scholarly journals Uridine Diphosphate (UDP) Promotes Rheumatoid Arthritis Through P2Y6 Activation

2020 ◽  
Author(s):  
Hongxing Wang ◽  
Hui Wu ◽  
Kehua Fang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Real Time ◽  
Proinflammatory Cytokines ◽  
Uridine Diphosphate ◽  
Synovial Fluids ◽  
Synovial Tissues ◽  
And Migration ◽  
G Protein Coupled ◽  
Fibroblast Like Synoviocytes

Abstract Background: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA).Methods: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) (n = 10) with samples from osteoarthritis (OA) as controls (n = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues (n = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls (n = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both (n = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid (n = 36) and peripheral blood (n = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flowResults：LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF) levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues.Conclusion: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.

scholarly journals Uridine Diphosphate Promotes Rheumatoid Arthritis Through P2Y6 Activation

2021 ◽  
Vol 12 ◽  
Author(s):  
Hongxing Wang ◽  
Hui Wu ◽  
Kehua Fang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Real Time ◽  
Proinflammatory Cytokines ◽  
Uridine Diphosphate ◽  
Synovial Fluids ◽  
Synovial Tissues ◽  
And Migration ◽  
G Protein Coupled ◽  
Fibroblast Like Synoviocytes

BACKGROUND: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA).METHODS: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) (n = 10) with samples from osteoarthritis (OA) as controls (n = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues (n = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls (n = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both (n = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid (n = 36) and peripheral blood (n = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flow.RESULTS: LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF)levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues.CONCLUSION: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.

scholarly journals Uridine Diphosphate (UDP) Promotes Rheumatoid Arthritis Through P2Y6 Activation

2020 ◽  
Author(s):  
Hongxing Wang ◽  
Hui Wu ◽  
Kehua Fang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Synovial Fluid ◽  
Real Time ◽  
Uridine Diphosphate ◽  
Collagen Induced Arthritis ◽  
Synovial Tissues ◽  
And Migration ◽  
G Protein Coupled ◽  
Fibroblast Like Synoviocytes

Abstract Background: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of the metabotropic pyrimidine and purine nucleotide receptor pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6).Methods: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in rheumatoid arthritis (RA) synovial fluid (SF) (n=10) with samples from osteoarthritis (OA) as controls (n=10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues and cultured with UDP or MRS2578, P2Y6 antagonist, and FLSs from OA was used as controls. Rats with collagen-induced arthritis (CIA) were established and injected with UDP or MRS2578 or both. P2Y6 expression was examined using real-time PCR, Western blotting and Immunohistochemistry. Cell proliferation, apoptosis and migration of FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, the wound healing assay and transwell assay. The concentration of UDP in culture medium, synovial fluid and peripheral blood RA and CIA rats was measured using a Transcreener UDP Assay, IL-6 was measured using ELISA and flow assay, and other pro-inflammatory cytokines was measured using Th1/Th2 Subgroup Detection Kit. Results: LC-MS analysis showed that the UDP level is not only higher in RA SF than in OA SF but also positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF) levels in RA. The increased UDP concentration was verified in the plasma and SF samples of RA patients (n=36) and healthy volunteers (n=36), and the levels were significantly correlated with RF and anti-CCP level in the samples. The study also found that UDP stimulated the cell proliferation, migration and interleukin-6 (IL-6) secretion of RA FLSs and inhibited their apoptosis in the culture. The P2Y6 antagonist MRS2578 inhibited this effect of UDP in the culture. UDP injection accelerated the development of collagen-induced arthritis (CIA) in a rat model and stimulated IL-6 production, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues and was unaltered by UDP treatment. UDP treatment and P2Y6 activity didn’t change levels of other proinflammatory cytokines in cultured FLSs and CIA rats.Conclusion: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.

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

2020 ◽  
Author(s):  
Yuejiao Wang ◽  
Kailin Zhang ◽  
Xiaowei Yuan ◽  
Neili Xu ◽  
Shuai Zhao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Real Time ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Proliferation And Apoptosis ◽  
Synovial Tissues ◽  
Proliferation Assays ◽  
Fibroblast Like Synoviocytes

Abstract Background miR-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.Methods Quantitative 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.Results miR-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.Conclusion miR-431-5p regulates cell proliferation, apoptosis，and cell cycle of RA FLSs by targeting XIAP, suggesting its potential in the treatment of RA.

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

2020 ◽  
Author(s):  
Yuejiao Wang ◽  
Kailin Zhang ◽  
Xiaowei Yuan ◽  
Neili Xu ◽  
Shuai Zhao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Real Time ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Proliferation And Apoptosis ◽  
Synovial Tissues ◽  
Proliferation Assays ◽  
Fibroblast Like Synoviocytes

Abstract 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.

