MicroRNA-15a/16/SOX5 axis promotes migration, invasion and inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes.
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ABSTRACT:
SUBMITTER: Wei H
PROVIDER: S-EPMC7425471 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dysregulation of circular RNAs (circRNAs) is involved in various human diseases. Fibroblast-like synoviocytes (FLSs), which form the lining of the joint, are epigenetically imprinted with an aggressive phenotype and contribute to joint destruction in rheumatoid arthritis (RA). In the present study, we identified a novel circRNA, Circ_0088194, which was upregulated in RA fibroblast-like synoviocytes (RA-FLSs) and correlated with the disease activity score in 28 joints. Overexpression of Circ_0088194 promoted RA-FLS migration and invasion, while inhibition of Circ_0088194 had the opposite effect. Mechanistically, Circ_0088194 acted as a miR-766-3p sponge to relieve the repressive effect of miR-766-3p on its target, MMP2 (encoding matrix metalloproteinase 2), thereby promoting migration and invasion. The expression level of Circ_0088194 was inversely correlated with that of miR-766-3p in RA-FLSs. Importantly, overexpression of miR-766-3p partially blocked the migration and invasion induced by Circ_0088194 overexpression. Collectively, this study identified a novel circRNA Circ_0088194 that promotes RA-FLS invasion and migration via the miR-766-3p/MMP2 axis. Circ_0088194 might represent a novel therapeutic target to prevent and treat RA.
| S-EPMC7937802 | biostudies-literature
Project description:IntroductionAnti-malarial drug artesunate can suppress inflammation and prevent cartilage and bone destruction in collagen-induced arthritis model in rats-suggesting it may be a potent drug for rheumatoid arthritis (RA) therapy. We aimed to investigate its effect on the invasive property of fibroblast-like synoviocytes (FLS) from patients with RA.MethodsSynovial tissues were obtained by closed needle biopsy from active RA patients, and FLS were isolated and cultured in vitro. RA-FLS were treated with artesunate at various concentrations, while methotrexate or hydroxychloroquine was employed as comparator drugs. Cell viability, proliferation, cell cycle, apoptosis, migration, invasion, and pseudopodium formation of RA-FLS were assessed by CCK-8 assays, EdU staining, Annexin V-FITC/PI staining, transwell assays, or F-actin staining, respectively. Further, relative changes of expressed proteases were analyzed by Proteome profiler human protease array and verified by quantitative real-time PCR (qPCR), Western blot, and ELISA. The expression of signaling molecules of MAPK, NF-κB, AP-1, and PI3K/Akt pathways were measured by qPCR and Western blot. PDK-1 knockdown by specific inhibitor AR-12 or siRNA transfection was used to verify the pharmacological mechanism of artesunate on RA-FLS.ResultsArtesunate significantly inhibited the migration and invasion of RA-FLS in a dose-dependent manner with or without TNF-α stimulation. The effect was mediated through artesunate inhibition of MMP-2 and MMP-9 production, and pre-treatment with exogenous MMP-9 reversed the inhibitory effect of artesunate on RA-FLS invasion. Artesunate had a stronger inhibitory effect on migration and invasion of RA-FLS as well as greater anti-inflammatory effect than those of hydroxychloroquine. Similar inhibitory effect was detected between artesunate and methotrexate, and synergy was observed when combined. Mechanistically, artesunate significantly inhibited PDK-1 expression as well as Akt and RSK2 phosphorylation-in a similar manner to PDK-1-specific inhibitor AR-12 or PDK-1 knockdown by siRNA transfection. This inhibition results in suppression of RA-FLS migration and invasion as well as decreased MMP-2 and MMP-9 expression.ConclusionsOur study demonstrates artesunate is capable of inhibiting migration and invasion of RA-FLS through suppression of PDK1-induced activation of Akt and RSK2 phosphorylation-suggesting that artesunate may be a potential disease-modifying anti-rheumatic drug for RA.
| S-EPMC6591920 | biostudies-literature
Project description:Objectives:Fibroblast-like synoviocytes (FLS) exhibit a unique aggressive phenotype in rheumatoid arthritis (RA). Increased FLS migration and subsequent invasion of the extracellular matrix are essential to joint destruction in RA. Our previous research reported that transcription factor SOX5 was highly expressed in RA-FLS. Here, the effects of SOX5 in RA-FLS migration and invasion will be investigated. Methods:The migration and invasion of RA-FLS were evaluated using a transwell chamber assay. The expression of several potential SOX5-targeted genes, including matrix metalloproteinases (MMP-1, 2, 3 and 9), chemokines (CCL4, CCL2, CCR5 and CCR2), and pro-inflammatory cytokines (TNF-? and IL-6), were examined in RA-FLS using SOX5 gain- and loss-of-function study. The molecular mechanisms of SOX5-mediated MMP-9 expressions were assayed by luciferase reporter gene and chromatin immunoprecipitation (ChIP) studies. The in vivo effect of SOX5 on FLS migration and invasion was examined using collagen-induced arthritis (CIA) in DBA/1J mice. Results:Knockdown SOX5 decreased lamellipodium formation, migration, and invasion of RA-FLS. The expression of MMP-9 was the only gene tested to be concomitantly affected by silencing or overexpressing SOX5. ChIP assay revealed that SOX5 was bound to the MMP-9 promoter in RA-FLS. The overexpression of SOX5 markedly enhanced the MMP-9 promoter activity, and specific deletion of a putative SOX5-binding site in MMP-9 promoter diminished this promoter-driven transcription in FLS. Locally knocked down SOX5 inhibited MMP-9 expression in the joint tissue and reduced pannus migration and invasion into the cartilage in CIA mice. Conclusion:SOX5 plays a novel role in mediating migration and invasion of FLS in part by regulating MMP-9 expression in RA.
