The Role And Mechanism Of TRIM3 And FoxO3a In Rheumatoid Arthritis

Rheumatoid arthritis is characterized by chronic arthromeningitis,persistent systematic inflammation and production of auto-antibody which especially include rheumatoid factors and cyclic citrullinated peptides.RA may lead to intraarticular cartilage injury,joint dysfunction,cardiovascular and pulmonary complication and which was worse,RA can induce disability.RA may be attribute to the complexity of interaction of gene and the environment,half the risk for development of RA is attributable to genetic factors while smoking accounts for the primary environmental risk.RA affects 0.5-1.0%adults in China,with 5-50 per 100000 new cases annually.Genome-wide research about RA showed that there were multiple genetically abnormal expression and dysfunction,highlighting that STAT4 and CD40 are markedly up-regulated in RA and other auto-immune diseases.Our research chose 30 active RA patients and 12 healthy controls which were only subjected to arthrotrauma.Real-time polymerase chain reaction and western blot were performed to determine the genetically abnormal expression,as a result,tripartite motif-containing protein 3 and fork-head box O3were found significantly down-regulated.Over-expression construction and shRNA of TRIM3 and Fox O3a were obtained and then transfected to fibroblast-like synoviocytes to study the impact of TRIM3 and Fox O3a on FLS function.pLVX-puro-TRIM3 was transfected to FLS,ELISA and Q-PCR were performed to measure the secretion of IL-1??IL-6 and TNF-?of FLS,and the results showed that the over-expression of TRIM3 induced reduction of IL-1?,IL-6 and TNF-?.CCK8 was performed to measure the proliferation of FLS,and the results showed that the over-expression of TRIM3 inhibited the proliferation of FLS.Western blot were performed to detect the protein level of CyclinD,PCNA,p53 and p21,and the results showed that the over-expression of TRIM3 inhibited the expression of CyclinD and PCNA,and promoted the expression of p53 and p21,The phosphorylation of p38 was inhibited after over-expression of TRIM3 and promoted after silencing of TRIM3.These data indicated that TRIM3 suppressed the proliferation of FLS via inhibiting the p38 pathway.To further study the impact of TRIM3 on the function of T cells and B cells,pLVX-puro-TRIM3 was transfected to T cells,ELISA and Q-PCR were perfoermed to measure the secretion of IL-2,IL4,IL-5,IFN-?,TGF-?and BAFF,the results showed that over-expression of TRIM3 inhibited the secretion of of IL-2,IL4,IL-5,IFN-?,TGF-?and BAFF in T cells.Auto-immune B cells which are characterized as CD19⁺CD27^-IgD⁺IgM^low/-B cells were obtained from the peripheral blood of RA patients by flow cytometry,pLVX-puro-TRIM3 were transfected to auto-immune B cells,CCK8 and Western blot were performed to detect the proliferation and activ ation of auto-immune B cells,the results showed that over-expression of pLVX-pur o-TRIM3 inhibited the proliferation and suppressed the activation of auto-immune B cells.These data suggest that TRIM3 inhibited the function of T cells and auto-im mune B cells.pLVX-puro-FoxO3a was transfected to FLS,the secretion of IL-1?,IL-6 and TNF-?were down-regulated,the proliferation of FLS was inhibited.Western blot were performed to detect the phosphorylation of related protein,the results showed that the phosphorylation of AKT were inhibited,the expression of NF-?B,CyclinD1and PCNA were up-regulated and the proliferation of FLS were promotedafter silencing of FoxO3a.These data indicate that FoxO3a inhibited the proliferation of FLS via the PI3K/AKT pathway.After transfection of p LVX-puro-FoxO3a,the proliferation of auto-immune B cells were inhibited while the secretion of IL-2,IL-4,IL-5,IFN-?,TGF-?and BAFF of T cells were unaffected.The gathered data showed that there were positive correlation between the mRNA level of FoxO3a and the mRNA level of TRIM3 in RA.FoxO3a over-expression led to up-regulation of TRIM3 in FLS.The total protein of FLS were extracted,pull-down and Western blot were performed to detect whether TRIM3 bound to FoxO3a each other,while the results showed that TRIM3 may not bind to Fox O3a each other.In conclusion,our research show that TRIM3 and FoxO3a are both down-regulated in RA.TRIM3 may inhibit the secretion of IL-1?,IL-6 and TNF-?of FLS,and suppress the proliferation of FLS via p38 pathway.TRIM3 can inhibit the secretion of IL-2,IL-4,IL-5,IFN-?,TGF-?and BAFF of T cells,and suppress the proliferation and activation of auto-immune B cells.FoxO3a could up-regulate the expression of TRIM3,and FoxO3a functions the similarly,Fox O3a could inhibit the secretion of IL-1?,IL-6 and TNF-?of FLS,and inhibit the proliferation of FLS via PI3K/AKT pathway.FoxO3a also could inhibit the proliferation of auto-immune B cells.These data show that TRIM3 and FoxO3a could negatively regulate the function of FLS,negatively affect the function of T cells and B cells as well,which lead to a changing complexity of the micro-environment of RA,and finally lead to persistent inflammation.Our research may apply logical proofs for the development of RA and prove that TRIM3 is a potential target for the treatment of RA.