Mechanism Of EB Virus-encoded IL-10 Promotes The Development Of Nasopharyngeal Carcinoma Through The JAK1-STAT3 Pathway

Objective(s):Nasopharyngeal carcinoma is a common nasopharyngeal malignant tumor in clinic.At present,some patients with nasopharyngeal carcinoma are related to EBV infection,but the molecular mechanism of v IL-10 secreted by EBV promoting the development of nasopharyngeal carcinoma is less studied.JAK1-STAT3 pathway is an important signal pathway in tumor development,and it is not clear whether it is regulated by v IL-10 secreted by EBV.In this paper,the potential target and mechanism of v IL-10 on nasopharyngeal carcinoma cells were discussed by sequencing the whole transcriptome.The effects of v IL-10 on the proliferation,migration,invasion and apoptosis of nasopharyngeal carcinoma cell lines CNE-1,CNE-2 and C666-1 were investigated by in vitro cell model.The effect of v IL-10 on the expression of key factors of JAK1-STAT3 signaling pathway in nasopharyngeal carcinoma cells was detected by western blot.This study provides a theoretical basis for the mechanism of v IL-10 secreted by EBV promoting the occurrence and development of nasopharyngeal carcinoma.Methods:1.Whole transcriptomic sequencing was performed after 100ng/m L of v IL-10 was treated with nasopharyngeal carcinoma CNE-2 cell line for 48 h to obtain potential targets and signaling pathways of v IL-10 for nasopharyngeal carcinoma.2.The effects of v IL-10 on the proliferation,migration,invasion and apoptosis of human nasopharyngeal carcinoma cells CNE-1,CNE-2 and C666-1 were determined by elisa,immunofluorescence,Ki67 staining,cell colony formation,transwell migration experiment,transwell invasion experiment and Hoechst33258 staining,and the potential effects of v IL-10 on nasopharyngeal carcinoma cells were verified from the cellular level.3.The changes of key factors of JAK1-STAT3 signaling pathway in nasopharyngeal carcinoma cells treated with v IL-10 were detected by Western blot.Results:1.Complete transcriptome gene sequencing was performed on nasopharyngeal carcinoma cell line CNE-2 before and after the treatment of v IL-10.The results indicated that the gene expression profile of nasopharyngeal carcinoma cells had characteristic changes.5899 differentially expressed genes were found,including3136 up-regulated genes and 2763 down-regulated genes,among which: JAK1 and STAT3 were up-regulated genes,and JAK-STAT signaling pathway showed enrichment of differential genes.2.The concentration of v IL-10 secreted by EBV-positive C666-1 NPC cells was significantly higher than that of EBV-negative CNE-2 NPC cells by Elisa,suggesting that EBV-positive NPC cells could secrete v IL-10.3.Immunofluorescence experiment: after 100ng/m L v IL-10 acted on nasopharyngeal carcinoma cells CNE-1,CNE-2 and C666-1 24 h hours,the fluorescence intensity of IL-10R1 in the cells was detected.The experimental results indicated that v IL-10 could effectively promote the expression of IL-10R1 in nasopharyngeal carcinoma cells.4.Western blot experiment: the expression of IL-10R1 protein in nasopharyngeal carcinoma cells CNE-1,CNE-2,and C666-1 was detected after treated with100ng/m L v IL-10 for 24 h and 48 h.The results indicated that the expression of IL-10R1 protein could be up-regulated with the prolonged treatment time of v IL-10.5.Cell phenotype experiments(proliferation,migration,invasion,apoptosis).5.1 Ki67 immunofluorescence assay: the fluorescence intensity of Ki67 factor in nasopharyngeal carcinoma cells CNE-1,CNE-2 and C666-1 was significantly higher than that in the control group after being treated with 100ng/m L v IL-10 for 24 h and48h,suggesting that v IL-10 can promote the proliferation of nasopharyngeal carcinoma cells.5.2 Cell colony formation experiment: after being treated with 100ng/m L v IL-10 for 24h and 48 h,nasopharyngeal carcinoma cells CNE-1,CNE-2,and C666-1 were cultured in a 5% CO2 incubator at 37℃ for 2 weeks.The number of cell colonies increased with the extension of the treatment time of v IL-10,suggesting that v IL-10 could promote the proliferation of nasopharyngeal carcinoma cells.5.3 Transwell migration experiment: after 24 h treatment with 100ng/m L v IL-10,the number of nasopharyngeal carcinoma cells CNE-1,CNE-2,and C666-1 passing through Transwell micropores was significantly higher than that of the control group,suggesting that v IL-10 promoted the longitudinal migration of nasopharyngeal carcinoma cells.5.4 Transwell invasion assay: after treatment with 100ng/m L v IL-10 for 36 h,the number of CNE-1,CNE-2,and C666-1 cells passing through the matrix gel and Transwell micropores was significantly higher than that of the control group,suggesting that v IL-10 promoted the invasion ability of nasopharyngeal carcinoma cells.5.5 Hoechst33258 Dyeing: nasopharyngeal carcinoma cells CNE-1,CNE-2 and C666-1 were treated with 100ng/m L v IL-10 for 24 h and 48 h,and then stained with Hoechst33258 dye.The number of bright blue nuclei in the experimental group was less than that in the control group,suggesting that the number of apoptotic cells in the experimental group was less than that in the control group.6.Western blot: after being treated with 100ng/m L v IL-10 for 24 h and 48 h,the protein expressions of related factors p-STAT3,STAT3,p-JAK1 and JAK1 in the JAK1-STAT3 signaling pathway were detected.The results suggested that v IL-10 could promote the expression of p-STAT3 and p-JAK1,while the total expression of STAT3 and JAK1 remained unchanged.Conclusions:1.After the effect of v IL-10 on nasopharyngeal carcinoma cell CNE-2,whole transcriptome gene sequencing results showed that v IL-10 could effectively activate JAK-STAT signaling pathway and up-regulate the expression of JAK1 and STAT3.2.The expression of IL-10R1 in nasopharyngeal carcinoma cells can be promoted by the action of v IL-10 on CNE-1,CNE-2 and C666-1,and the expression is mainly in the cytoplasm.3.Virus interleukin-10 can inhibit the apoptosis of nasopharyngeal carcinoma cells CNE-1,CNE-2 and C666-1,enhance the migration and invasion ability of nasopharyngeal carcinoma cells,and further promote the proliferation of nasopharyngeal carcinoma cells.4.Virus interleukin-10 can promote the development of nasopharyngeal carcinoma by regulating the JAK1-STAT3 signaling pathway.