Li Hualong, Zhang Lei, Cai Nan, Zhang Bing, Sun Shaomei
Department of Gastroenterology, Yantai Affiliated Hospital of Binzhou Medical University, Yantai, Shandong Province, 264100, China.
Department of Gastroenterology, Yantai Affiliated Hospital of Binzhou Medical University, Yantai, Shandong Province, 264100, China.
Ann Hepatol. 2021 Jul-Aug;23:100305. doi: 10.1016/j.aohep.2021.100305. Epub 2021 Jan 9.
Alcoholic hepatitis (AH) is characterized by high morbidity and mortality. MicroRNA-494-3p is possibly involved in the regulation of cancers, but its role in AH has been rarely studied.
AH mice model and primarily cultured mice hepatic stellate cells (HSCs) model were constructed. Levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed by ELISA. Expressions of miRNAs, HSC activation-related proteins and fibrosis-related protein were analyzed by qRT-PCR and Western blot. Cell counting kit, colony formation and flow cytometry assays were used to detect cell viability, proliferation and apoptosis, respectively. The relationship between TNF receptor-associated factor 3 (TRAF3) and miR-494-3p was predicted and verified by TargetScan and dual-luciferase assay, respectively. Results of the above experiments were verified by rescue experiments using TRAF3.
Liver damage and miRNA expression were observed in AH mice, and AST and ALT levels were increased in serum of AH mice. MiR-494-3p was reduced in AH liver tissues, and it decreased the levels of α-SMA and fibrosis-related proteins. HSCs were isolated, and activating HSCs or upregulating miR-494-3p had a regulatory effect on the levels of miR-494-3p, HSC activation-related proteins and fibrosis-related proteins as well as cell viability, proliferation and apoptosis. In addition, miR-494-3p targeted TRAF3 and inhibited TRAF3 expression, while overexpressed TRAF3 promoted TRAF3 expression and rescued the regulatory effect of miR-494-3p on the levels of related proteins as well as cell viability, proliferation and apoptosis.
This study provided a novel mechanistic comprehension of the anti-fibrotic effect of miR-494-3p.
酒精性肝炎（AH）具有高发病率和高死亡率的特点。微小RNA-494-3p可能参与癌症的调控，但其在AH中的作用鲜有研究。
构建AH小鼠模型和原代培养的小鼠肝星状细胞（HSCs）模型。通过酶联免疫吸附测定（ELISA）分析天冬氨酸转氨酶（AST）和丙氨酸转氨酶（ALT）水平。通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法分析微小RNA、HSC激活相关蛋白和纤维化相关蛋白的表达。分别使用细胞计数试剂盒、集落形成和流式细胞术检测细胞活力、增殖和凋亡。分别通过TargetScan预测和双荧光素酶测定验证肿瘤坏死因子受体相关因子3（TRAF3）与miR-494-3p之间的关系。上述实验结果通过使用TRAF3的拯救实验进行验证。
在AH小鼠中观察到肝损伤和微小RNA表达，AH小鼠血清中AST和ALT水平升高。MiR-494-3p在AH肝组织中降低，并且它降低了α-平滑肌肌动蛋白（α-SMA）和纤维化相关蛋白的水平。分离出HSCs，激活HSCs或上调miR-494-3p对miR-494-3p水平、HSC激活相关蛋白和纤维化相关蛋白水平以及细胞活力、增殖和凋亡具有调节作用。此外，miR-494-3p靶向TRAF3并抑制TRAF3表达，而过表达TRAF3促进TRAF3表达并挽救miR-494-3p对相关蛋白水平以及细胞活力,增殖和凋亡的调节作用。
本研究为miR-494-3p的抗纤维化作用提供了新的机制理解。
