scholarly journals MiRNA-506 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targetting TLR4

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Dan Li ◽  
Qingchen Zhou ◽  
Gaojian Hu ◽  
Gang Wang
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Counting ◽  
Luciferase Assay ◽  
Qrt Pcr ◽  
Cycle Arrest ◽  
Tnf Α ◽  
Proliferation And Apoptosis ◽  
Synovial Tissues ◽  
Induced Apoptosis ◽  
Fibroblast Like Synoviocytes

Abstract Fibroblast-like synoviocytes (FLSs) play a crucial role in rheumatoid arthritis (RA) pathogenesis. While miRNA (miR)-506 has been implicated in the progression of multiple diseases, its role in RA remains to be explored. The present study evaluated the function of miR-506 in the regulation of RA-FLSs. FLSs were prepared from RA and healthy synovial tissues. The expression of miR-506 was measured by quantitative real time PCR (qRT-PCR). The effects of miR-506 on RA-FLSs proliferation and apoptosis were detected by cell counting Kit-8 and flow cytometry assays, respectively. The determination of TNF-α, IL-6, and IL-1β concentrations in RA-FLSs supernatant were done by ELISA. The levels of miR-506 were detected to be significantly lower in the synovial tissues and FLSs of RA than in the synovial tissues and FLSs of healthy controls. The miR-506 up-regulation in RA-FLSs significantly inhibited the proliferation and promoted cell cycle arrest at the G0/G1 phase. The overexpression of miR-506 induced apoptosis, along with an increase in activities of caspase-3 and -8. A target gene Toll-like receptor 4 (TLR4) under the direct regulation of miR-506 was identified through the luciferase assay, qRT-PCR and western blot analysis. Forced overexpression of TLR4 in the rescue experiments showed that TLR4 effectively reversed the effect on proliferation and apoptosis in miR-506-overexpressing RA-FLSs. Thus, miR-506 may be a potential target for RA prevention and therapy of RA.

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

2020 ◽  
Author(s):  
Suxian Lin ◽  
Zhiyong Zhang ◽  
Shengnan Wang ◽  
Yang Lu ◽  
Meilv Yang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Protein Expression ◽  
Caspase 3 ◽  
Healthy Controls ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr ◽  
Tnf Α ◽  
Proliferation And Apoptosis ◽  
Synovial Tissues ◽  
Fibroblast Like Synoviocytes

Abstract Background: Growing data have indicated that fibroblast-like synoviocytes (FLS) 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. Methods: The expression of miR-6089 was measured by quantitative real time PCR (qRT-PCR). The RA-FLSs were transfected with si-CCR4 plasmids or miR-6089 mimic, and subjected to CCK-8 and flow cytometry to analyze proliferation and apoptosis. The concentrations of MMP-1, TNF-α and IL-6 in RA-FLSs supernatant were detected using ELISA. The protein expression of caspase-3, -8 and -9 was detected using western blot.Results: The levels of miR-6089 were detected to be significantly lower in the synovial tissues and FLSs of RA than in the synovial tissues and FLSs of healthy controls. The miR-6089 up-regulation in RA-FLSs significantly inhibited the proliferation and promoted cell apoptosis accompany with an increase protein expression of caspase-3, -8 and -9. Furthermore, CCR4 was determined to directly target miR-6089, and its expression was significantly increased in the synovial tissues of RA than in the synovial tissues of healthy controls. Moreover, CCR4 overexpression effectively reversed the effect on proliferation and apoptosis induced by miR-6089 in RA-FLSs. Conclusion: Our results revealed that miR-6089 may be a potential target for RA prevention and therapy of RA.

scholarly journals Down-regulation of microRNA-142-3p inhibits the aggressive phenotypes of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting nuclear factor-κB signaling

2019 ◽  
Vol 39 (7) ◽  
Author(s):  
Jianhong Qiang ◽  
Tingting Lv ◽  
Zhenbiao Wu ◽  
Xichao Yang
Keyword(s):  
Rheumatoid Arthritis ◽  
B Cell Lymphoma ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Down Regulation ◽  
Qrt Pcr ◽  
Tnf Α ◽  
Synovial Tissues ◽  
Fibroblast Like Synoviocytes

