MicroRNA-372-3p impairs fatty acid metabolism in hepatocellular carcinoma cells by targeting CPT1A and ACSL4.

Abstract

Introduction: Hepatocellular carcinoma (HCC) remains a major cause of cancer mortality, and effective therapeutic options are limited. MicroRNA‑372‑3p (miR‑372‑3p) has been implicated in HCC, yet its exact role is unclear.

Methods: We established miR‑372‑3p‑overexpressing HCC cell lines (HepG2, SNU‑449, JHH‑4) via lentiviral transduction. Malignant phenotypes were assessed with MTT, transwell migration/invasion, and colony‑formation assays. Transcriptomic changes were analyzed by RNA‑sequencing followed by Gene Set Enrichment Analysis. Lipid metabolism was examined using BODIPY/Oil Red O staining, triglyceride quantification, FAOBlue assays, and organelle colocalization imaging. Candidate targets of miR‑372‑3p were computationally predicted and validated by dual‑luciferase reporter assays.

Results: miR‑372‑3p overexpression significantly reduced cell proliferation by more than 50%, migration by over 30%, invasion by over 30%, and colony formation by more than 50%, supporting its tumor-suppressive role. Transcriptomic analysis identified 1,759 downregulated genes, significantly enriched in pathways associated with fatty acid oxidation (FAO). miR‑372‑3p‑overexpressing cells exhibited increased lipid droplet accumulation, with triglyceride levels elevated by more than 50% and an approximate 50% reduction in FAO activity, indicating defective use under glucose-deprived conditions. High-resolution organelle imaging further revealed diminished physical contacts between lipid droplets and mitochondria, as well as a similar disruption in lipid droplet-lysosome interactions. Dual-luciferase reporter assays confirmed CPT1A and ACSL4 as direct targets of miR‑372‑3p.

Conclusion: miR‑372‑3p functions as a tumor suppressor in HCC by directly downregulating CPT1A and ACSL4, thereby inhibiting FAO and disrupting lipid metabolism. Targeting this miRNA and FAO axis may offer a novel therapeutic strategy for HCC.