FOXS1 Promotes Liver Fibrosis Through TGF-β1/Smad Signaling: Targeting a Novel miR-212-5p/FTO Axis

Liver fibrosis is a progressive pathological condition characterized by excessive extracellular matrix (ECM) deposition, primarily driven by hepatic stellate cell (HSC) activation. Although Forkhead box S1 (FOXS1) has been implicated in fibrotic and neoplastic processes, its functional role in hepatic fibrosis and its regulatory mechanisms remain unclear.

FOXS1 expression was analyzed in human fibrotic liver tissues, CCl₄-induced, and methionine-choline-deficient (MCD) diet-induced nonalcoholic steatohepatitis (NASH) mouse models and activated HSCs. CCl₄-induced and MCD mouse models of liver fibrosis and TGF-β1-stimulated LX-2 and HSC-T6 cells and H-HSCs and M-HSCs were used to investigate FOXS1 function in vivo and in vitro. TGF-β1-induced fibrosis models were used to explore the FOXS1-TGF-β1/Smad axis in HSC activation. Furthermore, functional assays, luciferase reporter, RNA pulldown, MeRIP, and RIP assays were conducted to elucidate the regulatory relationships among FOXS1, miR-212-5p, and m6A demethylase FTO.

FOXS1 expression was elevated in fibrotic human and mouse liver tissues and positively correlated with fibrosis severity. FOXS1 knockdown in mice ameliorated fibrosis, reduced ECM protein expression, and improved liver function. In HSCs, FOXS1 enhanced TGF-β1/Smad2/3 signaling in a positive feedback loop, promoting HSC activation, migration, and resistance to apoptosis. Mechanistically, miR-212-5p directly targeted FOXS1 and suppressed fibrogenesis, but its maturation was inhibited by FTO-mediated m6A demethylation of pri-miR-212. Additionally, FTO knockdown promoted miR-212-5p processing, downregulated FOXS1, and disrupted the TGF-β1/Smad loop, thereby exerting antifibrotic effects.

FOXS1 acts as a novel driver of hepatic fibrosis that promotes HSC activation through TGF-β1/Smad-dependent feedback loop involving FTO-mediated m6A modification and miR-212-5p. Targeting the FTO/miR-212-5p/FOXS1 axis offers a promising therapeutic strategy for liver fibrosis.