小干扰RNA靶向VEGF基因体内外抑制乳腺癌细胞MCF-7的增殖

 血管生成与肿瘤生长、侵袭、转移密切相关.血管内皮生长因子能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用.通过RNAi抑制VEGF表达的抗血管生成疗法可有效应用于肿瘤治疗.本研究采用化学修饰的siRNA在体内外抑制VEGF基因表达,探讨化学修饰的siRNA介导的RNA干扰技术在乳腺癌基因治疗的可行性和特异性.选用阳离子脂质体Lipofectamine^TM2000作为转染试剂，将针对人VEGF基因的小干扰RNA(small interfering RNA,siRNA)转染人类乳腺细胞株MCF-7和裸鼠移植瘤，在体内外诱导RNAi.采用四甲基偶氮唑蓝（MTT）法，逆转录聚合酶链反应(RT-PCR)，蛋白印迹实验等检测siRNA治疗组和对照组VEGF基因表达及细胞增殖变化.体外实验结果显示：靶向VEGF基因siRNA转染乳腺癌MCF-7细胞后，细胞生长抑制率达52.5%；VEGF的mRNA和蛋白表达水平显著降低（P<0.01）；裸鼠体内实验结果显示：siRNA治疗组瘤组织的增长受到明显抑制；RT-PCR结果同时表明治疗组VEGF表达下调.体内外对照组各指标无显著变化.化学修饰的siRNA介导的RNAi在体内外均能成功下调靶基因VEGF的表达，抑制MCF-7细胞增殖,是潜在的肿瘤治疗新方法.

Small Interfering RNAs Directed Against VEGF Gene Inhibit Proliferation of Breast Cancer Cells in Vitro and in Vivo

Angiogenesis, the formation of blood vessels, is a vital process in tumor growth, invasion and metastasis. Vascular endothelial growth factor (VEGF), which can especially promote endothelial cell division, proliferation, and migration, plays a critical role in tumor angiogenesis. Inhibiting the expression of VEGF by RNAi was efficiently applicable to the treatment of cancers as an antiangiogenic therapeutic. In this study,we utilized chemically modified siRNA to inhibit VEGF gene expression in vitro and in vivo,to investigated the feasibility and specificity of gene therapy for breast cancer . The VEGF siRNA was transfected into MCF-7 cells and transplanted tumors in nude mice to induce RNAi by using cationic liposome Lipofectamine TM 2000 as transfecting agent. The changes of VEGF gene expression and MCF-7 cell proliferation in both siRNA treatment groups and control groups were measured by MTT assay, RT-PCR, and Western blotting in vitro and in vivo. Experiments in vitro showed that siRNA directed against VEGF gene effectively inhibited the proliferation of MCF-7 cells, the inhibitory rate was 52.5%, and down-regulated the expression of VEGF in the level of mRNA and protein (P<0.01). The growth of tumor was visibly suppressed in vivo. Furthermore, RT-PCR results indicated VEGF mRNA expression reduced in excised tumors. In contrast, there were no obvious changes in control groups. RNAi mediated by chemically modified siRNA markedly decrease VEGF gene expression and inhibited cellular proliferation, and may have the potential as a therapeutic modality to treat human cancer.