Exploring the effect of LncRNA DANCR to regulate the Keap1-Nrf2/ARE pathway on oxidative stress in rheumatoid arthritis.
Aberrant expression of long noncoding RNAs (LncRNAs) can regulate oxidative stress in rheumatoid arthritis (RA). This study focused on investigating the effects of LncRNA differentiation antagonizing nonprotein coding RNA (DANCR) regulation of Keap1-Nrf2/ARE pathway on inflammation and oxidative stress in RA.
The levels of LncRNA DANCR/miR-486-3p/Keap1 in peripheral blood of 30 RA groups and 30 normal subjects were examined, and the association of LncRNA DANCR with inflammatory indicators of RA was investigated. We stimulated fibroblast-like synoviocytes (FLS) from RA patients with tumor necrosis factor α and subsequently performed in vitro cellular assays to construct overexpression plasmids and small interfering RNAs of LncRNA DANCR to investigate the relationship between LncRNA DANCR and FLSs viability and migration in RA, as well as the effects on cellular oxidative stress factors and Keap1-Nrf2/ARE pathway; molecular biology analysis was used to predict microRNAs that can bind LncRNA DANCR, and luciferase verified the binding sites of LncRNA DANCR with Keap1 and miR-486-3p; to further refine the gene and protein expression results, we used reverse transcription-quantitative polymerase chain reaction and immunoblotting assays.
In both groups of peripheral blood mononuclear cells, the expression levels of LncRNA DANCR and Keap1 messenger RNA were higher in the RA group than in the normal control group, and the opposite was true for miR-486-3p; LncRNA DANCR was positively correlated with total antioxidant capacity (TAOC), IL6, IL17, malondialdehyde (MDA), but not with IL11, rheumatoid factor, cyclic citrullinated peptide, superoxide dismutase (SOD), with 28-joint disease activity score, reactive oxygen species, C-reactive protein, and erythrocyte sedimentation rate were negatively correlated; overexpression of LncRNA DANCR stimulated the Keap1-Nrf2/ARE pathway, decreased the expression of IL10, SOD, TAOC, and increased the expression levels of MDA, IL11, IL-17, PD-L1, and silencing of LncRNA DANCR was the opposite, but knockdown of miR-486-3p or overexpression of keap1 reversed the expression of the above-mentioned inflammatory and oxidative factors. In addition, pcDNA-DANCR clearly showed stronger cell invasion and migration ability and exacerbated its inflammatory response, which may be related to the regulatory role of miR-486-3p and Keap1-Nrf2/ARE signaling pathway, and we verified their targeting relationship using dual luciferase, showing that DANCR could regulate Keap1-Nrf2/ARE through miR-486-3p modulates the Keap1-Nrf2/ARE pathway and affects inflammatory and oxidative responses in RA patients.
The low-expressed LncRNA DANCR may regulate the Keap1-Nrf2/ARE pathway and suppress the inflammatory and oxidative responses in RA patients.
Cai S
，Sun Y
，Wang Y
，Lin Z
... -
《Immunity Inflammation and Disease》
WTAP-Mediated m6A Modification of TRAIL-DR4 Suppresses MH7A Cell Apoptosis.
N6-methyladenosine (m6A) is one of the most conserved internal RNA modifications, which has been implicated in many biological processes, such as apoptosis and proliferation. Wilms tumor 1-associating protein (WTAP), as a key component of m6A methylation, is a nuclear protein that has been associated with the regulation of proliferation and apoptosis. Rheumatoid arthritis (RA), a systemic, infiltrating autoimmune disease, is characterized by synovial hyperplasia. However, little is known about the precise role of WTAP in RA. This study investigated the role of the WTAP-mediated m6A modification of TNF-related apoptosis-inducing ligand death receptor 4 (TRAIL-DR4) in RA.
Methyltransferase WTAP overexpression plasmids and small interfering RNAs were constructed and transfected into MH7A cells. Immunofluorescence (IF) staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blot were used to detect changes in the expression of WTAP, the B-cell lymphoma 2 (BCL2) gene family, BCL2-associated X (BAX) and TRAIL-DR4 expression, and the effects of WTAP overexpression on cell viability, cell cycle, apoptosis, and proliferation were assessed by a cell counting kit-8 (CCK-8), flow cytometry, and transmission electron microscopy (TEM). The m6A modification of TRAIL-DR4 was verified by m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) and its stability was assessed by an actinomycin D assay.
Overexpression of WTAP not only increased the levels of WTAP and BCL2, and decreased the levels of BAX and TRAIL-DR4, but also significantly inhibited MH7A cell apoptosis and promoted cell viability and proliferation, while WTAP silencing led to the opposite trend. The SRAMP online database predicted that TRAIL-DR4 has multiple potential methylation-binding sites, and fluorescence in situ hybridization (FISH) combined with IF showed that WTAP and TRAIL-DR4 were mainly expressed in both the nucleus and cytoplasm. MeRIP-qPCR and actinomycin D analysis experiments revealed that WTAP could promote the m6A level of TRAIL-DR4, decrease the stability of TRAIL-DR4 mRNA, and subsequently inhibit apoptosis.
This study suggests that WTAP-mediated m6A modification of TRAIL-DR4 suppresses MH7A cell apoptosis. This discovery offers a new focus and avenue for the clinical treatment of RA, while also extending our understanding of the pathophysiology of RA from the standpoint of m6A alteration.
Cui X
，Xu F
，Pang X
，Fan C
，Jiang H
... -
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ResearchGate
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钛学术
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