RNA干扰对VEGF在人卵巢癌细胞中表达及细胞增殖的影响

Influence of RNA interference on expresssion of vascular endothelial growth factor in human ovarian cancer cells and on cell proliferation

目的:探讨小发夹RNA(shRNA)载体介导抑制人卵巢癌细胞中血管内皮生长因子(vascular endothelial growth fac-tor,VEGF)的表达和对卵巢癌细胞增殖的影响。方法::采用脂质体介导的方法将抗VEGF小发夹状双链RNA真核基因表达载体转染到人卵巢癌细胞(HO-8910)中,用G418筛选获得阳性克隆。用Western印迹法、RT-PCR检测VEGF基因在卵巢癌细胞中的表达;通过MTT及细胞周期分析观察其对卵巢癌细胞增殖的影响。结果:转染特异性小发夹RNA质粒表达载体的HO-8910细胞中VEGF表达量明显减少;HO-8910细胞生长减慢,阻滞于G1期的增多,S期细胞减少。结论:VEGF小发夹RNA质粒载体可显著抑制HO-8910细胞中的VEGF基因的表达,抑制卵巢癌细胞的增殖,该研究为应用RNAi进行肿瘤的基因治疗提供了实验依据。

Objective：To investigate the inhibitory effect of vector-based RNA interference（RNAi） on the expression of vascular endothelial growth factor（VEGF） protein in ovarian cancer cells and on proliferation of overian cancer cells, Methods： A vector for transcribing specific small hairpin RNA（shRNA） targeting VEGF gene was constructed, introduced into ovarian cancer HO-8910 cells by Lipofectamine 2000 and the positive clones were screened by G418. The VEGF protein and mRNA expression level of HO-8910 cells were detected by Western blot and semi-quantitative RT-PCR. MTT method and FCM analysis were used to observe the effect of RNAi on proliferation of ovarian cancer cells, Results： The vector of RNA interference was successfully constructed and VEGF expression was descreased significantly in HO-8910-H1-VEGF cells. The growth of ovarian cancer ceils transfected with specific small hairpin RNA expression vector was blocked, with G1 phase ceils increased and S phase cells decreased. Conclusion： The specific small hairpin RNA targeting VEGF mRNA can inhibit the expression of VEGF and the proliferation of ovarian cancer cells, which provides an experimental basis for treating human tumors with anti-anglogenesis method using RNAi.