Functional Role Of MicroRNA-203 On The Activation Of Wnt/β-catenin Signaling By Targeting APC In Fibroblast-like Synoviocytes Of Adjuvant-induced Arthritis Rats

Rheumatoid arthritis(RA) is a chronic and progressive systemic inflammatory arthropathy characterized by synovial hyperplasia, inflammation, cartilage loss and bone destruction. As we know, aberrant activation of fibroblastlike synoviocytes(FLS) play an important role in both initiation and perpetuation of synovitis and joint destruction in the progression of RA. Accroding to the report and the known attributes of Wnt/β-catenin pathway, one might rationalize that untimely activation of Wnt/β-catenin pathway in RA FLS is partly responsible for driving RA FLS activation and RA pathogenesis. Wnt signaling is modulated at different levels by a wide range of evolutionarily conserved effectors. In the absence of Wnt signaling, Adenomatous polyposis coli(APC) functions in a proteolytic destruction complex to control the turnover of β-catenin, a transcriptional coactivator of the Wnt pathway。The loss of these “normal” functions due to mutation or epigenetics(DNA methylation and micro RNAs) have found in a lot of human desease.MicroRNAs(mi RNA) are a class of 19-23 nucleotide-long short endogenous, single-stranded, noncoding RNAs and control numerous basic biologic functions including cell cycle, cellular differentiation proliferation, and apoptosis. In the last few years, some up-regulated and down-regulated mi RNA have also been identified in RA, but the direct target involved in this mechanism is still unknown.In this study, we choose the adjuvant arthritis(AA) instead of RA as animal model for the study and FLS as cell research object.To detect the relationship between mi RNA-203 and APC that is well known as an essential intracellular negative regulator of the canonical Wnt signaling pathway and the role of mi RNA-203 and APC dysregulation in RA.The main contents are summarized as follows: 1. Down-regulation of APC and the Wnt/β-catenin pathway is activated in FLSobtained from the synovial tissues of AA rats.Adult male SD rats were treatment with complete Freund’s adjuvant by paw injection.at the left paw to induce AA rat models and found there were untimely activation of Wnt/β-catenin pathway that the expression of β-catenin, c-myc and cyclin D1 increased and decreased adenomatous polyposis coli(APC) in the FLS isolated from the synovium of AA rats. 2. The functional role of APC during activation of FLS.To elucidate the functional role of APC during activation of FLS, si RNA specific for rat APC(APC-si RNA) was used to knockdown the APC expression. The results showed that downregulation of APC promote cell proliferation and the expression of β-catenin who is the key of Wnt/β-catenin pathway was higher in FLS transfected with APC-si RNA than control and the scrambled-si RNA transfected cells in protein level. Downregulation of APC also increased the expression of C-myc and Cyclin D1 in m RNA and protein levels. These data suggested that the blockade of APC promoted FLS proliferation and decreased the proteolysis of β-catenin, responsible for the activation of Wnt/β-catenin signaling pathway in normal FLS. 3. The relationship between mi RNA-203 and APC.Using different mi RNA target gene prediction algorithms, we observed the APC gene,which is the intracellular negative regulator of the canonical Wnt signaling pathway, has a mi R-203 binding site in its 3,-untranslated region(3,UTR). In addition,we observed that the expression of mi R-203 increased significantly in the FLS isolated from the synovium of AA rats compared with the FLS isolated from the synovium of normal rats. The data suggested that overexpression of mi R-203 in AA FLS is like to take part in the pathogenesis of AA by APC-mediated signaling.4. The functional role of mi R-203 during activation of FLS.In order to investigate the roles of mi R-203 in regulating FLS proliferation and to illuminate the relationship between mi R-203 and APC. First we ectopic expression of mi R-203 in normal FLS by transfect with Mir-203 mimic. The MTT assay showed that introduction of mi R-203 caused a significant promotion of cell proliferation in normal FLS, whereas inhibition of mi R-203 by transfect with Mir-203 inhibitor in AA FLS reduced this effect. Furthermore, mi R-203 upregulation in normal FLS suppresse significantly the expression of APC and this down-regulation of APC expression decreased the proteolysis of β-catenin triggered activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus causing transcriptional activation of target genes. The expression of C-myc and Cyclin D1 were higher in FLS transfected with mi R-203 mimics both in m RNA and protein levels. When transfect AA FLS with Mir-203 inhibitor can increase the expression of APC and inhibited the activation of Wnt/β-catenin signaling pathway. However, down-regulation of mi R-203 has no influence on inhibit the activation of Wnt/β-catenin signaling pathway in case of silence APC by transfected with si APC in AA FLS. Our results suggest that up-regulated mi R-203 plays an important role in promoting the proliferation and activation of AA FLS by targeting APC-mediated activation of Wnt/β-catenin signaling and take part in the pathogenesis of AA.In summary, our findings in the present study suggested that APC may play a pivotal role during FLS activation by inhibit β-catenin for ubiquitin-mediated proteolysis and activation of Wnt/β-catenin signaling pathway. Moreover, it appeared that the expression of APC was directly regulated by mi R-203 in FLS, indicating the potential of APC and mi R-203 as the therapeutic target for RA.