Abstract The present study aimed to investigate the regulatory roles of miR-142-3p on the aggressive phenotypes of rheumatoid arthritis (RA) human fibroblast-like synoviocytes (RA-HFLSs), and reveal the potential mechanisms relating with nuclear factor-κB (NF-κB) signaling. miR-142-3p expression was detected in RA synovial tissues and RA-HFLSs by quantitative real-time PCR (qRT-PCR) and Northern blot analysis. RA-HFLSs were transfected with miR-142-3p inhibitor and/or treated with 10 µg/l tumor necrosis factor α (TNF-α). The viability, colony formation, apoptosis, migration, invasion, and the levels of interleukin (IL)-6, and matrix metalloproteinase 3 (MMP-3) were detected. The mRNA expressions of B-cell lymphoma-2 (Bcl-2), Bax, Bad, IL-6, and MMP-3 were detected by qRT-PCR. Moreover, the expression of Bcl-2, IL-1 receptor-associated kinase 1 (IRAK1), Toll-like receptor 4 (TLR4), NF-κB p65, and phosphorylated NF-κB p65 (p-NF-κB p65) were detected by Western blot. The interaction between IRAK1 and miR-142-3p was identified by dual luciferase reporter gene assay. MiR-142-3p was up-regulated in RA synovial tissues and RA-HFLSs. TNF-α activated the aggressive phenotypes of RA-HFLSs, including enhanced proliferation, migration, invasion, and inflammation, and inhibited apoptosis. miR-142-3p inhibitor significantly decreased the cell viability, the number of cell clones, the migration rate, the number of invasive cells, the contents and expression of IL-6 and MMP-3, and increased the apoptosis rate and the expressions of Bax and Bad, and decreased Bcl-2 expression of TNF-α-treated RA-HFLSs. MiR-142-3p inhibitor significantly reversed TNF-α-induced up-regulation of IRAK1, TLR4, and p-NF-κB p65 in TNF-α-treated RA-HFLSs. Besides, IRAK1 was a target of miR-142-3p. The down-regulation of miR-142-3p inhibited the aggressive phenotypes of RA-HFLSs through inhibiting NF-κB signaling.

scholarly journals Knockdown of nrf2 exacerbates TNF-α-induced proliferation and invasion of rheumatoid arthritis fibroblast-like synoviocytes through activating JNK pathway

2020 ◽  
Author(s):  
Yu Du ◽  
Qian Wang ◽  
Na Tian ◽  
Meng Lu ◽  
Xian-Long Zhang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Western Blot ◽  
Nuclear Translocation ◽  
Cell Counting ◽  
Related Factor ◽  
Jnk Pathway ◽  
Tnf Α ◽  
Synovial Tissues ◽  
Proliferation And Invasion ◽  
Fibroblast Like Synoviocytes

Abstract Background. Fibroblast-like synoviocytes (FLS) in the synovial tissue of rheumatoid arthritis (RA) exhibit over-proliferative and aggressive phenotypes, which participate in the pathophysiology of RA. In RA, little is known about the non-antioxidant effect of nuclear factor erythroid 2-related factor 2 (nrf2), master regulator of redox homeostasis. In this study, we explored the expression and upstream regulatory factors of nrf2, and revealed its functions in modulating the proliferation and invasion in RA-FLS. Methods. FLS were isolated from RA and osteoarthritis patients. Expression of nrf2 in the synovial tissues and FLS was analyzed by immunohistochemistry, real-time PCR, western blot, and immunofluorescence. Cell proliferation was examined by Cell Counting Kit-8, and cell invasion was tested by transwell assay. Phosphorylation of JNK was determined by Western blot. Results. Nrf2 expression in the RA synovial tissues was upregulated. TNF-α promoted expression and nuclear translocation of nrf2 in RA-FLS, and increased the intracellular reactive oxygen species (ROS) level. Nrf2 nuclear translocation was blocked by ROS inhibitor N-acetylcysteine. Both knockdown of nrf2 by siRNA and inhibition of nrf2 by ML385 significantly promoted the TNF-α-induced proliferation and invasion of RA-FLS. Activation of nrf2 by sulforaphane (SFN) profoundly inhibited the TNF-α-induced proliferation and invasion of RA-FLS. Knockdown of nrf2 also enhanced the TNF-α-induced matrix metalloproteinases (MMPs) expression and phosphorylation of JNK in RA-FLS. Proliferation and invasion of RA-FLS incubated with TNF-α and nrf2 siRNA was inhibited by pre-treatment with JNK inhibitor SP600125. Conclusion. Taken together, nrf2 is over-expressed in synovial tissues of RA patients, which may be induced by TNF-α and ROS levels. Increased nrf2 may suppress TNF-α-induced proliferation, invasion, and MMPs expression in RA-FLS through inhibiting JNK activation, indicating that nrf2 plays a protective role to relieve the severity of synovitis in RA.

