人乳头瘤病毒16型E6/E7基因shRNA真核表达载体构建及其对人宫颈癌SiHa细胞增殖及迁移能力的影响

Construction of shRNA Eukaryotic Expression Vector Targeting Human Papillomavirus Type 16 E6/E7 Gene and Its Effects on Proliferation and Migration of SiHa Cells

目的构建针对人乳头瘤病毒16型E6/E7基因的shRNA真核表达载体,获得稳定表达干扰质粒的人宫颈癌SiHa细胞系并探讨其对SiHa细胞增殖及迁移能力的影响。方法合成3条特异性干扰HPV16 E6/E7基因的shRNA片段并定向插入psilencer 2.1-U6 hygro载体,构建重组质粒psilencer 2.1-U6hygro-shE6/E7并转染入人宫颈癌SiHa细胞,Real-time PCR检测转染后细胞E6/E7 mRNA的表达,选择沉默效应最好的重组质粒并用潮霉素B稳筛,获得稳定表达重组质粒的SiHa细胞,并用real timePCR和Western blot方法进行鉴定;分别运用CCK-8细胞增殖实验和细胞划痕愈合实验检测细胞的增殖及迁移能力。结果测序证实针对HPV16 E6/E7的shRNA真核表达载体psilencer 2.1-U6 hygro-shE6/E7构建正确;转染psilencer 2.1-U6 hygro-shE6/E7的SiHa细胞HPV16 E6/E7表达明显受抑,同时其增殖及迁移能力也明显受抑制。结论 psilencer 2.1-U6 hygro-shE6/E7真核表达载体的成功构建并获得稳定沉默表达HPV16 E6/E7的人宫颈癌SiHa细胞系,证实沉默表达HPV16 E6/E7的SiHa细胞增殖及迁移能力会明显受到抑制,这为进一步研究HPV 16 E6/E7基因在宫颈癌发生发展过程中的功能奠定了实验基础。

Objective To construct the short hairpin RNA(shRNA) eukaryotic expressing vector targeting human papillomavirus type 16 E6/E7 to establish stable transfected SiHa cell line and to observe its effects on cell proliferation and migration. Methods Three pairs of shRNA fragments which could specifically inhibit HPV16 E6/E7 genes were synthesized and cloned into the shRNA vector psilencer2.1-U6 hygro. The recombinant vectors were transfected into SiHa cells by Lipo2000 and E6/E7 mRNA expression was validated by real-time PCR. The stable psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line was selected with hygromycin B and validated by real-time PCR and Western blot.The CCK-8 cell proliferation assay and cell scratch wound healing assay were performed to examine cell proliferation and migration.Results Sequencing suggested that shRNA eukaryotic expression vector targeting HPV16 E6/E7 gene, psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line, was established successfully with a significantly inhibition of HPV16 E6/E7 expression, cell proliferation and migration.Conclusion Inactivation of HPV16 E6/E7 in cervical cancer cells could reduce cell proliferation and migration.