Bcl-XL siRNA对肺腺癌细胞A549/DDP药物敏感性及凋亡的影响

Bcl-XL siRNA sensitisizes cisplatin-resistant human lung adenocarcinoma cells A549 to cisplatin

目的:探讨小干扰RNA沉默Bcl-XL对肺腺癌耐药细胞株A549/DDP药物敏感性的影响及其凋亡机制。方法:将Bcl-XLsiRNA质粒载体转染至A549/DDP细胞,MTT、流式细胞仪检测细胞药物敏感性的改变;丫啶橙/溴化乙锭(AO/EB)染色法检测细胞凋亡;RT-PCR检测Bcl-XLmRNA水平;Western-Blot检测Bcl-XL、caspase-3蛋白表达的变化。结果:分别用0.2、2、20、200μg/mL顺铂处理细胞48 h,MTT显示转染Bcl-XLsiRNA细胞组A570吸光度值较阴性siRNA细胞组和未转染对照组明显降低,细胞生长抑制率明显增高;用20μg/mL顺铂处理细胞48 h,流式细胞仪检测,转染Bcl-XLsiRNA组凋亡率较转染阴性siRNA组和未转染组增加;AO/EB染色显示转染Bcl-XLsiRNA的细胞较转染阴性siRNA及未转染者凋亡率增加;转染Bcl-XLsiRNA降低了Bcl-XLmRNA和蛋白水平,升高了caspase-3活性形式蛋白表达。结论:Bcl-XLsiRNA特异性下调A549/DDP细胞Bcl-XL的表达,活化caspase-3蛋白,促进A549/DDP细胞凋亡,增强该肺腺瘤细胞对顺铂的敏感性。

AIM： To investigate the effect of Bcl-XL siRNA on apoptosis and drug sensitization in cisplatin-resistance human lung adenocarcinoma cell line A549/DDP and to determine the inhibitory effect of the Bcl-XL siRNA on the expression of Bcl-XL gene in cells A549/DDP. METHODS： Bcl-XL siRNA and negative siRNA plasmid vector were stably transfected into A549/DDP cells. Drug sensitivity of the cells to eisplatin （DDP） was analyzed with MTT and flow cytometry. Spontaneous apoptosis of cells was detected by AO/EB dyeing. RT-PCR and Westem-blot were used to detect the target gene expression. RESULTS： MTT results showed that Bcl-XL siRNA transfectants had a higher cell inhibition rate than negative siRNA or untreated cells after treated with 0.2, 2, 20, 200 μg/mL DDP. Moreover, flow eytometry results demonstrated that Bcl-XL siRNA cells had increased apoptosis rate after addition of 20 μg/mL DDP. Spontaneous apoptosis of cells were significantly increased in Bcl-XL siRNA stable transfectants. The mRNA and protein expression level of Bcl-XL in Bcl-XL siRNA stable transfectants were obviously reduced compared with negative siRNA transfectants or untreated cells, moreover, Bcl-XL siRNA increased the activity of active caspase-3. CONCLUSION： siRNA targeting Bcl-XL gene can specifically down-regulate Bcl-XL expression in A549/DDP cells, increase cell spontaneous apoptosis and sensitize cells to DDP. Bcl-XL siRNA may be a potential therapy agent against human lung adenocarcinoma.