title: MicroRNA-26b inhibits cell proliferation and cytokine secretion in human RASF cells via the Wnt/GSK-3β/β-catenin pathway.
abstract: 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, 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.
  
  
  RESULTS
  GSK-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).
  
  
  CONCLUSIONS
  MiR-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.
  The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9063056861547150.
authors.affiliation: Nursing Office, Linyi People's Hospital, Linyi, 276000, P. R. China. sunjiling0515@163.com.
authors.forename: Jiling
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Rheumatology, Linyi People's Hospital, Linyi, 276000, P. R. China. yanpeng4978@163.com.
authors.forename: Peng
authors.initials: P
authors.lastname: Yan
authors.affiliation: Department of Burn and Plastic Surgery, Linyi People's Hospital, Linyi, 276000, P. R. China. chenyuanzheng798@163.com.
authors.forename: Yuanzheng
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Orthopedics, Linyi People's Hospital, North of Yimeng Road, Lanshan District, Linyi, 276000, P. R. China. chenyangcy789@163.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Orthopedics, Linyi People's Hospital, North of Yimeng Road, Lanshan District, Linyi, 276000, P. R. China. yangjianxun0515@163.com.
authors.forename: Jianxun
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Orthopaedics, Nanjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, P. R. China. xuguangyue0623@163.com.
authors.forename: Guangyue
authors.initials: G
authors.lastname: Xu
authors.affiliation: Department of Orthopaedics, Nanjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, P. R. China. maohaijun0623@163.com.
authors.forename: Haijun
authors.initials: H
authors.lastname: Mao
authors.affiliation: Department of Orthopaedics, Nanjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, P. R. China. qiuyong0623@163.com.
authors.forename: Yong
authors.initials: Y
authors.lastname: Qiu
mesh_terms: D017209:Apoptosis; D001172:Arthritis, Rheumatoid / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D019938:Cyclin D1; D016207:Cytokines; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D035683:MicroRNAs; D051176:beta Catenin; C530104:CCND1 protein, human; C495270:CTNNB1 protein, human; C528163:MIRN26 microRNA, human; C000605506:GSK3B protein, human