Twist1-Induced miR-199a-3p Promotes Liver Fibrosis by Suppressing Caveolin-2 and Activating TGF-β Pathway

Background: The activation of hepatic stellate cells (HSCs) participates in liver fibrosis, and emerging evidences indicate that microRNAs (miRNAs) are abnormally expressed during HSC activation. However, the potential roles of miRNAs in liver fibrosis still remain elusive. Methods: The differential expression of miRNAs between the quiescent and activated HSCs was measured by miRNA microarray analysis, which was further validated by RT-qPCR in animal models and patients. The effect of miR-199a-3p on CAV2 expression was examined by Western blotting using miRNA mimics or inhibitor. Induction of miR-199a-3p expression by TWIST1 was explored by the luciferase assay and RT-qPCR. The role of miR-199a-3p in liver fibrosis was systematically investigated in vitro and in vivo. Findings: miR-199a-3p expression was dramatically upregulated during HSC activation in vitro, and during liver fibrogenesis in CCl4-treated rats, and was increased in livers from high-fat-diet-induced mice or the patients with cirrhosis. The expression of miR-199a-3p was driven by Twist1 which could be further induced by TGF-β treatment. Functional studies showed that inhibition of miR-199a-3p in both human LX2 cells and rat HSCs significantly decreased the expression of fibrotic markers, whereas the forced expression of miR-199a-3p exhibited opposite effects, demonstrating the role of miR-199a-3p in promoting HSCs activation. Mechanistically, miR-199a-3p plays an important role in TGF-β signalling pathway activation through targeting CAV2 that negatively regulates the expression of transforming growth factor-beta receptor type I (TGFβRI). Importantly, administration of antagomiR-199a-3p in the CCl4 treated mice significantly ameliorated hepatic fibrosis. Interpretation: Twist1-induced miR-199a-3p mediates the activation of HSCs by suppressing CAV2 expression and subsequently increasing TGFβRI expression to promote TGF-β pathway. Our findings highlight the therapeutic potential of miR-199a-3p for hepatic fibrosis. Funding Statement: This study was supported by National Natural Science Foundation of China (81570554 and 81770568 to LY; 81572739 to YP), and by National Key R&D Program of China (2016YFA0502204 to YP). TS is supported by a joint PhD studentship by King’s College London, UK and China Scholarship Council, China. Declaration of Interests: None of the authors has any potential conflict of interests to disclose. Ethics Approval Statement: All animal studies were approved by the Medical Ethics Committee of the West China Hospital of Sichuan University. All specimens were collected upon obtaining informed consent from all patients, and the study was approved by the Human Ethics Committee of Sichuan University.