miRNA-322/424 通过靶向 CUL2/HIF-1α 通路调节血管生成促进肝纤维化。
microRNA-322/424 promotes liver fibrosis by regulating angiogenesis through targeting CUL2/HIF-1α pathway.
机构信息
Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China; College of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; E-Institute of Shanghai Municipal Education Committee, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
College of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
出版信息
Life Sci. 2021 Feb 1;266:118819. doi: 10.1016/j.lfs.2020.118819. Epub 2020 Dec 14.
AIMS
To investigate the effects and mechanism of miR-322/424 in liver fibrosis.
MAIN METHODS
miR-322/424 expression in liver cirrhosis patients, mouse and rat liver fibrosis was determined by qPCR. Mice liver fibrosis was established by CCl, and intervened by miR-322/424 agomir or antagomir. Liver hydroxyproline content and Sirius red staining were used to evaluate collagen deposition. CD31 expression was used to evaluate liver microvessel density. In vitro, the effects of miR-322/424 mimic or inhibitor on human hepatic sinusoidal endothelial cells (HHSECs) migration and tube formation were investigated. A dual luciferase reporter assay was performed to confirm the direct interaction between miR-322/424 and Cullin2. mRNA expression of elongin B/C, Cullin2, and RBX1 was determined by qPCR. HIF-1α protein expression was determined by Western blotting.
KEY FINDINGS
miR-322/424 level in liver cirrhosis patients, mouse liver fibrosis induced by CCl and BDL, and rat liver fibrosis induced by CCl and dimethylnitrosamine was increased. miR-322/424 agomir exacerbated CCl-induced mouse liver fibrosis, whereas the opposite effect was observed for miR-322/424 antagomir. miR-322/424 agomir significantly upregulated liver CD31 expression; opposite effects occurred with miR-322/424 antagomir. In vitro, miR-322/424 mimic significantly promoted tube formation and cell migration, and increased von Willebrand factor expression, whereas miR-322/424 inhibitor had the opposite effect. Dual-Luciferase Reporter Assay identified Cullin2 as miR-322/424 target. miR-322/424 decreased the mRNA expression of elongin B/C, Cullin2, and RBX1 and increased HIF-1α protein expression in HHSECs.
SIGNIFICANCE
miR-322/424 plays a central role in the pathogenesis of liver fibrosis by targeting Cullin2, and enhancing HIF-1α-mediated hepatic angiogenesis.
目的
研究 miR-322/424 在肝纤维化中的作用及机制。
主要方法
通过 qPCR 检测肝硬化患者、小鼠和大鼠肝纤维化中 miR-322/424 的表达。采用 CCl 建立小鼠肝纤维化模型，并通过 miR-322/424 agomir 或 antagomir 进行干预。用肝羟脯氨酸含量和天狼猩红染色评估胶原沉积。用 CD31 表达评估肝微血管密度。在体外，通过 miR-322/424 模拟物或抑制剂研究其对人肝窦内皮细胞（HHSECs）迁移和管形成的影响。通过双荧光素酶报告基因实验证实 miR-322/424 与 Cullin2 的直接相互作用。通过 qPCR 测定 elongin B/C、Cullin2 和 RBX1 的 mRNA 表达。通过 Western blot 测定 HIF-1α 蛋白表达。
主要发现
肝硬化患者、CCl 诱导的小鼠肝纤维化、BDL 诱导的小鼠肝纤维化以及二甲基亚硝胺诱导的大鼠肝纤维化中 miR-322/424 水平升高。miR-322/424 agomir 加重 CCl 诱导的小鼠肝纤维化，而 miR-322/424 antagomir 则产生相反的效果。miR-322/424 agomir 显著上调肝 CD31 表达；miR-322/424 antagomir 则产生相反的效果。在体外，miR-322/424 模拟物显著促进管形成和细胞迁移，并增加血管性血友病因子表达，而 miR-322/424 抑制剂则产生相反的效果。双荧光素酶报告基因实验鉴定 Cullin2 为 miR-322/424 的靶标。miR-322/424 在 HHSECs 中降低 elongin B/C、Cullin2 和 RBX1 的 mRNA 表达，并增加 HIF-1α 蛋白表达。
意义
miR-322/424 通过靶向 Cullin2 并增强 HIF-1α 介导的肝血管生成，在肝纤维化发病机制中发挥核心作用。
Zotero 插件