| Rheumatoid Arthritis (RA) is an autoimmune, systemic and multi-joint disease, and fibroblast-like synoviocytes (FLS) display important roles in the pathogenesis of RA disease. Wnt signaling is involved in the pathogenesis of RA, and plays an important role in RA pathology. Masala and his colleagues found that in myelodysplasia marrow cells, secreted frizzled-related protein4(SFRP4) was modified by epigenetic, leading to its expression inhibition and inducing the Wnt signaling. Whether epigenetic modifications are involved in SFRP4gene in RA pathogenesis, causing the SFRP4inhibition and affecting the RA pathogenesis through the Wnt signaling? Adjuvant arthritis (AA) has similar feature of RA pathology and immunology, and is an ideal animal model for RA reserch. In this experiment, AA rats also were used as RA animal model to study the SFRP4expression in synovium from control rats and AA rats, and its effect on Wnt signaling. The SFRP4epigenetic modifications were deeply investigated to reveal the effect of methyl CpG binding protein2(MeCP2) and DNA methyltransferase1(DNMT1) on the SFRP4expression. In addition, the important role of microRNA (miR)-152in this process was also investigated. All the work will provide us new ideas for the study of RA. The study includes the following five parts.1. The SFRP4expression in AA rats and controls, and its relationship with the Wnt signaling.The SFRP4expression in synovium from control rats and AA rats was detected by immunohistochemistry, RT-PCR and Western blotting. FLS from control rats and AA rats were isolated and cultured, and RT-PCR and Western blotting were used to detect the SFRP4expression in FLS from control rats and AA rats. The results showed that the SFRP4expression was significantly decreased in AA rats compared with controls, suggesting that SFRP4may play a regulatory role in the pathogenesis of AA. In order to investigate the regulated role of SFRP4in Wnt signaling, FLS from control rats were isolated and cultured. Culture was added with SFRP4antibodies, real time qPCR and Western blotting were used to detect the effect of SFRP4antibodies on the β-catenin expression in Wnt signaling pathway in control FLS. The results showed that SFRP4antibody significantly up-regulated the β-catenin expression in control FLS. In order to further verify the effect of SFRP4on the Wnt signaling, FLS from AA rats were cultured with SFRP4recombinant protein, Western blotting detection found that SFRP4recombinant protein can significantly reduce the expression of β-catenin and fibronectin, indicating that SFRP4may affect the pathological change of AA by regulating the Wnt signaling.2. The effect of DNA methylation inhibitor5-aza-2’-deoxycytidine (5-azadC) on the SFRP4expression in AA rats.The effect of5-azadC stimulation on the SFRP4expression in AA FLS was detected by RT-PCR, Western blotting and MTT. The result showed that2.5μM,5.0μM,7.5μM doses of5-azadC could significantly increase the SFRP4expression in FLS from AA rats respectively.5μM5-azadC cultured FLS for24,48,72h also up-regulated the SFRP4expression respectively, indicating that SFRP4down-regulation in AA FLS might be caused by DNA methylation in SFRP4gene.5μM5-azadC cultured AA FLS for24,48,72h significantly inhibited the expression of canonical Wnt sigaling key gene β-catenin, ccndl respectively. In addition,5-azadC could effectively inhibit the fibronectin expression and FLS proliferation. These results suggest that the SFRP4inhibition in AA may be due to DNA methylation in SFRP4gene,5-azadC may increase the SFRP4expression, inhibit the expression of β-catenin, ccndl and fibronectin, and SFRP4may influence the AA pathogenesis through the Wnt signaling pathway. 3. MeCP2affects the SFRP4expression in AA rats.RT-PCR and Western blotting were used to detect the MeCP2expression in synovium from control rats and AA rats. The results showed that MeCP2expression was significantly elevated in synovium from AA rats compared with controls. RT-PCR and Western blotting analysis further found that MeCP2expression was significantly higher in FLS from AA rats compared with controls. siMeCP2was transfected to AA FLS by LipofectamineTM2000transfection reagent, and the effect of MeCP2on the SFRP4expression in FLS was detected. The data showed that MeCP2inhibition up-regulated the SFRP4expression significantly. The above results suggest that increased MeCP2in AA pathogenesis may inhibit the SFRP4expression.4. DNMT1affects the SFRP4expression in AA rats.The DNMT1expression in synovium from control rats and AA rats was detected by real time qPCR and Western blotting. The results showed that DNMT1expression was significantly up-regulated in the synovium from AA rats compared with controls. Real time qPCR and Western blotting analysis further found that the DNMT1expression was significantly higher in FLS from AA rats compared with controls. After the DNMT1expression was significantly inhibited in AA rats, the SFRP4expression was up-regulated. The above results suggest that increased DNMT1in AA pathogenesis may inhibit the SFRP4expression.5. The role of miR-152in SFRP4expression and its signaling pathwayReal time qPCR and Western blotting were used to detect the differentially expressed miR-152in AA rats and controls, to investigate the effect of overexpressed miR-152on the expression of DNMT1, SFRP4and the Wnt signaling activation. The results showed that miR-152expression was significantly lower in AA rats, overexpressed miR-152inhibited the DNMT1expression, up-regulated the SFRP4expression, inhibited the canonical Wnt signaling, and inhibited AA FLS abnormal proliferation. The data suggests that miR-152has an ability to inhibit the AA pathogenesis, and this inhibited role is achived by inhibiting the DNMTl expression and the activation of the Wnt signaling.In summary, the SFRP4down-regulation in AA rats may be caused by epigenetic modifications, leading to the Wnt signaling activation and AA development. Increased DNMT1and MeCP2in AA rats may inhibit the SFRP4expression, and low expression of miR-152may be associated with the high expression of DNMT1. DNMT1, MeCP2and miR-152display important roles in the decreased SFRP4in AA rats. |
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