路的DNA损伤修复功能,逆转A549/DDP细胞对顺铂的耐药性;两个基因共沉默对顺铂耐药性的逆转作用更为明显。

Objective： To investigate the effect of co-silencing of ERCC1 and BRCA2 genes on reversing cisplatin resistance in resistant lung cancer cell A549/DDP. Methods： Small interference RNAs （siRNAs） targeting ERCC1 and BRCA2 genes （ERCC1- and BRCA2-siRNA） were transfected into A549/DDP cells using lipofectamine 2000. Transfection efficacy was verified by protein expressions of ERCC1 and BRCA2 detected with Western blotting. BRCA1 and RAD51 proteins expressions as well as the FANCD2 monoubiq- uitination induced by cisplatin were determined by Western blotting. The cell proliferation and apoptosis rate in A549/DDP ceils pre- and post-treatment with cisplatin were measured by CCK-8 assay and flow cy- tometry using Annexin V/PI strain, respectively, before and after transfection. The formation of FANCD2 and RADS1 nuclear foci were detected using immunofluorescence staining. Results： Cisplatin-induced FANCD2 monoubiquitination and FANCD2 nuclear loci formation were significantly decreased after transfection of ERCCI-siRNA. The expressions of BRCA1 and RADS1 proteins and RAD51 nuclear foci induced by cisplatin were markedly reduced after transfection of BRCA2-siRNA （P 〈 0.05 ）. Individual transfection of ERCCI-siRNA or BRCA2-siRNA suppressed cell proliferation and promoted cell apoptosis produced by cisplatin. Co-transfection of the two siRNAs further enhanced the cytotoxicity of cisplatin to ASd9/DDP cells, as compared to transfection of ERCCI-siRNA or BRCA2-siRNA alone. Conclusion： Silencing of ERCC1 and BRCA2 gene could reverse the resistance of resistant lung cancer cells to cisplatin through inhibition of the FA pathway and the HR pathway respectively, and the reversion effect of cisplatin resistance was further potentiated by co-silencing of ERCC1 and BRCA2 genes.