Transcriptome sequencing and characterization of differentially expressed miRNAs in exosomes from mice macrophages infected by Listeria monocytogenes
[Objective]The paper aimed to isolate and identify the Exosomes(Exos)of macrophages infected with Listeria monocytogenes(LM)in monocyte proliferation mice,and analyze their miRNA expression profile,laying a solid research foundation for revealing the regu-latory mechanism of macrophage Exos miRNA in LM infection.[Method]Macrophages infected with LM were used as the experimental group,while macrophages not infected with LM were used as the control group.Exos in the supernatant of macrophages were isolated and ex-tracted using ultracentrifugation.The morphology,size and surface marker characteristics of Exos were identified by transmission electron mi-croscopy,nanoparticle tracking technology and Western blot.Small RNA-seq sequencing of miRNA in macrophage Exos from the two groups was performed using the Illumina SE50 sequencing platform to screen for significantly differentially expressed miRNA.Targetscan,miRDB and miRWalk databases were used to predict the target genes of differentially expressed miRNA.GO and KEGG-Pathway functional enrich-ment analysis were conducted on the different miRNA target genes.The miRNA-mRNA regulatory network diagram of the top 20 Hub genes was drawn using Cytoscape software.[Result]The diameter of Exos ranged from 30 to 150 nm,with a typical double-layer membrane struc-ture,and the surface marker molecules CD9,CD63 and TSG101 were positively expressed.High-throughput sequencing results showed that compared to the control group,9 significantly differentially expressed miRNA were screened in the experimental group Exos,including 8 sig-nificantly down-regulated genes(mmu-miR-7a-5p,mmu-miR-365-2-5p,mmu-miR-122-5p,mmu-miR-122b-3p,mmu-let-7i-5p,mmu-miR-151-3p,mmu-miR-182-5p and mmu-miR-1198-5p)and 1 significantly up-regulated gene(mmu-miR-192-5p),with a total of 1064 predic-ted miRNA-regulated target genes.GO enrichment analysis results showed that the different miRNA target genes were mainly enriched in processes such as axon formation,cell junction assembly,actin binding and metal ion transmembrane transport;KEGG analysis showed that the target genes were significantly enriched in the cGMP-PKG signaling pathway and the FcγR-mediated phagocytosis pathway.[Conclusion]The miRNA expression profile of macrophage Exos in mice infected with LM underwent significant changes,and the potential target genes regulated by them were mainly involved in signaling pathways related to infection and immune response.
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