| Objective:Rheumatoid arthritis(RA)is a common disease in the Rheumatology and Immunology Department.It is a chronic,systemic autoimmune disease,characterized by erosive joint destruction and chronic synovial inflammation,and mainly affects hands and feet.The pathological features of RA are synovial tissue hyperplasia and pannus formation,and can finally cause joint destruction and loss of function.Moreover,there will be other organs affected,such as heart,lung and kidney.RA is often companied with long duration and unhealed.At present,some patients still cannot achieve total disease remission,and the disease progresses to disability and disability in the later stage,which seriously affects the quality of the life of patients.The etiology of RA is not fully understood.In addition to recognized genetic and environmental factors,factors such as infection and lifestyle may be involved.The existing researches on the pathogenesis of RA havefocused on the cellular,molecular,and genetic levels.RA-related pathogenic factors have been continuously discovered,providing some new ideas for the treatment of RA,and improving the quality of life of patients in some extent.However,the cause of RA has not yet been elucidated.In order to seek targeted therapy for RA to achieve complete remission of the disease,it is particularly important to continue to explore the pathogenesis of RA.Previous studies have shown that excessive synovial tissue hyperplasia is the initial event of RA,and it plays a role in the early onset of RA.The excessive proliferation of synovial cells is the main cause of synovial hyperplasia through promoting proliferation and inhibiting apoptosis;and many inflammatory cytokines in the joint microenvironment also promote the proliferation of synovial cells,resulting in a sustained inflammatory response.Therefore,systematically studying the mechanism of regulating the abnormal biological behavior of synovial cells is of great value for elucidating the etiology of RA and developing more effective targeted therapies.In recent years,non-coding RNA has emerged as a research hotspot of many diseases,among which micro RNA is the most-studied non-coding RNA.In our previous experiments,we found that mi R-410-3p is down-regulated in synovial tissue of patients with RA,suggesting that mi R-410-3p may play a role in RA.It was predicted by the micro RNA target gene website,such as Target Scan,that the transcription factor Yin Yang 1(YY1)is a downstream target gene of mi R-410-3p.There are no reports on the secretion,proliferation and apoptosis of inflammatory cytokines in YY1 and fibroblast-like synovial cells.This study is intended to be an entry point to investigate whether there is a direct interaction between mi R-410-3p and YY1,and the mechanism by which mi R-410-3p regulates the secretion of inflammatory cytokines,proliferation and apoptosis in synovial cells.The NF-κB signaling pathway is a classical inflammatory and proliferative-related pathway in RA.It is mainly induced by TNF-α or IL-1β binding to cell membrane surface receptors,and the synovial cell signaling abnormality is one of the important mechanisms of RA.The positive feedback regulation between NF-κB signal and TNF-α causes persistent inflammatory response,and RA targeted therapy for NF-κB signaling pathway is very promising in the future.This study also explored the effect of mi R-410-3p on the expression of IκBα,p-IκBα,p65 and p-p65 in synoviocytes,and studied the relationship between mi R-410-3p and NF-κB signal transduction pathway.Methods: 1.Real-time quantitative PCR was used to detect the expression of mi R-410-3p in synovial tissue and rheumatoid arthritis fibroblast-like synoviocytes(HFLS-RA)in patients with rheumatoid arthritis;The HFLS-RA cell line was transfected with mi R-410-3p mimics,inhibitor,negative control sequences and blank groups.Real-time quantitative PCR was used to detect transfection efficiency,and the effects of mi R-410-3p on the secretion of TNF-α,IL-1β,IL-6 and MMP-9 in HFLS-RA was detected by ELISA.CCK-8 assay and flow cytometry detect the effectsof mi R-410-3p on Proliferation,apoptosis and cell cycle in HFLS-RA.2.Transfection of mi R-410-3p mimics and inhibitor in HFLS-RA cells,protein expression of IκBα,p-IκBα,p65 and p-p65 were examined by Western Blot;NF-κB activation nuclear translocation