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目的:研究针对VEGF基因的siRNA(small interference RNA)对乳腺癌MCF-7细胞细胞周期的影响。方法:依据Promega公司在网上提供的设计软件,设计针对VEGF基因的siRNA,合成DNA模板,体外转录合成siRNA。脂质体转染法将合成的siRNA转染入MCF-7细胞,以未转染细胞以及错义序列siRNAscr转染细胞为对照。用细胞计数法检测siRNA对MCF-7细胞增殖的影响。流式细胞法检测细胞周期变化,RT-PCR法比较转染前后p21、CyclinD1表达水平的变化,Westernblot检测转染前后磷酸化ERK的表达。结果:细胞计数法结果显示,转染24h后siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。siRNA转染后能有效地抑制MCF-7细胞的增殖,阻滞细胞周期于G0/G1期,S期细胞明显减少,G0/G1期细胞比例逐渐增多;p21mRNA表达显著上调,抑制Cyclin D1 mRNA及磷酸化ERK蛋白的表达。结论:体外转录合成的siRNA可能通过上调细胞周期蛋白激酶抑制剂p21的表达,下调Cyclin D1及磷酸化ERK的表达,将细胞周期阻滞于G0/G1期,从而显著抑制MCF-7细胞的增殖。
Objective: To investigate the effect of siRNA targeting small interfering RNA on the cell cycle of breast cancer MCF-7 cells. Methods: According to the design software provided by Promega on the internet, siRNA targeting VEGF gene was designed, DNA template was synthesized and transcribed into siRNA in vitro. The siRNA was transfected into MCF-7 cells by lipofection method. The untransfected cells and siRNAscr transfected cells with missense sequence were used as controls. The effect of siRNA on the proliferation of MCF-7 cells was detected by cell counting. The changes of cell cycle were detected by flow cytometry. The expression of p21 and CyclinD1 was detected by RT-PCR. The expression of phosphorylated ERK was detected by Western blot. Results: The results of cell counting showed that siRNA significantly inhibited the proliferation of MCF-7 cells 24h after transfection, and the inhibition efficiency was stable 48h after transfection. siRNA transfection can effectively inhibit the proliferation of MCF-7 cells, arrest the cell cycle in G0 / G1 phase, S phase cells was significantly reduced, G0 / G1 phase cells gradually increased; p21mRNA expression was significantly increased, inhibition of Cyclin D1 mRNA and Phosphorylated ERK protein expression. Conclusion: siRNA synthesized in vitro may inhibit the proliferation of MCF-7 cells by up-regulating the expression of p21, down-regulating the expression of Cyclin D1 and phosphorylation ERK, and arresting the cell cycle in G0 / G1 phase .