ABSTRACT

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

摘要

巨自噬/自噬是一种自我降解过程，可调节代谢稳态以应对各种压力条件，是非酒精性脂肪肝的治疗靶点。我们发现，由于自噬相关基因（如Ulk1）在小鼠模型和脂肪肝患者中的表达显着降低，自噬活性受到抑制。这种下调是由肝细胞中Mir214-3p水平升高和Hnf4a/Hnf4α mRNA 水平降低引起的。Mir214-3p通过直接结合 3' 非翻译区序列抑制Ulk1表达。Hnf4a直接激活Ulk1的转录. 我们研究了用抗Mir214-3p治疗的小鼠肝脏中的脂质积累和自噬相关基因的表达。通过锁定核酸介导的Mir214-3p沉默，肝脏脂肪变性得到缓解，并且Ulk1 mRNA 水平显着增加。此外，自噬体形成和 MAP1LC3/LC3-II 蛋白水平增加，表明自噬活性增加。有趣的是，抑制Mir214-3p并没有改善Ulk1抑制下的脂肪肝，这表明降低的 Mir214-3p水平通过上调Ulk1减轻肝脂肪变性。这些结果表明抑制Mir214-3p表达通过增加 Ulk1 的表达增加自噬活性来改善脂肪肝疾病。因此，Mir214-3p是非酒精性脂肪病的潜在治疗靶点。

缩写：AMPK：一磷酸腺苷活化蛋白激酶；ATG：自噬相关；ChIP：染色质免疫沉淀；CTSB：组织蛋白酶 B；CTSL：组织蛋白酶L；CQ：氯喹；HFD：高脂肪饮食；HNF4A：肝细胞核因子 4，α；IF：免疫荧光；IHC：免疫组化；LDs：脂滴；Leup：亮肽素；LFD：低脂饮食；LNA：锁核酸；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；miRNA：微小RNA；MTOR：雷帕霉素激酶的机制靶点；NAFLD：非酒精性脂肪肝；NASH：非酒精性脂肪性肝炎；PCR：聚合酶链式反应；TEM：透射电子显微镜；TF：转录因子；TLDA：TaqMan 低密度阵列；ULK1：unc-51 样激酶 1；UTR：未翻译区域