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.

中文翻译：

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受 GRLH2 调控的 MicroRNA 122 可保护小鼠和患者的肝脏免受乙醇诱发的肝病

背景与目标

长期过量饮酒会导致酒精性肝病 (ALD)，其特征是脂肪变性、炎症，最终导致肝硬化。肝细胞特异性 microRNA 122 (MIR122) 调节肝细胞分化和代谢。我们研究了酒精引起的 MIR122 水平降低是否有助于 ALD 的发展。

方法

我们从 12 名 ALD 合并肝硬化患者和 9 名健康个体（对照）中获取肝脏样本，并通过组织学和免疫组织化学进行分析。C57Bl/6 小鼠接受 Lieber-DeCarli 流质饮食，其中喂食乙醇 8 周，作为 ALD 模型或对照饮食。这些小鼠还被注射了 CCl4，以增加肝纤维化，持续 8 周。第 28 天，患有乙醇诱导的肝病和晚期纤维化的小鼠以及对照小鼠被注射重组腺相关病毒 8 载体，该载体表达初级 miR-122 转录物（pri-MIR122，在肝细胞中过度表达 MIR122）或载体（控制）。在喂食乙​​醇前两周，一些小鼠被注射了表达抗 MIR122 的载体，以抑制其表达。收集血清和肝组织；通过组织学、免疫印迹和共聚焦显微镜对肝细胞和肝单核细胞进行分析。我们进行了计算机分析，以确定 Huh-7 细胞中 MIR122 的靶点和染色质免疫沉淀定量聚合酶链反应分析。

结果

与对照组相比，ALD 患者和接受 Lieber-DeCarli 饮食的小鼠肝脏样本中的 MIR122 水平降低。与注射对照载体的小鼠相比，在患有乙醇诱导的肝病和晚期纤维化的小鼠的肝细胞中转基因表达 MIR122 显着降低了丙氨酸氨基转移酶 (ALT) 的血清水平以及肝脏脂肪变性和纤维化。饲喂乙醇通过增加小鼠肝组织中转录因子粒状头样转录因子 2 (GRHL2) 剪接形式的表达来减少 pri-MIR122 的表达。与对照组相比，ALD 患者肝组织中 GRHL2 的水平也有所增加；人类肝脏中 MIR122 水平的降低与此相关。与给予对照载体的小鼠相比，在乙醇喂养前注射抗 MIR122 的小鼠脂肪变性、炎症和血清丙氨酸氨基转移酶水平增加。与对照组相比，ALD 患者和小鼠肝组织中缺氧诱导因子 1 α ( HIF1α ) mRNA（MIR122 的靶标）水平升高。肝细胞特异性Hif1α破坏的小鼠 在给予乙醇、注射抗 MIR122 或两者同时注射后，出现较轻的肝损伤。

结论

与对照组相比，ALD 患者和患有乙醇诱发肝病的小鼠肝脏中 MIR122 水平降低。MIR122 的转录受到 GRHL2 的抑制，GRHL2 在小鼠和 ALD 患者的肝脏中增加。抗 MIR122 的表达加剧了小鼠乙醇喂养后肝损伤的严重程度。MIR122 似乎可以通过降低 HIF1α 水平来保护肝脏免受乙醇引起的损伤。可以操纵这些过程来减轻患者 ALD 的严重程度。