Expression Profile Analysis Of Proteins,lncRNAs And MiRNAs In Exosomes Released From Resting And Degranulated Mast Cells

Objective: The aim of this research is to construct the expression profiles of proteins,lncRNAs and miRNAs in resting and degranulated mast cells derived exosomes(Rest-Exo and Sti-Exo),screen the differentially expressed genes(DEGs)between mast cells and exosomes,explore the biological characteristics of Rest-Exo and Sti-Exo,and provide a basic database and the platform of methodology for mast cells derived exosomes.Methods:(1)Murine bone marrow-derived mast cells(BMMC)were obtained by stimulating the bone marrow cells with SCF and IL-3 in vitro.The particles in the cytoplasm of the cell were analyzed using toluidine blue staining,and the expression of CD117 and Fc?RI on the cell surface was detected by flow cytometry.DNP-HSA activated BMMC by DNP-Ig E sensitization.Electron microscopy and nanoparticle tracking analysis technique were used to identify exosomes from Rest-Exo and Sti-Exo by differential centrifugation;(2)TMT labelling quantitative proteomic analysis were applied to analyze protein content of Rest-Exo and Sti-Exo,and the differential expression proteins(DEPs)were screened by bioinformatics methods.Biochemical pathways were analyzed by GO annotation and KEGG pathway on the online tool DAVID.The proteins identified from these bioinformatics analyses were verified by Western blot;(3)The GSE25330 array dataset was downloaded from the Gene Expression Omnibus(GEO)database.DEGs were screened by bioinformatics method and the enrichment analysis was performed.The genes identified from these bioinformatics analyses were verified by q RT-PCR in mast cells and exosomes;(4)The expression profiles of lncRNAs and miRNAs in RestExo and Sti-Exo were detected byRNA-seq and smallRNA-seq,and filtered the DEGs.q RT-PCR was preformed to validate DEGs.Results:(1)BMMC were successfully induced in vitro.The vesicles in the supernatant of resting and degranulated mast cells extracted by differential centrifugation are really exosomes,and the amount of exosomes released from degranulation is more than that at rest;(2)A total of 1988 proteins were identified in Rest-Exo and Sti-Exo by TMT labelling quantitative proteomic analysis,of which 415 DEPs(286 up-regulated and 129 downregulated).Enrichment pathway mainly involved in lysosomes,the process of neurological disease,and endoplasmic reticulum protein processing.The results of Western blot were consistent with that of mass spectrometry;(3)We identified 2121 DEGs(843 up and 1278 down-regulated genes)in HMC-1 cell-derived exosomes and HMC-1 cells from GEO database,and the chemokine receptor CCR1 was screened as a hub gene in mast cell derived exosomes.The results of q RT-PCR were similar to that of bioinformatics analyses.(4)A total of 397 lncRNAs were identified in Rest-Exo and Sti-Exo byRNA-seq,including 99 antisense lncRNAs,181 lincRNAs,97 processed transcript lncRNAs,19 sense intronic lncRNAs,1 sense overlapping lncRNA,and 61 differentially expressed lncRNAs.A total of 272 miRNAs were detected resting and degranulated mast cells derived exosomes by smallRNA-seq.In the expression analysis,47 differentially expressed miRNAs(11 up-regulated and 36 down-regulated)were identified.The results of q RT-PCR were consistent with the highthroughput sequencing.Conclusion:This is the first study to analyze the expression profiles of proteome and transcriptome and differential expression profiles of RestExo and Sti-Exo,and explored the differential gene expression between mast cells and exosomes.It is an important reference for a comprehensive understanding of the biological characteristics of and mast cells and their exosomes.