Role of mitochondria-targeting miRNAs in non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) comprises a range of liver lesions from simple steatosis to non-alcoholic steatohepatitis (NASH) and remains a major cause of mortality when progressing to cirrhosis and hepatocellular carcinoma (HCC). Although major risk factors relate with the metabolic syndrome, including cross-talk between the liver and the skeletal muscle, the biological mechanisms of disease are not entirely known. Therefore, a better understanding of NAFLD pathogenesis may help in finding novel targeted therapies for patients with liver damage. In that regard, we have shown that morbid obese patients with NAFLD exhibit increased microRNA-34a (miRNA/miR-34a) expression, p53 acetylation and apoptosis, as well as decreased Sirtuin-1 expression, in more severe disease stages, suggesting that miRNAs actively contribute to disease progression. In turn, further evidence also supports a role for mitochondrial dysfunction in NAFLD pathogenesis. In particular, mitochondrial fusion protein mitofusin 2 (Mfn2) is decreased in human NASH, with its liver-specific ablation in mice leading to a NASH-like phenotype. The main goal of the work presented in this thesis was to dissect the role of mitochondria-targeting miRNAs in human and experimental models of NAFLD, elucidating their functional role in disease progression and potential as therapeutic targets. First, we explored the role of miR-34a in skeletal muscle dysfunction associated to NASH development. Our results showed that miR-34a is activated in skeletal muscle of both human and experimental models of NASH, leading to inhibition of Sirtuin-1 and AMP-activated protein kinase (AMPK). This resulted in impairment of insulin signalling and dysfunctional mitochondrial dynamics, including abnormally downregulated levels of Mfn2. Further, functional studies established a direct association between miR-34a- and palmitic acid (PA)-induced muscle cell deregulation, with downstream AMPK activation being able to restore muscle homeostasis. Finally, muscle miR-34a expression and Mfn2 protein levels correlated with hallmarks of human NAFLD and its progression. We next sought to identify miRNAs directly targeting liver Mfn2, which we showed to be consistently downregulated in multiple, complementary diet-induced NAFLD mice models. miR-222-3p was identified and validated as a direct Mfn2-binding miRNA, in vitro. Strikingly, miR-222-3p inhibition in two complementary diet-induced NASH mice models led to significantly increased Mfn2 expression levels, paralleling decreased hepatic steatosis, inflammation, liver injury and oxidative stress. Finally, we evaluated expression levels of both miR-34a and miR-222-3p in the liver of a large cohort of NAFLD patients, which progressively increased with disease severity and, further, correlated with the grades of steatosis, lobular inflammation and fibrosis. Overall, our results highlight the key function of mitochondria-targeting miRNAs in metabolic syndrome-associated NAFLD. In particular, muscle miR-34a- and liver miR-222-3p-induced mitochondrial dysfunction, involving Mfn2 downregulation, appear amenable to effective therapeutic targeting. This is particularly relevant considering that both miR-34a and miR-222-3p associate to a worse disease prognosis in NAFLD patients