BAMBI Inhibits Migration And Invasion By Regulating TGF-β1/Smads Signaling Pathway In Fibroblast-Like Synoviocytes Of Rheumatoid Arthritis

BackgroundRheumatoid arthritis(RA)is recognized as a major refractory disease with prominent pathological features of synovial hyperplasia and erosion of articular cartilage and bone,which ultimately leads to joint destruction.The proliferation of fibroblast like synoviocyte(FLS)with a large number of inflammatory cell infiltration and neovascularization is the basis of RA synovial hyperplasia.In this pathological process,RA-FLS is characterized by malignant proliferation,migration,and invasion,and is characterized by tumor-like transformed cells(called active hyperplasia),which is closely related to advanced joint damage in RA.TGF-β1 is involved in the regulation of cell proliferation,apoptosis,differentiation,migration and immune response.The study found that TGF-β1 was significantly elevated in synovial and synovial fluid of patients with RA compared with osteoarthritis(OA)or healthy controls.Epithelial-mesenchymal transition(EMT)is a biological process that allows epithelial cells to acquire mesenchymal cell phenotypes,enhance cell migration and invasion,and increase cell anti-apoptotic ability.TGF-β1 is closely related to this process.The transmembrane inhibitor of bone morphogenetic protein and activin(BAMBI)acts as a"pseudo-receptor" of TGF-β1 and can affect the functional state of cells by regulating the physiological action of TGF-β1.Based on the above theory,we studied the pathological mechanism of RA-FLS in four parts.Part Ⅰ The relationship between phenotypic changes of RA-FLS and epithelial-mesenchymal transitionObjective:To provide evidences that the pathological phenotypic changes of RA-FLS are related to epithelial-mesenchymal transition.Method:The expression of epithelial-mesenchymal transition markers in RA-FLS was detected by immunofluorescence;thereafter,the expression of EMT markers derived from RA-FLS and OA-FLS were analyzed by Western blot.Results:(1)N-cadherin,E-cadherin,α-SMA,Vimentin were positively expressed in RA-FLS;(2)Compared with OA-FLS,the expression of E-cadherin and Vimentin decreased in RA-FSL,while the expression of N-cadherin and α-SMA increased.Conclusion:RA-FLS phenotypic changes are associated with epithelial-mesenchymal transition.However,the expression of Vimentin is decreased,and this change may be related to the specificity of RA.Part Ⅱ TGF-β1 promotes cell migration and invasion by inducing epithelial-mesenchymal transition in RA-FLSObjective:To investigate the relationship between TGF-β1 and RA-FLS phenotype transformation and functional changes.Method:The cells were treated with 0.1,0.1,10 ng/ml of TGF-β1 for 48 hours,and the expressions of N-cadherin,E-cadherin,α-SMA,Vimentin,MMP9 and MMP2 were detected by immunoblotting;After treating with 10 ng/ml of TGF-β1,the expression levels of N-cadherin,E-cadherin,α-SMA,Vimentin,MMP9 and MMP2 were detected by immunoblotting at 12,24 and 48 hours.Cells were treated with 10 ng/ml TGF-β1 for 48 hours,Transwell was used to detect cell migration and invasiveness.Results:(1)The expression levels of N-cadherin,MMP2 and α-SMA were increased with the increase of TGF-β1 concentration,and the expression levels wer the highest in 10 ng/ml.The expression level of E-cadherin was the lowest in 10 ng/ml;the expression levels of MMP9,Vimentin remain unchanged.(2)The expression levels of N-cadherin,MMP2 and α-SMA were increased with the prolongation of TGF-β1,and the expression levels were the highest at 48h.The expression level of E-cadherin was reversed.The expression level was the lowest at 48h,while the expression levels of Vimentin and MMP9 remained unchanged.(3).RA-FLS was treated with TGF-βl at a concentration of 10 ng/ml After 48 hours,the cell invasion and migration ability was significantly enhanced.Conclusion:TGF-β1 can induce epithelial-mesenchymal transition in RA-FLS and enhance cell migration and invasion.However,TGF-β1 did not affect the expression levels of MMP9,Vimentin.Part Ⅲ Interfering with Smad2/3 reverses RA-FLS phenotypic transformation and blocks TGF-β1-induced epithelial-mesenchymal transitionObjective:To investigate the role of Smad2/3 in the phenotypic transformation of RA-FLS,and to further elucidate the mechanism by which TGF-β1 regulates cell phenotype and function.Method:The cells were treated with 10 ng/ml TGF-β1 for 48 hours.The activation level of Smad2/3 and the expression level of Snail were detected by immunofluorescence and Western blotting.The expression of Smad2/3 was interfered with siRNA,then expression levels of N-cadherin,E-cadherin,α-SMA,Vimentin,MMP9,MMP2 were detected by immunoblotting;cells were treated with Smad2/3 siRNA or control siRNA for 48 hours,then treated with TGF-β1 for 48 hours,expression levels of N-cadherin,E-cadherin,α-SMA,Vimentin,MMP9,MMP2 were detected by immunoblotting,and cell migration and invasion were dected by Transwell assay.Results:(1)In RA-FLS,TGF-β1 induces the expression of Snail by activating Smad2/3;(2)Interfering with Smad2/3 can reverse the expression of N-cadherin,E-cadherin andα-SMA,but has no effect on the expression levels of Vimentin,MMP9 and MMP2;(3)Interference with Smad2/3 blocked TGF-β1-induced epithelial-mesenchymal transition and inhibited RA-FLS migration and invasion.Conclusion:Activation of Smad2/3 plays an important role in the phenotypic changes of RA-FLS.TGF-β1 can promote cell migration and invasion by activating TGF-β1/Smads signaling pathway.Part Ⅳ BAMBI affects the migration and invasion of RA-FLS by regulating the TGF-β1/Smads pathwayObjective:To explore the relationship between BAMBI and TGF-β1/Smads pathway in RA-FLS,and the mechanism by which BAMBI regulates cell migration and invasion.Method:Immunofluorescence and immunoblotting were used to detect the expression of BAMBI.RA-FLSs were treated with TGF-β1,and the expression level of BAMBI was detected by immunoblotting.The expression levels of N-cadherin,E-cadherin,α-SMA,Vimentin,MMP9 and MMP2 were detected by immunoblotting after BAMBI was overexpressed by lentivirus,cell migration and invasion were detected by Transwell.After BAMBI was overexpressed by lentivirus,cells were treated with 10 ng/ml TGF-β1 for 48 hours.Immunoblotting was used to detect N-cadherin,E-cadherin,α-SMA,Vimentin,MMP9 and MMP2,cell migration and invasion were detected by transwell.Results:(1)Immunofluorescence showed that BAMBI was positively expressed in RA-FLS;(2)Immunoblotting showed that the expression level of BAMBI in RA-FLS was lower than that of OA-FLS;(3)TGF-β1 treatment of RA-FLS inhibits the expression of BAMBI;(4)Overexpression of BAMBI reverses the expression level of the cell epithelial-mesenchymal transition marker and inhibits RA-FLS migration and invasion;(5)Overexpression of BAMBI inhibits TGF-β1-induced epithelial-mesenchymal transition and inhibits cell migration and invasion.Conclusion:BAMBI is involved in the phenotypic changes of RA-FLS,which blocks epithelial-mesenchymal transition and inhibits cell migration and invasion through the TGF-β1/Smads signaling pathway.