Extracellular vesicles miRNome profiling reveals a close liaison between circulating miRNAs and lipid metabolism derangements in patients with NAFLD

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease that encompasses a broad spectrum of liver diseases ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) with or without fibrosis. Insulin resistance, obesity, and other features of metabolic syndrome are linked to NAFLD, however, the aetiology of the disease remains largely unknown, particularly with respect to NAFL/NASH progression. MicroRNAs (miRNAs) are regulatory molecules involved in numerous metabolic conditions. Altered levels of miRNAs encapsulated in Extracellular Vesicles (EVs) showed the potential for minimally invasive tracking of NAFLD. In a cohort of 228 deeply-characterized NAFLD patients we aimed at identifying: i) specific miRNAs associated with the presence of NASH and significant (F≥2) fibrosis; and ii) miRNAs relevant to the deranged metabolic pathways in NAFLD by tracers studies and lipid analysis. Serum EV miRNAs were profiled by the small RNA sequencing analysis. In vivo metabolic studies were performed in a subgroup of 54 patients by tracer infusion ([6,6-2H2]glucose and [2H5]glycerol) to assess glucose clearance and indexes of insulin resistance (IR) in the adipose tissue and in the liver. Circulating free fatty acids (FFAs) composition was determined by GC-MS. Overall, 23 and 31 miRNAs were significantly differentially expressed according to the diagnosis of non-alcoholic steatohepatitis (NASH) and the presence of significant fibrosis (F ≥ 2), respectively. In patients that underwent metabolic studies, miR-122 and miR-375 showed a significant correlation with FFAs plasma levels and adipose tissue IR, while let-7d and let-7f showed an inverse correlation with the same parameters. Interestingly, miR-122 up-regulation was proportional to plasma levels of palmitic acid, the most abundant FFA responsible for hepatocytes lipoapoptosis, (rs = 0.429, p = 0.006), while let-7d and let-7f were inversely correlated (rs = -0.405, p = 0.011; rs = -0.467, p = 0.003, respectively). Our results suggest an intertwined connection of miR-122 and let-7 in the counter-regulation of lipid derangements in patients with severe NAFLD, revealing potential therapeutic targets for the treatment of NASH and liver fibrosis.