Cervical Cancer Cell Derived Exosomes Regulate The Differentiation Of Th17 Cells In Microenvironment

Background:Cervical cancer is the most common gynecological malignant tumor,the invasion and metastasis are the most important causes of death in cervical cancer patients.Tumor microenvironment is a special environment suitable for tumor growth,which consisted of various components such as tumor cells,extracellular matrix and immune cells.Tumor immune microenvironment plays a key role in tumor growth and metastasis.As an important part of the tumor microenvironment,the role of immune cells in tumor progression cannot be ignored.Among them,the increased proportion of Th17 cells in the tumor immune microenvironment can increase the invasive ability of cervical cancer cells,thereby promoting the malignant progression of cervical cancer.Exosomes refer to the small vesicles containing complex RNA and proteins,which are secreted by a variety of cells to the outside of the cell for intercellular signal transmission,and are important signaling molecules for the interaction between tumor cells and immune cells in the microenvironment,directly or indirectly affect the development and differentiation of immune cells.However,the regulation of cervical cancer-derived exosomes in Th17 cell differentiation and its related mechanisms have not been studied.Objective:The aim of this present study was to explore the molecular mechanism of cervical cancer cell derived exosomes regulating the differentiation of Th17 cells in tumor immune microenvironment,and provide a theoretical basis for further searching for new ideas and targets for cervical cancer treatment.Methods:(1)We down-regulated HPSE expression in cervical cancer cells with siRNA,then the exosomes derived from cervical cancer cells were extracted and identified by transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and Western Blot assays.The exosomes were labeled with fluorescent dyes SytoRNA and Dil and co-cultured with human CD4~+T cells.The immunofluorescence technique was used to detect whether CD4~+T cells can effectively absorb fluorescently labeled in cervical cancer cell-derived exosomes;Flow cytometry and enzyme-linked immunosorbent assays were used to detect the differentiation ratio of Th17 cells,the cell proliferation and cytokine secretion ability of CD4~+T cells co-cultured with cervical cancer cell-derived exosomes;(2)Clariom D-chip was used to sequence the lncRNA of exosomes derived from the cervical cancer cell.The target genes of differentially expressed lncRNA were subjected to GO clustering and KEGG pathway enrichment analysis to screen out the lncRNA that may be involved in the regulation of Th17;(3)Separate CD4~+T lymphocytes in human peripheral blood,using overexpression vector or siRAN to intervene in the expression of ENST00000610044 and JAK3 genes in CD4~+T cells;The differentiation ratio of Th17 cells in each group was detected by flow cytometry;The expression of Th17 cell-specific transcription factor Rorc and Th17 specific secretion factor IL-17A in each group was detected by Real-time PCR;The secretion of IL-17A was detected by ELISA;The level of JAK3/STAT3 signaling pathway was detected by Western Blot.Results:(1)Exosomes derived from cervical cancer cells were successfully isolated and identified:TEM morphology clearly showed the vesicular structure of the exosomes;Nanoparticle tracking analysis showed that the extracted exosomes conformed to the size of30-150 nm particle size;Western Blot showed that the vesicles contained CD63 and CD81marker proteins.CD4~+T cells can effectively absorb cervical cancer cell-derived exosomes;Cervical cancer cell-derived exosomes can promote Th17 cell differentiation and IL-17A secretion(P<0.05),however,it was not associated with CD4~+T cell proliferation and secretion of other cytokines(IFN-γ,IL-4,TGF-β)(P>0.05).(2)We used the gene chip to analyze the lncRNAs in the exosomes derived from normal and HPSE down-regulated SiHa cells,and screened a variety of differential expressed lncRNAs.In the functional analysis of lncRNA,KEGG signaling pathway enrichment analysis showed that the differential lncRNAs were mainly involved in mTOR signaling pathway,Th17 cell differentiation signaling pathway,immune system regulation and so on.Among the many differentially expressed lncRNAs,ENST00000610044 on chromosome 1 satisfied the following characteristics:ENST00000610044 level in SiHa derived exosomes was significantly higher than that of HPSE down-regulated SiHa cells;Up-regulated the expression of ENST00000610044 in CD4~+T cells,Th17 differentiation proportion was significantly increased;ENST00000610044 target genes were related to T cell differentiation,such as JAK3 and SMAD4.(3)Compared with the control group,the expression of Rorc and IL-17A significantly increased after ENST00000610044 was up-regulated in CD4~+T cells(P<0.05),and the secretion of IL-17A was also significantly increased(P<0.05).Western Blot detected a significant increase in JAK3/STAT3phosphorylation levels(P<0.05).However,the down regulation of the JAK3 gene has the opposite effect,the expression of Rorc and IL-17A was significantly decreased(P<0.05);the secretion of cytokine IL-17A was significantly decreased(P<0.05);the phosphorylation levels of JAK3/STAT3 were also significantly reduced(P<0.05).Conclusion:Exosomes derived from cervical cancer cells could be effectively internalized by CD4~+T cells,the lncRNA-ENST00000610044 entering CD4~+T cells activated the JAK3 kinase,which recruited and phosphorylated STAT3 protein,activated STAT3 protein into nucleus,enhanced transcriptional activity of transcription factor RORC,promoted differentiation of Th17 cells,then promoted the invasion and metastasis of cervical cancer cell.This study provided an experimental basis for further understanding the mechanism of invasion and metastasis of cervical cancer and new treatment targets.