Long Noncoding RNA MEG3 Regulates Rheumatoid Arthritis By Targeting NLRC5

Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease that mainly invades joints.Because of its high morbidity and mortality,we need to find a better treatment.Fibroblast synovial cells(FLSs)play an important role in the occurrence and development of rheumatoid arthritis.Inhibiting the proliferation of FLSs has become one of the important strategies in the treatment of rheumatoid arthritis.It has been shown that NOD like receptor(NLRs)is a pattern recognition receptor family in cytoplasm,which is associated with inflammatory diseases and could promote the rapid clearance of invasive lesions.NLRC5 is the largest protein in the NLRs family.There are significant expressions in immune cells and immune-related tissues such as B cells and liver,which can regulate inflammation and cell death.Some studies have shown that NLRC5 and its family members NLRP3 play an important role in inflammation and autoimmunity,and play a pivotal role in the development of rheumatoid arthritis.In recent years,long non-coding RNA(lnc RNA)has been considered to be involved in a variety of biological processes.The maternally expression gene 3(MEG3)is a widely studied lnc RNA with tumor suppressor function,and the expression of MEG3 in the tumor is associated with the high methylation of the promoter region.The activated FLSs have shown the same aggressive behavior as the tumor cells.Nonetheless,whether the MEG3 is involved in the formation of rheumatoid arthritis and whether the mechanism is related to the expression of NLRC5.Therefore,the study has carried on the related experiment research for the question.In order to study the pathogenesis and mechanism of rheumatoid arthritis(RA)induced by complete Freund’s adjuvant(CFA)in rats,we studied the mechanism of MEG3 targeting NLRC5 in the regulation of rheumatoid arthritis.The study consists of the following five parts.1.The expression of MEG3 and NLRC5 in synovial tissues and FLSsSynovial tissues and FLSs of RA rats were collected and the expression of MEG3 and NLRC5 in synovial tissues and FLSs were detected by Western Blot,q RT-PCR,immunohistochemistry and immunofluorescence assay.The results showed that the expression of MEG3 in synovial tissues and FLSs of RA rats was significantly lower than that in normal rats,while the expression of NLRC5 in synovial tissues and FLSs of RA rats was significantly higher than that in normal rats.The expression of inflammatory cytokines in synovial tissue and FLSs and proliferation of FLSs in RA rats was significantly higher than that in normal group.2.The effect of overexpression MEG3 on NLRC5 expression and cell proliferationIn order to investigate the effect of overexpression MEG3 on the expression of NLRC5 and cell proliferation,Western Blot and q RT-PCR carried out after overexpression MEG3.The results showed that the expression of NLRC5 after overexpression MEG3 was significantly lower than that in the control group.The expression of TNF-α and IL-6 significantly downregulation compared with the control group.MTT assay and cell cycle assay showed that the proliferation of FLSs inhibited significantly after overexpression MEG3.These results suggestted that overexpression MEG3 inhibited the expression of NLRC5 and the proliferation of FLSs.3.The effect of silencing NLRC5 on inflammatory cytokines and cell proliferationIn order to study the effect of silencing NLRC5 on inflammatory cytokines and cell proliferation,small interfering RNA of NLRC5 was transfected in primary FLSs.The results showed that the level of TNF-α and IL-6 decreased significantly after silencing NLRC5 compared with the control group.The results of MTT assay and cell cycle assay showed that the proliferation of FLSs inhibited after silencing NLRC5.These results suggested that silencing NLRC5 could inhibit the level of inflammatory cytokines and the proliferation of FLSs.4.The methylation of MEG3 in synovial tissues and FLSs of RA ratsThe methylation of MEG3 in synovial tissues and FLSs was detected by MSP.The results showed that the promoter region of the MEG3 gene in the synovial tissues and FLSs of RA rats highly methylated and the methylation inhibitor 5-azad C could inhibit the hypermethylation status.In addition,after the stimulation of 5-azad C,the expression of MEG3 was up-regulated and NLRC5 was down-regulated.Futhermore,the level of TNF-α and IL-6 was significantly lower in FLSs that stimulated with 5-azad C.MTT and cell cycle analysis showed that 5-azad C could significantly inhibit the proliferation of primary FLSs.5.The expression of DNMT1 in synovial tissues and FLSsSynovial tissues and FLSs of RA rats were collected and the expression of DNMT1 in synovial tissue and FLSs was detected by Western Blot,q RT-PCR and immunohistochemistry assay.The results showed that the expression of DNMT1 in synovial tissues and FLSs of RA rats was significantly higher than that in normal rats.Overexpression of MEG3 and use of methylation inhibitor 5-azad C could decrease the expression of DNMT1.In summary,we found that MEG3 involved in the occurrence and development of rheumatoid arthritis by targeting NLRC5.