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 / 中华微生物学和免疫学杂志

ABSTRACT

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-α and 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.