人类乳头瘤病毒16 E7真核表达载体构建及对A431皮肤鳞癌细胞增殖和侵袭的影响

Construction of human papilloma virus 16 E7 eukaryotic expression vector and its effect on proliferation and invasion of A431 cells

目的构建HPV16 E7(E7)真核表达载体,观察E7过表达对A431细胞增殖和侵袭的影响。方法从Caski细胞中抽提总RNA,针对E7序列设计特异引物,进行逆转录和聚合酶链式反应扩增得到E7cDNA。应用基因工程方法将得到的PCR片段克隆至真核表达载体pEGFP-N1的多克隆位点。重组质粒经双酶切鉴定和测序后,使用LipofectamineTM 2000转染A431细胞。Western blot检测E7在细胞中的表达。WST-1和Transwell系统分别检测细胞增殖和侵袭能力。流式细胞术检测转染前后细胞周期的变化。ELISA检测TGF-β分泌情况。结果经酶切、测序鉴定,E7基因正确插入到pEGFP-N1多克隆位点,获得pEGFP-E7;Western blot检测结果显示:与未转染组相比,转染组E7蛋白高表达。过表达E7的A431细胞增殖和侵袭能力均显著增强,细胞周期中S期细胞数量增多,G1期细胞数量相对减少。TGF-β分泌增多。结论E7过表达可改变细胞周期,提高皮肤鳞癌细胞的增殖和侵袭能力,而分泌增多的TGF-β并不能抑制肿瘤的增殖和侵袭。

Objective To construct an eukaryotic expression vector encoding human papilloma virus E7, and investigate the effect of E7 overexpression on the proliferation and invasion of human epithelial carcinoma cell line A431. Methods After total RNA from Caski cells was obtained by Tripurer, eDNA was amplified by RT-PCR and then cloned into pEGFP-N1 vector using gene engineering technology. Recombinant expression vector pEGFP-E7 was confirmed by restriction digestion and sequencing, and then transfected into A431 cells by liposome. Expression of E7 protein was detected by Western blotting. WST-1 assay and Transwell system were used to observe the growth and invasion of A431 cells. Cell cycle was measured by flow eytometry, and the expression of TGF-β in the supernatant was measured by ELISA. Results The recombinant expression vector pEGFP-E7 was successfully constructed. Western blotting indicated that E7 protein was overexpressed in A431 cells. Overexpression of E7 significantly increased cell proliferation and invasion. Flow cytometry showed there were more cells arresting in S phase and lesser in G1 phase. Overexpression of E7 significantly improved the secretion of TGF-β in A431 cells. Conclusion Overexpression of E7 enhances tumor cell proliferation and invasion, although TGF-β secretion is increased at the same time.