| Objective:LncRNA play an important regulatory role in gene expression,and LncRNA in microglia-derived exosomes are involved in the development and progression of central neuroinflammation.In this study,we intend to investigate the differential expression of LncRNA in different phenotypes of MG-Exos and predict the biological functions of differentially expressed LncRNA in different phenotypes of MG-Exos.Methods:1.Experimental cell isolation and culture: C57BL/6 neonatal mice(0-2 days old)were isolated from primary microglia for in vitro culture,and the successful extraction of primary microglia was confirmed by immunofluorescence.2.Experimental cell model establishment: primary microglia were cultured in DMEM/F12 culture medium in a constant temperature incubator,and a portion of the cells were induced with LPS(100 ng/m L)+ IFN-γ(20 ng/m L)for 24 h to activate primary microglia to M1 type,and another portion was induced with IL-4(20 ng/m L)for 24 h to activate primary microglia to M2 type.Western Blot,immunofluorescence and RT-q PCR were performed to verify the M1 microglia markers CD32 and i NOS,and the M2 microglia markers CD206 and Arg-1 to ensure successful cell activation for subsequent experiments.3.Extraction of exosomes andRNA: In the supernatant of the three phenotypic microglia,exosomes were isolated and extracted,and validated by transmission electron microscope(TAE),Nanoparticle Tracking Analysis(NTA),Western Blot to verify the exosome analysis.RNA in exocytosis was extracted usingRNA lysate,and the concentration and purity ofRNA were detected.4.Bioinformatics analysis of differentially expressed genes: Three phenotypic microglia-derived exosomes were subjected to high-throughput gene sequencing to screen for differentially expressed lncRNA and mRNA,and target gene prediction by GO enrichment analysis,KEGG signaling pathway analysis and Reactome database enrichment analysis.Construction of lncRNA-mRNA interaction network was performed using Cytoscape 3.8.0.Results:1.Primary microglia specifically express Iba-1 and verified by immunofluorescence.2.M1-type microglia specifically express CD32 and i NOS,and M2-type microglia specifically express CD206 and Arg-1,which were verified by q PCR,WB,and immunofluorescence.3.Extraction and identification of exosomes: after successful extraction of exosomes from cell supernatants,the extracts were analyzed by transmission electron microscopy to show obvious membrane structures with diameter sizes ranging from 30 to 150 nm;NTA particle size analysis of extracts was consistent with exosome structure and particle size;exosome-specific proteins CD9,CD63,Hsp70 were banded and abundantly expressed.4.Exosomal lncRNA and mRNA bioinformatics analysis: M1-exos compared with M0-exos,634 significantly differentially expressed LncRNA were screened,of which 612 were up-regulated LncRNA and 22 were down-regulated LncRNA.m2-exos compared with M0-exos,89 significantly differentially expressed LncRNA were screened,of which 1 M2-exos compared with M1-exos,screened 1377 significantly differentially expressed LncRNA,of which 27 were up-regulated LncRNA and 1350 were downregulated LncRNA.differentially expressed genes were subjected to GO enrichment analysis,KEGG signaling pathway analysis and Reactome database enrichment analysis..5.LncRNA-mRNA interaction network prediction and construction: Based on the differential co-expression results,we predicted the binding of co-expression candidate lncRNA to mRNAs at the nucleic acid level and used Cytoscape 3.8.0 for the construction of lncRNA-mRNA interaction network.Conclusion:1.Screening for differentially expressed lncRNA and mRNAs in primary microgliaderived exosomes of different phenotypes;2.Differentially expressed lncRNA and mRNAs may be involved in information exchange in microglia-derived exosomes through mechanisms such as HIF-1 signaling pathway,AGE-RAGE signaling pathway,and formation of neutrophil exocytosis to regulate CNS inflammation and thus participate in the development of various CNS diseases. |
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