Zuo Zhihua, Li Yiqin, Zeng Chuyi, Xi Yuge, Tao Hualin, Guo Yongcan
Department of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University, Luzhou, China.
Department of Clinical Laboratory, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, China.
Front Med (Lausanne). 2021 Dec 7;8:767584. doi: 10.3389/fmed.2021.767584. eCollection 2021.
Alcoholic liver disease (ALD) is one of the most common chronic liver diseases worldwide. However, the potential molecular mechanism in ALD development remains unclear. The objective of this work was to identify key molecules and demonstrate the underlying regulatory mechanisms. RNA-seq datasets were obtained from Gene Expression Omnibus (GEO), and key molecules in ALD development were identified with bioinformatics analysis. Alcoholic liver disease mouse and cell models were constructed using Lieber-DeCarli diets and alcohol medium, respectively. Quantitative real-time PCR and Western blotting were conducted to confirm the differential expression level. Dual-luciferase reporter assays were performed to explore the targeting regulatory relationship. Overexpression and knockdown experiments were applied to reveal the potential molecular mechanism in ALD development. Between ALD patients and healthy controls, a total of 416 genes and 21 microRNAs (miRNAs) with significantly differential expression were screened. A comprehensive miRNA-mRNA network was established; within this network, the miR-182-5p/FOXO1 axis was considered a significant pathway in ALD lipid metabolism. Mouse and cell experiments validated that miR-182-5p was substantially higher in ALD than in normal livers, whereas the expression of FOXO1 was dramatically decreased by alcohol consumption ( < 0.05). Next, dual-luciferase reporter assays demonstrated that miR-182-5p directly targets the binding site of the FOXO1 3'UTR and inhibits its mRNA and protein expression. In addition, miR-182-5p was found to promote hepatic lipid accumulation via targeting the FOXO1 signaling pathway, and inhibition of the miR-182-5p/FOXO1 axis improved hepatic triglyceride (TG) deposition in ALD by regulating downstream genes involved in lipid metabolism. In summary, key molecules were identified in ALD development and a comprehensive miRNA-mRNA network was established. Meanwhile, our results suggested that miR-182-5p significantly increases lipid accumulation in ALD by targeting FOXO1, thereby providing novel scientific insights and potential therapeutic targets for ALD.
酒精性肝病（ALD）是全球最常见的慢性肝病之一。然而，ALD发生发展的潜在分子机制仍不清楚。本研究的目的是鉴定关键分子并阐明其潜在的调控机制。从基因表达综合数据库（GEO）获取RNA测序数据集，通过生物信息学分析鉴定ALD发生发展中的关键分子。分别使用Lieber-DeCarli饮食和酒精培养基构建酒精性肝病小鼠和细胞模型。采用定量实时PCR和蛋白质免疫印迹法确认差异表达水平。进行双荧光素酶报告基因检测以探索靶向调控关系。通过过表达和敲低实验揭示ALD发生发展的潜在分子机制。在ALD患者和健康对照之间，共筛选出416个基因和21个差异表达显著的微小RNA（miRNA）。建立了一个综合的miRNA- mRNA网络；在该网络中，miR-182-5p/FOXO1轴被认为是ALD脂质代谢中的一条重要通路。小鼠和细胞实验证实，ALD中miR-182-5p显著高于正常肝脏，而饮酒使FOXO1的表达显著降低（P<0.05）。接下来，双荧光素酶报告基因检测表明，miR-182-5p直接靶向FOXO1 3'UTR的结合位点并抑制其mRNA和蛋白质表达。此外，发现miR-182-5p通过靶向FOXO1信号通路促进肝脏脂质积累，抑制miR-182-5p/FOXO1轴可通过调节脂质代谢相关下游基因改善ALD中的肝甘油三酯（TG）沉积。总之，在ALD发生发展过程中鉴定出关键分子并建立了一个综合的miRNA- mRNA网络。同时，我们的结果表明，miR-182-5p通过靶向FOXO1显著增加ALD中的脂质积累，从而为ALD提供了新的科学见解和潜在治疗靶点。
