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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 expression of eukaryotic translation initiation factor 4E (EIF 4E) gene in breast cancer MDA-MB-231 cells and its effect on breast cancer cells Effects of proliferation and growth cycle. Methods: siRNA expression plasmid targeting EIF 4E gene was constructed and transfected into human breast cancer MDA-MB-231 cells by lipofectamine 2000. The expression of pGPU6 / GFP / Neo-EIF4E was detected by RT-PCR, The effect of EIF4E siRNA transfection on the expression of EIF4E and cyclinD1 mRNA and protein in MDA-MB-231 cells was detected by cytochemistry. The proliferation of MDA-MB-231 cells was detected by MTT assay, plate clone formation assay and FCM. Formation rate and cell cycle. Results: The EIF4E siRNA expression plasmid was successfully constructed. The results of RT-PCR, Western blotting and immunocytochemistry showed that the mRNA and protein expressions of EIF4E and cyclinD1 in MDA-MB-231 cells were significantly decreased (P <0.05). The cell growth of EIF4E siRNA group was slow and the number of colony formation was significantly reduced, compared with the blank control group and the transfected empty vector group, the difference was statistically significant (P <0.05). FCM results showed that the percentage of cells in G1 phase of EIF4E siRNA transfected group was (71.30 ± 0.47)%, which was significantly higher than that of blank control group (53.10 ± 0.43)%, while the percentage of S phase cells was (12.53 ± 0.13) )%, Which was significantly lower than that of the control group (26.47 ± 0.38)% (P <0.05). Conclusion: EIF4E siRNA can down-regulate the expression of EIF4E gene in breast cancer MDA-MB-231 cells and inhibit the proliferation of breast cancer cells to a certain extent, which may be one of the targets of clinical treatment of breast cancer.