Proliferation Of Rheumatoid Arthritis Fibroblast-like Synoviocytes Research Articles

Novel mechanistic study of HDAC6 regulation of rheumatoid arthritis via CMA: exploring potential therapeutic targets.

Rheumatoid arthritis (RA) is a systemic autoimmune disease. Its pathogenesis has not yet been clarified, so it is urgent to explore therapeutic targets. Here, we clarified the role of HDAC6 in the mechanism of action of RA through mediating chaperone-mediated autophagy (CMA) to provide a clinical treatment of RA. We used rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and collagen-induced arthritis mice (CIA mice) as models of RA and pharmacological inhibitors as well as genetic interference with adeno-associated viruses to reduce the expression of HDAC6. We explored the influence of CAY10603 on RA-FLS proliferation and inflammation, as well as the expression of proteins related to the CMA signaling pathway. CIA model was constructed using DBA/1J mice. Arthritis symptoms in CIA mice were evaluated, and the expression and localization of CMA-related proteins in mouse ankle joints were examined. CAY10603 inhibited proliferation as well as the level of the molecular chaperone autophagy in RA-FLS. HDAC6 shRNA significantly reduced the clinical signs of arthritis in CIA mice, as did the expression of HDAC6 in the serum and ankle synovial tissues of CIA mice. Finally, it significantly inhibited the level of Hsc70 and LAMP-2A, which are involved in the CMA signaling pathway, in ankle joint tissues. Downregulation of HDAC6 may inhibit CMA and thereby ameliorate RA.

Shikonin suppresses rheumatoid arthritis by inducing apoptosis and autophagy via modulation of the AMPK/mTOR/ULK-1 signaling pathway

BackgroundThe overproliferation of fibroblast-like synoviocytes (FLS) contributes to synovial hyperplasia, a pivotal pathological feature of rheumatoid arthritis (RA). Shikonin (SKN), the active compound from Lithospermum erythrorhizon, exerts anti-RA effects by diverse means. However, further research is needed to confirm SKN's in vitro and in vivo anti-proliferative functions and reveal the underlying specific molecular mechanisms. PurposeThis study revealed SKN's anti-proliferative effects by inducing both apoptosis and autophagic cell death in RA FLS and adjuvant-induced arthritis (AIA) rat synovium, with involvement of regulating the AMPK/mTOR/ULK-1 pathway. MethodsSKN's influences on RA FLS were assessed for proliferation, apoptosis, and autophagy with immunofluorescence staining (Ki67, LC3B, P62), EdU incorporation assay, staining assays of Hoechst, Annexin V-FITC/PI, and JC-1, transmission electron microscopy, mCherry-GFP-LC3B puncta assay, and western blot. In AIA rats, SKN's anti-arthritic effects were assessed, and its impacts on synovial proliferation, apoptosis, and autophagy were studied using Ki67 immunohistochemistry, TUNEL, and western blot. The involvement of AMPK/mTOR/ULK-1 pathway was examined via western blot. ResultsSKN suppressed RA FLS proliferation with reduced cell viability and decreased Ki67-positive and EdU-positive cells. SKN promoted RA FLS apoptosis, as evidenced by apoptotic nuclear fragmentation, increased Annexin V-FITC/PI-stained cells, reduced mitochondrial potential, elevated Bax/Bcl-2 ratio, and increased cleaved-caspase 3 and cleaved-PARP protein levels. SKN also enhanced RA FLS autophagy, featuring increased LC3B, reduced P62, autophagosome formation, and activated autophagic flux. Autophagy inhibition by 3-MA attenuated SKN's anti-proliferative roles, implying that SKN-induced autophagy contributes to cell death. In vivo, SKN mitigated the severity of rat AIA while also reducing Ki67 expression, inducing apoptosis, and enhancing autophagy within AIA rat synovium. Mechanistically, SKN modulated the AMPK/mTOR/ULK-1 pathway in RA FLS and AIA rat synovium, as shown by elevated P-AMPK and P-ULK-1 expression and decreased P-mTOR expression. This regulation was supported by the reversal of SKN's in vitro and in vivo effects upon co-administration with the AMPK inhibitor compound C. ConclusionSKN exerted in vitro and in vivo anti-proliferative properties by inducing apoptosis and autophagic cell death via modulating the AMPK/mTOR/ULK-1 pathway. Our study revealed novel molecular mechanisms underlying SKN's anti-RA effects.

