CAJ | 학술논문

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。
목적：이용shRNA침묵E3범소련접매Iduna기인，관찰Iduna대비소세포폐암세포계A549세포주기급기상관단백표체적영향。방법채용반전록취합매련반응（RT-PCR）화단백인적법사선Iduna고표체세포계。구건shRNA-Iduna진핵표체재체，순시전염A549세포，단백인적법검측전염효솔。류식세포의검측세포주기변화。단백인적법검측세포주기상관단백적표체。결과단백인적법사선Iduna고표체세포계현시A549화SPC세포중Iduna mRNA화단백수평고우기타세포계。shRNA-Iduna전염A549세포계후，류식세포의검측세포주기현시간우조G0/G1기비례증가，S기비례상대감소（ P ＜0.05）。단백인적법검측여세포주기상관적G1/S기조공단백[세포주기단백（cyclin）A、cyclin D1、cyclin E、CDK2、CDK4、CDK6、P21、P27、P53등]적표체정황，결과현시간우조cyclin D1、cyclin E화CDK4단백표체감소（ P ＜0.05）。결론 Iduna가능통과조공cyclin D1、cyclin E화CDK4단백적표체，사세포조체우G0/G1기，종이조절폐암세포주기진정。
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.