shRNA干扰VEGF的表达对宫颈癌SiHa细胞凋亡的影响

Inhibition of VEGF expression by shRNA and effect on the apoptosis of cervical cancer SiHa cell line

目的利用RNA干扰技术抑制S iHa细胞中血管内皮生长因子(VEGF)的基因表达,探讨其对肿瘤细胞凋亡的诱导作用。方法设计针对VEGF的两种短发夹RNA(shRNA),分别构建PGPU6/GFP/Neo-VEGF重组质粒表达载体,命名为pv1、pv2。利用脂质体将质粒转染入人宫颈癌S iHa细胞,采用RT-PCR方法检测VEGF mRNA的变化情况。筛选出抑制率较高的质粒进行下一步实验;转染后,用流式细胞术检测细胞凋亡。结果 pv1、pv2对VEGF mRNA均有显著的抑制作用,抑制率分别为(82.17±4.45)%和(64.55±3.80)%,显著高于阴性对照组的(7.63±4.13)%(P<0.05)。pv1转染组的细胞凋亡率为(47.85±4.65)%,明显高于阴性对照组的(17.76±2.12)%和空白对照组的(19.66±3.74)%(P<0.01)。结论 RNA干扰技术可以有效沉默人宫颈癌细胞株S iHa中VEGF mRNA的表达,并可诱导宫颈癌细胞凋亡。

Objective To study the induction of apoptosis by RNA interference-mediated inhibition of VEGF expression in SiHa cells.Methods Two types of short hairpin RNA were designed and prepared.The recombinant plasmid PGPU6/GFP/Neo-VEGF were constructed respectively,named pv1 and pv2,which were transfected into SiHa cells using LipofectamineTM 2000.RT-PCR was performed to detect the expression of VEGF mRNA.The most efficient shRNA was selected according to the expression of VEGF mRNA.The cell apoptosis was studied by flow cytometry.Results pv1 and pv2 inhibited the expression of VEGF mRNA in transfected group,and inhibition rates were（82.17±4.45）% and（64.55±3.80）% respectively,with significant differences from that in the negative control group（P0.05）.The apoptosis rate of SiHa cells in pv1 group was（47.85±4.65）%,which was higher than that in negative control group（17.76±2.12）% and in blank control group（19.66±3.74）%,showing significant differences（P0.01）.Conclusion RNAi technology can silence the expression of VEGF mRNA in SiHa cells and induce apoptosis,suggesting that the technology may be an effective therapy in cervical cancer.