MicroRNA-322/424 promotes liver fibrosis by regulating angiogenesis through targeting CUL2/HIF-1α pathway

Abstract

AimsTo investigate the effects and mechanism of miR-322/424 in liver fibrosis. Main methodsmiR-322/424 expression in liver cirrhosis patients, mouse and rat liver fibrosis was determined by qPCR. Mice liver fibrosis was established by CCl4, 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 findingsmiR-322/424 level in liver cirrhosis patients, mouse liver fibrosis induced by CCl4 and BDL, and rat liver fibrosis induced by CCl4 and dimethylnitrosamine was increased. miR-322/424 agomir exacerbated CCl4-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. SignificancemiR-322/424 plays a central role in the pathogenesis of liver fibrosis by targeting Cullin2, and enhancing HIF-1α-mediated hepatic angiogenesis.