The Role Of Plasma And Muscle MiRNAs In Idiopathic Inflammatory Myopathy

Objective:Idiopathic inflammatory myopathies(IIMs)are a group of systemic autoimmune diseases characterized by multiple organ involvement and predominated by progressive weakness and reduced endurance of the proximal skeletal muscles.IIMs can be divided into dermatomyositis(DM),polymyositis(PM),antisynthetase syndrome(ASS),sporadic inclusion body myositis(s IBM),immune-mediated necrotizing myopathy(IMNM)and overlapping myositis based on patients’symptoms and pathological changes.Myositis-specific autoantibodies(MSAs)are closely associated with IIM-specific clinical phenotypes.The MSA-based classification of IIM effectively combined its serological and clinical characteristics,and provided a new method for disease management.The pathogenesis of IIM is unclear,the immunological mechanism dominated immune cells and related molecules may have an essential role.In recent years,non-immunological mechanism mediated by muscle cells,such as endoplasmic reticulum stress,have been increasingly recognized in IIM.Micro RNAs(miRNAs)are a class of relatively stable small RNAs that can induce the degradation of target gene m RNAs or inhibit their translation.In IIM,circulating and muscle miRNAs are differentially expressed.In this study,we analyzed the plasma miRNA profiles in different MSA-positive IIM subtypes and identified their correlation with the clinical characteristics and treatment response of IIM,aiming to explore their potential value as biomarkers of subtype diagnosis and disease monitoring,and involvement of probable pathways;we also analyzed the muscle miRNAs regulated by endoplasmic reticulum stress(ER stress)in DM and the specific downstream mechanisms,aiming to identify potential novel therapeutic targets for the drug development.Methods:1.Plasma miRNA profiles were detected by miRNA sequencing in a discovery cohort including 54 untreated MSA-positive IIM patients and11 healthy controls(HCs).R software package DESeq2 was used to identify the differentially expressed(DE)miRNAs in 28 DM patients(anti-Mi2+(n=6),anti-TIF1γ+(n=7),anti-MDA5+(n=10),and anti-NXP2+(n=5)),19 ASS patients(anti-JO1+(n=7),anti-PL12+(n=6),and anti-EJ+(n=6)),7 with anti-SRP+IMNM.Using DE miRNAs,ten machine learning models(linear support vector machine(SVM),adaptive boosting(Ada Boost),Gaussian process,nearest neighbors,random forest,neural network,decision tree,radial basis function(RBF)SVM,Gaussian na(?)ve Bayes and quadratic discriminant analysis(QDA))were constructed,and the recursive feature elimination(RFE)algorithm was applied to identify the top 10 most valuable DE miRNAs for classifying each IIM subtype.2.The plasma expression of the most specific DE miRNAs were validated by real-time qPCR in an expended cohort of 84 patients(including 49 follow-up patients)and 15 HCs.The correlations between plasma miRNAs and clinical features were analyzed.3.GO analysis was performed on the predicted target genes of DE miRNAs in each IIM subtype,and the specific and common biological processes were selected.4.The sequencing data of GSE143323 in the NCBI GEO database was used to analyze the expression of ER stress-related genes in DM muscles,and the results were validated in 45 DM patients and 19 HC by real-time qPCR,immunofluorescence or immunohistochemical staining.5.The miRNA changes of human skeletal muscle cells after tunicamycin stimulation were identified by sequencing and then tested in DM muscles by real-time qPCR.The miRNA mimic was transfected into human skeletal muscle cells,and its regulated genes were also detected by sequencing and further validated by real-time qPCR,as well as in DM muscles.The pathways regulated by differentially expressed miRNAs were analyzed by KEGG and GO.6.The human skeletal muscle cells were stimulated by tunicamycin with or without the transfection of miRNA mimic/inhibitor or si RNA,and the expression of miRNA,target gene and/or signaling molecules were detected by real-time