| Interstitial lung disease(ILD)is the most common clinical manifestation of lung involvement in rheumatoid arthritis(RA)contributing to significantly increased morbidity and mortality.The high resolution computed tomography(HRCT)has emerged as an important tool in the evaluation of patients with RA-ILD,which leads to a delay in disease progression for some RA-ILD patients who have HRCT contraindications,and HRCT is relatively expensive.At the same time,early RA-ILD patients often lack specific symptoms and signs,which can easily lead to misdiagnosis and missed diagnosis.Matrix metalloproteinase 3(MMP3)is a convenient and effective indicator for monitoring disease activity and prognosis in RA,and its role has received increased research attention in fibrotic pulmonary diseases over recent years.However,the involvement of MMP3 in the development of RA-ILD remains unclear.Given that transforming growth factor-β(TGF-β)is a known and key pro-fibrotic factor,the Wnt/β-catenin signaling pathway is a key signaling pathway that causes epithelial-mesenchymal transition(EMT)in pulmonary fibrosis,and the interaction between TGF-βand the Wnt/β-catenin signaling pathway has been gradually revealed in fibrotic pulmonary diseases.Therefore,we hypothesize that MMP3-mediated interaction between TGF-β and Wnt/β-catenin signaling pathway in the development of RA-ILD?The deliberation of above questions will allow us to screen non-invasive biomarkers that can predict risk of lung injury early and efficiently and understand the molecular mechanisms of RA-ILD.Therefore,below four specific aims will be included in this proposal:(1)To Screen the target protein of RA-ILD by bioinformatics:Firstly,the differentially expressed genes(DEGs)were screened from GSE128813 and GSE47460 using bioinformatics techniques.Secondly,differentially expressed proteins(DEPs)were screened by proteinomics.Moreover,the co-DEP MMP3 was obtained based on transcriptomics and proteomics,and the expression of total MMP3 and active MMP3 protein in RA-FLS and THP-1 supernatants of the RA-ILD cell model were further verified.Meanwhile,the GeneCards database was used to retrieve the subcellular localization and major enriched signaling pathways of the MMP3,and the PHAROS database was used to predict the correlation between the MMP3 and RA-ILD.(2)Clinical correlation between plasma MMP3 protein and RA-ILD:The concentrations of plasma MMP3 were determined by enzyme-linked immunosorbent assays(ELISAs)in RA,idiopathic pulmonary fibrosis(IPF)and RA-ILD,and the concentrations of synovial fluid MMP3 were also detected in RA,osteoarthritis(OA)and RA-ILD.At the same time,association analysis of plasma MMP3 and clinical routine indexes were analyzed.(3)To explore the effect of MMP3 on the interstitial transformation of MRC-5 cells in vitro:Firstly,to investigate the potential impacts of endogenous MMP3 on MRC-5 cells into myofibroblasts transformation by western blotting and immunofluorescence,MRC-5 cells were generated with lentiviral-mediated MMP3 overexpression or silence.Secondly,in the presence of exogenous MMP3 protein,MMP3 inhibitor and TGF-β protein,the potential impacts of exogenous MMP3 protein on MRC-5 cells into myofibroblasts transformation,the proliferation and reactive oxygen species(ROS)production of MRC-5 cells were analyzed by western blotting,immunofluorescence,fibroblast-containing gel contraction capability,EdU(5-Ethynyl-2’-deoxyuridine)and ROS detection,(4)To explore the mechanism of MMP3 regulating the interstitial transformation of MRC-5 cells in vitro:Firstly,secreted TGF-β1 protein was detected in MRC-5 cells stimulated by exogenous MMP3 protein at eight different time points.Furthermore,the levels of Wnt/β-catenin signaling pathway in cytoplasm and nucleus,MMP2 and MMP9 in nucleus were determined in MRC-5 cells exposed to exogenous MMP3 protein,TGF-β protein and combined MMP3 with TGF-β protein using western blotting and immunofluorescence assays.Finally,while Wnt/β-catenin signaling pathway was activated or inhibited by Wnt3a cell supernatant or XAV939,the levels of mesenchymal transition markers,TGF-β,MMP2 and MMP9 in response to MMP3 protein exposure were examined in MRC-5 cells