| Rheumatoid arthritis(RA)is a commonly occurring chronic autoinflammatory disease.Its main pathobiological characteristic is synovitis,accompanied by erosion and damage of articular cartilage and bone.RA fibroblast-like synoviocytes(RA-FLS)are the main cell types of pathologically proliferative synovium.Their uncontrolled proliferation,accumulation,migration and invasion are the main causes of chronic inflammation,joint injury and destruction.Activated RA-FLS can produce a large number of pro-inflammatory cytokines and chemokines that over-recruit,maintain,and activate cells of the immune system,leading to chronic inflammation and progressive joint destruction.In recent years,activated transcription factor 3(ATF3)has been shown to be abnormally expressed in several diseases and to impair cell growth and metabolism.Therefore,in this work,we analyzed the relationship between ATF3 and RA using bioinformatics,investigated the expression of ATF3 in RA synovial tissue and its effects on the biological function of RA-FLS,and explored its molecular mechanism.Objective:Part Ⅰ: To investigate the correlation between ATF3 and RA and the association with drug treatment response and immune infiltration characteristics using bioinformatics methods.Part Ⅱ: Validation of ATF3 expression in tissues and its effect on cell biological functions.Part Ⅲ: To observe the effect of ATF3 on the clinical manifestation,pathology,and inflammatory factors of the CIA mouse model.Part Ⅳ: Exploring the molecular mechanism of the effect of ATF3 on the progression of RA-FLS.Methods:Part Ⅰ:(1)The microarray associated with RA is selected from the GEO database,and the WGCNA method is used to explore the GEO database.The selected differential gene set and the clinically related modules are overlapped and the target genes are determined.(2)The relationship between the clinical parameters of ATF3 and RA was analyzed using the dataset.(3)The dataset was used to analyze the relationship between ATF3 expression and gene set enrichment and immune infiltration analysis.Part Ⅱ:(1)Collection of RA and health control synovial tissue and extraction of the primary RA-FLS.(2)ATF3 expression in synovial tissue was observed by HE,immunohistochemistry,and again verified by q RT-PCR and Western Blot.(3)RA-FLS cells were identified by cellular immunofluorescence,and stable cell lines of Sh-ATF3 and ATF3-OE were constructed and infection efficiency was verified.(4)q RT-PCR and Western Blot to verify the expression of ATF3 in RA-FLS.(5)CCK-8 method to detect proliferation,flow cytometry to detect cell cycle and apoptosis of RA-FLS after infection.(6)Transwell to observe the migration and invasion ability of RA-FLS after infection.(7)To detect the secretion and expression of inflammatory factors by q RTPCR and enzyme-linked immunosorbent assay(ELISA).Part Ⅲ:(1)Construct the CIA mouse model and package the lentivirus.(2)Lentivirus was injected into the knee joint lumen,and q RT-PCR was validated.(3)ELISA to assay serum inflammatory factor secretion.(4)HE observation of knee joint pathology in mice.(5)Saffron-O-fixed green was used to observe the cartilage erosion of knee joint.(6)Synovial cell apoptosis was observed by TUNEL fluorescence method.Part Ⅳ:(1)m RNA sequencing was conducted using stable ATF3 knockdown cell lines and knockdown controls,and the differential genes were analyzed for KEGG and GO enrichment using bioinformatics methods.(2)Protein expression was detected by Western Blot to explore the relationship between ATF3 in PI3K/AKT/mTOR pathway and cellular autophagy with RA-FLS.Results:Part Ⅰ:(1)Download and merge the microarray datasets GSE12021,GSE55235,GSE55457,and GSE55584 into the GPL96 platform database.Download microarray datasets GSE48780 and GSE77298 and merge them into GPL570 platform database.(2)The GO analysis showed that differential genes in the GPL96 platform database significantly enhanced immunity and inflammation-related functions.KEGG analysis showed that differential genes were all remarkably enriched in inflammatory-associated pathways,consistent with the inflammatory properties of RA.(3)The characteristic heat map showed 106 tissue samples in GPL570 platform database(99 RA,7 normal)and 74 samples in GPL96 platform database(45 RA,29 normal).(4)The modulefeatured heat map of the GPL570 platform shows that the negative correlation between the ME-greenyellow module and RA synovial tissue is the highest.The module-feature related heatmap of GPL96 platform shows that the negative correlation between MEsalmon module and RA sample is strongest.