Investigation Of Long Noncoding RNAs In Multiple Sclerosis

Aims: Long noncoding RNAs(lncRNAs)play a key role in regulating immunological functions.Their impact on the chronic inflammatory disease multiple sclerosis(MS),however,remains unknown.We investigated the expression of lncRNAs in peripheral blood mononuclear cells(PBMCs)of patients with MS and try to explain their possible role in the process of MS.Methods: For this study,we recruited 26 MS patients according to the revised McDonald Criteria.Then we chosen 6 patients for microarray analysis randomly.Microarray assays identified outstanding differences in lncRNA expression,which were verified through real-time PCR.LncRNA functions were annotated for target genes using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses,and regulatory relationships between lncRNAs and target genes were analyzed using the “cis” and “trans” model.Results: There were 2353 upregulated lncRNAs,389 downregulated lncRNAs,1037 upregulated mRNAs,and 279 downregulated mRNAs in MS patients compared with healthy control subjects(fold change > 2.0).Real-time PCR results of six aberrant lncRNAs were consistent with the microarray data.The co-expression network comprised 864 lncRNAs and 628 mRNAs.Among differentially expressed lncRNAs,10 lncRNAs were predicted to have 10 cis-regulated target genes,and 33 lncRNAs might regulate their trans target genes.Conclusions: We identified a subset of dysregulated lncRNAs and mRNAs,the differentially expressed lncRNAs may importent in the process of MS.However,the specific molecular mechanisms and biological functions of these lncRNAs in the pathogenesis of MS need further exploration.PURPOSE: Here,we strove to substantiate the ability of linc-MAF-4 to act as a regulator of pathogenesis during multiple sclerosis(MS).PROCEDURE: We recruited 34 patients diagnosed with MS according to the revised Mc Donald Criteria.Six of these MS patients and 5 healthy volunteers contributed peripheral blood mononuclear cells(PBMCs)for microarray analysis.Subsequent knockdown and overexpression of linc-MAF-4 in na?ve CD4~+ T cells from the additional 28 MS patients were performed to track changes in CD4~+ T cell subsets and their function and to confirm results from the prior microarray analysis.FINDINGS: The expression of linc-MAF-4 increased significantly in the PBMCs of patients with MS compared to those of control subjects.Additionally,linc-MAF-4 regulated encephalitogenic Th1-cell differentiation in MS patients.Transfection of synthetic linc-MAF-4 into na?ve CD4~+ T cells facilitated Th1-cell differentiation and inhibited Th2-cell differentiation by directly inhibiting MAF,which is a Th2-cell transcription factor.Linc-MAF-4 also promoted the activation of CD4~+ T cells from MS patients.The expression level of linc-MAF-4 correlated with the annual relapse rate in MS patients.CONCLUSION: Our results suggest that linc-MAF-4 is involved in the pathogenesis of MS,specifically through the regulation of encephalitogenic T cells.Inflammation that complicates several autoimmune diseases,including multiple sclerosis,has been linked to abnormal differentiation of Th17 cells.However,the reasons that promote Th17-driven autoimmunity are still unclear.Here,we sought evidence that DDIT4 and its associated long noncoding RNA DDIT4(lnc DDIT4)inhibit Th17 cell differentiation.We recruited 36 patients,whose MS was diagnosed according to the revised Mc Donald Criteria.Six MS patients and 5 healthy volunteers(controls)contributed peripheral blood mononuclear cells(PBMCs)as material for microarray analysis.Microarray assays of lnc DDIT4 and DDIT4 RNA expression identified outstanding differences between MS and control subjects,which were verified with real-time q PCR.We then interrupted the expression of lnc DDIT4 and DDIT4 m RNA in MS patients' na?ve CD4~+ T cells and observed the resulting changes in Th17 cells.The expression of Lnc DDIT4 and DDIT4 m RNA were higher both in PBMCs and CD4~+ T cells of MS patients than in healthy controls.DDIT4(2.79-fold upregulation)was then recognized as a candidate for the cis-regulated target of lnc DDIT4(4.32-fold upregulation).Isolation of naive CD4~+ T cells revealed increased levels of lnc DDIT4 and DDIT4 following stimulation with Th17-inducing cytokines but not following Th1,Th2,or Treg induction.Overexpression of lnc DDIT4 in naive CD4~+ T cells reduced IL17 transcription through increased activation of DDIT4 and decreased activation of the DDIT4/m TOR pathway.Consistently,silencing lnc DDIT4 in naive CD4~+ T cells enhanced Th17 differentiation through increased activation of the DDIT4/m TOR pathway.However,these results vanished when DDIT4 was silenced.This outcome suggests that lnc DDIT4 regulates Th17 cell differentiation by directly targeting DDIT4.These results suggest that lnc DDIT4 and DDIT4 have potential as a therapeutic strategy for alleviating MS and other Th17-driven autoimmune diseases.