Mir214-3p and Hnf4a/Hnf4α reciprocally regulate Ulk1 expression and autophagy in nonalcoholic hepatic steatosis

Macroautophagy/autophagy, a self-degradative process, regulates metabolic homeostasis in response to various stress conditions and is a therapeutic target for nonalcoholic fatty liver disease. We found that autophagic activity was inhibited as a result of a significant reduction in the expression of autophagy-related genes such as Ulk1 in a mouse model and patients with fatty liver. This downregulation was caused by increased Mir214-3p levels and decreased Hnf4a/Hnf4α mRNA levels in hepatocytes. Mir214-3p suppressed Ulk1 expression through direct binding at a 3′ untranslated region sequence. Hnf4a directly activated transcription of Ulk1. We investigated lipid accumulation and the expression of autophagy-related genes in the livers of mice treated with anti-Mir214-3p. Hepatic steatosis was alleviated, and Ulk1 mRNA levels were significantly increased by locked nucleic acid-mediated Mir214-3p silencing. Additionally, autophagosome formation and MAP1LC3/LC3-II protein levels were increased, indicating an increase in autophagic activity. Interestingly, suppression of Mir214-3p did not ameliorate fatty liver under Ulk1 suppression, suggesting that reduced Mir214-3p levels mitigate hepatic steatosis through upregulation of Ulk1. These results demonstrate that inhibition of Mir214-3p expression ameliorated fatty liver disease through increased autophagic activity by increasing the expression of Ulk1. Thus, Mir214-3p is a potential therapeutic target for nonalcoholic fatty disease. Abbreviations: AMPK: adenosine monophosphate-activated protein kinase; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; CTSB: cathepsin B; CTSL: cathepsin L; CQ: chloroquine; HFD: high-fat diet; HNF4A: hepatocyte nuclear factor 4, alpha; IF: immunofluorescence; IHC: immunohistochemistry; LDs: lipid droplets; Leup: leupeptin; LFD: low-fat diet; LNA: locked nucleic acid; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; miRNA: microRNA; MTOR: mechanistic target of rapamycin kinase; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PCR: polymerase chain reaction; TEM: transmission electron microscopy; TF: transcription factor; TLDA: TaqMan low-density array; ULK1: unc-51 like kinase 1; UTR: untranslated region