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

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 time-PCR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。

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

Objective To construct the short hairpin RNA(shRNA) eukaryotic expressing vector targetinghuman papillomavirus type 16 E6/E7 to establish stable transfected SiHa cell line and to observe its effectson cell proliferation and migration. Methods Three pairs of shRNA fragments which could specificallyinhibit HPV16 E6/E7 genes were synthesized and cloned into the shRNA vector psilencer2.1-U6 hygro. Therecombinant vectors were transfected into SiHa cells by Lipo2000 and E6/E7 mRNA expression was validatedby real-time PCR. The stable psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line was selected with hygromycin Band validated by real-time PCR and Western blot.The CCK-8 cell proliferation assay and cell scratch woundhealing assay were performed to examine cell proliferation and migration.Results Sequencing suggestedthat shRNA eukaryotic expression vector targeting HPV16 E6/E7 gene, psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line, was established successfully with a signifi cantly inhibition of HPV16 E6/E7 expression, cellproliferation and migration.Conclusion Inactivation of HPV16 E6/E7 in cervical cancer cells could reducecell proliferation and migration.