Discovery and validation of anti-arthritic ingredients and mechanisms of Qingfu Juanbi Tang, a Chinese herbal formulation, on rheumatoid arthritis

Ethnopharmacological relevanceQingfu Juanbi Tang (QFJBT), a novel and improved Chinese herbal formulation, has surged in recent years for its potential in the therapy of rheumatoid arthritis (RA). Anti-arthritic effects and underlying molecular mechanisms of QFJBT have increasingly become a focal point in research. Aim of the studyThis study utilized network pharmacology, molecular docking, and experimental validation to elucidate effective ingredients and anti-arthritic mechanisms of QFJBT. Materials and methodsTargets associated with QFJBT and RA were identified from relevant databases and standardized using the Uniprot for gene nomenclature. A “QFJBT-ingredient-target network” and a “Venn diagram of QFJBT and RA targets” were created from the data. The overlap in the Venn diagram highlighted potential targets of QFJBT in the treatment of RA. These targets were subjected to PPI network, GO, and KEGG pathway analysis. The findings were subsequently confirmed through molecular docking and pharmacological experiments to propose the mechanism of action of QFJBT. ResultsThe study identified 236 active ingredients in QFJBT, with 120 predicted to be effective against RA. Molecular docking showed high binding affinity of key targets (JUN, PTGS2, and TNF-α) with bioactive compounds (rhein, sinomenine, calycosin, and paeoniflorin) of QFJBT. Pharmacodynamic evaluation demonstrated the effects of QFJBT at the dose of 4.56 g/kg in ameliorating symptoms of AIA rats and in reducing levels of JUN, PTGS2, and TNF-α in synovial tissues. In vitro studies further exhibited that rhein, paeoniflorin, sinomenine, calycosin, and QFJBT-containing serum significantly inhibited abnormal proliferation of RA fibroblast-like synoviocytes. Interestingly, rhein and paeoniflorin specifically decreased p-JUN/JUN expression and TNF-α release, respectively, while sinomenine and calycosin selectively increased PTGS2 expression. Consistently, QFJBT-containing serum demonstrated similar effects as those active ingredients identified in QFJBT did. ConclusionsQFJBT, QFJBT-containing serum, and its active ingredients (rhein, paeoniflorin, sinomenine, and calycosin) suppress inflammatory responses in RA. Anti-arthritic effects of QFJBT and its active ingredients are likely linked to their modulatory impact on identified hub targets.

Circ_0011058 alleviates RA pathology through the circ_0011058/miR-335-5p/CUL4B signal axis

Our previous study found that Cullin 4B (CUL4B) inhibited rheumatoid arthritis (RA) pathology through glycogen synthase kinase-3beta (GSK3β)/canonical Wnt signalling pathway. In this work, pre-experiment and bioinformatics analysis suggested that circ_0011058 may lead to the up-regulation of CUL4B expression by inhibiting miR-335-5p. Therefore, we studied whether circ_0011058 can promote the expression of CUL4B through sponging the miR-335-5p and further promote the pathological development of RA. Bioinformatics prediction, real-time quantitative PCR (RT-qPCR), western blot (WB), double luciferase reporter gene and other relevant methods were used to study the inhibition of circ_0011058 on RA pathology and its molecular mechanism. Results showed that the expression of circ_0011058 was significantly increased in adjuvant arthritis (AA) rats and RA fibroblast-like synoviocytes (FLS). The knockout of circ_0011058 inhibited the proliferation of AA FLS and RA FLS, decreased the levels of interleukin-1 beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), and inhibited the expression of matrix metalloproteinase 3 (MMP3), fibronectin, which showed that circ_0011058 had a strong role in promoting RA pathology. Furthermore, miR-335-5p expression was reduced in AA rats and RA FLS. The highly expressed circ_0011058 directly sponged the miR-335-5p, which led to the increase of CUL4B expression and promoted the activation of the GSK3β/canonical signalling pathway. Finally, we confirmed that miR-335-5p mediated the roles of circ_0011058 in promoting RA pathological development, which showed that the circ_0011058/miR-335-5p/CUL4B signal axis was involved in RA pathology. This work was of great significance for clarifying the roles of circ_0011058 in RA pathology, and further work was needed to establish whether circ_0011058 was a potential therapeutic target or diagnostic marker for RA.