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

2020 ◽  
Author(s):  
Yuejiao Wang ◽  
Kailin Zhang ◽  
Xiaowei Yuan ◽  
Neili Xu ◽  
Shuai Zhao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Real Time ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Proliferation And Apoptosis ◽  
Synovial Tissues ◽  
Proliferation Assays ◽  
Fibroblast Like Synoviocytes

Abstract Background miR-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. Methods Quantitative 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. Results miR-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. Conclusion miR-431-5p regulates cell proliferation, apoptosis，and cell cycle of RA FLSs by targeting XIAP, suggesting its potential in the treatment of RA.

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

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Yuejiao Wang ◽  
Kailin Zhang ◽  
Xiaowei Yuan ◽  
Neili Xu ◽  
Shuai Zhao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Real Time ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Proliferation And Apoptosis ◽  
Synovial Tissues ◽  
Proliferation Assays ◽  
Fibroblast Like Synoviocytes

Abstract Background miR-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. Methods Quantitative 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. Results miR-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. Conclusion miR-431-5p regulates cell proliferation, apoptosis, and cell cycle of RA FLSs by targeting XIAP, suggesting its potential in the treatment of RA.

scholarly journals Lnc RNA ZFAS1 regulates the proliferation, apoptosis, inflammatory response and autophagy of fibroblast-like synoviocytes via miR-2682-5p/ADAMTS9 axis in rheumatoid arthritis

2020 ◽  
Vol 40 (8) ◽  
Author(s):  
Shanshan Yang ◽  
Wei Yin ◽  
Yan Ding ◽  
Fan Liu
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Inflammatory Response ◽  
Large Subunit ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Behaviors ◽  
Synovial Tissues ◽  
Related Proteins ◽  
Fibroblast Like Synoviocytes

Abstract Backgrounds: Rheumatoid arthritis (RA) is a frequent autoimmune disease. Emerging evidence indicated that ZNFX1 antisense RNA1 (ZFAS1) participates in the physiological and pathological processes in RA. However, knowledge of ZFAS1 in RA is limited, the potential work pathway of ZFAS1 needs to be further investigated. Methods: Levels of ZFAS1, microRNA (miR)-2682-5p, and ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) were estimated using quantitative real-time polymerase chain reaction (qRT-PCR) assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to explore the ability of cell proliferation in fibroblast-like synoviocytes (FLS-RA). Cell apoptosis was measured via flow cytometry. Also, levels of ADAMTS9, apoptosis-related proteins, cleaved-caspase-3 (active large subunit), and autophagy-related proteins were identified adopting Western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the productions of inflammatory cytokines. Beside, the interrelation between miR-2682-5p and ZFAS1 or ADAMTS9 was verified utilizing dual-luciferase reporter assay. Results: High levels of ZFAS1 and ADAMTS9, and a low level of miR-2682-5p were observed in RA synovial tissues and FLS-RA. Knockdown of ZFAS1 led to the curbs of cell proliferation, inflammation, autophagy, and boost apoptosis in FLS-RA, while these effects were abolished via regaining miR-2682-5p inhibition. Additionally, the influence of miR-2682-5p on cell phenotypes and inflammatory response were eliminated by ADAMTS9 up-regulation in FLS-RA. Mechanically, ZFAS1 exerted its role through miR-2682-5p/ADAMTS9 axis in RA. Conclusion: ZFAS1/miR-2682-5p/ADAMTS9 axis could modulate the cell behaviors, inflammatory response in FLS-RA, might provide a potential therapeutic target for RA treatment.

scholarly journals Linc02381 Exacerbates Rheumatoid Arthritis Through Adsorbing miR-590-5p and Activating the Mitogen-Activated Protein Kinase Signaling Pathway in Rheumatoid arthritis-fibroblast-like synoviocytes

2020 ◽  
Vol 29 ◽  
pp. 096368972093802
Author(s):  
Jing Wang ◽  
Qing Zhao
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Signaling Pathway ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Synovial Tissues ◽  
Proliferation And Invasion ◽  
Fibroblast Like Synoviocytes

