RNAi抑制大肠癌HCT116细胞株VEGF-mRNA表达的实验研究

目的研究载体介导的RNAi(RNA interference) 技术对大肠癌细胞株HCT116中血管内皮生长因子(vascular endothelial growth factor, VEGF)mRNA表达的抑制作用.方法依据Tuschl等设计siRNA 的原则,以VEGF为靶基因设计并合成有小发夹结构的两条DNA序列,经退火成互补双链,再克隆到载体Pavu6 27中构建重组体 (Pavu6 27-VEGF siRNA).DOTAP脂质体法转染重组体至大肠癌HCT116细胞株,经G418筛选,以空质粒(Pavu6 27)转染为对照;半定量RT-PCR法观察HCT116细胞中VEGF-mRNA表达改变.结果①成功构建发卡样VEGF siRNA(small interfere RNA)真核表达载体(Pavu6 27-VEGF siRNA);② Pavu6 27-VEGF siRNA转染HCT116细胞后,VEGF-mRNA表达较空载体Pavu6 27转染的对照组显著下降.结论成功构建载体介导的VEGF靶向RNA干扰重组体,并能有效抑制HCT116细胞中VEGF-mRNA表达,为下一步利用RNAi技术抑制肿瘤血管形成以及局部侵袭和远处转移提供研究基础.

Inhibition effects of RNAi on VEGF-mRNA expression in HCT116 cells

Objective To investigate the effects of RNAi on VEGF-mRNA expression in HCT116 cells. Methods The Pavu6 27-VEGF siRNA expression vector was constructed by gene recombination, then transfected into the cultured HCT116 cells by DOTAP method. The positive cell clones were screened with G418. The stable transfection and expression of VEGF-mRNA in HCT116 were determined by semi-quantitative RT-PCR. The Pavu6 27 transfected plasmid was used as control. Results Pavu6 27-VEGF siRNA expression vector was successfully constructed. DOTAP-mediated gene transfection of Pavu6 27-VEGF siRNA expression vector into HCT116 cells down-regulated the mRNA expression level of VEGF gene, as compared with the control group. Conclusion The Pavu6 27-VEGF siRNA expression vector can inhibit the expression of VEGF-mRNA in HCT116 cells, which may be beneficial in searching new gene therapy of the tumors.