Exploring the effect of LncRNA DANCR to regulate the Keap1‐Nrf2/ARE pathway on oxidative stress in rheumatoid arthritis

Abstract

AbstractIntroductionAberrant 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.MethodsThe 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.ResultsIn 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.ConclusionThe low‐expressed LncRNA DANCR may regulate the Keap1‐Nrf2/ARE pathway and suppress the inflammatory and oxidative responses in RA patients.