| S-EPMC5906798 | biostudies-literature
Project description:BackgroundInterleukin-6 (IL-6) is involved in fibroblast-like synoviocyte (FLS) activation and promotes pannus formation and bone and cartilage destruction in rheumatoid arthritis (RA). Cysteine-rich 61 (Cyr61) protein regulates cell proliferation, migration, and differentiation. The aim of this study was to investigate the role of Cyr61 in RA-FLS migration and invasion after IL-6 stimulation.MethodsWestern blotting, immunohistochemistry, reverse transcription-polymerase chain reaction, and real time-polymerase chain reaction were used to examine protein and mRNA levels of Cyr61, matrix metalloproteinases (MMPs), and other signalling proteins. Knockdown of gene expression was performed with siRNA, and RNA sequencing was performed for differential gene analysis. Migration and invasion were assessed by wound healing and Boyden chamber assays.ResultsCyr61 levels were elevated in FLSs from RA patients compared to those in osteoarthritis patients. Control and IL-6-treated FLSs showed differential gene expression. IL-6 stimulated protein synthesis of Cyr61, which was attenuated by the extracellular signal-related kinase 1/2 (ERK 1/2) inhibitor, PD98059, and knockdown of early growth response 3 (EGR3), but not of JUN. IL-6-induced Cyr61 protein synthesis increased expression of MMP2. Cyr61 promoted FLS migration and invasion in an autocrine manner. Knockdown of CYR61 and a neutralising antibody attenuated Cyr61 synthesis and IL-6-induced FLS migration.ConclusionsBy modulating the ERK/EGR3 pathway, IL-6 stimulated Cyr61 production and in turn increased invasiveness of FLS. Our data suggest that Cyr61 might be a potential target to prevent the progression of joint damage in RA.
| S-EPMC7685583 | biostudies-literature
Project description:BackgroundLong non-coding RNAs (lncRNAs) have drawn increasing attention because they play a pivotal role in various types of autoimmune diseases, including rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), a prominent component of hyperplastic synovial pannus tissue, are the primary effector cells in RA synovial hyperplasia and invasion which can lead to joint destruction. In this study, we investigated whether lncRNAs could act as competing endogenous RNAs to regulate the pathological behaviors of RA-FLSs.MethodsLncRNA microarray was conducted to establish lncRNA expression profiles in FLSs isolated from RA patients and healthy controls (HCs). Differentially expressed lncRNAs were verified by quantitative real-time PCR (qRT-PCR) on RA-FLSs and synovial fluid. The functional role of lncRNA PICSAR downregulation was evaluated in RA-FLSs. We conducted molecular biological analysis to predict miRNAs which have a potential binding site for PICSAR and further refined the results by qRT-PCR. Luciferase reporter assay was adopted to validate the interaction of lncRNA PICSAR and miR-4701-5p. Western Blot and qPCR were used to identify the target gene and protein. The functional role of miR-4701-5p upregulation was examined in RA-FLSs.FindingsWe identified a long intergenic non-protein-coding RNA162 (LINC00162), also known as lncRNA PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA), has significantly higher expression in RA-FLSs and RA synovial fluid. The cell proliferation, migration, invasion and proinflammatory cytokines production of RA-FLSs showed significant alterations after the lncRNA PICSAR suppression. Mechanistically, lncRNA PICSAR functioned through sponging miR-4701-5p in RA-FLSs.InterpretationOur results reveal PICSAR may exert an essential role in promoting synovial invasion and joint destruction by sponging miR-4701-5p in RA and that lncRNA PICSAR may act as a biomarker of RA.