Periplogenin inhibits pathologic synovial proliferation and infiltration in rheumatoid arthritis by regulating the JAK2/3-STAT3 pathway

Rheumatoid arthritis (RA) is an autoimmune disease that affects joints, causing inflammation, synovitis, and erosion of cartilage and bone. Periplogenin is an active ingredient in the anti-rheumatic and anti-inflammatory herb, cortex periplocae. We conducted a study using a CIA model and an in vitro model of fibroblast-like synoviocytes (FLS) induced by Tumor Necrosis Factor-alpha (TNF-α) stimulation. We evaluated cell activity, proliferation, and migration using the CCK8 test, EDU kit, and transwell assays, as well as network pharmacokinetic analysis of periplogenin targets and RA-related effects. Furthermore, we measured inflammatory factors and matrix metalloproteinases (MMPs) expression using ELISA and qRT-PCR assays. We also evaluated joint destruction using HE and Safranin O-Fast Green Staining and examined the changes in the JAK2/3-STAT3 pathway using western blot. The results indicated that periplogenin can effectively inhibit the secretion of inflammatory factors, suppress the JAK2/3-STAT3 pathway, and impede the proliferation and migration of RA FLS. Thus, periplogenin alleviated the Synovial inflammatory infiltration of RA.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

ALKBH5-Mediated RNA m6 A Methylation Regulates the Migration, Invasion, and Proliferation of Rheumatoid Fibroblast-Like Synoviocytes.

Fibroblast-like synoviocytes (FLSs) are critical for promoting joint damage in rheumatoid arthritis (RA). N6 -methyladenosine (m6 A) modification plays key roles in various diseases, but its role in the pathogenesis of RA is largely unknown. Here, we investigate increased demethylase ALKBH5 promotion of proliferation, migration, and invasion of RA FLSs via regulating JARID2 expression. ALKBH5 expression in FLSs was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. 5-ethynyl-2'-deoxyuridine, scratch wound healing, and transwell assays were implemented to determine the role of ALKBH5 on RA FLS proliferation, mobility, and migration. Then, m6 A sequencing combined with RNA sequencing was performed to identify the potential targets of ALKBH5. RNA immunoprecipitation and RNA pulldown were then used to validate the interaction between the protein and messenger RNA (mRNA). Collagen-induced arthritis (CIA) and delayed-type hypersensitivity arthritis (DTHA) models were further established to assess the therapeutic potency of ALKBH5 in vivo. We demonstrated that ALKBH5 expression was increased in FLSs and synovium from RA. Functionally, ALKBH5 knockdown inhibited the proliferation, migration, and invasion of RA FLSs, whereas overexpression of ALKBH5 displayed the opposite effect. Mechanistically, ALKBH5 mediated m6 A modification in the JARID2 mRNA and enhanced its mRNA stability in cooperation with IGF2BP3. Intriguingly, the severity of arthritis was attenuated in mice with DTHA and ALKBH5 knockout or rats with CIA and intra-articular injection of ALKBH5 short hairpin RNA. Our findings suggest that ALKBH5-mediated m6 A modification is crucial for synovial hyperplasia and invasion in RA. ALKBH5 might be a potential therapeutic target for RA and even for dysregulated fibroblasts in a wide range of diseases.

Schisandrin treatment suppresses the proliferation, migration, invasion, and inflammatory responses of fibroblast-like synoviocytes from rheumatoid arthritis patients and attenuates synovial inflammation and joint destruction in CIA mice.

