VEGF基因特异性siRNA转染后对人乳腺癌细胞增殖和凋亡的影响

Effect of siRNA transfection targeting VEGF gene on proliferation and apoptosis of human breast cancer cells

目的:探讨利用干扰RNA(RNAi)抑制血管内皮生长因子(VEGF)基因表达对乳腺癌细胞MCF-7增殖和凋亡的影响。方法:设计针对VEGF基因的小干扰RNA(siRNA),合成DNA模板,体外转录siRNA。以脂质体转染法将双链siRNA导入MCF-7细胞后,用MTT比色法检测siRNA对MCF-7细胞增殖的影响。通过Hoechst33258染色观察MCF-7细胞的凋亡。用流式细胞术检测细胞周期的改变,RT-PCR检测VEGFmRNA表达的变化,免疫细胞化学法检测VEGF蛋白的表达。结果:所设计的两个靶位点siRNA,均能有效地抑制MCF-7细胞的增殖,诱导细胞凋亡,使细胞周期阻滞于G0/G1期,VEGFmRNA及其蛋白的表达明显减少;而作为阴性对照的错义序列组siRNASCR则没有上述效应。结论:体外转录合成的siRNA可抑制MCF-7细胞中VEGF基因的表达,抑制细胞增殖,促进细胞凋亡。

AIM： To explore the effect of RNA interference-mediated vascular endothelial growth factor （VEGF） gene silencing on proliferation and apoptosis of human breast cancer MCF-7 cells. METHODS： Small interfering RNA （siRNA） targeting VEGF gene was designed and DNA template was synthesized, then siRNA was obtained by in vitro transcription. After ds-siRNA was transfected into MCF-7 cells with Lipofectamine, the proliferation of MCF-7 cells was detected by MTT colorimetry, and the apoptosis was measured by Hoechst33258 staining. The change of cell cycle was analyzed by flow cytometry （FCM）. The expression of VEGF on mRNA and protein level was analyzed by RT-PCR and immunohistochemical technology respectively. RESULTS： The siRNA targeting human VEGF gene effectively inhibited the proliferation of MCF-7 cells, induced cell apoptosis, arrested the cell cycle in G0/G1 phase, and decreased the expression of VEGF on both mRNA and protein level. But these effects did not appear in scrambled siRNA （siRNASCR） control group. CONCLUSION： The siRNA targeting human VEGF gene could reduce VEGF expression, inhibit cellular proliferation and induce apoptosis in MCF-7 cells in vitro.