Nicotine promotes cardiac fibrosis by regulating miR-125b-5p maturation via HNRNPA2B1-mediated METTL14-dependent m⁶A methylation: therapeutic reversal by cinacalcet HCl

Abstract Background Cardiac fibrosis is a pivotal pathological process driving adverse cardiac remodeling and a defining feature of end-stage heart disease. Nicotine, a principal constituent of tobacco products, is now recognized as an independent risk factor for cardiovascular disease. However, its direct effects on cardiac fibroblasts (CFs) biology and the molecular mechanisms underlying nicotine-induced cardiac fibrosis remain incompletely understood. Methods Primary CFs and a rat model of nicotine exposure were used to access the pro-fibrotic effects of nicotine. Drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) were employed to identify the cellular targets of nicotine. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) and quantitative real-time PCR (qRT-PCR) were used to quantify m⁶A modification and microRNA biogenesis. miR-125b-5p overexpression or inhibition, heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1, abbreviated as A2B1 in all figures) silencing, and pharmacological inhibition with cinacalcet HCl were performed both in vitro and in vivo to evaluate the impact of this signaling axis on nicotine-induced fibrotic phenotypes. Collagen deposition, CFs proliferation, and activated transforming growth factor-β1 (TGF-β1)/Mitogen-Activated Protein Kinase (MAPK) signaling were assessed by histology, immunoblotting, and immunofluorescence. Results Nicotine promoted CFs proliferation and migration, myofibroblasts (MFs) transformation, and collagen accumulation. HNRNPA2B1 was identified as a potential binding target of nicotine. Mechanistically, nicotine up-regulated methyltransferase-like 14 (METTL14), thereby increasing m⁶A methylation of pri-miR-125b. HNRNPA2B1 recognized methylated pri-miR-125b, facilitated its interaction with DiGeorge Syndrome Critical Region 8 (DGCR8), and thereby accelerated miR-125b-5p maturation. Elevated miR-125b-5p suppressed p53 and activated the TGF-β1/MAPK axis, driving cardiac fibrosis. Knockdown of HNRNPA2B1 or treatment with cinacalcet HCl markedly reduced the levels of miR-125b-5p and ameliorated nicotine-induced cardiac fibrosis in vitro and in vivo. Conclusions Nicotine induces excessive maturation of miR-125b-5p through an m⁶A-dependent, HNRNPA2B1-mediated mechanism, thereby promoting cardiac fibrosis. Targeting this signaling pathway—either genetically or pharmacologically with cinacalcet HCl—effectively attenuates fibrotic remodeling, providing a novel mechanistic rationale and potential therapeutic strategy management of cardiac fibrosis. Graphical Abstract