qPCR,western blot,flow cytometry or immunofluorescence,to evaluate the relationships among ER stress,miRNA,target gene and signaling pathway.7.In DM muscle tissue,immunofluorescence was used to detect the co-localization between miRNA target gene or their ligand proteins and T cells.Results:1.There were 109,100,and 132 DE miRNAs in DM,ASS,and IMNM compared with HC,of which,36,33,and 78 were specific to each subtype and 30 were common in all three subtypes.Linear SVM performed best in distinguishing HC and IIM subtypes,with all accuracies over 80%.2.Plasma hsa-miR-21-5p and hsa-miR-6821-5p were specifically elevated in DM and ASS,respectively;the plasma level of hsa-miR-208b-3p,hsa-miR-206 and hsa-miR-499a-5p were obviously higher in IMNM than in other IIM subtypes;the expression of these 5miRNAs was significantly decreased after treatment and was associated with IIM specific rashes,muscle damage and inflammatory indexes.3.In DM,the unique biological processes of differential plasma miRNAs include CD4~+alpha-beta T cells differentiation and activation,megakaryocyte differentiation and B cell differentiation.In ASS,the unique biological processes of differential plasma miRNAs include Fc receptor signaling pathway,regulation of T cell proliferation and lymphocyte migration.In IMNM,the unique biological processes of differential plasma miRNAs include regulation of cytokine production and neutrophil activation involved in immune responses.The common biological processes of plasma miRNAs in all subtypes include response to hypoxia,response to endoplasmic reticulum stress,muscle cell proliferation/differentiation/development and endothelial cell proliferation/migration/tube morphogenesis.4.The ER stress was activated in the DM muscles,mainly mediated by IRE1αand PERK pathways.5.In myoblasts with ER stress,59 miRNAs were up-regulated and50 miRNAs were down-regulated.In myotubes with ER stress,19miRNAs were up-regulated and 44 miRNAs were down-regulated.A total of 17 miRNAs were changed in both cell types,and their target genes were mainly enriched in autophagy,TGFβsignaling pathway,cell adhesion molecules,T cell receptor signaling pathway,IκB kinase/NF-κB signaling.Among the 17 miRNAs,the expression of miR-20a-5p was also decreased in DM muscles.6.Overexpression of miR-20a-5p resulted in up-regulation of 319genes and down-regulation of 489 genes in myoblasts.The differentially expressed genes were mainly related to autophagy,NF-κB signaling pathway and TNF signaling pathway.EDA2R is potential target gene of miR-20a-5p and can be regulated by ER stress.The expression of EDA2R was increased in DM muscles.7.Overexpression of miR-20a-5p or knockdown of EDA2R blocked ER stress-induced activation of NF-κB pathway in myoblasts,and inhibiting miR-20a-5p has the opposite effect.8.In DM,more CD4~+and CD8~+T cells were infiltrated around the muscle cells with high EDA2R expression;CD8~+T cells were positive for EDA,the ligand of EDA2R.The expression of EDA on CD8~+T cells in peripheral blood of DM patients was higher than that of HC.Conclusions:1.MSA-positive DM,ASS,and IMNM have unique plasma miRNA profiles,which can be used to distinguish different IIM subtypes.The close correlation of miRNAs with the clinical characteristics and treatment response of IIM makes them good candidates for disease monitoring biomarkers.2.Target genes of specific differentially expressed miRNAs in DM,ASS and IMNM can be jointly enriched in endoplasmic reticulum stress,hypoxia and leukocyte adhesion,suggesting that these processes may be common factors in the pathogenesis of IIM.3.The pathways of ER stress are activated in DM muscles.Multiple miRNAs are differentially expressed in muscle cells undergoing endoplasmic reticulum stress.4.Muscle cells can actively participate in the muscle damage of DM through the ER stress-miR-20a-5p-EDA2R axis by activating the NF-κB signaling pathway.On the other hand,it promotes the interaction of a cluster of CD8~+T cells expressing higher levels of EDA.