人乳头瘤病毒早期癌蛋白E6/E7稳定表达细胞系的构建及其对Rab12表达的影响

目的 探究人乳头瘤病毒(HPV)编码的早期癌蛋白E6/E7对小GTP酶蛋白Rab12表达的影响。 方法 利用逆转录病毒包装系统获得含pBabe-Vector质粒和pBabe-16E7质粒的高滴度病毒颗粒,感染细胞,并用嘌呤霉素筛选获得稳定表达HPV16E7的UMSCC 10A-16E7细胞系,Western blotting检测E7相关蛋白p53和E2F1的表达变化,比较UMSCC 10A-Vector细胞系和UMSCC 10A-16E7细胞系的Rab12蛋白表达水平。用同样方法在HaCaT、RPE1细胞系中进一步验证。用siRNA干扰SiHa、Caski细胞(HPV-16阳性的宫颈癌细胞)中16E6/E7及HeLa细胞(HPV-18阳性的宫颈癌细胞)中18E6/E7的表达,通过Western blotting比较siRNA干扰前后E6和E7相关蛋白(E2F1、p53和Rab12)的表达差异。 结果 利用逆转录病毒包装系统成功构建稳定表达的细胞系,过表达E6/E7的细胞系中Rab12表达升高,干扰E6/E7的细胞系中Rab12的表达降低。 结论 HPV编码的早期癌蛋白E6/E7可调控Rab12的表达。

Construction of stable-expressed cell lines of human papillomavirus early oncoprotein E6/E7 and its effect on the expression of Rab12

Objective To explore the impact of early oncoprotein E6/E7, encoded by human papillomavirus(HPV), on the expression of Rab12 protein, a small GTP enzyme protein. Methods The high titer of virus particles, containing pBabe-Vector plasmid and pBabe-16E7 plasmid, were obtained with retroviruses packing system. UMSCC 10A cell line was infected with the virus particles and selected by puromycin to get UMSCC 10A-16E7cell line, which stably expressed HPV16E7. Western blotting was used to detect the expression of E7 associated protein, including p53 and E2F1, to verify the infection. The expression level of Rab12 in UMSCC 10A-Vector and UMSCC 10A-16E7 cell lines was compared. Further verification was conducted in HaCaT and RPE1 cell lines as above. The expression of 16E6/E7 in SiHa cell line and Caski cell line(HPV-16 positive cervical cancer cell line), and the expression of 18E6/E7 in Hela cell line(HPV-18 positive cervical cancer cell line)were interfered by siRNA. Western blotting was used to compare the expression level of E6 and E7 associated protein(E2F1, p53 and Rab12)after the interference. Results These stable-expressed cell lines were successfully built up with retroviruses packaging system. The expression of Rab12 increased in E6/E7 over-expression cells, and decreased in the E6/E7 interfere cells. Conclusion Early oncoprotein E6/E7 coded by HPV can regulate the expression of Rab12.