(5)The ME-greenyellow module gene and differentially expressed gene in GPL570 were crossed with the ME-salmon module gene and differentially expressed gene in GPL96,and the gene ATF3 was screened.(6)ATF3 expression was slightly higher in inflammatory infiltrative synovium than in noninflammatory synovium and was positively correlated with DAS-28 scores.Prior to treatment with tocilizumab or methotrexate,ATF3 expression was significantly higher in RA patients with good response than in those with limited response.(7)There was the most positive correlation between monocytes and live mast cells in the GPL96 platform,the most negative correlation between memory B cells and immature B cells,the most positive correlation between activated NK cells and resting mast cells in the GPL570 platform,and the most minus correlation between resting mast cells and activated mast cells.Part Ⅱ:(1)HE staining showed that a large number of inflammatory cells infiltrated in RA synovial tissue,synovial cell hypertrophy,and immunohistochemistry showed that ATF3 was highly expressed in RA,which was positively correlated with disease activity and negatively correlated with age.(2)Results of q RT-PCR and Western Blot showed high expression of ATF3 in RA synovial tissue.(3)Cellular immunofluorescence appraisals of isolated cultured RA-FLS,positive for Vimentin protein and negative for CD68.(4)Expression of Sh-ATF3 and ATF3-OE in RA-FLS was authenticated by q RT-PCR and Western Blot.(5)Compared with the empty virus control,Sh-ATF3 significantly inhibited the proliferation of RA-FLS,while ATF3-OE significantly promoted the proliferation of RA-FLS.(6)Knockdown of ATF3 blocked the RA-FLS cell cycle in G0/G1 phase,and overexpression of ATF3 increased the percentage of block in S and G2/M phases.(7)Inhibition of ATF3 expression increased the apoptosis of RA-FLS,while overexpression of ATF3 did the opposite.(8)Transwell results demonstrated that knockdown of ATF3 reduced the migratory and invasive ability of RA-FLS,and overexpression promoted this ability.(9)Test results of q RTPCR and ELISA showed that inhibition of ATF3 decreased IL-1β,IL-6 and IL-8secretion and expression in RA-FLS by giving TNF-α intervention stimulation,and overexpression of ATF3 promoted their secretion and expression.Part Ⅲ:(1)The arthritis score of the CIA mouse model was significantly higher than that of the normal group,and the arthritis index of the Sh-ATF3 group decreased compared with that of the Sh-Ctrl group after lentivirus injection in CIA mice.(2)Verification of the synovial membrane of the knee joint in mice by q RT-PCR revealed that the relative ATF3 m RNA expression was higher in the Sh-ATF3 and Sh-Ctrl groups than in the Normal group,and the expression was higher in the Sh-Ctrl group than in the Sh-ATF3 group.(3)The knee joints of Normal group mice were normal in structure,with no abnormal proliferation and inflammatory cell infiltration in the synovial membrane,while the CIA mice had an unsmooth cartilage surface,with excessive synovial membrane formation,massive inflammatory cell infiltration,and the formation of pannus.And the pathological score was reduced in the Sh-ATF3 group compared to the Sh-Ctrl group.(4)In CIA mice,the levels of TNF-α,IL-1β and IL-6expression in serum were significantly decreased in the Sh-ATF3 group compared with the Sh-Ctrl group.Part Ⅳ:(1)The m RNA sequencing was performed on the stable transfected cells of knockdown ATF3 and control group,and the differential genes were analyzed for enrichment analysis,and ATF3 was found to be closely related to the classical PI3 KAKT pathway.(2)Validation of the molecular mechanism of ATF3 in RA pathogenesis by Western Blot revealed that it could promote PI3K/AKT/mTOR pathway protein expression and reverse cellular autophagy.Conclusions:In this study,ATF3,a biomarker associated with the pathogenesis of RA,was successfully identified.High expression of ATF3 correlated positively with systemic inflammation and high disease activity at RA.In conjunction with the study of clinical tissue samples,cell experiments in vitro,CIA mice in vivo,and the molecular mechanism of RA-FLS,it was found that ATF3 can be overexpressed in the synovium of RA and CIA mice,promote the proliferation,migration,and invasion of RA-FLS,inhibit apoptosis,and stimulate the secretion and expression of inflammatory factors.The molecular mechanism explains that ATF3 can activate proteins of PI3K/AKT/mTOR signaling pathway and reverse autophagy,which affects the pathogenesis of RA.ATF3 may be a potential therapeutic target for intervention in RA. |
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