RNA干扰her2／neu基因表达抑制肺癌A549细胞增殖的研究

Inhibition of A549 cell proliferation by RNA interfered gene her2/neu

目的:HER2/neu表达对肺癌细胞生物学的影响尚不明了,文中运用RNA干扰技术抑制HER2/neu表达,并观察对肺癌A549细胞体外增殖的影响。方法:构建针对her2/neu基因的RNA干扰真核表达载体,运用脂质体介导转染A549细胞并用G418筛选出阳性克隆株;半定量RT-PCR和Western检测转染后HER2/neu mRNA及蛋白的表达水平;FCM检测细胞周期分布;MTT法绘制细胞生长曲线观察体外细胞的增值能力。结果:成功构建针对her2/neu基因的RNA干扰真核表达载体(命名为psilence3.1-HER2),转染后使A549细胞HER2/neu mRNA和蛋白的表达较未转染组和阴性对照质粒转染组均显著下调;细胞G0/G1期细胞增加,S期细胞减少;细胞生长曲线右移、增殖速度明显减慢。结论:psilence3.1-HER2能够稳定、高效、特异地抑制肺癌A549内her2/neu基因的表达,可显著抑制细胞增殖。针对her2/neu基因的RNAi策略有望成为肺癌的基因治疗的新选择。

Objective： The effect of HER2/neu expression on the proliferation of A549 cell is not clear. We inhibited the HER2/neu expression with RNA interference （RNAi） technique, and investigated the proliferation of A549 cell in this study. Methods： Firstly a recombinant RNAi expression plasmid targeting to her2/neu gene （ named psilence 3.1-HER2） was constructed. After RNAi plasmids being transfected to A549 cells with Lipofectamine, the positive clones were screened with G418. The expression changes of HER2/neu mRNA and protein were evaluated with RT-PCR and Western Blot respectively; Also FCM analysis, MTF method were applied to measure cell cycle, cell growth of A549 cell respectively after the transfection of psilence 3.1-HER2. Results： The psilence 3.1-HER2 was successfully constructed and can specially inhibit HER2/neu mRNA and protein expression after transfection. FCM results showed that cells transfected with psilence 3.1-HER2 increased accumulation of cells in G0/G1 phase with a decrease in the percentage of cells in S-phase, and the growth curve of cells exhibited slower proliferation. Conclusion： The psilence 3. 1-HER2 can knockdown her2/neu gene specially and efficiently, transfection of psilence 3.1-HER2 contributes to the depression of A549 cell proliferation. The data described here provided a proof of the concept that vector-based RNAi targeting to her2/neu gene could be used therapeutically for lung cancer.