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

Sun J, Yan P, Chen Y, Chen Y, Yang J, Xu G, Mao H, Qiu Y - Diagn Pathol (2015)

Bottom Line: Tumor Necrosis Factor-ɑ (TNF-ɑ), IL- 1β, and IL-6 levels were quantified by Enzyme-linked Immunosorbent Assay (ELISA).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).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.

View Article: PubMed Central - PubMed

Affiliation: Nursing Office, Linyi People's Hospital, Linyi, 276000, P. R. China. sunjiling0515@163.com.

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.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9063056861547150.

No MeSH data available.


Related in: MedlinePlus

Expression of green fluorescent protein (GFP) in RAFLS at 24 and 48 h post-transfection
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Fig1: Expression of green fluorescent protein (GFP) in RAFLS at 24 and 48 h post-transfection

Mentions: 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). After 24 h of transfection, GFP express was visible and this expression significantly increased after 48 h.Fig. 1


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

Sun J, Yan P, Chen Y, Chen Y, Yang J, Xu G, Mao H, Qiu Y - Diagn Pathol (2015)

Expression of green fluorescent protein (GFP) in RAFLS at 24 and 48 h post-transfection
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4472173&req=5

Fig1: Expression of green fluorescent protein (GFP) in RAFLS at 24 and 48 h post-transfection
Mentions: 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). After 24 h of transfection, GFP express was visible and this expression significantly increased after 48 h.Fig. 1

Bottom Line: Tumor Necrosis Factor-ɑ (TNF-ɑ), IL- 1β, and IL-6 levels were quantified by Enzyme-linked Immunosorbent Assay (ELISA).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).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.

View Article: PubMed Central - PubMed

Affiliation: Nursing Office, Linyi People's Hospital, Linyi, 276000, P. R. China. sunjiling0515@163.com.

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.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9063056861547150.

No MeSH data available.


Related in: MedlinePlus