反义HPV16 E6/E7-EGFP重组质粒的构建及其对宫颈癌SiHa细胞的促凋亡作用

Construction of EGFP-labled recombinant plasmid of antisense RNA targeted to human papillomavirus16 E6/E7 gene and its effect on apoptosis of SiHa cervical carcinoma cells

目的:构建HPV16早期基因E6/E7的反义重组质粒,探讨其对SiHa细胞的促凋亡作用。方法:将HPV16E6/E7基因片段反向克隆于真核表达载体pEGFP-C1并转染SiHa细胞,用RT-PCR方法检测转染后SiHa细胞E6、E7基因mRNA的表达,West-ernblot方法检测转染后E6/E7蛋白的表达,流式细胞仪检测转染后细胞的凋亡率。结果:成功构建携带HPV16E6/E7基因反义片段的真核表达载体,转染该质粒后,SiHa细胞E6、E7基因的mRNA和蛋白均明显下调;转染后细胞凋亡率为(59.3±11.3)%,明显高于转染空载体组[(9.4±1.8)%]和未转染组[(2.1±0.4)%](P<0.05)。结论:反义HPV16E6/E7基因可下调宫颈癌细胞中E6/E7癌基因的表达,诱导宫颈癌细胞凋亡,为宫颈癌的基因治疗提供了实验依据。

Objective： To construct recombinant EGFP-labled plasmid carrying anti-sense HPV16 E6/E7 gene fragment and investigate its effect on apoptosis induction in SiHa cervical carcinoma cells. Methods： The antisense HPV16 E6/E7 gene fragment was cloned into the eukaryotic expression vector pEGFP-C1 and the recombinant plasmid was transfected into SiHa cells harboring HPV16 genome. Western blot and RT-PCR were performed to document the changes of mRNA and protein of E6 and E7 genes. Flow cytometry was performed to detect the apoptosis of transfected SiHa cells. Results： The recombinant EGFP-labled plasmid carrying antisense HPV16 E6/E7 gene fragment was successfully constructed. The mRNA and protein of HPV E6 and E7 were sharply decreased in SiHa cells after transfection. The results of flow cytometry showed that the apoptosis rate of SiHa cells after transfection [（59.3±11.3）%] was significantly higher than that of SiHa cells transfected with blank vector [（9.4±1.8）%] and SiHa cell with no transfection [（2.1±0.4）%] （P 〈 0.05）. Conclusions： Delivery of anti-sense HPV16 E6/E7 decreases the expression of E6/E7 oncoprotein and induces apoptosis in SiHa cells. This may be potentially useful for gene therapy of HPV-associated cancer.