siRNA下调EIF4E基因对人乳腺癌MDA-MB-231细胞增殖和周期的影响

The effects of EIF4E gene down-regulation induced by siRNA on the proliferation and cell cycle of human breast cancer cell line MDA-MB-231

目的:应用小干扰RNA(small interfering RNA, siRNA)乳腺癌MDA-MB-231细胞中真核细胞翻译起始因子4E (eukaryotic translation initiation factor 4E, EIF 4E )基因的表达,探讨其对乳腺癌细胞增殖和生长周期的影响。方法:构建针对EIF 4E 基因的siRNA表达质粒,采用脂质体法将表达质粒pGPU6/GFP/Neo-EIF4E转染人乳腺癌MDA-MB-231细胞;分别采用RT-PCR、Western 印迹法和免疫细胞化学法检测转染EIF4E siRNA后, 对MDA-MB-231细胞中EIF4E及cyclinD1 mRNA和蛋白的表达水平的影响;MTT法、平板克隆形成实验和FCM检测MDA-MB-231细胞增殖活性、克隆形成率及细胞周期。结果:成功构建EIF4E siRNA表达质粒。RT-PCR、Western 印迹法和免疫细胞化学法检测结果显示,MDA-MB-231细胞中EIF4E及cyclinD1 mRNA和蛋白表达均明显下降(P <0.05)。转染EIF4E siRNA组细胞生长缓慢,集落形成数明显减少,与空白对照组和转染空载体组比较,差异有统计学意义(P<0.05)。FCM检测结果显示,EIF4E siRNA转染组G1期细胞所占百分比为(71.30±0.47)%, 较空白对照组的( 53.10±0.43)%明显增加,而S期细胞所占百分比为(12.53±0.13)%,较空白对照组的(26.47±0.38)%明显减少(P<0.05)。结论:EIF4E siRNA可显著下调EIF4E基因在乳腺癌MDA-MB-231细胞中的表达水平,在一定程度上抑制乳腺癌细胞的增殖,有可能成为临床治疗乳腺癌的靶点之一。

Objective：To study the effect of down-regulation of eukaryotic translation initiation factor 4E（EIF4E） induced by the siRNA expression plasmid on the proliferation and cell cycle of human breast cancer cell line MDA-MB-231.Methods：The siRNA expression plasmid pGPU6/GFP/Neo-EIF4E was constructed and then transfected into breast cancer MDA-MB-231 cells by LipofectAMINE2000.The expressions of EIF4E and cyclin D1 mRNAs and proteins were examined by RT-PCR,Western blotting and the immunocytochemistry method,respectively.The MTT method,plate colony formation assay and flow cytometry were performed to detect the proliferation ability,cell colony formation rate and cell cycle distribution of MDA-MB-231 cells transfected with siRNA expression plasmid.Results：The siRNA expression plasmid pGPU6/GFP/Neo-EIF4E was constructed successfully.The expressions of EIF4E and cyclin D1 mRNAs and proteins were all decreased markedly after transfection with pGPU6/GFP/Neo-EIF4E（P0.05）.The growth of MDA-MB-231 cells in the pGPU6/GFP/Neo-EIF4E-tranfected group was inhibited and the cell colony formation rate was statistically decreased（P0.05） as compared with those in the blank control and the pGPU6/GFP-transfected groups.The result of flow cytometry showed that the percentage of cells in G1 phase in the pGPU6/GFP/Neo-EIF4E-tranfected group was higher than that in the blank control group（71.30%±0.47% vs 53.10%±0.43%）;however,the percentage of cells in S phase was signifcantly lower（12.53%±0.13% vs 26.47%±0.38%,P0.05）.Conclusion：EIF4E siRNA can down-regulate EIF4E gene expression in human breast cancer MDA-MB-231 cells,suppress cell proliferation to some extent,and increase the number of cells in G1 phase with a decreased number of cells in S phase.EIF4E gene may become one of the important molecular targets to breast cancer.