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

Jiling Sun,Guangyue Xu,Haijun Mao,Yuanzheng Chen,Peng Yan,Yang Chen,Yong Qiu,Jianxun Yang

doi:10.1186/s13000-015-0309-x

Abstract

BackgroundRheumatoid 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.MethodsRAFLS were cultured in vitro and transfected with miR-26b mimics (experimental group) and negative sequence (control group). The protein levels of Wnt4, Wnt5ɑ, GSK-3β, CyclinD1, Ser9-GSK-3β and β-catenin were detected by western blot analysis. Tumor Necrosis Factor-ɑ (TNF-ɑ), IL- 1β, 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.ResultsGSK-3β and CyclinD1 expression levels were lower in miR-26b mimic group compared to Mock group and negative control (NC) group. Conversely, GSK-3β 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-3β and β-catenin in comparison to Mock and NC groups, while transfection of miR-26b inhibitors showed the opposite effect. In miR-26b mimic group, TNF-α, IL- 1β 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).ConclusionsMiR-26b regulates β-catenin and CyclinD1 levels by inhibiting GSK-3β expression, which in-turn alters the Wnt/GSK-3β/β-catenin pathway to lower RAFLS proliferation and elevate cell apoptosis and the secretion of TNF-α,IL-1β and IL-6 cytokines. Therefore, our results show that miR-26B plays a central role in inhibiting the inflammation associated with rheumatoid arthritis.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9063056861547150

Highlights

Rheumatoid arthritis (RA) is a chronic systemic auto- immune disease characterized by joint synovitis
Expression of green fluorescent protein (GFP) after Rheumatoid arthritis fibroblast-like synoviocyte (RAFLS) transfection in each group Following the transfection of RAFLS with various plasmids as indicated, the GFP marker expressed by the recombinant plasmid was used to determine transfection efficiency by fluorescence microscopy (Fig. 1)
Confirmation of miR-26b expression by real-time quantitative polymerase chain reaction (PCR) Figure 2 shows that the expression level of miR-26b in both Mock group and negative control (NC) group were 1.05 ± 0.05 and 1.04 ± 0.06, respectively (P > 0.05)

Summary

Introduction

Rheumatoid arthritis (RA) is a chronic systemic auto- immune disease characterized by joint synovitis. Synovial tissue from RA patients shows infiltration by macrophages, T cells, and B cells, proliferation of cells lining the synovium, and production of inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) [13, 14]. Inhibition of these cytokines ameliorates the clinical symptoms RA, strongly supporting the central role of cytokines in RA [15]. Rheumatoid arthritis synovial fibroblast (RASFs) activity promotes joint destruction and increased expression of proinflammatory pathways and secretion of matrixdestructive enzymes is a common feature associated with the disease [16]. Recent evidence suggests that miRNA dysregulation may contribute to RA etiopathogenesis and a better understanding of pathways regulated by miRNAs might shed light on RA pathogenesis and help identify effective RA treatments [17]

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Conclusion