| Rheumatoid arthritis(RA)is one of the most common autoimmune disease.it is characterized by chronic inflammation of the synovial tissue and hasgradually leads to the damage of cartilage even the corruption of bone and theimpairment of joints. The disease is often progressive and destructive, and greatlyaffected the life quality of the affected individuals. Although the cause of RA isstill unknown, studies in recent years showed that the activated fibroblast-likesynoviocytes(FLSs), as part of a complex cellular network, play an importantrole in the pathological process of rheumatoid arthritis. So theses cells hasaroused great concern of researchers in elucidating their specific features .For it's complex pathogenesis in advanced stage of RA disease,it's obviousthat singal signal passway can't interpret the RA pathogenesisi clearly andexhaustively. So we urgently need investigation into the complex signaltransduction network in RA FLS.The aim of our study is to detect the differencial gene expression in RAFLSs by genomics and proteomics approches and to discover the mechanism ofRA 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 firstused cDNA microarray to investigate the genes differently expressed in RA FLScompared with normal and OA FLS in the RNA level. We performed in vitroexperiments using the cultured synovial fibroblasts of passage 4.The total RNAwas extracted from RA,OA and normal synovium tissues and then purified. ThecDNA was obtained by reverse transcription polymerase chain reaction(RT-PCR), and then labeled with Cy5 and Cy3, which were hybridized withAgilent Human 1A gene expression microarray containing 21 073 human genes.Subsequently, the signal images were scanned and then analyzed by imageanalysis software.The results showed that among the 21 073 target genes, there are 1250 genesupregulated and 566 genes downregulated between RA and normal FLS. Amongthe upregulated genes, there are 17 genes rised more than 100 times, and amongthe upregulated genes there are several genes have more relation with tumoranalogous properties such as cell proliferation,adhesion,cell cycle andmigration.The result suggests that multiple genes take part in the pathogenesis of RA.These 1816 genes differentially expressed between RA and normal FLSs may beinvolved in the occurrence and development of RA. Further studying of thesegenes may be helpful for clarifying RA pathogenesis and finding moremoleculars involved in it.And then we used real-time PCR to verify the 8 up-regulated genes gettingfrom the gene chip result which may be related to RA pathogenesis,exceptMFAP4 and ITGA11,the other genes are all decreased in RA FLS.However, inthe type II collagen stimulated RA synovial cells, the expression levels of 8 genes were significantly upregulated. Real-time PCR results consistented with themicroarray gene expression shows that among individuals in different RApatients, the expression changes of genes have its own rules, embodied in RAFLS signal transduction molecules can change the development of disease. Forthe PCR results not consistent with the gene chip, we believe that this was due tothe chip and real-time PCR used in cell samples from different individuals withdifferent pathological states, more importantly, in vitro cultured cells ,which leftin vivo microenvironment that filled with a variety of immune media, cytokines,extracellular matrix and other pathological conditions of complex extracellularenvironment, will inevitably lead to the changes of many genes's expressionlevel, resulting in the PCR results and microarray results of the incomplete match.When we used collagen II to stimulate synovial cells, the cells partially reversedinto the invivo pathological state of RA, and we got the results which areconsistent with the chip .But in the normal synovial cells, when we stimulatedcells with collagen II , there are only half the number are upregulated .And thisillustrate that RA synovial cells have a unique response to the stimulation ofcollagen.And then we selected a gene called CYR61 from the microarray result, whichpromotes migration and proliferation in a variety of tumor cells. We cloned thefull sequence gene encoding CYR61 and successfully overexpressed CYR61 inprimary cultured in RA FLS using lentivirus expression system, and furtherexperiments proved that overexpression of CYR61 protein can promote theinvasion of RA FLS.The current study showed that RA articular cartilage damage is primarilycaused by matrix metalloproteinase secreted by synovial cells and synovial tissuewhich are excessivly proliferate. Matrix metalloproteinase degradate the main component of cartilage matrix: type II collagen, the fracture of collagen type IImay directly induce synovial cells to secret matrix metalloproteinase and matrixmetalloproteinase is of great significance for the lesions of cartilage and bonedestruction. So to clarify the key approach and moleculars in secretion of matrixmetalloproteinase mediated by type II collagen is the great breakthrough in RAtreatment and reduction of articular cartilage damage. However, as thecomplexity of rheumatoid arthritis in pathogenesis of late stage, we may find itdifficult to clarify its pathogenesis by a single signal transduction pathway , sothere's an urgent need to go deep into the complex signal transduction network ofthe proliferation of synovial cells and cartilage damage.We use gene chips to screen the differentially expressed genes after the stimulation by collagen type II cells in RA FLS, and there are 34 genes significantly different in expression. Among which there are 9 genes upregulated and 25 genes downregulated.Using real-time PCR to verify the result getting from gene chip, we found that except MRPGRD, the remainingeight genes's expression level are identical with the gene chip. The resultof real-time PCR demonstrates the high reliability of chip. In these genesinvolved in cell cycle regulation, cell receptors, adhesion and invasion, suggesting that type II collagen and its receptor-mediated RA signaling pathway may be the key to articular cartilage damage, but the detailed mechanism and the key elements of this process need further investigation.The research of miRNA related to RA pathological process has just started, there's already result shows that some miRNA expression in RA synovial tissue are abnormal.By detailed analysis using bioinformatics, we forecast the possible miRNA targeting the 3'-UTR and coding region of DDR2,MMP-1, MMP-13, STAT3 genes. The miRNA real-time PCR showed that miR-16-1, miR-143, miR-29a are downregulate after stimulation of collagen II in RA FLS, suggest that these miRNA might be involved in the pathogenesis of RA, and promoted collagen II-mediated RA progression. We will further explore these signal-regulating networks involved in RA pathology process.As we know,the phophoralation of protein is one of the most common and important post-translational protein modification. Protein phosphorylation occurs mainly in serine, threonine and tyrosine residues Although tyrosine phophoralation abundance is much lower than other proteins, they playvery important roles in both physiological and pathological process.such as proliferation,differentiation, apoptosis,etc. the disorder of tyrosine phosphoralation signal transduction network relate to many disease such as tumor,so the tyrosine phosphoralation proteomics can provide much information that genomics can't give.To explore the molecular basis for the activity of human synovial fibroblastsin RA pathological state, we for the first time applied differential proteomicstechnology to analyze differential tyrosine phosphoralation proteins of humansynovial fibroblasts from RA individuals before and after the stimulation ofcollagenII.And we found a differentially expressioned protein ANXA5 whichindeed take part in the tyrosine phosphorylation signal transduction pathwayactivation, but the exact mechanism needs further study. |
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