Rheumatoid arthritis (RA) is a systemic autoimmune disease causing joint dysfunction. As disease-modifying anti-rheumatic drugs (DMARDs) have poor efficacy in 20% to 25% of RA patients, additional novel RA medications are urgently needed. Schisandrin (SCH) has multiple therapeutic effects. However, whether SCH is effective against RA remains unknown. To investigate how SCH affects the abnormal behaviours of RA fibroblast-like synoviocytes (FLSs) and further elucidate the underlying mechanism of SCH in RA FLSs and collagen-induced arthritis (CIA) mice. Cell Counting Kit-8 (CCK8) assays were used to characterize cell viability. EdU assays were performed to assess cell proliferation. Annexin V-APC/PI assays were used to determine apoptosis. Transwell chamber assays were used to measure cell migration and invasion in vitro. RT-qPCR was used to assess proinflammatory cytokine and MMP mRNA expression. Western blotting was used to detect protein expression. RNA sequencing was performed to explore the potential downstream targets of SCH. CIA model mice were used to assess the treatment efficacy of SCH in vivo. Treatments with SCH (50, 100, and 200 μΜ) inhibited RA FLSs proliferation, migration, invasion, and TNF-α-induced IL-6, IL-8, and CCL2 expression in a dose-dependent manner but did not affect RA FLSs viability or apoptosis. RNA sequencing and Reactome enrichment analysis indicated that SREBF1 might be the downstream target in SCH treatment. Furthermore, knockdown of SREBF1 exerted effects similar to those of SCH in inhibiting RA FLSs proliferation, migration, invasion, and TNF-α-induced expression of IL-6, IL-8, and CCL2. Both SCH treatment and SREBF1 knockdown decreased activation of the PI3K/AKT and NF-κB signalling pathways. Moreover, SCH ameliorated joint inflammation and cartilage and bone destruction in CIA model mice. SCH controls the pathogenic behaviours of RA FLSs by targeting SREBF1-mediated activation of the PI3K/AKT and NF-κB signalling pathways. Our data suggest that SCH inhibits FLS-mediated synovial inflammation and joint damage and that SCH might have therapeutic potential for RA.

CX-4945 inhibits fibroblast-like synoviocytes functions through the CK2-p53 axis to reduce rheumatoid arthritis disease severity

Fibroblast-like synoviocytes (FLS) mediate many pathological processes in rheumatoid arthritis (RA), including pannus formation, bone erosion, and inflammation. RA FLS have unique aggressive phenotypes and exhibit several tumor cell-like characteristics, including hyperproliferation, excessive migration and invasion. Casein kinase 2 (CK2) is reportedly overexpressed in numerous tumor types, and targeted inhibition of CK2 has therapeutic benefits for tumors. However, the expression level of CK2 and its functions in RA FLS remain unclear. Herein, we aimed to elucidate whether CK2 is responsible for the aggressive phenotypes of RA FLS and whether targeted therapy can alleviate the severity of RA. We found that CK2 subunits were elevated in RA FLS compared with osteoarthritis FLS, and the activity of CK2 also markedly increased in RA FLS. Targeted inhibition of CK2 using CX-4945 suppressed RA FLS proliferation through cell cycle arrest. Cell migration and invasion were also inhibited by CX-4945 treatment. Moreover, CX-4945 reduced Interleukin-6 (IL-6), CC motif chemokine ligand 2 (CCL2) and Matrix metalloproteinase-3 (MMP-3) secretion in RA FLS. Further proteomic investigation revealed that p53 signaling pathway significantly changes after CX-4945 treatment in RA FLS. The siRNA-mediated p53 knockdown partly abolished the anti-proliferation and reduced IL-6, MMP-3 secretion effects of CX-4945. Furthermore, CX-4945 administration alleviates arthritis severity in CIA mice. Collectively, our results demonstrated the abnormal elevation of CK2 and its positive association with abnormal phenotypes in RA FLS. Our novel findings suggest the possible therapeutic potential of CX-4945 for RA.

Akuammiline alkaloid derivatives: divergent synthesis and effect on the proliferation of rheumatoid arthritis fibroblast-like synoviocytes.

