RUNX1-mediated repression of miR-24 promotes hepatic stellate cell activation and liver fibrosis by targeting the ALK4/Smad3 signaling pathway

BackgroundEpigenetic mechanisms, including microRNAs (miRNAs), are increasingly recognized as crucial regulators of organ fibrosis. In this study, we investigated the role of miR-24 in hepatic stellate cell (HSC) activation and liver fibrosis.MethodsmiR-24 expression was analyzed in carbon tetrachloride (CCl4)-induced liver fibrosis and activated HSCs using quantitative real-time PCR (qRT-PCR). Gain- and loss-of-function experiments of miR-24 were performed in vitro. Western blotting, qRT-PCR, 5-ethynyl-2′-deoxyuridine (EdU) staining, flow cytometry, luciferase reporter assays, bioinformatics analysis, and chromatin immunoprecipitation PCR (ChIP-PCR) were performed to examine the molecular mechanisms of miR-24. Serum miR-24 levels were measured in patients with liver cirrhosis and further analyzed by subgroup.ResultsWe observed significant downregulation of miR-24 in CCl4-induced liver fibrosis and activated HSCs. Functional assays showed that miR-24 overexpression markedly inhibited HSC activation and migration, whereas miR-24 inhibition had the opposite effects. Mechanistically, ALK4 was identified as a direct target of miR-24: miR-24 bound the 3′UTR of ALK4 mRNA, thereby suppressing Smad3 phosphorylation and downstream fibrosis-associated signaling pathways. Furthermore, the transcription factor RUNX1 was induced during HSC activation, and it transcriptionally repressed miR-24 expression. Clinically, serum miR-24 levels were significantly lower in patients with liver cirrhosis than in healthy controls and were negatively correlated with Child–Pugh grade.ConclusionOur findings suggest that the RUNX1/miR-24/ALK4 axis plays a crucial role in HSC activation and migration. miR-24 may serve as a biomarker for liver fibrosis screening, representing a potential therapeutic target for anti-fibrotic intervention.