scholarly journals Knockdown of nrf2 Exacerbates TNF-α-Induced Proliferation and Invasion of Rheumatoid Arthritis Fibroblast-Like Synoviocytes through Activating JNK Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Yu Du ◽  
Qian Wang ◽  
Na Tian ◽  
Meng Lu ◽  
Xian-Long Zhang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Western Blotting ◽  
Nuclear Translocation ◽  
Cell Counting ◽  
Related Factor ◽  
Jnk Pathway ◽  
Tnf Α ◽  
Synovial Tissues ◽  
Proliferation And Invasion ◽  
Fibroblast Like Synoviocytes

Fibroblast-like synoviocytes (FLS) in the synovial tissue of rheumatoid arthritis (RA) exhibit over-proliferative and aggressive phenotypes, which participate in the pathophysiology of RA. In RA, little is known about the nonantioxidant effect of nuclear factor erythroid 2-related factor 2 (nrf2), the master regulator of redox homeostasis. In this study, we aimed to explore the expression and upstream regulatory factors of nrf2 and revealed its functions in modulating the proliferation and invasion in RA-FLS. FLS were isolated from RA and osteoarthritis patients. Expression of nrf2 in the synovial tissues and FLS was analyzed by immunohistochemistry, real-time PCR, Western blotting, and immunofluorescence staining. Cell proliferation was examined by Cell Counting Kit-8. Cell invasion was tested by transwell assay. Phosphorylation of JNK was determined by Western blotting. The results showed that nrf2 expression in the RA synovial tissues was upregulated. TNF-α promoted expression and nuclear translocation of nrf2 in RA-FLS and increased the intracellular reactive oxygen species (ROS) level. Nrf2 nuclear translocation was blocked by ROS inhibitor N-acetylcysteine. Both knockdown of nrf2 by siRNA and inhibition of nrf2 by ML385 significantly promoted the TNF-α-induced proliferation and invasion of RA-FLS. Activation of nrf2 by sulforaphane (SFN) profoundly inhibited the TNF-α-induced proliferation and invasion of RA-FLS. Knockdown of nrf2 also enhanced the TNF-α-induced matrix metalloproteinases (MMPs) expression and phosphorylation of JNK in RA-FLS. Proliferation and invasion of RA-FLS incubated with TNF-α and nrf2 siRNA were inhibited by pretreatment with JNK inhibitor SP600125. In conclusion, nrf2 is overexpressed in synovial tissues of RA patients, which may be promoted by TNF-α and ROS levels. Activation of nrf2 may suppress TNF-α-induced proliferation, invasion, and MMPs expression in RA-FLS by inhibiting JNK activation, indicating that nrf2 plays a protective role in relieving the severity of synovitis in RA. Our results might provide novel insights into the treatment of RA.

scholarly journals Mitomycin C Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via a Mitochondrial-Mediated Pathway

2015 ◽  
Vol 35 (3) ◽  
pp. 1125-1136 ◽  
Author(s):  
Chuqi Yan ◽  
Dechao Kong ◽  
Dong Ge ◽  
Yanming Zhang ◽  
Xishan Zhang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Viability ◽  
Mitomycin C ◽  
Apoptotic Cell ◽  
Chronic Inflammatory Disease ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Treatment ◽  
Induced Apoptosis ◽  
Fibroblast Like Synoviocytes

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway.

