MicroRNA-21 from bone marrow mesenchymal stem cell-derived extracellular vesicles targets TET1 to suppress KLF4 and alleviate rheumatoid arthritis.

BACKGROUND: Accumulating evidence has demonstrated that bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles (EVs) can be used effectively to transfer drugs and biomolecules to target lesions. Meanwhile, BMSCs have been reported to be beneficial in the treatment of rheumatoid arthritis (RA). In this study, we employ gain- and loss-of-function experiments to determine how BMSCs-derived EVs alleviate RA in vitro and in vivo. METHODS: We isolated EVs from BMSCs and characterized them by transmission electron microscopy and western blot analysis. The regulatory relationship between miR-21 and TET1 was predicted by bioinformatics analysis and validated by dual luciferase assay. Next, we utilized bisulfite sequencing PCR to decipher how TET1 promoted KLF4 transcription. Then, we established an RA mouse model and determined the role of miR-21 in RA progression. Functional assays were used to validate the role the miR-21-TET1-KLF4 regulatory axis in controlling mouse fibroblast-like synoviocytes (mFLS) cell proliferation and inflammatory cytokines secretion in vitro. RESULTS: RT-qPCR results revealed that miR-21 was highly expressed in BMSCs-derived EVs, and confirmed that BMSCs-derived EVs transferred miR-21 into mFLS cells. Bioinformatic analysis predicted that TET1 was the directly downstream target of miR-21, which was further validated by dual luciferase assay. TET1 promoted KLF4 promoter methylation to increase its expression. Collectively, BMSCs-derived EVs relieved RA by delivering miR-21, while the exosomal miR-21 alleviated RA through targeting the TET1/KLF4 regulatory axis. CONCLUSION: miR-21 from BMSCs-derived EVs suppresses KLF4 to relive RA by targeting TET1.

Long non-coding RNA PVT1 knockdown suppresses fibroblast-like synoviocyte inflammation and induces apoptosis in rheumatoid arthritis through demethylation of sirt6.

BACKGROUND: As a type of chronic autoimmune joint disease, rheumatoid arthritis (RA) is a disorder, characterized by a variety of physical symptoms as well as RA fibroblast-like synoviocyte (RA-FLS) proliferation. More recently, long non-coding RNAs (lncRNAs) have been implicated in the progression of various diseases including the progression of RA. Hence, the aim of the current study was to investigate the role by which the lncRNA, plasmacytoma variant translocation 1 (PVT1), influences RA-FLSs and its ability to modulate the methylation of sirtuin 6 (sirt6). METHODS: RA rat models were initially established to determine the expression of PVT1 and sirt6 in synovial tissues and RA-FLSs. Elevation or depletion of PVT1 or sirt6 was achieved by means of transformation with plasmids in order to investigate their effects on RA-FLS proliferation, inflammation and apoptosis. The localization of PVT1 and its binding ability to the sirt6 promoter region were also explored in an attempt to elucidate the correlation between PVT1 and sirt6 methylation. RESULTS: High expression of PVT1 and low expression of sirt6 were detected in the synovial tissues and RA-FLSs of the rat models. RA-FLSs treated with sh-PVT1 or oe-sirt6 exhibited suppressed cell proliferation, inflammation and induced apoptosis. PVT1 was predominately localized in the nucleus while evidence was obtained indicating that it could bind to the sirt6 promoter to induce sirt6 methylation, thus inhibiting sirt6 transcription. PVT1 knockdown was observed to restore sirt6 expression through decreasing sirt6 methylation, thereby alleviating RA. CONCLUSION: The key findings of the study provide evidence suggesting that, PVT1 knockdown is able to restrain RA progression by inhibiting sirt6 methylation to restore its expression.

MALAT1-Driven Inhibition of Wnt Signal Impedes Proliferation and Inflammation in Fibroblast-Like Synoviocytes Through CTNNB1 Promoter Methylation in Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLSs) participate in the pathogenesis of rheumatoid arthritis (RA). Emerging evidence has highlighted the role of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) and its potential involvement in RA. In this study, we test the hypothesis that the MALAT1 might inhibit proliferation and inflammatory response of FLSs in RA. The expression of MALAT1 was examined in synovial tissues from patients with RA. The effect of MALAT1 on cultured FLSs was analyzed by introducing overexpressed MALAT1 or short hairpin RNA (shRNA) against MALAT1. To validate whether methylation of CTNNB1 promoter was affected by MALAT1 alternation, we assessed the recruitment of DNA methyltransferases to CTNNB1 promoter. In cultured FLSs with shRNA-mediated CTNNB1 knockdown or activated Wnt signaling, we found the interaction between CTNNB1 and Wnt signaling. MALAT1 expression was reduced in synovial tissues of RA. MALAT1 could bind to CTNNB1 promoter region and recruit methyltransferase to promote CTNNB1 promoter methylation, thereby inhibiting CTNNB1. Notably, MALAT1 could suppress the transcription and expression of CTNNB1, thereby modulating the Wnt signaling pathway. Silenced MALAT1 stimulated the nucleation of ß-catenin and the secretion of inflammatory cytokines including interleukin-6, interleukin-10, and tumor necrosis factor-α. Additionally, shRNA-mediated MALAT1 silencing elevated proliferation and suppressed apoptosis of FLSs accompanied. These findings provide evidence for the inhibitory effect of MALAT1 on proliferation and inflammation of FLSs by promoting CTNNB1 promoter methylation and inhibiting the Wnt signaling pathway. Therefore, this study provides a candidate therapeutic target for RA.

Inhibition of NF-κB signaling pathway induces apoptosis and suppresses proliferation and angiogenesis of human fibroblast-like synovial cells in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is the most common inflammatory arthritis and is a major cause of disability. The nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway has been reported to be involved in the pathogenesis of RA with unclear mechanisms. Therefore, this study aims to explore the effect of NF-κB pathway on proliferation, apoptosis, and angiogenesis of human fibroblast-like synovial cells (HFLS) in RA. METHODS: Normal HFLS and RA-HFLS were selected as the normal and control groups, respectively. RA-HFLS were treated by BAY11-7082 (an inhibitor of NF-κB) in different concentrations, namely 2.5 µmol/L BAY11-7082, 5 µmol/LBAY11-7082 and 10 µmol/L BAY11-7082. MTT assay was employed to detect cell proliferation. Cell apoptosis was determined by flow cytometry at 24, 48, and 72 hours after culture. Western blot analysis was employed to detect the expressions of NF-κB, angiogenesis-related factors (VEGF, Ang1, and Ang2). RESULTS: Initially, we found that BAY11-7082 inhibited NF-κB expression in a concentration-dependent manner. According to the findings of MTT assay and flow cytometry, we understood that RA-HFLS treated by BAY11-7082 (an inhibitor of NF-κB), the inhibition of NF-κB pathway, suppressed RA-HFLS proliferation and induced RA-HFLS apoptosis in a concentration and time-dependent manner. Furthermore, RA-HFLS treated by BAY11-7082 presented decreased VEGF, Ang1 and Ang2 expressions in a concentration-dependent manner. CONCLUSION: The study concluded that inhibition of NF-κB pathway induced cell apoptosis and suppressed proliferation and angiogenesis of RA-HFLS, which could serve as a novel target in the treatment of RA.