fluorescence method NF-κB activation was detected after transfection of HFLS-RA cells;BAY 11-7082(NF-κB inhibitor)was treated with HFLS-RA cells,and protein expression of p65 and p-p65 was detected by Western blot;Expressions of TNF-α,IL-1β,IL-6 and MMP-9 in supernatants of HFLS-RA cells between mi R-410-3p inhibitor,inhibitor negative control and inhibitor +BAY 11-7082 were compared.3.Construction of YY1 dual luciferase reporter gene vector was to detect the direct binding of mi R-410-3p to YY1;construct the YY1 overexpression vector,and then transfect HFLS-RA,real-time quantitative PCR and Western Blot detect the expression of YY;the proliferation,apoptosis and cell cycle of HFLS-RA were compared between mi R-410-3p mimics,mimics negative control and mimics +YY1 overexpression.Results: 1.The expression of mi R-410-3p in synovial tissue of rheumatoid arthritis patients is 1/10 of normal human synovial tissue(p<0.05);The expression of mi R-410-3p in HFLS-RA was 1/3 of HFLS(p < 0.001).2.After transfection48 hin HFLS-RA,mi R-410-3p was up-regulated approximately 1200 folds in the mimics group(p<0.0001)and down-regulated in the inhibitor group to 1/5(p <0.0001).3.The expression of TNF-α,IL-1β,IL-6 and MMP-9 in the mi R-410-3p mimics group was significantly decreased after transfection(p<0.05),the expression of TNF-α,IL-1β,IL-6 and MMP-9 in the inhibitor group was significantly increased(p<0.05).4.The proliferation of HFLS-RA was detected at 24,48 and 72 hours after transfection of mi R-410-3p.The cell proliferation of mi R-410-3p mimics group was inhibited(p<0.05),and the inhibitor promoted proliferation(p<0.05);Apoptosis of HFLS-RA was detected at 48 hours after transfection of mi R-410-3p,apoptosis was promoted in mi R-410-3p mimics group(p<0.05),apoptosis in inhibitor group was inhibited(p<0.05);HFLS-RA in mi R-410-3p mimics group were arrested in S phase(p<0.05).The expression of IκBα,p-IκBα,p65 and p-p65 in HFLS-RA was detected 72 hours after transfection of mi R-410-3p,and the expression of p-IκBα and p-p65 protein in the mimics group was decreased(p<0.05).There was no difference in total IκBα and total p65 protein levels.The expression of p-IκBα and p-p65 protein in the inhibitor group was increased(p<0.05),and there was no difference in total IκBα and total p65 protein levels;72 hours after transfection of mi R-410-3p NF-κB activation nuclear translocation in HFLS-RA was detected.NF-κB activation was inhibited in the mimics group,and NF-κB activation was promoted in the inhibitor group.After the treatment of HFLS-RA with BAY 11-7082 for 72 hours,the protein expression of p65 and p-p65 was detected.The p-p65 of BAY 11-7082 group was significantly decreased(p<0.0001),and the total p65 was unchanged;the concentration of TNF-α,IL-1β,IL-6 and MMP-9 secreted by HFLS-RA was increased significantly in the inhibitor group(p <0.05),then decreased after BAY 11-7082 co-treatment(p<0.05).7.Luciferase reporter gene assay showed that luciferase activity was significantly decreased(p<0.05)after co-transfection of mi R-410-3p mimics and YY1 gene 3’-UTR wild-type recombinant reporter plasmid,and there was no change in luciferase activity after co-transfection of the mutant recombination reporter plasmid,confirming that YY1 is the target of mi R-410-3p.8.At 48 and 72 hours after transfection of YY1 overexpression plasmid,m RNA and protein expression levels were detected,and m RNA and protein of YY1 was increased(p<0.05);The proliferation of HFLS-RA was inhibited,apoptosis was promoted in the mimics group,while YY1 co-transfection group partially rescued the regulatory effect of mi R-410-3p on proliferation and apoptosis of HFLS-RA(p<0.05),suggesting that mi R-410-3p regulates proliferation and apoptosis of HFLS-RA via targeting YY1.Conclusion: 1.mi R-410-3p acts as an inflammatory suppresser in rheumatoid arthritis and is down expressed in RA synovial tissues and cells;mi R-410-3p inhibits the secretion of inflammatory cytokines and proliferation of HFLS-RA,inhibitapoptosis and arrest HFLS-RA in the S phase.2.mi R-410-3p binds directly to YY1.3.mi R-410-3p inhibits the secretion of TNF-α,IL-1β,IL-6 and MMP-9 of HFLS-RA by regulating NF-κB signaling pathway.4.mi R-410-3p regulates proliferation and apoptosis of HFLS-RAvia targeting YY1. |
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