UbcH10基因沉默联合紫杉醇干预对NCI-H226细胞增殖活性和凋亡的影响

摘要

目的 观察小干扰RNA(siRNA)介导的UbcH10基因沉默联合紫杉醇处理对人肺鳞癌细胞株NCI-H226细胞增殖活性和凋亡的影响.方法 化学合成针对UbcH10基因的siRNA-UbcH10序列,脂质体转染siRNA至NCI-H226细胞(siRNA转染组),转染后24 h,采用Real-Time PCR和Western blotting分别检测UbcH10 mRNA和蛋白的表达水平,以未予任何转染细胞作为空白对照,以阴性序列转染细胞作为阴性对照.以紫杉醇(1μmol/L)处理siRNA转染及未转染NCI-H226细胞(siRNA+紫杉醇组和紫杉醇组),分别于转染后24、48 h收集细胞,MTT比色法检测细胞增殖；转染后24 h收集细胞,流式细胞仪检测细胞凋亡；以未经紫杉醇处理的siRNA转染、阴性序列转染和未予转染的NCI-H226细胞作为siRNA转染组、阴性对照组和空白对照组.结果 Real-Time PCR和Western blotting检测结果显示:转染后24 h,siRNA转染组UbcH10 mRNA和蛋白的表达较空白对照组和阴性对照组显著下调(P＜0.01).MTT比色法检测结果显示,siRNA转染组细胞增殖抑制率显著高于紫杉醇组和对照组(P＜0.05),而siRNA+紫杉醇组细胞增殖抑制率显著高于siRNA转染组(P＜0.05).siRNA转染组细胞凋亡率显著高于紫杉醇组和对照组(P＜0.05),而siRNA+紫杉醇组细胞凋亡率显著高于siRNA转染组(P＜0.05),紫杉醇组与对照组细胞凋亡率比较差异无统计学意义(P＞0.05).结论 UbcH10基因沉默可显著增强NCI-H226细胞对于化疗药物紫杉醇的敏感性.%Objective To investigate the effects of small interfering RNA ( siRNA)-mediated UbcH10 gene silencing combined with paclitaxel treatment on proliferation and apoptosis of human lung squamous carcinoma cell line NCI-H226. Methods siRNA-UbcH10 sequence targeting UbcH10 gene was synthesized chemically, and was transfected into NCI-H226 cells by lipofectin ( siRNA transfection group) . Twenty-four hours after transfection, the expression of UbcH10 mRNA and protein was detected by Real-Time PCR and Western blotting. Cells without transfection were served as blank controls, and those transfected with negative sequence as negative controls. NC1-H226 cells transfected with or without siRNA were treated with paclitaxel ( 1 μmol/L) ( siRNA + paclitaxel group or paclitaxel group) , cell proliferation was detected by MTT assay 24 h and 48 h after transfection, and cell apoptosis was determined by flow cytometry 24 h after transfection. NCI-H226 cells without paclitaxel treatment while with siRNA transfection, negative sequence transfection and no transfection were served as siRNA transfection group, negative control group and blank control group respectively. Results Real-Time PCR and Western blotting revealed that the expression of UbcH10 mRNA and protein in siRNA transfection group was significantly lower than that in blank control group and negative control group (P <0.01) . MTT assay indicated that the cell proliferation inhibition rate in siRNA transfection group was significantly higher than those in paclitaxel group and control group (P<0.05), while the cell proliferation inhibition rate in siRNA + paclitaxel group was significantly higher than that in siRNA transfection group (P<0.05). The cell apoptosis rate in siRNA transfection group was significantly higher than those in paclitaxel group and control group (P < 0. 05), and the cell apoptosis rate in siRNA + paclitaxel group was significantly higher than that in siRNA transfection group (P <0. 05) , while there was no significant difference in the cell apoptosis rate between paclitaxel group and control group ( P > 0. 05 ) . Conclusion UbcH10 gene silencing can significantly increase the sensitivity of NCI-H226 cells to the chemotherapy drug paclitaxel.