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MiR-19 suppresses fibroblast-like synoviocytes cytokine release by targeting toll like receptor 2 in rheumatoid arthritis.

ABSTRACT: Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis (RA) through participating in joint tissue inflammation and joint damage. MicroRNAs are a kind of small non-coding RNAs that can regulate gene expression in the transcription level to affect cell behaviors. This study intended to investigate the expression of miR-19 in FLS from RA patients and related mechanism. A total of 126 RA patients were selected in this study. MiR-19 expression in FLS was detected by qRT-PCR. Toll like receptor 2 (TLR2) protein expression was tested by Western blot. MiR-19 target genes were confirmed by bioinformatics analysis and luciferase reporter assay. The impact of miR-19 on the expression of TLR2, interleukin 6 (IL-6), and matrix metalloproteinase 3 (MMP-3) in FLS were analyzed by cell transfection and Western blot. MiR-19 expression in FLS from RA patients was significantly downregulated compared with control (P < 0.05), while TLR2 level was increased (P < 0.05). Bioinformatics analysis and luciferase reporter assay confirmed that TLR2 was the target gene of miR-19. Transfection of miR-19 mimic or miR-19 inhibitor obviously suppressed or increased TLR2 expression, and reduced or promoted release of cytokines IL-6 and MMP-3 in FLS, respectively. In conclusion, MiR-19 expression was downregulated in FLS from RA patients, leading to increased TLR2 expression and enhanced cytokines release.

SUBMITTER: Li Z

PROVIDER: S-EPMC5209502 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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MiR-19 suppresses fibroblast-like synoviocytes cytokine release by targeting toll like receptor 2 in rheumatoid arthritis.

Li Zongyu Z Cai Jinfang J Cao Xuecheng X

American journal of translational research 20161215 12

Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis (RA) through participating in joint tissue inflammation and joint damage. MicroRNAs are a kind of small non-coding RNAs that can regulate gene expression in the transcription level to affect cell behaviors. This study intended to investigate the expression of miR-19 in FLS from RA patients and related mechanism. A total of 126 RA patients were selected in this study. MiR-19 expression in FLS was ...[more]

PMID: 28078022

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Project description:BackgroundFibroblast-like synoviocytes (FLS) have cancer cell-like characteristics, such as abnormal proliferation and resistance to apoptosis, and play a pathogenic role in rheumatoid arthritis (RA). Hyperproliferation of RA-FLS that can be triggered by the activation of interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling destructs cartilage and bone in RA patients. Chrysoeriol is a flavone found in medicinal herbs such as Chrysanthemi Indici Flos (the dried capitulum of Chrysanthemum indicum L.). These herbs are commonly used in treating RA. Chrysoeriol has been shown to exert anti-inflammatory effects and inhibit STAT3 signaling in our previous studies. This study aimed to determine whether chrysoeriol inhibits hyperproliferation of RA-FLS, and whether inhibiting STAT3 signaling is one of the underlying mechanisms.MethodsIL-6/soluble IL-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS were used to evaluate the effects of chrysoeriol. CCK-8 assay and crystal violet staining were used to examine cell proliferation. Annexin V-FITC/PI double staining was used to detect cell apoptosis. Western blotting was employed to determine protein levels.ResultsChrysoeriol suppressed hyperproliferation of, and evoked apoptosis in, IL-6/sIL-6R-stimulated RA-FLS. The apoptotic effect of chrysoeriol was verified by its ability to cleave caspase-3 and caspase-9. Mechanistic studies revealed that chrysoeriol inhibited activation/phosphorylation of Janus kinase 2 (JAK2, Tyr1007/1008) and STAT3 (Tyr705); decreased STAT3 nuclear level and down-regulated protein levels of Bcl-2 and Mcl-1 that are transcriptionally regulated by STAT3. Over-activation of STAT3 significantly diminished anti-proliferative effects of chrysoeriol in IL-6/sIL-6R-stimulated RA-FLS.ConclusionsWe for the first time demonstrated that chrysoeriol suppresses hyperproliferation of RA-FLS, and suppression of JAK2/STAT3 signaling contributes to the underlying mechanisms. This study provides pharmacological and chemical justifications for the traditional use of chrysoeriol-containing herbs in treating RA, and provides a pharmacological basis for developing chrysoeriol into a novel anti-RA agent.

