A Preliminary Study Of MiR-200b On Secondary Brain Injury After Intracerebral Hemorrhage In Mice

Objective: To screen the differentially expressed mRNA and miRNA after intracerebral hemorrhage.Validation the expression of mir-200 b and Lpar1 in mouse cerebral hemorrhage model and LPS induced microglia inflammation model,and to explore the relationship between mir-200 b and Lpar1 and between mir-200 b and pro-inflammatory factors.Methods: C57BL/6 mice were randomly divided into normal operation group(normal group)and cerebral hemorrhage group(ICH group)to establish the model of secondary brain injury after intracerebral hemorrhage,and the brain tissues of the ipsilateral basal ganglia of 3 mice in the normal group and 3 mice in the ICH group were sequenced by the second generation sequencing method.Using R software to find out the differentially expressed mRNA and miRNA between the two groups.Using the STRING database to construct the interaction network of up-regulated differentially expressed gene coding proteins,the relationship between protein interactions in the network was analyzed,and the key genes of key clusters in the network were screened by Cytoscape software and the correlation network was constructed.The upstream miRNA of key genes was predicted by miRDB and Target Scan database.At the same time,the key genes identified in this study were analyzed by person correlation with the differentially expressed miRNA,and finally the miRNA with negative correlation with the key genes were intersected.In order to verify the relevant results of previous bioinformatics analysis,we verified it by establishing the inflammation model of BV-2 microglia in vitro and the model of cerebral hemorrhage on the 1st,3rd and 7th day.Finally,the relative expression levels of key genes lpar1,upstream miR-200 b and IL-6,IL-1 β and TNF-α were detected by q PCR.Results: Through bioinformatics analysis.A total of 969 differentially expressed mRNA and 106 differentially expressed miRNA were obtained in ICH group and normal group.Compared with normal,545 down-regulated expression and 424 up-regulated expression mRNA,55 down-regulated expression and 51 up-regulated expression miRNA were found in ICH group.Through the construction of the protein interaction network of up-regulated differentially expressed genes and the identification of the key gene clusters in the interaction network by using the "MODE" plug-in in Sytoscape software,the up-regulated Lpar1 was determined to be the key gene of interest.Through the prediction of miRDB and Target Scan database,it was found that the down-regulated miR-200 b may be the targeted binding with Lpar1.In vivo and in vitro models,compared with normal group and control group,in ICH group and LPS treated microglia,the relative expression level of Lpar1 increased and the relative expression level of miR-200 b decreased(P < 0.05).The relative expression of miR-200 b in ICH group 1,3,and 7 days was 0.52±0.07,0.38±0.06 and0.40±0.04,Lpar1 relative expression was 2.05±0.12,2.97±0.11 and 2.96±0.09,the miR-200 b and Lpar1 relative expression of microglial inflammatory models were 0.55±0.06 and2.93±0.38,respectively.There was a negative correlation between the expression level of Lpar1 and miRNA-200 b.The relative expression levels of IL-6,IL-1 β and TNF-α were decreased after overexpression miR-200 b compared with control group in the microglia inflammatory model.Their relative expression were 2.16±0.26,2.62,0.39 and 2.27±0.31,respectively(P < 0.05).Conclusion: Lpar1 is high expressed and miR-200 b low expressed in ICH of mice.In BV-2 microglia inflammation model,overexpression of miR-200 b can inhibit microglia inflammation.