MiR-496 Regulates Fibroblast-like Synoviocytes Via Targeting MMP10 In Human Rheumatoid Arthritis

[Objective]Rheumatoid arthritis(RA)is a common chronic autoimmune disease featured by synovial inflammation.However,the cause and underlying molecular events of RA are not clear.Here,we applied bioinformatics analysis to identify key genes and miRNAs involved in RA.Cumulative evidences have indicated that miRNAs can regulate the pathogenesis of RA through various biological processes.miR-496 has been found to be an important molecule in multiple diseases.Nevertheless,its role as well as underlying mechanism in RA has not yet been elucidated.[Methods]The expression profile of GSE72564 was downloaded from the Gene Expression Omnibus(GEO)database,which contained 8 samples,including 4 cases of RA samples and 4 cases of OA samples,and the differentially expressed miRNAs(DEMs)between control and RA samples were screened using GEO2R tool.Next,all the DEMs with p<0.05 and |log2(fold change)|>1 were identified.Interactions between key miRNA and its target genes were theoretically predicted by using the miRanda,miRWalk,TargetScan,and miRDB databases.The data from GSE29746 were compared using R software limma package to determine the differentially expressed genes(DEGs)with a threshold of P<0.05 and |log2(fold change)|?1.Functional and pathway enrichment analyses were performed for common DEGs using the DAVID database,and the protein-protein interaction(PPI)network of common DEGs was constructed by the STRING database and visualized with Cytoscape software.In addition,the hub genes in the network and module analysis of the PPI network were performed using Cytoscape and plugin Molecular Complex Detection(MCODE).Finally,the key genes were obtained by taking the intersection of the predicted target genes and the differentially expressed genes.the mRNA expression levels of key genes were validated in the tissue samples from GSE29746 dataset.Gene Expression Omnibus(GEO)datasets provided the clinical samples utilized to analyze miR-496 and MMP10 expressions in RA.In vitro model of RA was established using IL-1?(10 ng/mL)-induced MH7A cells.Cell counting kit 8(CCK-8)and flow cytometry experiments were implemented to assess the MH7A cell viability and apoptosis rate.TargetScan was applied to identify the potential targets of miR-496 and dual-luciferase reporter gene assay was employed to witness the relationship between miR-496 and its target in RA.qRT-PCR and western blot analyses were conducted to examine the expression of indicated genes,including miR-496,MMP10 and NF-?B pathway-related markers.[Results]A total of 23 miRNAs were identified as DEMs,of which 17 miRNAs were upregulated and 6 miRNAs were downregulated.A total of 180 target genes were predicted to combine with the miR-496.A total of 418 DEGs were obtained,comprising 206 upregulated genes and 212 downregulated genes.GO analysis showed that the biological functions of DEGs focused primarily on positive regulation of gene expression,response to cAMP,homophilic cell adhesion via plasma membrane adhesion molecules,negative regulation of platelet activation,negative regulation of endopeptidase activity,response to stimulus,positive regulation of synapse assembly,T-helper 1 type immune response,subpallium development,and cell adhesion.The main cellular components include proteinaceous extracellular matrix,extracellular matrix,apical plasma membrane,cell junction,endocytic vesicle membrane,integral component of membrane,extracellular region,extracellular space,plasma membrane,and integral component of plasma membrane.The molecular functions include extracellular matrix structural constituent,receptor binding,delayed rectifier potassium channel activity,Wnt-protein binding,G-protein coupled serotonin receptor activity,growth factor activity,serine-type endopeptidase inhibitor activity,inward rectifier potassium channel activity,actin monomer binding,and calcium ion binding.Genes are mainly involved in the KEGG pathway termed Graft-versus-host disease,Phagosome,Asthma,Inflammatory bowel disease(IBD),Morphine addiction,Staphylococcus,aureus infection,Rheumatoid arthritis,Toxoplasmosis,Amoebiasis and Cell adhesion molecules(CAMs).In order to screen the important pathways related to the development of RA,we constructed an interaction network between DEGs and the signaling pathway using the ClueGO plug-in of Cytoscape platform.The results showed that NF-B pathway may be involved in the pathogenesis of RA through TLR4 and other interactive genes.Seven key genes ST6GALNAC5,LIMCH1,MMP10,CDH2,PTPRB,PAPPA,and PDE3A were obtained.Among all the key genes,MMP10 was distinctively high expressed in RA tissue.miR-496 was obviously reduced in RA tissues and MH7A cells after IL-1?treatment.Overexpression of miR-496 could inhibit IL-1?-treated MH7A cell viability,while miR-496 knockdown accelerated cell proliferation.MMP10 was identified as a downstream factor of miR-496 and its expression was negatively modulated by miR-496.The effects of miR-496 on MH7A cell proliferation and apoptosis were reversed by MMP10.The activity of NF-?B pathway was associated with the miR-496/MMP10 axis in IL-1?-stimulated MH7A cells.[Conclusion]This study indicates that screening for Key Genes and miRNAs in RA using bioinformatics analysis could help us understand the mechanism of development of RA.Besides,miR-496/MMP10 axis might be participated in the pathogenesis of RA through mediating NF-?B pathway.In vitro model,this study demonstrated that miR-496 can impair the proliferative ability and facilitate the apoptosis of IL-1?-treated MH7A through directly targeting MMP10.And this regulatory mechanism might be linked with the inactivation of NF-?B signaling pathway in RA.