Hepatic miR-149-5p upregulation fosters steatosis, inflammation and fibrosis development in mice and in human liver organoids

Background & aimsThe incidence of metabolic dysfunction – associated steatotic liver disease (MASLD) is increasing worldwide. Alterations of hepatic microRNA (miRNA) expression/activity significantly contribute to the development and progression of MASLD. Genetic polymorphisms of miR-149 are associated with an increased susceptibility to develop MASLD in humans. Aberrant expression of miR-149 was also associated with metabolic alterations in several organs, but the impact of hepatic miR-149-5p deregulation in MASLD remains poorly characterized.MethodsMiR-149-5p was downregulated in the liver of mice by in vivo transduction with hepatotropic adeno-associated virus 8 (AAV8) harboring shRNAs specific for miR-149-5p (shmiR149) or scrambled shRNAs (shCTL). MASLD was then induced in mice with a methionine/ choline deficient (MCD, n=7 per group) or a fructose, palmitate and cholesterol-enriched (FPC, n=8-12 per group, per protocol) diet. The impact of miR-149-5p modulation on MASLD development was assessed in vivo and in vitro using multi-lineage 3D human liver organoids (HLOs) and in Huh7 cells.ResultsMiR-149-5p expression was strongly upregulated in mouse liver from different models of MASLD (2-4 fold increase in ob/ob, db/db mice, high-fat and FPC fed mice). In vivo downregulation of miR-149-5p led to an amelioration of diet-induced hepatic steatosis, inflammation/fibrosis, and to increased whole-body fatty acid consumption. In HLOs, miR-149-5p overexpression promoted lipid accumulation, inflammation and fibrosis. In vitro analyses of human Huh7 cells overexpressing miR-149-5p indicated that glycolysis and intracellular lipid accumulation was promoted, while mitochondrial respiration was impaired. Translatomic analyses highlighted deregulation of multiple miR-149-5p potential targets in hepatocytes involved in MASLD development.ConclusionsMiR-149-5p upregulation contributes to MASLD development by affecting multiple metabolic/inflammatory/fibrotic pathways in hepatocytes. Our results further demonstrate that human liver organoids are a relevant 3D in vitro model to investigate hepatic steatosis and inflammation/fibrosis development.Impact and implicationsThis study delves into the escalating global concern of metabolic dysfunction-associated steatotic liver disease (MASLD). Our research shows compelling evidence that miR-149-5p plays a pivotal role in the disease's development and progression. By employing in vivo and innovative in vitro models using multi-lineage human liver organoids (HLOs), we demonstrate that miR-149-5p upregulation significantly impacts hepatocyte energy metabolism, exacerbating hepatic steatosis and inflammation/fibrosis by modulating a wide network of target genes. These findings not only shed light on the intricate miR-149-5p-dependent molecular mechanisms underlying MASLD, but also underscore the importance of HLOs as valuable 3D in vitro models for studying the disease's pathogenesis.