| Rheumatoid arthritis（RA）is one of the most common autoimmune disease. it ischaracterized by chronic inflammation of the synovial tissue and has gradually lead to thedamage of cartilage even the corruption of bone and the impairment of joints. The diseaseis often progressive and destructive, and greatly affect the life quality of the affectedindividuals. Although the cause of RA is still unknown, studies in recent years showed thatthe activated fibroblast-like synoviocytes（FLSs）, as part of a complex cellular network,play an important role in the pathological process of rheumatoid arthritis. So these cellshas aroused great concern of researchers in elucidating their specific features.For it’s complex pathogenesis in advanced stage of RA disease，it’s obvious thatsingal signal passway can’t interpret the RA pathogenesis clearly and exhaustively. So weurgently need investigation into the complex signal transduction network in RA FLS.The aim of our study is to detect the differencial gene expression in RA FLSs bygenomics and proteomics approches and to discover the mechanism of RA pathogenesis as much as possible.Gene expression microarray， as a new tool in biotechnology, it allowssimultinously monitoring thousands of gene expression levels in cells. So we first usedcDNA microarray to investigate the genes differently expressed in RA FLS compared withnormal and OA FLS in the RNA level. We performed in vitro experiments using thecultured synovial fibroblasts of passage4.The total RNA was extracted from RA，OA andnormal synoviocytes and then purified. The cDNA was obtained by reverse transcriptionpolymerase chain reaction (RT-PCR), and then labeled with Cy5and Cy3, which werehybridized with Agilent Human1A gene expression microarray containing21073humangenes. Subsequently, the signal images were scanned and then analyzed by image analysissoftware.The results showed that among the21073target genes, there are1250genesupregulated and566genes downregulated between RA and normal FLS. Among theupregulated genes, there are17genes rised more than100times, and among theupregulated genes there are several genes have more relation with tumor analogousproperties such as cell proliferation、adhesion、cell cycle and migration.The result suggests that multiple genes take part in the pathogenesis of RA. These1816genes differentially expressed between RA and normal FLSs may be involved in theoccurrence and development of RA. Further studying of these genes may be helpful forclarifying RA pathogenesis and finding more moleculars involved in it.Then we used bioinformatics for further analysis, of the intention of mininginformation from these gene expression profiles reflecting the essential characteristics fordisease. The results suggest that most tumor-related genes belong to the incresed group ofRA vs OA and normal. In these genes involves genes associating cancer pathways, cellcycle regulation, cell receptor and extracellular matrix interaction. It is expected to carryout further analysis in order to clear the pathogenesis of RA mechanisms and to find thenew disease-related key molecules.Then we selected14genes according to thebioinformatics results to verify their expression level by real-time PCR.Except CDKN1A,the remaining13genes were consistent with the microarray results. Indicating that our experiments have good reproducibility and credibility. According to the microarrayresults and bioinformatics analysis, we believe that the expression profiles of RA andnormal and OA synovial cells have obvious differences.And part of these moleculescontribute to the different biological function of RA synovial cell of and the incidence andevolution of RA disease. The next further in-depth study of the key elements will help usfor a better understanding of RA pathological process and possibly as a potential drugtarget to guide clinical treatment.The current study showed that RA articular cartilage damage is primarily caused bymatrix metalloproteinase secreted by synovial cells and synovial tissue which areexcessivly proliferate. Matrix metalloproteinase degradate the main component ofcartilage matrix: type II collagen, the fracture of collagen type II may directly inducesynovial cells to secret matrix metalloproteinase and matrix metalloproteinase is of greatsignificance for the lesions of cartilage and bone destruction. So to clarify the keyapproach and moleculars in secretion of matrix metalloproteinase mediated by type IIcollagen is the great breakthrough in RA treatment and reduction of articular cartilagedamage. However, as the complexity of rheumatoid arthritis in pathogenesis of late stage,we may find it difficult to clarify its pathogenesis by a single signal transduction pathway,so there’s an urgent need to go deep into the complex signal transduction network of theproliferation of synovial cells and cartilage damage.According to the results of the chips and the previous works by our group, wepropose a DDR2-CYR61-MMPs pathway may play an important role in the articularcartilage damage in RA. The results confirm that the activation of DDR2increase CYR61expression, and thus increase the expression of MMP-13and promote the invasion ofRA FLS. And confirm DDR2deletion mutation in the mouse model can inhibit theexpression of CYR61and collagen-induced joint damage. Then we confirmed thatdownregulation of CYR61effectively reduce the joint damage in CIA rats. Subsequentlywe also confirmed CYR61promote the proliferation of RA FLS.These results provideimportant clues for the pathogenesis of RA.The research of miRNA related to RA pathological process has just started, there’s already result shows that abnormal miRNA expression in RA synovial tissue.By detailedanalysis using bioinformatics, we forecast the possible miRNA targeting the3’-UTR andcoding region of DDR2, MMP-1, MMP-13, STAT3genes. The miRNA real-time PCRshowed that miR-16-1, miR-143, miR-29a are downregulate after stimulation of collagenII in RA FLS, suggest that these miRNA might be involved in the pathogenesis of RA, andpromote collagen II-mediated RA progression. We will further explore thesesignal-regulating networks involved in RA pathology process.As we know，the phosphorylation of protein is one of the most common andimportant post-translational protein modification. Protein phosphorylation occurs mainlyin serine, threonine and tyrosine residues Although tyrosine phophoralation abundance ismuch lower than other amino acids, they play very important roles in both physiologicaland pathological process，such as proliferation,differentiation, apoptosis,etc. The disorderof tyrosine phosphoralation signal transduction network relate to many disease such astumor,so the tyrosine phosphoralation proteomics can provide much information thatgenomics can’t give.To explore the molecular basis for the activity of human synovial fibroblasts in RApathological state, we for the first time applied differential proteomics technology toanalyze differential tyrosine phosphoralation proteins of human synovial fibroblasts fromRA individuals before and after the stimulation of collagenII.And we found a differentiallyexpressioned protein ANXA5which indeed take part in the tyrosine phosphorylationsignal transduction pathway activation, but the exact mechanism needs further study. |
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