miR-302 protects the liver from glucolipotoxicity-induced lipid accumulation through activating AMPK and suppressing Elovl6 in HepG2 cells and also in mice

Introduction: Metabolic dysfunction–associated steatotic liver disease (MASLD) is driven by glucolipotoxicity, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and insulin resistance. Although microRNAs regulate metabolic homeostasis, the role of the miR-302 cluster in hepatic lipid dysregulation and MASLD pathogenesis remains unclear. Methods: In HepG2 cells, glucolipotoxicity was induced using high glucose plus free fatty acids (HG + FFA). miR-302 cluster was overexpressed to assess protective effects. Lipid accumulation, triglycerides (TG), AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, Elovl6 expression, long-chain fatty acids (LCFA) content, ER stress markers, stemness factors, mitochondrial function, and insulin signaling were evaluated using biochemical assays, qPCR, and Western blotting. In db/db mice fed a high-fat diet (HFD), AAV8-mediated hepatic miR-302 delivery was used to evaluate steatosis and AMPK/Akt signaling. Results: HG + FFA induced lipid droplet accumulation, elevated neutral lipids and triglycerides, increased stearoyl-CoA desaturase 1 (SCD1) mRNA and LCFAs with Elovl6 upregulation, suppressed AMPK/ACC phosphorylation, activated ER stress, impaired mitochondrial function, elevated reactive oxygen species (ROS), and reduced insulin-stimulated Akt activation. miR-302 overexpression reversed these effects, restoring AMPK signaling, suppressing Elovl6 expression and SCD1 mRNA induction, reducing LCFAs, ROS, rescuing mitochondrial polarization, and normalizing insulin signaling. miR-302 also preserved stemness-related proteins, an effect abolished by AMPK inhibition. In db/db-HFD mice, miR-302 reduced hepatic steatosis, suppressed Elovl6, and restored hepatic AMPK and Akt phosphorylation. Conclusion: miR-302 protects hepatocytes from glucolipotoxic metabolic injury by activating AMPK and suppressing the Elovl6–LCFA axis, thereby improving mitochondrial, ER, and insulin-signaling homeostasis. These findings position miR-302 as an RNA therapeutic for MASLD.