高危型人乳头瘤病毒16 E6蛋白通过外泌体传递热休克蛋白70调控巨噬细胞介导宫颈癌细胞增殖、侵袭和迁移

High-risk human papillomavirus 16 E6 protein regulates tumor-associated macrophages through exosomal heat shock protein 70 to promote cervical cancer cell C33A proliferation,invasion and migration

目的初步探讨高危型人乳头瘤病毒16(human papillomavirus 16,HPV16)E6蛋白促进宫颈癌细胞增殖、侵袭和迁移的潜在免疫调控分子机制。方法构建HPV16 E6蛋白过表达慢病毒并感染C33A细胞,获得C33A-E6稳定细胞系,采用qRT-PCR和Western blot法检测C33A和C33A-E6组细胞内HSP70的表达。放线菌素D(ActD)和放线菌酮(CHX)处理细胞后不同时间采用qRT-PCR和Western blot法分别检测HSP70 mRNA和蛋白质降解速率。超速离心法提取两组细胞外泌体,酶联免疫吸附试验(ELISA)法检测外泌体中HSP70含量。提取C33A和C33A-E6细胞干扰HSP70表达后的外泌体,ELISA检测经外泌体处理的THP-1细胞培养液中TNF-α、IL-1β、IL-6和IL-10的表达水平。将外泌体处理的THP-1细胞与C33A细胞共培养,CCK-8法检测C33A细胞增殖。Transwell小室评估C33A细胞侵袭和迁移状态。结果与C33A细胞相比,C33A-E6细胞HSP70 mRNA水平显著增高(P<0.05),但蛋白质水平没有明显变化(P>0.05)。经ActD/CHX处理不同时间的两组细胞中HSP70 mRNA和蛋白质降解速率差异无统计学意义(P>0.05),但C33A-E6组分泌的外泌体中HSP70含量明显增高(P<0.001)。C33A-E6细胞分泌的外泌体可增强THP-1细胞分泌IL-6、TNF-α和IL-10的水平(P<0.001),并且外泌体处理后的THP-1细胞可促进C33A细胞增殖、迁移和侵袭(P<0.001);而干扰C33A-E6细胞中HSP70的表达可显著降低外泌体诱导的THP-1细胞分泌IL-6和IL-10及THP-1细胞对C33A细胞增殖、迁移和侵袭的促进作用(P<0.001)。结论高危型HPV16 E6蛋白可上调宫颈癌细胞外泌体中HSP70的表达,进而促进巨噬细胞分泌IL-6和IL-10,并介导宫颈癌细胞增殖、迁移和侵袭。

Objective To investigate the possible molecular mechanism of high-risk human papillomavirus(HPV)16 E6 protein in promoting the proliferation,invasion and migration of cervical cancer cells.Methods C33A cells were infected with HPV E6 protein overexpression lentivirus to construct a stable C33A-E6 cell line.The expression of heat shock protein 70(HSP70)was detected by qRT-PCR and Western blot.Degradation rates of HSP70 at mRNA and protein levels were detected by qRT-PCR and Western blot at different time points after actinomycin D(ActD)and actinomycin ketone(CHX)treatment.Exosomes were extracted by hypervelocity centrifugation and the concentrations of HSP70 in exosomes were detected by enzyme-linked immunosorbent assay(ELISA).The concentrations of TNF-α,IL-1β,IL-6 and IL-10 in the supernatants of THP-1 cell culture that treated with exosomes isolated from C33A and C33A-E6 cells after interfering HSP70 expression were detected by ELISA.C33A cells were co-cultured with exosome-treated THP-1 cells to evaluate the changes in cell proliferation with CCK-8 method.The invasion and migration of C33A cells were assessed by Transwell assay.Results Compared with the C33A cells,the expression of HSP70 at mRNA level in C33A-E6 cells was significantly increased(P<0.05),but no significant change was observed at the protein level(P>0.05).No significant difference in the degradation rate of HSP70 at mRNA or protein level was detected between C33A and C33A-E6 groups after treating with ActD and CHX for different times,but the concentration of HSP70 in exosomes was significantly increased in the C33A-E6 group(P<0.001).The exosomes secreted by C33A-E6 cells could enhance the secretion of IL-6,TNF-αand IL-10 by THP-1 cells(P<0.001),and the THP-1 cells treated with the exosomes could promote the proliferation,migration and invasion of C33A cells(P<0.001).However,interfering the expression of HSP70 in C33A-E6 cells could significantly reduce the secretion of IL-6 and IL-10 by exosome-treated THP-1 cells,and inhibit the THP-1 cell-induced proliferation,migration and invasion of C33A cells(P<0.001).Conclusions High-risk HPV 16 E6 protein upregulated the level of HSP70 in the exosomes secreted by cervical cancer cells,thereby promoting the secretion of IL-6 and IL-10 by macrophages and enhancing the macrophage-induced proliferation,migration and invasion of cervical cancer cells.