During the past decades, rheumatoid arthritis had become a serious problem, torturing millions of patients because of unclear pathogenesis and no ideal therapies. Natural products remain an important source of medicines to treat various major diseases such as rheumatoid arthritis (RA) given their excellent biocompatibility and structural diversity. Herein, we have developed a versatile synthetic method for constructing various skeletons of akuammiline alkaloid analogs based on our previous research on the total synthesis of the related indole alkaloids. We have also evaluated the effect of these analogs on the proliferation of RA fibroblast-like synoviocytes (FLSs) in vitro and analyzed the corresponding structure-activity relationship (SAR). Among these analogs, compounds 9 and 17c have demonstrated a promising inhibitory effect on the proliferation of RA-FLSs, with IC50 values of 3.22 ± 0.29 μM and 3.21 ± 0.31μM, respectively. Our findings provide a solid foundation for future pharmacological studies on akuammiline alkaloid derivatives and inspiration for the development of anti-RA small molecule drugs derived from natural products.

Sarsasapogenin, a principal active component absorbed into blood of total saponins of Anemarrhena, attenuates proliferation and invasion in rheumatoid arthritis fibroblast-like synoviocytes through downregulating PKM2 inhibited pathological glycolysis.

Increased glycolytic in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) not only contributes to early-stage disease pathogenesis but leads to sustained proliferation of FLS. Given the importance of PKM2 in glycolysis and apoptosis, PKM2 is considered a potential therapeutic and drug discovery target in RA. Total saponins of anemarrhena (TSA), a class of steroid saponins, originated from Anemarrhena asphodeloides Bge. In this study, we verified that 200 mg/kg TSA could significantly alleviate inflammation and the pathological characteristics of RA and inhibit synovial hyperplasia in AA rats. We confirmed that sarsasapogenin (SA) was the principal active ingredient absorbed into the blood of TSA by the UPLC/Q Exactive MS test. Then we used TNF-α-induced MH7A to get the conclusion that 20 μM SA could effectively inhibit the glycolysis by inhibiting the activity of PKM2 tetramer and glucose uptake. Moreover, 20 μM SA could suppress proliferation, migration, invasion, and cytokine release of FLS, interfere with the growth cycle of FLS, and induce FLS apoptosis by depressing the phosphorylation of PKM2. At last, In-1, a potent inhibitor of the PKM2 was used to reverse verify the above results. Taken together, the key mechanisms of SA on RA treatment through downregulating the activity of PKM2 tetramer and phosphorylation of PKM2 inhibited pathological glycolysis and induced apoptosis to exert inhibition on the proliferation and invasion of RA FLS.

Epigenetic Regulation of Nutrient Transporters in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

Since previous studies indicate that metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), we undertook this study to determine if changes in the genome-wide chromatin and DNA states in genes associated with nutrient transporters could help to identify activated metabolic pathways in RA FLS. Data from a previous comprehensive epigenomic study in FLS were analyzed to identify differences in genome-wide states and gene transcription between RA and osteoarthritis. We utilized the single nearest genes to regions of interest for pathway analyses. Homer promoter analysis was used to identify enriched motifs for transcription factors. The role of solute carrier transporters and glutamine metabolism dependence in RA FLS was determined by small interfacing RNA knockdown, functional assays, and incubation with CB-839, a glutaminase inhibitor. We performed 1 H nuclear magnetic resonance to quantify metabolites. The unbiased pathway analysis demonstrated that solute carrier-mediated transmembrane transport was one pathway associated with differences in at least 4 genome-wide states or gene transcription. Thirty-four transporters of amino acids and other nutrients were associated with a change in at least 4 epigenetic marks. Functional assays revealed that solute carrier family 4 member 4 (SLC4A4) was critical for invasion, and glutamine was sufficient as an alternate source of energy to glucose. Experiments with CB-839 demonstrated decreased RA FLS invasion and proliferation. Finally, we found enrichment of motifs for c-Myc in several nutrient transporters. Our findings demonstrate that changes in the epigenetic landscape of genes are related to nutrient transporters, and metabolic pathways can be used to identify RA-specific targets, including critical solute carrier transporters, enzymes, and transcription factors, to develop novel therapeutic agents.