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. New evidence suggested that linc02381 suppressed colorectal cancer progression by regulating PI3 K signaling pathway, but the role of linc02381 in other diseases, such as RA, remains unclear. This study aimed to reveal the mechanism of linc02381 in RA progression. In vivo and in vitro, we found that linc02381 was upregulated in RA synovial tissues or RA fibroblast-like synoviocytes (RA-FLSs, P < 0.01), which were detected by quantitative real-time polymerase chain reaction. Cell Counting Kit-8, EDU, and Transwell assays revealed that linc02381 overexpression enhanced cell proliferation and invasion, and linc02381 knockdown inhibited cell proliferation and invasion in FLSs. Moreover, the results of bioinformatics analysis, luciferase reporter gene assay, and pull-down assay verified that linc02381 could directly bind with miR-590-5p. MiR-590-5p was downregulated in RA-FLSs, and overexpression of linc02381 suppressed expression of miR-590-5p that post-transcriptionally suppressed the expression of mitogen-activated protein kinase kinase 3 (MAP2K3), and overexpression of miR-590-5p reversed the effect of linc02381 overexpression on MAP2K3 expression. MiR-590-5p inhibitor reversed the inhibition effect of linc02381 knockdown on proliferation and invasion of FLSs, which enhanced expression of MAP2K3, and activation of p38 and AP-1 in the MAPK signaling pathway. In summary, linc02381 was upregulated in RA synovial tissues and RA-FLSs, and it exacerbated RA by adsorbing miR-590-5p to activate the MAPK signaling pathway.

scholarly journals miR-653-5p suppresses the viability and migration of fibroblast-like synoviocytes by targeting FGF2 and inactivation of the Wnt/beta-catenin pathway

2022 ◽  
Vol 17 (1) ◽  
Author(s):  
Peilong Dong ◽  
Xiaobo Tang ◽  
Jian Wang ◽  
Botao Zhu ◽  
Zhiyun Li
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Viability ◽  
Luciferase Reporter ◽  
Systemic Autoimmune Disease ◽  
Beta Catenin ◽  
Protein Levels ◽  
Rho Family ◽  
Synovial Tissues ◽  
And Migration ◽  
Fibroblast Like Synoviocytes

Abstract Background Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Several studies reported that fibroblast-like synoviocytes (FLSs) and miRNAs are associated with RA pathogenesis. This study explored the function of miR-653-5p in the regulation of human fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) cells. Methods The mRNA and protein levels of genes were measured by RT-qPCR and western blot, respectively. MTT, wound healing, and invasion assays were used to evaluate the viability and metastasis of FLSs. Luciferase reporter and RNA pull-down assays were employed to determine the interaction between miR-653-5p and FGF2. Results RT-qPCR results demonstrated that miR-653-5p expression was decreased and FGF2 level was increased in synovial tissues and FLSs of RA. Moreover, the viability and metastasis of FLSs were accelerated by miR-653-5p addition, which was restrained by miR-653-5p suppression. Furthermore, we demonstrated that levels of Rac1, Cdc42, and RhoA were decreased after miR-653-5p addition. Besides, luciferase reporter and RNA pull-down assays implied that miR-653-5p targeted the 3′-UTR of FGF2. Functional assays showed that FGF2 overexpression neutralized the suppressive effects of miR-653-5p addition on HFLS-RA cell viability, metastasis, and the levels of Rho family proteins. Meanwhile, the levels of β-catenin, cyclin D1, and c-myc were declined by miR-653-5p supplementation, but enhanced by FGF2 addition. Conclusion In sum, we manifested that miR-653-5p restrained HFLS-RA cell viability and metastasis via targeting FGF2 and repressing the Wnt/beta-Catenin pathway.

The Protective Role of Inhibition of Keratin on Rheumatoid Arthritis

2019 ◽  
Vol 9 (6) ◽  
pp. 797-803
Author(s):  
Yan Zhao ◽  
Lanxiu Yang ◽  
Jihong Pan ◽  
Huan Zhang ◽  
Guodong Sun ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometric Analysis ◽  
Protective Role ◽  
Invasion And Migration ◽  
Flow Cytometric ◽  
Migratory Capacity ◽  
Synovial Tissues ◽  
And Migration ◽  
Fibroblast Like Synoviocytes

Objective: Rheumatoid arthritis (RA) is a common inflammatory disease. Studies showed that keratin type II cuticular Hb4 (KRT84) was highly expressed in the synovial membrane of patients with RA. However, the function and mechanism of KRT84 in RA is still unclear. Methods: In this study, we isolated fibroblast-like synoviocytes (FLS) from the mixed knee joint synovial tissues from five patients with RA and the cells were treated with KRT84 siRNA. After transfection of 24 h and 36 h, the knockdown efficiency and expression of relevant genes was detected by RT-PCR. MTT assay, transwell assay, wound scratch assays, flow cytometric analysis and ELISA were used to assess cell proliferation, invasive and migratory capacity. Results: We found that the invasion and migration of RAFLS were significantly decreased after transfection of KRT84 siRNA. ELISA showed a remarkably decrease in TNF-α secretion after KRT84 knockdown. We also explore stimulatory factors for high expression of KRT84 in RA. The inhibitors of ERK, STAT3 and NF-κ B pathways were employed. Our results showed that the expression of KRT84 in RAFLS was evidently increased after treatment with ERK and STAT3 pathway inhibitor. Conclusions: These results imply a protective role of KRT84 knockdown on RA and lay a foundation for further studies on the pathogenetic mechanisms of RA.

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