Project description:BackgroundRheumatoid arthritis is a progressive and systemic autoimmune disease seriously compromises human health. Fibroblast like synoviocytes are the major effectors of proliferation and inflammation in rheumatoid arthritis synovial tissue. Shikonin has anti-inflammatory and immunomodulatory activities. But, its role on synovitis of rheumatoid arthritis is unknown.MethodsThe DBA/1 male mice were randomly divided into the following three groups (n = 6): (1) the normal control group of mice, (2) the CIA (collagen-induced arthritis) group in which mice suffered from arthritis induced by collagen, (3) the SKN (shikonin) group of mice which got arthritis and given intragastrically with shikonin 4 mg/kg per day continuously for 20 days,(4) the MTX (methotrexate) group of mice which got arthritis and orally administration with shikonin 0.5  mg/kg once two days continuously for 20 days. The therapeutic effect of shikonin on collagen induced arthritis mice was tested by arthritis incidence rate, arthritis score and inflammatory joint histopathology. The invasion, adhesion and migration of fibroblast like synoviocytes induced by tumor necrosis factor-α were applied to measure the anti-synovitis role of shikonin. The effect of shikonin on expression of interleukin-6, interleukin-1β and tumor necrosis factor-α was measured by enzyme linked immunosorbent assay. The interaction between shikonin and suppressor of cytokine signaling 1 was verified by molecular docking. The signaling pathways activated by shikonin were measured by western blot.ResultsShikonin decreased the arthritis score and arthritis incidence, and inhibited inflammation of inflamed joints in collagen induced arthritis mice. And shikonin reduced the number of vimentin+cells in collagen induced arthritis mice inflamed joints. Meanwhile, shikonin suppressed tumor necrosis factor-α-induced invasion, adhesion and migration of fibroblast like synoviocytes and reduced the expression of interleukin-6, interleukin-1β and tumor necrosis factor-α. And we found that shikonin targeted suppressor of cytokine signaling 1. More interestingly, shikonin blocked the phosphorylation of Janus kinase 1/signal transducer andactivator of transcription 1/signal transducer andactivator of transcription 6 in synovial tissues and in fibroblast like synoviocytes.ConclusionShikonin represents a promising new anti-rheumatoid arthritis drug candidate that has anti-synovitis effect in collagen induced arthritis mice and inhibits tumor necrosis factor-α-induced fibroblast like synoviocytes by targeting suppressor of cytokine signaling 1/ Janus kinase/signal transducer andactivator of transcription signaling pathway. These findings demonstrate that shikonin has anti-synovitis effect and has great potential to be a new drug for the treatment of rheumatoid arthritis.

Project description:ObjectiveUp-regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast-like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage.MethodsSynovium and FLS were obtained from patients with RA and patients with osteoarthritis (OA). The rate of glycolysis after stimulation of FLS with lipopolysaccharide and platelet-derived growth factor BB was measured using glycolysis stress test technology. FLS function was evaluated using a glycolysis inhibitor, 2-deoxy-d-glucose (2-DG). After stimulation of the FLS, a migration scratch assay, MTT assay, and enzyme-linked immunosorbent assay were performed to measure the effect of 2-DG on FLS migration, viability of the FLS, and cytokine secretion, respectively. IRDye 800CW 2-DG was used to assess glucose uptake in the arthritic joints and stromal cells of mice after K/BxN mouse serum transfer. The mice were injected daily, intraperitoneally, with 3-bromopyruvate (BrPa; 5 mg/kg) to assess the effect of inhibition of glycolysis in vivo.ResultsCompared to human OA FLS, the balance between glycolysis and oxidative phosphorylation was shifted toward glycolysis in RA FLS. Glucose transporter 1 (GLUT1) messenger RNA (mRNA) expression correlated with baseline functions of the RA FLS. Glucose deprivation or incubation of the FLS with glycolytic inhibitors impaired cytokine secretion and decreased the rate of proliferation and migration of the cells. In a mouse model of inflammatory arthritis, GLUT1 mRNA expression in the synovial lining cells was observed, and increased levels of glucose uptake and glycolytic gene expression were detected in the stromal compartment of the arthritic mouse joints. Inhibition of glycolysis by BrPa, administered in vivo, significantly decreased the severity of arthritis in this mouse model.ConclusionTargeting metabolic pathways is a novel approach to understanding the mechanisms of disease. Inhibition of glycolysis may directly modulate synoviocyte-mediated inflammatory functions and could be an effective treatment strategy for arthritis.

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MiR-19 suppresses fibroblast-like synoviocytes cytokine release by targeting toll like receptor 2 in rheumatoid arthritis.

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MiR-19 suppresses fibroblast-like synoviocytes cytokine release by targeting toll like receptor 2 in rheumatoid arthritis.

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