shRNA介导的Iduna沉默对肺癌A549细胞系细胞周期及其相关蛋白表达的影响

Effect of Iduna gene silencing by shRNA on lung cancer cell line A549 cell cycle and related regulatory proteins expression

目的利用shRNA沉默E3泛素连接酶Iduna基因,观察Iduna对非小细胞肺癌细胞系A549细胞周期及其相关蛋白表达的影响。方法采用反转录聚合酶链反应(RT-PCR)和蛋白印迹法筛选Iduna高表达细胞系。构建shRNA-Iduna真核表达载体,瞬时转染A549细胞,蛋白印迹法检测转染效率。流式细胞仪检测细胞周期变化。蛋白印迹法检测细胞周期相关蛋白的表达。结果蛋白印迹法筛选Iduna高表达细胞系显示A549和SPC细胞中Iduna mRNA和蛋白水平高于其他细胞系。shRNA-Iduna转染A549细胞系后,流式细胞仪检测细胞周期显示干扰组G0/G1期比例增加,S期比例相对减少(P<0.05)。蛋白印迹法检测与细胞周期相关的G1/S期调控蛋白[细胞周期蛋白(cyclin)A、cyclin D1、cyclin E、CDK2、CDK4、CDK6、P21、P27、P53等]的表达情况,结果显示干扰组cyclin D1、cyclin E和CDK4蛋白表达减少(P<0.05)。结论 Iduna可能通过调控cyclin D1、cyclin E和CDK4蛋白的表达,使细胞阻滞于G0/G1期,从而调节肺癌细胞周期进程。

Objective To study the effects of short hairpin RNA （shRNA） mediated gene silencing of Iduna on non-small cell lung cancer （NSCLC） cell line A549 cell cycle and related regulatory proteins expression .Meth-ods RT-PCR and Western blotting were used to screen NSCLC cell line with high Iduna expression at mRNA and protein levels .shRNA-Iduna eukaryotic expression vector was constructed and transiently transfected into A 549 cells .The expression of Iduna gene was evaluated by Western blotting .The cell cycle was detected by flow cytom-etry .Cell cycle related proteins expression was measured by Western blotting .Results The cell cycle was ana-lyzed by flow cytometry ,and the results demonstrated that knockdown of Iduna in A549 cells increased the per-centage of cells in the G0/G1 phase and decreased the percentage of cells in S stage（ P 〈0.05） .Western blotting results revealed correlations between the levels of Iduna in lung cancer cells and the expression of cell cycle-related regulatory proteins including cyclin D1 ,cyclin E and CDK4 .Conclusion Iduna may regulate cell cycle progression by inducing the G0/G1-S transition and regulating the expression of cyclin D 1 and cyclin E and CDK4.