by western blotting and immunofluorescence assays.Results:(1)Firstly,43 overlapping DEGs from the GSE128813 and GSE474460 datasets were filtered as objective genes.Secondly,a total of 33 DEPs in the RA-FLS and THP-1 supernatants of the RA-ILD cell model were identified by proteomics.Moreover,the intersection of the transcriptomic and proteomic results identified one co-DEP,MMP3.Meanwhile,the levels of total MMP3 and active MMP3 protein were elevated in(RA-FLS+)-CM and(RA-FLS++THP-1)-CM of the RA-ILD cell model compared to(RA-FLS)-CM and(RA-FLS+THP-1)-CM by wstern blotting and immunofluorescence in vitro,respectively.The GeneCards database showed that MMP3 was mainly distributed in the extracellular matrix and involved in epithelial-esmenchymal transition(EMT)and collagen catabolic process.The PHAROS database further predicted a correlation between the MMP3 and RA-ILD disease,with few published studies currently available.(2)Clinical data research showed that the level of MMP3 in the plasma of RA-ILD patients was significantly higher than that of the RA group and IPF group.In addition,the level of MMP3 in the plasma of RA-ILD patients was positively correlated with disease activity.Meanwhile,the level of MMP3 in the plasma of RA-ILD patients who underwent drug treatment and showed significant improvement was significantly lower than that of patients who were not treated with drugs before hospitalization.It is worth noting that a prediction model with good discrimination and accuracy for predicting the occurrence of RA-ILD can be constructed by combining clinical routine monitoring indicators(gender,smoking,RF,CRP)and plasma MMP3.In addition,a prediction model with good discrimination for predicting the risk of death in RA-ILD can be constructed by combining radiological UIP type and plasma MMP3.(3)The overexpression of MMP3 could significantly increased ECM marker,Collagen I,as well as myofibroblasts markers,such as Vimentin and N-cadherin,of MRC-5 cells by western blotting and immunofluorescence assays,whereas MMP3 siRNA knocked down Vimentin,and N-cadherin levels.In consistence,exogenous MMP3 protein also significantly increased the levels of Vimentin and N-cadherin,and addition of a specific MMP3 inhibitor significantly decreased the level of Vimentin of MRC-5 by western blotting,immunofluorescence and RT-PCR assays.Fibroblast-containing gel contraction capability results further showed that exogenous MMP3 protein promoted collagen contraction of MRC-5 cells.In other words,exogenous MMP3 protein promoted fibroblast-to-myofibroblast transformation.EdU and ROS results showed that exogenous MMP3 protein promoted the proliferation and ROS production of MRC-5 cells.(4)The level of secreted TGF-β was the highest in MMP3-stimulated MRC-5 cells at one hour.Western blotting and immunofluorescence results further showed that exogenous MMP3 protein and TGF-β protein synergistically in up-regulating the transition of MRC-5 cells into myofibroblasts.In addition,the results showed that culture supernate from Wnt3a cell significantly induced the transformation of MRC-5 into myofibroblasts,and the expression of TGF-β,MMP2 and MMP9.Moreover,MMP3 futher synergistically promoted the activation of Wnt/β-catenin signaling pathway by culture supernate from Wnt3a cell,the transformation of MRC-5 into myofibroblasts,and the expression of TGF-β,MMP2 and MMP9.In conclusion,the target protein-MMP3 of RA-ILD was screened by bioinformatics methods firstly.Moreover,the gender of male,current smoking state,levels of circulating RF,CRP,and plasma MMP3 were identified as risk factors for the construction of the predictive model of ILD in RA patients.UIP and level of plasma MMP3 were identified as risk factors for the construction of the risk of death in RA-ILD patients.In addition,the effect of MMP3 on the interstitial transformation of MRC-5 cells was analyzed.Finally,MMP3-mediated cross-talk between TGF-β and Wnt/β-catenin signaling pathways played a regulatory role in promoting esmenchymal transition of MRC-5 cells.The above results indicates that MMP3 may serve as a novel target for RA-ILD and may provide a new direction for exploring the delay of lung involvement. |
