MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease.
BACKGROUND & AIMS
Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD.


METHODS
We obtained liver samples from 12 patients with ALD and cirrhosis and 9 healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8 weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8 weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative polymerase chain reaction analyses in Huh-7 cells.


RESULTS
Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared with mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in liver tissues from mice. Levels of GRHL2 also were increased in liver tissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of alanine aminotransferase compared with mice given a control vector. Levels of hypoxia-inducible factor 1 alpha (HIF1α) mRNA, a target of MIR122, were increased in liver tissues from patients and mice with ALD, compared with controls. Mice with hepatocyte-specific disruption of Hif1α developed less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both.


CONCLUSIONS
Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1α. These processes might be manipulated to reduce the severity of ALD in patients.
['Satishchandran|Abhishek|A|', 'Ambade|Aditya|A|', 'Rao|Sitara|S|', 'Hsueh|Ying-Chao|YC|', 'Iracheta-Vellve|Arvin|A|', 'Tornai|David|D|', 'Lowe|Patrick|P|', 'Gyongyosi|Benedek|B|', 'Li|Jia|J|', 'Catalano|Donna|D|', 'Zhong|Li|L|', 'Kodys|Karen|K|', 'Xie|Jun|J|', 'Bala|Shashi|S|', 'Gao|Guangping|G|', 'Szabo|Gyongyi|G|']
D000328:Adult D000368:Aged D000818:Animals D016022:Case-Control Studies D004268:DNA-Binding Proteins D004195:Disease Models, Animal D005260:Female D022781:Hepatocytes D006801:Humans D008108:Liver Diseases, Alcoholic D008297:Male D051379:Mice D035683:MicroRNAs D008875:Middle Aged D014157:Transcription Factors
Ethanol miR-122 AAV Gene Expression Mouse Model