| Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by synovitis,accompanied by the destruction of articular cartilage and subchondral bone erosion.Activation of RA synovial blasts(RASFs)is an important participant in the above pathological process.There is evidence that the Wnt/?-catenin signaling pathway is activated in RA and is involved in the activation of RASFs.The activated SFs have the characteristics of proliferation,enhanced invasiveness,abnormal secretion of inflammatory factors and tumor-like growth.In recent years,the transcription factor Forkhead box C1(Fox C1)has been found to be abnormally expressed in a variety of tumor pathogenesis and plays an important role in the proliferation,migration and invasion of tumor cells.Although Fox C1 has been reported in tumors,there are few studies on the expression and function of Fox C1 in RA.Therefore,this study focused on the expression of Fox C1 in the synovial tissues of RA and RASFs,as well as the related molecular mechanisms of Fox C1 influence on the proliferation,migration,invasion and secretion of inflammatory factors of RASFs.This experiment is divided into four parts.1.Fox C1 and ?-catenin are highly expressed in RA synovial tissues and RASFsSynovial tissues of patients with RA and normal trauma group(Control)were obtained and the expressions of Fox C1 and ?-catenin were detected by immunohistochemistry(IHC),western blot(WB)and real-time fluorescence quantitative reverse transcription polymerase chain reaction(q RT-PCR).The results showed that the expression level of Fox C1,?-catenin gene,and protein in RA synovial tissue was significantly higher than that in control patients.In addition,the primary SFs were extracted from synovial tissue for detection,and the results also showed that Fox C1 and ?-catenin were higher in RASFs than in Control SFs.Immunohistochemistry of synovial tissues and immunofluorescence of SFs showed that Fox C1 and ?-catenin were expressed in both cytoplasm and nucleus of SFs,and the expression of Fox C1 and ?-catenin in the nucleus was significantly higher than that of the Control group.These results suggest that Fox C1 and ?-catenin are abusually expressed in RA pathology.2.Fox C1 regulates the proliferation,migration,invasion,and secretion ofinflammatory cytokines in RASFsIn order to further explore the function of Fox C1 in RA,primary RASFs were extracted and transfected with lentiviruses that overexpressed Fox C1 to observe their effects on the proliferation,migration,invasion,and secretion of inflammatory factors of RASFs.The results showed that the proliferation,migration and invasion of RASFs that overexpressed Fox C1 in vitro were significantly enhanced.Enzyme-linked immunosorbent assay(ELISA)showed that overexpression of Fox C1 increased the secretion of interleukin-1?(IL-1?),interleukin-6(IL-6),and tumor necrosis factor-?(TNF-?)expression in RASFs supernate.At the same time,the secretion of interleukin-10(IL-10)was decreased.Si RNA fragments that inhibited Fox C1 expression were transfected to observe their effects on RASFs proliferation,migration,invasion and secretion of inflammatory factors.The results showed that the proliferation,migration and invasion ability of Fox C1 inhibiting RASFs were significantly decreased in vitro.ELISA showed that inhibition of Fox C1 decreased the secretion of IL-1?,IL-6 and TNF-? in RASFs supernate,and promoted the secretion of IL-10.3.Fox C1 affects the proliferation,migration,invasion,and inflammatory factorsecretion of RASFs by regulating ?-catenin activation of the Wnt/?-cateninsignaling pathwayCombined with these results,we hypothesized that Fox C1 regulates the proliferation,migration,invasion,and secretion of inflammatory factors in RASFs,most likely by regulating the Wnt/?-catenin signaling pathway.Therefore,we applied WB and q RT-PCR methods to detect the genes related to Wnt/?-catenin pathway and RA pathology.The results showed that overexpression of Fox C1 in RASFs could promote the expression of ?-catenin,an important signaling molecule of the Wnt/?-catenin pathway,and the expression of cyclin D1,c-Myc,and related genes fibronectin and matrix metalloproteinase 3(MMP3)increased.However,this phenomenon had the opposite effect after inhibiting Fox C1 expression.In order to further clarify that Fox C1 may affect the Wnt/?-catenin pathway and achieve the above functions by regulating ?-catenin.We do a positive and negative verification experiment.Firstly,in the case of overexpression of Fox C1,the expression of cyclin D1,c-Myc,fibronectin and MMP3 was decreased.The proliferation,migration,invasion and secretion of pro-inflammatory cytokines(IL-1?,IL-6,and TNF-?)and anti-inflammatory cytokines(IL-10)of RASFs were inhibited.Secondly,we overexpressed ?-catenin with Fox C1 inhibition and found that the expression levels of cyclin D1,c-Myc,fibronectin and MMP3 were all increased.The proliferation,migration and invasion of RASFs and the secretion of pro-inflammatory cytokines(IL-1?,IL-6 and TNF-?)increased,while the secretion of anti-inflammatory factors(IL-10)decreased.Finally,through co-immunoprecipitation(COIP),we found that Fox C1 protein was bound to ?-catenin protein in RASFs.Furthermore,through dual-luciferase assay and chromatin immunoprecipitation(CHIP),it was found that Fox C1 was bound to ?-catenin promoter in RASFs,promoting the transcription of ?-catenin.4.Inhibition of Fox C1 in vivo could reduce the progression of arthritis incollagen-induced arthritis(CIA)ratsFourteen days after the CIA model was established,we injected Fox C1-specific si RNA that inhibited Fox C1 expression into the left posterior ankle of the rats.In vivo imaging was used to determine that the drug was effective for one week in vivo.After 3 weeks of intervention,the therapeutic effects of Fox C1-specific si RNA on synovium,bone,and cartilage in CIA rats were evaluated by HE staining,immunohistochemistry,micro-CT and red solid green staining,respectively.Our results showed that Fox C1-specific si RNA reduced inflammatory progression in CIA rats.To sum up,our study comprehensively investigated the expression of Fox C1 in RASFs and the effects of Fox C1 on the proliferation,migration,invasion and inflammatory factors of RASFs from both in vitro and in vivo systems.Fox C1 was found to play these roles by regulating the important signaling molecule ?-catenin in the Wnt/?-catenin pathway.Finally,we demonstrated that local inhibition of Fox C1 expression alleviated the progression of arthritis in CIA rats.The discovery of Fox C1 provides important clues for the understanding and exploration of the pathological mechanism of RA and provides new ideas for finding new therapeutic targets for RA. |
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