MicroRNA-26b inhibits cell proliferation and cytokine secretion in human RASF cells via the Wnt/GSK-3[beta]/[beta]-catenin pathway

Background Rheumatoid arthritis (RA) is a chronic systemic auto- immune disease characterized by joint synovitis. Recent evidence suggests that rheumatoid arthritis synovial fibroblasts (RASFs) promote joint destruction. In this study, we investigated the role of microRNA-26b (miR-26b) in cell proliferation and inflammatory cytokine secretion using patient-derived Rheumatoid arthritis fibroblast-like synoviocyte (RAFLS) to understand pathways influencing rheumatoid arthritis. Methods RAFLS were cultured in vitro and transfected with miR-26b mimics (experimental group) and negative sequence (control group). The protein levels of Wnt4, Wnt5É, GSK-3[beta], CyclinD1, Ser9-GSK-3[beta] and [beta]-catenin were detected by western blot analysis. Tumor Necrosis Factor-É (TNF-É), IL- 1[beta], and IL-6 levels were quantified by Enzyme-linked Immunosorbent Assay (ELISA). RAFLS proliferation and apoptosis were measured by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Results GSK-3[beta] and CyclinD1 expression levels were lower in miR-26b mimic group compared to Mock group and negative control (NC) group. Conversely, GSK-3[beta] and CyclinD1 expression levels were markedly higher in the miR-26b inhibitor group compared to Mock and NC group (P < 0.05). Transfection of miR-26b mimics significantly increased the, levels of Ser9-GSK-3[beta] and [beta]-catenin in comparison to Mock and NC groups, while transfection of miR-26b inhibitors showed the opposite effect. In miR-26b mimic group, TNF-[alpha], IL- 1[beta] and IL-6 levels were lower than the Mock and NC groups, while in miR-26b inhibitor group, these cytokine levels were higher than the Mock and NC groups (P < 0.05). Transfection of miR-26b mimics significantly reduced the cell proliferation of RAFLS, compared to the Mock and NC groups, and miR-26b inhibitors increased the proliferative capacity of RAFLS compared to Mock and NC groups (P < 0.05). The miR-26b mimic group exhibited higher RAFLS apoptosis rate compared to Mock and NC group and miR-26b inhibitor group showed significantly lower RAFLS apoptosis rate compared to Mock and NC groups (P < 0.05). Conclusions MiR-26b regulates [beta]-catenin and CyclinD1 levels by inhibiting GSK-3[beta] expression, which in-turn alters the Wnt/GSK-3[beta]/[beta]-catenin pathway to lower RAFLS proliferation and elevate cell apoptosis and the secretion of TNF-[alpha],IL-1[beta] and IL-6 cytokines. Therefore, our resul