HPV6E7介导TLR通路调控CA角质形成细胞凋亡、焦亡、增殖作用

Effect of HPV6E7 Mediated TLR Pathway Regulation on Apoptosis,Pyroptosis and Proliferation of CA Keratinocytes

目的:探讨HPV6E7基因介导TLR通路调控对尖锐湿疣(Condyloma acuminate,CA)角质形成细胞凋亡、焦亡和增殖的作用。方法:应用CRISPR/case9系统敲除Caspase-3和Caveolin-1基因。角质形成细胞(HaCaT细胞、HDF-a细胞)分组:分为Con组、sh HPV6E7组、HPV6E7OE组。TLR抑制剂Chloroquine干预,Re-al time PCR和Western blot定量检测角质形成细胞HPV6E7、TLR、焦亡标志性基因NLRP3 mRNA及蛋白。染色质免疫共沉淀(CHIP)检测角质形成细胞中HPV6E7与NLRP3结合。iTRAQ标记液相色谱-串联质谱(LC-MS/MS)分析。功能性实验:(1)CCK-8检测细胞增殖;(2)FCM检测细胞凋亡。结果:Chloroquine干预后,sh HPV6E7组和HPV6E7OE组HPV6E7、TLR、NLRP3 mRNA及蛋白显著下调,sh HPV6E7和HPV6E7OE组增殖上升,凋亡率下降,差异有统计学意义(P<0.05)。CHIP验证HPV6E7与NLRP3直接结合。LC-MS/MS分析出43个显著差异蛋白质。结论:HPV6E7是引起尖锐湿疣发病的重要基因,Chloroquine干预对尖锐湿疣角质形成细胞凋亡、焦亡和增殖作用和影响显著。

Objective:To investigate the role of HPV6E7 gene-mediated TLR pathway regulation on apoptosis,py-roptosis and proliferation of Condyloma acuminatum(CA)keratinocytes.Methods:The Caspase-3 and Caveolin-1 genes were knocked down using the CRISPR/case9 system.Keratinocytes(HaCaT cells,HDF-a cells)were grouped into:a Con group,a sh HPV6E7 group and a HPV6E7OE group.With intervention of TLR inhibitor Chlo-roquine,the mRNA and protein of HPV6E7,TLR and pyroptosis signature gene NLRP3 in keratinocytes were quantified by Real time PCR and Western blot.The binding of HPV6E7 to NLRP3 in keratinocyte was detected by Chromatin coimmunoprecipitation(CHIP).The quantitative proteomic analysis of HDF-a cells was performed by iTRAQ-labeled liquid chromatography-tandem mass spectrometry(LC-MS/MS).Functional experiments:(1)Cell proliferation was measured by CCK 8;(2)Cell apoptosis was determined by FCM.Results:After Chloro-quine intervention,in sh HPV6E7 and HPV6E7OE groups,the HPV6E7,TLR,NLRP3 mRNA and protein were sig-nificantly reduced,the proliferation was increased,the apoptosis rate was decreased,and the differences were sta-tistically significant(P<0.05).The CHIP verified that HPV6E7 binds directly to NLRP3.The LC-MS/MS analy-sis revealed 43 significant different proteins.Conclusion:HPV6E7 is an important gene for the pathogenesis of condyloma,and Chloroquine intervention has significant effect on the apoptosis,pyroptosis and proliferation of condyloma acuminatum keratinocytes.