HPV16 E6、E7通过糖原合成酶激酶-3β通路影响宫颈鳞癌细胞生物学行为

HPV16 E6 and E7 affect the biological behaviors of cervical cancer cells through the GSK3β pathway

目的:探讨人乳头瘤病毒16(HPV16)的致癌基因E6、E7对宫颈鳞癌细胞C33A生物学行为的影响及其机制。方法:构建HPV16 E6、E7及E6/E7重组质粒,流式细胞术检测转染前后细胞周期的变化;划痕实验和Transwell实验检测细胞迁移及侵袭能力;克隆形成实验检测细胞的增殖能力;qPCR和Western blot检测相关基因和蛋白表达水平。结果:HPV16 E7重组质粒可引起宫颈鳞癌细胞S期比例显著增加(P<0.01),HPV16 E6、E7和E6/E7重组质粒能显著增强宫颈鳞癌细胞C33A的迁移能力(P<0.05),同时细胞的克隆形成能力也显著增强(P<0.05)。HPV16 E6重组质粒可显著上调细胞糖原合成酶激酶-3β(GSK3β)mRNA水平(P<0.05);HPV16 E6、E7重组质粒可在24 h内引起GSK3β磷酸化水平升高,同时调控下游靶基因Cyclin D1和β-catenin在不同时间点的表达发生变化。结论:HPV16 E6、E7在较短时间(24 h内)可通过GSK3β蛋白磷酸化激活,发挥对GSK3β下游靶基因Cyclin D1、β-catenin基因的调控作用,从而促进宫颈鳞癌细胞的增殖、迁移及侵袭。

Objective：To investigate the inhibition and mechanism of human papilloma virus HPV16 oncogene E6 and E7 on the biological behaviors of cervical cancer C33A cells.Methods：The recombinant plasmids of HPV16 E6,E7 and E6/E7 were constructed and the cell cycle distribution was detected by flow cytometry.The cell migration and invasion abilities were detected by scratch test and Transwell assay.The proliferation ability of the cells was detected by clone forming test.qPCR and Western blot were used to detect the expression of related genes and proteins.Results：HPV16 E7 recombinant plasmid can significantly increase the ratio of the cells in S phase.The recombinant plasmid of HPV16 E6,E7 and E6/E7 could significantly enhance the migration ability of C33A cells and the clone formation ability of the cells was also significantly increased.In addition,HPV16 E6 recombinant plasmid could significantly up-regulate the expression of GSK3β mRNA,and HPV16 E6 and E7 recombinant plasmid could induce the phosphorylation of GSK3β in 24 h,and regulate the expression of downstream genes CyclinD1 and β-catenin at different time points.Conclusion：Human papilloma virus HPV16 E6,E7 can activate the phosphorylation of GSK3β protein in short time （24 h）,and they also play a role in the regulation of GSK3β downstream target gene CyclinD1 and β-catenin,thereby promoting the proliferation,migration and invasion of cervical cancer cells.