E2F2 transcription factor promotes a cholestatic MASH phenotype by regulating hepatobiliary metabolism through miR-34a-5p

Background & Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) affects a heterogeneous group of patients. Among them, those with cholestatic profile show worse outcomes. Here, we investigated whether E2F2 is involved in MASLD-associated cholestasis and, if so, the role of miRNAs. Methods: E2f2-knockout (E2f2 -/- ) and wild-type (WT) mice were fed a choline-deficient high-fat diet (ChD-HFD) or a HFD after injection of diethylnitrosamine (DEN-HFD) to induce metabolic dysfunction-associated steatohepatitis (MASH). E2F2 was overexpressed in liver by AAV8. Cholestasis was induced by bile duct ligation or by a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-enriched diet. microRNA sequencing was performed. Two biopsy-proven MASLD patient cohorts were used. Results: E2F2 deficiency resulted in increased synthesis and excretion of cholesterol, phosphatidylcholine and bile acids, reducing their storage in the liver while increasing their presence in feces. This was consistent with increased expression of genes involved in biliary lipid metabolism, reduced inflammation and fibrosis, and the generation of a distinct miRNA profile, thereby preventing MASH. Liver-specific induction of E2F2 in vivo hampered the transcriptional program involved in biliary lipid metabolism and upregulated miR-34a-5p, which was downregulated in E2f2 -/- mice. The protective effects observed in E2f2 -/- mice were lost when a miR-34a-5p mimic was used. Hepatic miR-34a-5p levels were elevated in patients with advanced fibrosis, inflammation, steatosis score, cholelithiasis, and increased serum bile acids and biliary lipids. E2f2 deficiency conferred protection against cholestatic liver injury. Conclusions: E2F2 deficiency protects against MASH and cholestasis preventing cholesterol accumulation, fibrosis, and inflammation through modulation of miR-34a-5p, which could provide therapeutic benefits for patients with cholestatic-MASH.