Identification of Exosomes from Supernatant of IPEC-J2 Cells Before and After Infected with Salmonella,and Expression and Transcriptome Analysis of m6A Related Proteins
[Objective]The aim of this study was to investigate the effect and molecular mechanism of exosomes in regulating the porcine small intestinal epithelial cells(IPEC-J2 cells)during Salmonella infection.[Method]The distribution of Salmonella antibody in IPEC-J2 cells after Salmonella infection was observed by indirect immunofluorescence assay.The exosomes in the cell supernatant of Salmonella-infected IPEC-J2 cells were observed by supercentrifugation.The morphology of the exosomes was observed by transmission electron microscopy(TEM),the particle size of the exosomes was measured by nanoparticle tracking analysis(NTA),and the molecular markers CD9,CD63,CD81 and TSG101 were detected by Western blotting.Additionally,the expression of N6-methyladenosine(m6 A)related proteins was detected by Real-time quantitative PCR and Western blotting.Besides,the differentially expressed genes of exosomes in supernatant were screened by transcriptome sequencing.[Result]Indirect immunofluorescence assay results showed that Salmonella adhered to IPEC-J2 cells,which indicated that Salmonella was able to infect IPEC-J2 cells.TEM and NTA analysis showed that the exosomes had bilayer membrane vesicles with a diameter of 30-150 nm,and four exosomal protein markers CD9,CD63,CD81 and TSG101 were expressed in the purified exosomes by Western blotting.The expression of m6 A modification-related genes and proteins ALKBH5,YTHDC2 and YTHDF1 was significantly or extremely significantly up-regulated in exosomes after Salmonella infection(P＜0.05 or P＜0.01).Transcriptome sequencing results showed that there were a total of 1 524 differentially expressed genes(DEGs)in exosomes of IPEC-J2 cells after Salmonella infection,including 842 down-regulated genes and 682 up-regulated genes.Go function enrichment analysis showed that Salmonella infection mainly affected the immune system process,cell junction and enzyme regulator activity and other biological processes.KEGG pathway enrichment analysis revealed that differentially expressed genes were closely related to intestinal immune network for IgA production,T cell receptor signaling pathway,Toll-like receptor signaling pathway.[Conclusion]In this study,exosomes were successfully isolated and identified in the supernatant of Salmonella infected IPEC-J2 cells,and it indicated that m6A modification could regulate Salmonella infection through the activation of immune signaling pathways,and this study could provide a reference for the subsequent study of exosome-induced release of exosomes from IPEC-J2.
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