Regulation Of TAGLN2 On The Immune Response And Cell Function In Retinal Microglia

Objective:A stable silencing BV2 microglia cells of transgelin-2（TAGLN2）gene was established with lentiviral transfection.Gene expression change were analyzed with RNA-seq to investigate the regulation of TAGLN2 on immune response and cell function of retinal microglia.It provides some experimental basis for the treatment disease related to retinal inflammation.Methods:Three TAGLN2 si RNAs were designed and synthesized according to the TAGLN2 promoter sequence.The BV2 microglia were transiently transfected with Lipofectamine 3000.The si RNA with the highest silencing efficiency was screened by Real-time quantitative PCR（RT-PCR）.TAGLN2 sh RNA lentiviral vector and BV2 cell lines with silenced TAGLN2 gene were constructed,cell lines were screened by purinomycin and selected for monoclonal amplification.RT-PCR and Western Blot were used to detect the silencing effect of TAGLN2 in the TAGLN2 silencing group（sh TAGLN2）and the control group（Con）.RNA-seq and bioinformatics technology was performed for the two groups to screen significantly differentially expressed genes.In which,cluster analysis,KEGG pathway enrichment analysis and GO function enrichment were performed for the significantly up-regulated and down-regulated genes among the screened differentially expressed genes.Protein-protein interaction network analysis（PPI）was performed by String database and Cytoscape software,and the top 10 key genes with significant difference in expression were screened by Cytohubba plug-in.Key genes were verified by RT-PCR.In addition,cell growth was detected by CCK-8 and multifunctional real-time Cell Analysis.Cell cycle detection kit and flow cytometry were used to detect cell cycle,and cell apoptosis was detected by flow cytometry.Cell migration was detected by scratch test.Results:After using TAGLN2-si RNA to silence the TAGLN2 gene in microglia cells,RT-PCR was used to detect the silencing effect of TAGLN2.Results showed that TAGLN2-si RNA2 had the highest knockout efficiency（above 75%）.After TAGLN2 sh RNA lentivirus was constructed and infected BV2 cells,the expression level of TAGLN2 was detected by RT-PCR and Western-blot.Results showed that the m RNA and protein expression of TAGLN2 in the TAGLN2 silencing group（sh TAGLN2）were significantly decreased compared with the control group（Con）（P<0.001）,which proved that the BV2 cell line with silenced TAGLN2 gene was successfully constructed.Transcriptome sequencing analysis of sh TAGLN2group and Con group,a total of 16149 genes were detected.According to the|log₂（Fold Change）|≥1,P≤0.05 standard selected 635 genes,there were 456 up-regulated and 179 down-regulated genes with significant differences.The GO function enrichment analysis of differentially expressed genes showed that,the significantly up-regulated differential genes were concentrated in the chemotaxis of neutrophils,macrophages and thymocytes,the preparation and presentation of MHCⅡantigen,and the regulation of microglia activation and other immune system processes.Significant down-regulation of differential genes are mainly enriched in immune system process,virus defense response,response to bacteria,response to IFN-α,IFN-β,lipopolysaccharide,and innate immune response process.Significantly up-regulated in KEGG pathway enrichment analysis that the first five pathways,including Jak-STAT signaling pathway,Th17 cells differentiation,Th1 and Th2 cells differentiation,cytokine-cytokine receptor interaction,and T cell receptor signaling pathway which related to immune system process.The significantly down-regulated KEGG pathway was mainly concentrated in NOD like receptor signaling pathway,RIG-Ⅰlike receptor signaling pathway,Toll-like receptor signaling pathway,C-type lectin receptor signaling pathway and other pattern recognition receptor signaling pathways as well as IL-17,TNF signaling pathways.The results of protein interaction network analysis showed that 16 KEGG enrichment pathways were associated with immune-related PPI networks of 134 genes.Among 635 significantly differentially expressed genes in sh TAGLN2 group and Con group,eight transcription factors were screened out,including Myc,Maf,Esr1,Egr2,Fos,Foxa1,Irf4 and Irf7.The top 10 key genes were IFN-γ,Ccl5,Ccr7,Sell,Fn1,Mmp9,Cxcl10,Myc,Irf7 and Isg15,which at the core of PPI network,involved in inflammatory response,cellular immune response,cell migration,proliferation,and regulation of cell growth and differentiation.After TAGLN2gene was silenced,the growth of BV2 microglia cells increased significantly at 24h to 48h（P<0.05）.sh TAGLN2 group cells were blocked in G2/M phase,and multinucleated giant cells formed.There were no significant changes in cell apoptosis and cell migration.Compared with Con group,the expressions of chemokines Ccl2,Ccl3 and Ccl5 in sh TAGLN2 group were significantly increased at m RNA level（P<0.01）.Conclusions:Silencing of TAGLN2 gene can activate the Jak-STAT signaling pathway,helper T cells differentiation（Th17,Th1,Th2）and cytokine-cytokine receptor interaction,and inhibit the pattern recognition receptor signaling pathway.TAGLN2 can inhibit or reduce the expression of inflammatory cytokines and chemokines in retinal microglia and inhibit microglia activation.TAGLN2 can regulate the growth and cycle of microglia.TAGLN2 can inhibit or reduce the activation and inflammation of retinal microglia cells,so as to protect the normal physiological function and structure of the retina for reducing the occurrence of pathological changes and maintain retinal homeostasis.