A novel C/EBPα–miR-335-5p–PRKAA2 regulatory axis drives hepatic lipid accumulation in MASLD

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a rapidly growing global health concern. However, the molecular mechanisms underlying its onset and progression remain incompletely understood. This study aimed to investigate the molecular mechanism by which the transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates hepatic lipid accumulation through the miR-335-5p–mediated PRKAA2/AMPK signaling pathway. MASLD models were established both in high-fat diet (HFD)-fed mice and in free fatty acid (FFA)-treated hepatocytes. Gene expression, lipid accumulation, and related signaling pathways were analyzed using quantitative RT–PCR, Western blotting, Oil Red O staining, dual-luciferase reporter assays, and rescue experiments. We identified a novel regulatory axis in which C/EBPα transcriptionally activates miR-335-5p, leading to suppression of PRKAA2, the catalytic subunit of AMPKα2, and subsequent inhibition of AMPK signaling. This cascade promotes the expression of lipogenic genes and enhances hepatic lipid deposition. Inhibition of either C/EBPα or miR-335-5p restored PRKAA2/AMPK activity, reduced intracellular triglyceride and cholesterol levels, and alleviated hepatic steatosis in both cellular and murine models. Conversely, PRKAA2 silencing reproduced the lipogenic phenotype, while its overexpression rescued miR-335-5p-induced lipid accumulation. Our findings reveal a previously unrecognized C/EBPα/miR-335-5p/PRKAA2 axis that drives hepatic lipogenesis and steatosis in MASLD. Targeting this signaling pathway—by inhibiting C/EBPα or miR-335-5p, or activating PRKAA2/AMPK—may provide a promising therapeutic strategy for MASLD.