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目的探讨HPV16E6对细胞中Rap1GAP蛋白水平的影响,为阐明宫颈癌发生发展的分子机制提供实验依据。本研究组前期实验显示,宫颈癌石蜡切片组织中Rap1GAP蛋白水平下降,且与高危型HPV16/18感染相关。本文将进一步探讨HPV16 E6是否导致Rap1GAP蛋白下调的原因。方法通过将HPV16 E6基因插入pGEX-KG的BamHI和Hind Ⅲ酶切位点构建GST标记的HPVE6质粒,采用脂质体转染法将其转染入HeLa细胞中,Westernblot方法观察GST-tagged-HPV16 E6在HeLa细胞中的表达,并观察其对HeLa细胞中内源性Rap1GAP蛋白水平的影响。结果测序表明成功构建GST标记的HPV16E6质粒;Western blot检测表明HPV16E6在HeLa细胞中成功表达;并且发现HeLa细胞中过表达HPV16 E6后,Rap1GAP蛋白相对含量(0.602±0.205)明显低于未转染组(1.130±0.163),差异具有统计学意义(P<0.05)。结论HPV16 E6下调Rap1GAP蛋白水平。
Objective To investigate the effect of HPV16E6 on the expression of Rap1GAP in cells and provide experimental evidence for elucidating the molecular mechanism of cervical carcinogenesis. In our previous study, Rap1GAP protein levels in paraffin-embedded cervical tissue decreased, and were associated with high-risk HPV16 / 18 infection. This article will further explore whether HPV16 E6 causes the down-regulation of Rap1GAP protein. Methods GST-tagged HPVE6 plasmid was constructed by inserting the HPV16 E6 gene into BamHI and Hind Ⅲ sites of pGEX-KG. The plasmid was transfected into HeLa cells by lipofection method. The expression of GST-tagged-HPV16 E6 in HeLa cells and observe its effect on endogenous Rap1GAP protein level in HeLa cells. The results of sequencing showed that GST-tagged HPV16E6 plasmid was successfully constructed. The results of Western blot showed that HPV16E6 was successfully expressed in HeLa cells. The relative content of Rap1GAP protein in HeLa cells was significantly lower than that in untransfected cells (0.602 ± 0.205) (1.130 ± 0.163), the difference was statistically significant (P <0.05). Conclusion HPV16 E6 downregulates Rap1GAP protein level.