The Role And Mechanism Of MicroRNA-181c In Th17 Cell Differentiation And The Progression Of Experimental Autoimmune Encephalomyelitis

Objective:Experimental autoimmune encephalomyelitis(EAE)is characterized by demyelination of the central nervous system(CNS),impaired nerve conduction,and paralysis.EAE is a typical autoimmune neurodegenerative disease and is a mature animal model of multiple sclerosis(MS).Although the exact pathogenesis of MS remains unidentified,it is highly believed that autoimmunity-related CD4~+T cells within the central nervous system(CNS)contribute to the most important components.T helper(Th)cell subsets are differentiated from naive CD4~+T cells and IL-17-producing Th17 cells are closely associated with the development of autoimmunity.The miR-181 family is another regulator of the Th cell subset population.miR-181c is one of the miR-181 family members,however,whether miR-181c can influence the pathogenesis of experimental autoimmune encephalomyelitis by regulating Th17 cell differentiation remains unknown.Therefore,our study intends to explore the role and mechanism of miR-181c in Th17cell differentiation and the progression of EAE.Methods:By evaluating the expression of miR-181c at different stages during EAE development,the relationship between miR-181c and the development of the disease was determined.Lentivirus was used to construct miR-181c-knockdown mice,and the efficiency of knockdown and infection was verified by RT-PCR and GFP immunofluorescence staining.The EAE mice was induced and divided into groups followed by demonstrating the effect of knocking down the expression of miR-181c on the development of the disease.The inflammatory cell infiltration and demyelination in the CNS was evaluated by H.E.and Fast blue staining of spinal cord sections.Peripheral lymphocytes and CNS mononuclear cells were isolated at the time of onset and peak stage of EAE development,and the proportion of helper T cell subsets(Th1 cells,Th17 cells,and Treg cells)which were reported to be highly correlated with EAE was measured by flow cytometry.The in vitro directed differentiation assay of primary T cells was performed to detect the expression of miR-181c in different Th cell subsets.Flow cytometry and RT-PCR were used to detect the effect of miR-181c knockdown on Th cell differentiation.The signaling pathways and the genes targeted by miR-181c were predicted via bioinformatics analysis.The predicted signaling pathway and the potential target gene were further functional verificated by Western blot and luciferase reporter experiments.In addition,we searched Gene Expression Omnibus for raw data from the miRNA expression profiles of MS patients and healthy control subjects and analyzed the differential expression of miR-181c.Results:In this study,we found that miR-181c-knockdown EAE mice exhibited delayed onset and reduced clinic scores.By analysis of the isolated peripheral and CNS lymphocytes,it was found that the proportion of Th17 cells was significantly reduced in vivo in miR-181c-knockdown EAE mice.By in vitro experiments,we found that miR-181c is highly expressed specifically in Th17 cells,and Smad7,which is the negative regulator of the TGF-βsignaling pathway,is a potential target gene of miR-181c identified by luciferase reporter experiments.miR-181c knockdown can block the activation of Smad2/3 induced by TGF-βin T cells and increase the expression of IL-2,which a negative regulator of Th17 cell differentiation.In addition,through analysis of published microRNA expression profiles from the Gene Expression Omnibus database,increased miR-181c and deceased Smad levels were found in the peripheral blood from MS patients.Conclusion:miR-181c knockdown can alleviate EAE progression and limit inflammatory cell infiltration and demyelination in the CNS.miR-181c is highly expressed specifically in Th17 cells,and miR-181c downregulated Smad7 expression and thereby rendered T cells more sensitive to TGF-β-mediated Smad signaling,limiting the inhibitory effects of autocrine IL-2 and thus contributing to Th17 cell differentiation and induction of EAE.In addition,the analysis of published miRNA expression profiles exhibited that miR-181c level is relatively high in MS patients.Our results identified a novel miRNA promoting Th17 cell differentiation and autoimmunity,suggesting that miR-181c may serve as a valuable target for clinical applications in patients with MS.