XAF1基因对顺铂诱导肺腺癌A549细胞凋亡作用的研究

Effect of XAF1 gene on lung adenocarcinoma A549 cell apoptosis induced by cisplatin

目的:通过顺铂诱导人肺腺癌细胞A549的凋亡,研究X染色体连锁凋亡抑制蛋白相关因子1(X-linked inhibitor of apoptosis protein associated factor-1,XAF1)基因在A549细胞中的表达情况,初步探讨该基因在凋亡中的作用。方法:采用4μg/mL浓度的顺铂处理A549细胞,MTT法检测细胞的增殖抑制率;AnnexinⅤ/7-AAD检测A549细胞凋亡情况;RT-PCR和蛋白质印迹法分别检测A549凋亡后XAF1mRNA和蛋白的表达;Caspase-3试剂盒检测A549细胞凋亡后Caspase-3的活性。结果:用4μg/mL浓度顺铂处理A549细胞2、12和24h后,A549细胞增殖抑制率分别为(3.40±0.65)%、(8.31±0.73)%和(14.72±0.24)%,与对照组(1.03±0.28)%相比均明显增高,P值分别为0.004、0.000和0.000,细胞增殖抑制率随时间增加而增强;A549细胞凋亡率分别为(4.35±0.95)%、(9.69±2.60)%和(22.35±1.24)%,与对照组(1.39±0.21)%相比均明显增加,P值分别为0.006、0.005和0.000,细胞凋亡率随顺铂作用时间延长而增加;实验组XAF1mRNA表达水平分别为(0.199±0.029)、(0.654±0.093)和(1.216±0.101),对照组为(0.091±0.020),XAF1mRNA表达水平随作用时间的延长呈增加趋势,P值分别为0.006、0.001和0.000;实验组XAF1蛋白表达水平分别为(0.322±0.041)、(0.508±0.014)和(0.901±0.014),对照组为(0.124±0.007),XAF1蛋白表达水平随作用时间的延长呈增加趋势,P值分别为0.001、0.000和0.000;Caspase-3活性(A405nm)分别为(34.745±3.781)、(69.524±3.096)和(94.787±5.429),对照组为(21.914±1.962),Caspase-3活性随顺铂作用时间的延长而增加。结论:顺铂诱导A549细胞凋亡可以引起XAF1表达增加,且随细胞凋亡水平的提高而增加,提示XAF1可能参与顺铂诱导人肺腺癌A549细胞的凋亡过程。

OBJECTIVE：Based on the apoptosis process of A549cells induced by cisplatin,we researched the expression and functions of the XAF1gene.METHODS：The proliferation inhibition rate after A549cells treated with the cisplatin of 4μg/mL was detected by MTT.The A549cell apoptosis was detected by AnnexinⅤ/7-AAD.The XAF1mRNA and protein expression was monitored by the RT-PCR and western blotting.The Caspase-3activity in apoptotic A549cells was detected by the Caspase-3assay.RESULTS：The cell proliferation inhibition rate increased with the cisplatin treating duration.The cell proliferation inhibition rates of the experiment group were 2h（3.40±0.65）%,12h（8.31±0.73）%,and 24h（14.72±0.24）%respectively.Compared with the cell proliferation inhibition rate（1.03±0.28）%of the control group,Pvalues were 0.004,0.000,and 0.000respectively.The cell apoptosis rate increased with the cisplatin treating duration.The apoptosis rates were 2h（4.35±0.95）%,12h（9.69±2.60）%,and 24h（22.35±1.24）% respectively.Compared with the cell apoptosis rate（1.39±0.21）% of the control group,Pvalues were 0.006,0.005,and 0.000respectively.The mRNA expressions of the experiment group were 2h（0.199±0.029）,12h（0.654±0.093）,and 24h（1.216±0.101）respectively,while the mRNA expression of the control group was（0.091±0.020）.The XAF1mRNA expression increased with the cell apoptosis rate.Compared with the mRNA expression of the control group,Pvalues were0.006,0.001,and 0.000respectively.The XAF1protein expressions of the experiment group were 2h（0.322±0.041）,12h（0.508±0.014）,and 24h（0.901±0.014）respectively,while the XAF1protein expression of the control group was（0.124±0.007）.The protein expression of the experiment group was increased with the cell apoptosis rate.Compared with the XAF1protein expression of the control group,Pvalues were 0.001,0.000,and 0.000respectively.The Caspase-3activity increased with the cisplatin treating duration.The Caspase-3activity values of the experiment group were 2h（34.745±3.781）,12h（69.524±3.096）,and 24h（94.787±5.429）respectively.The Caspase-3activity value of the control group were（21.914±1.962）.CONCLUSIONS：A549cell apoptosis induced by cisplatin may increase the XAF1gene expression.The expression level increases according to the improvement of A549cell apoptosis level,which indicates that the XAF1gene takes effects in the apoptosis process of A549cells induced by cisplatin.