HPV16 E6E7癌蛋白通过TWEAK/Fn14信号途径对细胞增殖和凋亡的影响

The effect of HPV16 E6E7 oncoproteins on cell proliferation and apoptosis via TWEAK / Fn14 signal pathway

目的:探讨HPV16 E6E7对TWEAK/Fn14信号途径及细胞增殖和凋亡的影响。方法:制备表达HPV16 E6E7的逆转录病毒,感染原代角质形成细胞。用流式细胞仪分析可诱导纤维母细胞生长因子14(Fn14)表达,Real-time RT-PCR和Western blot法检测调解活化正常T细胞表达和分泌的趋化因子(RANTES)、单核细胞趋化蛋白-1(MCP-1)、干扰素-γ诱导蛋白10水平(IP10)和肿瘤坏死因子(TNF)受体相关因子(TRAF2)表达,生物化学法检测Ras GTpase活性,TUNEL检测细胞的凋亡,MTT法检测细胞增殖。结果:表达HPV16 E6E7的逆转录病毒感染角质形成细胞后,Fn14高表达,RANTES、MCP-1、IP10和TRAF2表达增加,Ras GTpase活性升高。与未感染表达HPV16 E6E7逆转录病毒的细胞比较,表达E6E7感染的角质形成细胞对TNF-α刺激不敏感,细胞增殖显著增加(P<0.05)。结论:表达HPV16 E6E7细胞激活TWEAK/Fn14信号途径,从而上调RANTES和TRAF2表达,导致细胞增殖,这可能是高危型HPV的一个致病机制。

Objective: To investigate the effect of HPV16 E6E7 oncoproteins on TWEAK / Fn14 signal pathway,proliferation and apoptosis. Methods: Recombinant retrovirus expressing HPV16 E6E7 was prepared and infect primary keratinocytes. Fn14 expression was determined using FACS. RANTES,MCP-1,IP10 and TRAF2 expressions were measured using real-time RT-PCR and ELISA assays. The activity of Ras GTPase was detected using biochemical method. Cell apoptosis and proliferation were detected using TUNEL and MTT assay. Results: The results showed that Fn14 was highly expressed upon infection and accompanied by an increase in RANTES,MCP-1,IP10 and TRAF2 expression levels,as well as Ras GTPase activity. Moreover,keratinocytes infected by the Recombinant retrovirus expressing HPV16 E6E7 infected keratinocytes weakened apoptotic response to TNF-α stimuli compared with the control without HPV16 E6E7 expression. Significant increase in proliferation but not apoptosis was seen in E6E7 positive keratinocytes. Conclusions: HPV16 E6E7 expression activates TWEAK / Fn14 signal pathway,and up-regulates Ras and TRAF-2,leading to cell proliferation,not apoptosis,suggesting that it is another pathogenic mechanism of high-risk HPV infection.