| Objective: High-throughput sequencing technology was used to detect mRNAs and miRNAs expression in mice after ICH,and bioinformatics analysis was used to screen differentially expressed mRNA-Egr2 and mi R-200a-3p with its potential interaction.In vivo and In vitro models of ICH were established to observe the expression of mi R-200a-3p and Egr2,and to further explore the biological regulation effect of mi R-200a-3p on secondary inflammatory injury of peripheral tissues of posterior ICH through Egr2,so as to explore a potential therapeutic target for the treatment of ICH.Methods: 1.Screen the target mRNA and miRNA: Established mice ICH models,prepared tissue samples,and constructed a sequencing library using high-throughput sequencing technology.Using bioinformatics technology to analyze the sequencing results,screen out the differentially expressed mRNA-Egr2 and mi R-200a-3p that may have a mutual regulatory relationship with it.2.In vitro experiment: BV2 cells were stimulated by LPS,and the optimal induction concentration and time were selected to establish an in vitro inflammatory injury model,and the expression of mi R-200a-3p,Egr2 were detected.The subcellular localization of Egr2 in BV2 cells was determined by immunofluorescence staining.Through transient transfection of mi R-200a-3p mimics or inhibitors,the expression of Egr2 and the downstream inflammatory cytokines IL-1β,IL-6,TNF-α in each group were detected after overexpression or inhibition.Statistical analysis of the relationship between the expression of mi R-200a-3p and Egr2 and the above-mentioned various inflammatory factors.The binding sites of mi R-200a-3p and Egr2 were predicted using the Target Scan database and verified by dual luciferase reporter gene assay.3.In vitro experiment: The ICH models of mice were established,and the expression of mi R-200a-3p and Egr2 in the perihematoma tissues were detected at 1,3 and 7 days after modeling.The mice models of ICH with down-regulation of mi R-200a-3p were established by injecting mi R-200a-3p adeno-associated virus into the lateral ventricle.Changes in expression of Egr2 and inflammatory factors were detected,and neurological injuries such as brain edema and behavioral changes of the mice were observed.Results: 1.High-throughput sequencing showed that there were 969 differentially expressed mRNAs(545 down-regulated and 424 up-regulated)and 106 differentially expressed miRNAs(55 down-regulated and 51 up-regulated)in the mice models of ICH.Bioinformatics analysis screened out significantly down-regulated gene Egr2 and significantly up-regulated mi R-200a-3p.2.In vitro experiments showed that the cell activity was the best when the concentration of LPS was 1ug/ml.The expression of TNF-α in the medium reached the peak after continuous treatment for 24 h.Compared with the Control group,the mi R-200a-3p in the LPS group was significantly increased,while the Egr2 was significantly decreased.Immunofluorescence results showed that Egr2 was mainly expressed in the nucleus of BV2 cells.In BV2 cells transfected with mi R-200a-3p mimics,the expression of Egr2 was decreased,and the IL-1β,IL-6 and TNF-α were increased.The transfection of mi R-200a-3p inhibitors increased the expression of Egr2,and decreased the IL-1β,IL-6,and TNF-α.Dual-luciferase reporter assay showed that the 3′-UTR of Erg2 mRNA contained conserved mi R-200a-3p binding sites.3.In vivo experiments showed that compared with the Sham group,the mi R-200a-3p was significantly increased on the 1st day after ICH and gradually decreased in the following 1 week.The Egr2 decreased significantly on the 1st day after ICH and gradually increased in the following 1 week.The injection of mi R-200a-3p silencing adeno-associated virus into the lateral ventricle of mice with ICH could significantly increase the expression of Egr2,reduce the inflammatory factors,reduce the degree of brain edema,and improve the neurological function injury.Conclusion: In the perihematoma tissues of mice after ICH,the highly expressed mi R-200a-3p may promote microglia to secrete inflammatory cytokines IL-1β,IL-6 and TNF-α through targeted inhibition of Egr2,which aggravated the secondary neuroinflammatory injury after ICH.Inhibition of mi R-200a-3p may exert neuroprotective effects by alleviating secondary neuroinflammatory injury. |
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