Quantitative Proteomic Analysis of Synovial Tissue Reveals That Upregulated OLFM4 Aggravates Inflammation in Rheumatoid Arthritis.

Tandem mass tag (TMT)-coupled liquid chromatography coupled with tandem mass spectrometry is a powerful method to investigate synovial tissue protein profiles in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Protein was isolated from synovial tissue samples of 22 patients and labeled with a TMT kit. Over 500 proteins were identified as the differential expression protein on comparing RA and OA synovial tissue, including 239 upregulated and 271 downregulated proteins. Data are available via ProteomeXchange with identifier PXD027703. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that the majority participated in the developmental processes and protein processing in the endoplasmic reticulum. Olfactomedin 4 (OLFM4), a secreted glycoprotein, in joint inflammation of RA was explored. OLFM4 was upregulated in RA synovial tissue samples. In fibroblast-like synoviocytes (FLS), inflammation cytokines, TNF-α, interleukin (IL)-1β, and LPS can upregulate OLFM4. After OLFM4 knockdown under TNF-α stimulation, RA FLS proliferation was inhibited and the expression of CXCL9, CXCL11, and MMP-1 was decreased. Overall, the RA synovial tissue protein expression profile by proteomic analysis shows some unique targets in RA pathophysiology, and OLFM4 in FLS plays an important role in RA joint inflammation. OLFM4 can be a promising therapeutic target in RA synovial tissue.

Retraction: MicroRNA-506 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targeting TLR4

Retraction: MicroRNA-506 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targeting TLR4

Retraction: MicroRNA-506 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targeting TLR4.

Retraction: MicroRNA-506 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targeting TLR4.

Cyclic mechanical stimulation inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation via cell cycle arrest

The imbalance between proliferation and apoptosis of fibroblast-like synoviocytes (FLSs) has been the main cause of rheumatoid arthritis (RA) synovial hyperplasia. Our previous study confirmed that the cyclic mechanical stimulation (CMS) inhibited the proliferation of RA FLSs, but the underlying mechanisms are still unclear. This study aimed to investigate these underlying mechanisms. The in vitro cultured human RA FLSs were subjected to CMS (6%, 1.0 Hz). Cell cycle was detected by flow cytometry. The expression of cyclin D1, cyclin E1, CDK-2 and p27 was detected by reverse transcription-polymerase chain reaction (RT-PCR). MTS assay was used to detect cell viability. Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) levels in RA FLSs were detected by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that CMS significantly inhibited the cell cycle transformation of RA FLSs from G1 phase to S phase, which significantly decreased the cell proliferation index. Meanwhile, both cyclin E1 and CDK-2 gene expressions were significantly decreased, p27 gene expression was increased, and no significant change was observed in the expression of cyclin D1. The inhibition of COX-2/PGE2 pathway in RA FLSs by celecoxib treatment showed no effect on the inhibition of RA FLSs proliferation by CMS. In conclusion, CMS inhibited the proliferation of RA FLSs by modulating the expression of cell cycle-related molecules such as cyclin E1, CDK2 and p27 to arrest cell cycle transformation, which is independent of COX-2/PGE2 signaling pathway.

Therapeutic Effects of (5R)-5-Hydroxytriptolide on Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via lncRNA WAKMAR2/miR-4478/E2F1/p53 Axis.

Rheumatoid arthritis (RA) is an autoimmune disease. Fibroblast-like synoviocytes (FLS) serve a major role in synovial hyperplasia and inflammation in RA. (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative, shows promising therapeutic effects for RA and is now in phase II clinical trials in China. However, the underlying mechanism of LLDT-8 is still not fully understood. Here, we found that LLDT-8 inhibited proliferation and invasion of RA FLS, as well as the production of cytokines. Microarray data demonstrated that LLDT-8 upregulated the expression of long non-coding RNA (lncRNA) WAKMAR2, which was negatively associated with proliferation and invasion of RA FLS, as well as the production of pro-inflammatory cytokines. Knockdown of WAKMAR2 abolished the inhibitory effects of LLDT-8 on RA FLS. Mechanistically, WAKMAR2 sponged miR-4478, which targeted E2F1 and downstreamed p53 signaling. Rescue experiments indicated that the inhibitory effects of LLDT-8 on RA FLS were dependent on WAKMAR2/miR-4478/E2F1/p53 axis.