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.

The Function and Molecular Mechanism of MicroRNA-429 in Rheumatoid Arthritis

2020 ◽  
Vol 10 (7) ◽  
pp. 945-950
Author(s):  
Pengdong Zhang ◽  
Bailong Yu ◽  
Bin Lei ◽  
Changlin Li ◽  
Xiaoqiang Yuan
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Molecular Mechanism ◽  
Synovial Tissue ◽  
Luciferase Assay ◽  
Rt Pcr ◽  
Tnf Α ◽  
Tlr4 Gene ◽  
Synovial Tissues ◽  
D Model

Objective: To explain the function and molecular mechanism of miRNA-429 in Rheumatoid Arthritis development. Methods: Collecting synovial tissue of 36 RA patients and 36 traumatic amputation patients, the miRNA-429 and TLR4 gene expressions were measured by RT-PCR. The SD rats were divided into NC, 14 d Model and 28 d Model groups. The IL-1β and TNF-α concentrations of serum were measured by Elisa assay in difference rats groups; The synovial tissue pathology was evaluated by HE staining; the miRNA-429 gene expression of rats groups were measured by RT-PCR, the TLR4 and NF-κB proteins expressions of rats groups were evaluated by IHC staining; the correlation between miRNA-429 and TLR4 were evaluated by Double luciferase assay. Results: Compared with normal synovial tissues, the miRNA-429 and TLR4 gene expression of synovial tissues were significantly difference in RA patients. In rats vivo study, we found that IL-1 and TNF-α concentrations were significantly up-regulation with time increasing (P < 0 05, respectively); inflammation degree was serious by HE staining and miRNA-429 gene expression was significantly reduced (P < 0.05, respectively); TLR4 and NF-κB proteins expressions were significantly up-regulation (P < 0.05, respectively) with time increasing; TLR4 was the target gene of miRNA-429 by Double luciferase assay. Conclusion: miRNA-429 over-expression stimulated RA development.

scholarly journals CircASH2L facilitates tumor-like biologic behaviours and inflammation of fibroblast-like synoviocytes via miR-129-5p/HIPK2 axis in rheumatoid arthritis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xia Li ◽  
Meiting Qu ◽  
Jie Zhang ◽  
Kuanyin Chen ◽  
Xianghui Ma
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Cycle Progression ◽  
Circular Rna ◽  
Cycle Progression ◽  
Qrt Pcr ◽  
Cycle Arrest ◽  
Fibroblast Like Synoviocytes

Abstract Background Previous study showed that circular RNA Absent-Small-Homeotic-2--Like protein (circASH2L) was higher in rheumatoid arthritis (RA) patients. However, the roles and mechanisms of circASH2L in RA progression remain unclear. Methods Levels analysis was conducted using western blot and qRT-PCR. The proliferation, apoptosis, cell cycle progression, migration, invasiveness, and inflammation of RA fibroblast-like synoviocytes (RA-FLSs) were determined via MTT, flow cytometry, western blot, transwell, and ELISA assays. Results CircASH2L knockdown in RA-FLSs suppressed cell proliferative, migratory, and invasive capacities, triggered cell cycle arrest, promoted apoptosis, and inhibited inflammation. Mechanistically, circASH2L targeted miR-129-5p, and repression of miR-129-5p abolished the functions of circASH2L silencing on the growth, motility, and inflammation of RA-FLSs. Besides, miR-129-5p was found to directly target HIPK2, and suppressed the tumor-like biologic behaviors and inflammation of RA-FLSs via regulating HIPK2. Importantly, we proved that circASH2L could modulate HIPK2 expression via miR-129-5p. Conclusion CircASH2L promoted RA-FLS growth, motility, and inflammation through miR-129-5p/HIPK2 axis.

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.

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