| S-EPMC6921299 | biostudies-literature
Project description:BACKGROUND:Reactive oxygen species (ROS) regulate the migration and invasion of fibroblast-like synoviocytes (FLS), which are key effector cells in rheumatoid arthritis (RA) pathogenesis. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) induces ROS generation and, consequently, enhances cell migration. Despite the important interrelationship between RA, FLS, and ROS, the effect of NOX4 on RA pathogenesis remains unclear. METHODS:FLS isolated from RA (n?=?5) and osteoarthritis (OA, n?=?5) patients were stimulated with recombinant interleukin 17 (IL-17; 10?ng/ml) and tumor necrosis factor alpha (TNF-?; 10?ng/ml) for 1?h. Cell migration, invasion, adhesion molecule expression, vascular endothelial growth factor (VEGF) secretion, and ROS expression were examined. The mRNA and protein levels of NOX4 were analyzed by RT-qPCR and western blotting, respectively. The NOX4 inhibitor GLX351322 and NOX4 siRNA were used to inhibit NOX4 to probe the effect of NOX4 on these cellular processes. RESULTS:Migration of RA FLS was increased 2.48-fold after stimulation with IL-17 and TNF-?, while no difference was observed for OA FLS. ROS expression increased in parallel with invasiveness of FLS following cytokine stimulation. When the expression of NOX was examined, NOX4 was significantly increased by 9.73-fold in RA FLS compared to unstimulated FLS. Following NOX4 inhibition, cytokine-induced vascular cell adhesion molecule 1 (VCAM1), VEGF, and migration and invasion capacity of RA FLS were markedly decreased to unstimulated levels. CONCLUSION:NOX4 is a key contributor to cytokine-enhanced migration and invasion via modulation of ROS, VCAM1, and VEGF in RA FLS.
| S-EPMC7227051 | biostudies-literature
Project description:Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.
| S-EPMC5953939 | biostudies-literature
Project description:LncRNA NEAT1 functions as an oncogene in multiple human cancers. However, its expression and role in fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) remain unclear. Thus, we investigated the expression of NEAT1 in synovial tissues and FLSs in RA, to determine its role in the development of RA. Quantitative real-time polymerase chain reaction was used to measure the expression of NEAT1. FLS proliferation was evaluated using cell proliferation assays. Flow cytometry was used to analyze cell cycle progression and apoptosis in FLSs. Binding between NEAT1 and miR-410-3p was demonstrated by dual-luciferase assays. We found that NEAT1 was upregulated in synovial tissues and FLSs in RA. Upregulation of NEAT1 promoted cell proliferation, induced S-to G2/M phase transition, and suppressed apoptosis in RA FLSs. NEAT1 directly bound to and negatively modulated miR-410-3p expression, while positively regulating YinYang 1(YY1; a miR-410-3p target). Inhibiting miR-410-3p and upregulating YY1 partially restored the inhibitory role in cell viability induced by the depletion of NEAT1 in RA FLSs. Besides pro-proliferative and anti-apoptotic roles, upregulation of NEAT1 promoted migration, invasion, and inflammatory cytokines secretion in RA FLSs. Taken together, our results suggest that the NEAT1 may serve as a novel diagnostic and therapeutic target for patients with RA.
| S-EPMC7485383 | biostudies-literature
Project description:Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary effectors. We previously reported that the ability of RA FLS to degrade extracellular matrix (ECM) components depends on the formation of actin-rich membrane protrusions, called invadosomes, through processes that remain elusive. 14-3-3η belongs to a family of scaffolding proteins involved in a wide range of cellular functions, and its expression is closely related to joint damage and disease activity in RA patients. In this study, we sought to assess the role of 14-3-3η in joint damage by examining its contribution to the invadosome formation phenotype of FLS. Using human primary FLS, we show that 14-3-3η expression is closely associated with their ability to form invadosomes. Furthermore, knockdown of 14-3-3η using shRNAs decreases the level of invadosome formation in RA FLS, whereas addition of the recombinant protein to FLS from healthy individuals promotes their formation. Mechanistic studies suggest that 14-3-3η regulates invadosome formation by increasing Snail expression, a mechanism that involves nuclear exclusion of the transcription repressor FOXO3. Our results implicate the 14-3-3η-FOXO3-Snail axis in promoting the aggressive ECM-degrading phenotype of RA FLS, and suggest a role for this scaffolding protein in cartilage degradation.
| S-EPMC8745703 | biostudies-literature
Project description:To probe the role of protein arginine methyltransferase 5 (PRMT5) in regulating inflammation, cell proliferation, migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). FLSs were separated from synovial tissues (STs) from patients with RA and osteoarthritis (OA). An inhibitor of PRMT5 (EPZ015666) and short interference RNA (siRNA) against PRMT5 were used to inhibit PRMT5 expression. The standard of protein was measured by Western blot or immunofluorescence. The excretion and genetic expression of inflammatory factors were, respectively, estimated by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Migration and invasion in vitro were detected by Boyden chamber assay. FLSs proliferation was detected by BrdU incorporation. Increased PRMT5 was discovered in STs and FLSs from patients with RA. In RA FLSs, the level of PRMT5 was up-regulated by stimulation with IL-1? and TNF-?. Inhibition of PRMT5 by EPZ015666 and siRNA-mediated knockdown reduced IL-6 and IL-8 production, and proliferation of RA FLSs. In addition, inhibition of PRMT5 decreased in vitro migration and invasion of RA FLSs. Furthermore, EPZ015666 restrained the phosphorylation of I?B kinase? and I?B?, as well as nucleus transsituation of p65 as well as AKT in FLSs. PRMT5 regulated the production of inflammatory factors, cell proliferation, migration and invasion of RA FLS, which was mediated by the NF-?B and AKT pathways. Our data suggested that targeting PRMT5 to prevent synovial inflammation and destruction might be a promising therapy for RA.
| S-EPMC5345686 | biostudies-literature