The Abnormal Expression Of MiRNA At CD4~+T Iymphocyte In A Murine Model Of Asthma And MiRNA-regulation Mechanism During The Th2Polarization

ObjectiveTo screen the miRNAs associated with CD4~+T lymphocyte differentiation in amurine model of asthma, and identify the regulation mechanism of the miRNA.Methods(1) Microarray-based miRNA profiling of spleen CD4~+T lymphocytes in a murinemodel of asthma was performed, so did it in the normal control group.The differentlyexpressed miRNAs were found out. Predicted mRNA targets for each differentiallyexpressed miRNA were identified using three databases: mirbase,miranda andtargetscan. A pathway enrichment analysis (KEGG pathways) was carried out takinginto account both modulated miRNAs and predicted mRNA targets.(2) Balb/c mice were divided into3groups: the normal control group, asthmagroup and the dexamethasone group. The total number of the inflammatory cells and thenumber of eosinophils in the BALF were compared among the three groups, so did theexpression levels of IL-4and IFN-γ in the BALF. Using quantitative real-timepolymerase chain reaction (qRT-PCR), the roles of five microRNAs (microRNAs-181a,-146a,-146b-155and-150) in Th2inflammation of asthma were analyzed.(3) Using electroporation, miRNA-181a mimic and inhibitor were transfected into CD4~+T lymphocyte of asthma model in vitro respectively. The expressions levels ofmiRNA-181a and DUSP6mRNA were determined by qRT-PCR, Cell proliferation wasmeasured by BrdU Elisa, and the expression levels of IL-4and IFN-γ in the supernantfluid of the CD4~+T cell were tested by Elisa. The percentage of Th1or Th2cell wasdetermined by detecting the intracellular IFN-γ or IL-4production in CD4~+Tlymphocytes with flow cytometry.Results(1)48differently expressed miRNAs were identified for each miRNAsignificantly up-or downregulated above1.5fold. The numbers of upregulatedmiRNAs were39, and these miRNAs included miRNA-29b, miRNA-451andmiRNA-146b; The numbers of downregulated miRNAs were9, and these miRNAsincluded miRNA-1843-3p, miRNA-378/miRNA-378b and miRNA-150。The numbersof the overlapping genes predicted by three databases (mirbase,miranda and targetscan)were773, and these genes included IL-13, FCER1A, MAP2K3, MAPK10, MAPK12, PIK3CD, etc. KEGG pathway analysis showed these predicted genes was involved inthe metabolic pathway such as Fc epsilon RI pathway,MAPK pathway and ubiquitinmediated proteolysis pathway,which were involved in cell proliferation and celldifferentiation. miRNA-181a, miRNA-146a, miRNA-146b and miRNA-150, whichwere reported to be associated with the differentiation of T lymphocytes, were alsoup-regulated at CD4~+T lymphocytes in asthma(P <0.05).(2) The level of IL-4at the BALF in the asthma group was higher than in thenormal control group at Day32, so did the total number of the inflammatory cells or thenumber of eosinopheals. The total number of the inflammatory cells or the number ofeosinopheals in the dexamethasone group was less than in the asthma group. There wasno significant difference of IL-4between the dexamethasone group and the normalcontrol group, or the asthma group. At Day37, the number of the inflammatory cells orthe number of eosinopheals in the asthma group was more than in the normal controlgroup. There was no significant difference of IL-4between the asthma group and thenormal control group, The results of qRT-PCR showed that the expression levels ofmicroRNAs-181a,-146a,-146b and-150, were higher in the asthma group compared tothe normal control group in the beginning of the disease, and after5days dropped to thenormal control group levels because there was no new airway challenge. Moreover,miRNA-146a expression was down-regulated by treatment with dexamethasone.MicroRNA-181a had a positive linear correlation with the total number of inflammatorycells or the number of the eosinophils in the BALF by spearman correlation analysis, sodid miRNA-146a and miRNA-146b.(3) The expression level of miRNA-181a in CD4~+T lymphocyte was significantlyup-regulated after the miRNA-181a mimic was electroporated into CD4~+T lymphocyteof the asthma model. The expression level of IL-4was also upregulated. The expressionlevel of DUSP6mRNA was down-regulated at the same time. On the contrary,miRNA-181a inhibitor could up-regulate the expression level of DUSP6mRNA,accompanied with down-regulation of miRNA-181a. The percentage of Th2cell wasdecreased after the miRNA-181a inhibitor treatment.Conclusion(1) There are many kinds of abnormally expressed miRNA at CD4~+Tlymphocytes in asthma, whose predicted target genes are involved in the cellproliferation and cell differentiation of CD4~+T lymphocytes. It is noteworthy thatmiRNA-181a, miRNA-146a, miRNA-146b and miRNA-150may take part in the Th2 polarization of CD4~+T lymphocytes in asthma.(2) These observations suggest that microRNAs-181a,-146a and-146b areproinflammatory factors in asthma, and that down-regulation of miRNA-146a maypartially account for the anti-inflammatory effect of dexamethasone.(3) miRNA-181a effectively induces Th2polarization of CD4~+T cell partially bytargeting DUSP6mRNA in CD4~+T lymphocytes in a murine model of asthma.miRNA-181a can be used as a new intervention target during the treatment of Th2inflammation in asthma.