MiR-431-5p regulates cell proliferation and apoptosis in fibroblast-like synoviocytes in rheumatoid arthritis by targeting XIAP

BackgroundmiR-431-5p is dysregulated in various cancers and plays an important function in the development of cancer. However, its role in fibroblast-like synoviocytes (FLSs) in patients with rheumatoid arthritis (RA) remains to be understood.MethodsQuantitative real-time polymerase chain reaction was used to detect the relative expression of miR-431-5p in synovial tissues and FLSs. Cell proliferation assays helped examine RA FLS proliferation. Flow cytometry was performed to determine apoptosis and cell cycle progression in RA FLSs. We used dual-luciferase assays to determine the correlation between miR-431-5p and its putative target, X-linked inhibitor of apoptosis (XIAP). Quantitative real-time PCR and western blotting were used to measure XIAP levels in synovial tissues and transfected RA FLSs.ResultsmiR-431-5p was downregulated in synovial tissues and FLSs of patients with RA. Upregulation of miR-431-5p prohibited cell proliferation and the G0/G1-to-S phase transition but promoted apoptosis in RA FLSs, while miR-431-5p inhibition showed the opposite results. miR-431-5p directly targeted XIAP in RA FLSs and reversely correlated with XIAP levels in synovial tissues. Notably, XIAP silencing partially restored the effects of miR-431-5p inhibition in RA FLSs.ConclusionmiR-431-5p regulates cell proliferation, apoptosis, and cell cycle of RA FLSs by targeting XIAP, suggesting its potential in the treatment of RA.

MiR-483-3p promotes cell proliferation and suppresses apoptosis in rheumatoid arthritis fibroblast-like synoviocytes by targeting IGF-1

Accumulating evidence suggests that miR-483−3p is implicated in maintaining biological properties in human cancers. However, its biological roles in rheumatoid arthritis (RA) remain unknown. miR-483−3p levels in synovial tissue samples and fibroblast-like synoviocytes (FLSs) were determined using quantitative real-time PCR. The CCK-8 assay and EdU staining were performed to assess cell proliferation in RA FLSs after transfection with miR-483−3p mimics or inhibitor. Flow cytometry with Annexin V-FITC staining or PI staining was performed to assess apoptosis or cell cycle progression in RA FLSs, respectively. miR-483−3p was upregulated in RA, which markedly promoted cell proliferation, induced the G0/G1-to-S phase transition, and suppressed apoptosis in RA FLSs, whereas miR-483−3p silencing yielded opposite results. Moreover, insulin growth factor 1 (IGF-1) was detected as a direct miR-483−3p target. IGF-1 silencing partially restored cell proliferation, the G0/G1-to-S phase transition, and apoptosis suppression in RA FLSs via miR-483−3p inhibition. Our results showed that miR-483−3p promotes RA FLSs proliferation by targeting IGF-1, suggesting a potential strategy for diagnostic and treatment strategy for RA.

MiRNA-6089 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targeting CCR4

Several studies have suggested that fibroblast-like synoviocytes (FLSs) and miRNAs are implicated in the pathogenesis of rheumatoid arthritis (RA). This study was aimed to evaluate the function of miR-6089 in the regulation of RA-FLSs. The levels of miR-6089 were detected to be significantly lower in the synovial tissues and FLSs of RA than in the healthy synovial tissues and FLSs. The miR-6089 up-regulation in RA-FLSs significantly inhibited the proliferation and promoted cell apoptosis accompany with an increase protein expression of cleaved-Caspase-3, -8 and -9. Furthermore, CCR4 was determined to target miR-6089 directly, and its expression was significantly increased in the synovial tissues of RA than in the healthy synovial tissues. The overexpression of CCR4 reversed the effect of miR-6089 on proliferation and apoptosis in RA-FLSs effectively. In conclusion, our study suggests that the miR-6089 may be a potential target for prevention and treatment of RA.