Preliminary Study On The Role Of MiRNA-467b In The Regulation Of Th Cells Development In EAE

Multiple sclerosis?MS?and its animal model experimental autoimmune encephalomyelitis?EAE?are neuroinflammatory autoimmune disease characterized by the axonal loss,demyelination and neurodegeneration of the central nervous system.Overactivation of CD4⁺T cells,especially the Th1 and Th17 subsets migrate into central nervous system secreting inflammatory mediator and destroy the contact between neurons with activated macrophage together,which can lead to a series of neurocognitive and motor deficits.miRNAs were recently found to be a class of endogenous non-protein coding small single-stranded RNA.A great number of researches have verified that miRNAs could specifically modulate gene expression by binding with the 3'UTR.Several recent studieshave found miRNAs participating in Th cells development and playing an important role in the development and function of the immune system through regulating several signaling pathways.Its dysregulation could result in a series of pathological process,including allergies,autoimmune diseases and so on.This study was intended to explore the role of miRNA-467b in regulating Th cells development in EAE.EAE model was established by MOG_35-55 peptide in the presence of adjuvant immunized on C57BL/6 mice.Firstly,we dectect the proportion of Th subsets by Flow cytometry and the mRNA expression of Th subsets related transcription factors and cytokines by Real-time PCR in peripheral immune organs and the brain,spinal cord of mice.Secondly,we detected the differently expressed miRNAs in EAE by miRNA array and verifiedmiRNA-467b and its predicted target genes expression by Western Blot,Real-time PCR and Dual Luciferase Reporter system.Further,we transfected miRNA or with its target gene together into CD4⁺T cell in vitro and explored their effects on the differentiation and function of Th subsets.Last,miRNA-467b mimics was injected into EAE mouse through caudal vien and we explored its influence on the disease process.The results showed that the proportion of Th1 and Th17 cells were highly increased whereas the function of regulatory T cells were damaged in EAE mice.Then we found the expression of miRNA-467b was upregulated in normal mice and IFN-?-treatment EAE mice compared with EAE mice by miRNA array.Further,we verified that the level of miRNA-467b was downregulated within LN and CNS at EAE peak and found that miRNA-467bcould suppress the proportion of Th17 cell and its differentiation through targeting eIF4E.Last,injecting miRNA-467b mimics into EAE through caudal vien could alleviatethediseaseprocess.Theaboveresultsshowedthatthe decreased expression of miRNA-467b in EAE could increase the proportion of Th17 cell and promote its differentiation in vitro by promoting eIF4E expression,which can aggravate the disease progress.This indicated that miRNA-467b can regulate the differentiation and function of Th17 cells by targeting eIF4E,thereby alleviating EAE disease.