Studies On The Mechanism Of MiR-101and MiR-30Regulating Epithelial-to-mesenchymal Transition In Hepatocyte

Epithelial-to-mesenchymal transition (EMT) describes a transdifferentiation process during which epithelial cells lose their epithelial cell characteristics and acquire the structural and functional characteristics of mesenchymal cells. EMT is a fundamental process in embryonic development and is actively involved in tumor invasion, tissue homeostasis, wound healing and fibrosis. The most observed character of EMT is that the cells turn to be spindle-like morphology from the compact and well-arranged epithelial structure. Increase in cell migration, associated with down-regulation of the adherens junction protein E-cadherin. MicroRNAs (miRNAs) are short20-22nucleotides that exhibit a high degree of structural and functional conservation throughout metazoan species. Binding of miRNA to3’UTR of the target mRNAs with perfect or near perfect complementarity induces either translation suppression or degradation of mRNA, making them powerful regulators of gene expression in a variety of eukaryotic organisms. Recent reports have highlighted the importance of miR-200as a powerful regulator of EMT in cancer cells. miR-200family members directly target the3’UTRs of E-cadherin transcriptional repressors ZEB1and ZEB2.The EMT in hepatocyte play an important role in HCC, metastasis and liver fibrosis and other pathological processes. In vivo, TGF-beta are key factors induce EMT in hepatocyte. Snail1and ZEB1are the well-known E-cadherin-transcriptional repressor that is significantly upregulated during the TGF-β1-induced EMT in hepatocyte. We first discovered that the expression of miR-30and miR-101exhibited significant downregulation in the TGF-(31-induced EMT in AML12murine hepatocytes, accompanied by the expression of Snail1and ZEB1significantly increased. Computational microRNA target predictions detected a conserved sequence matching to the seed region of miR-30in the3’UTR of Snail1mRNA. Also, we find the sequence matching to the seed region of miR-101in the3’UTR of ZEB1mRNA. Our results include Luciferase, Q-PCR and Western-blot demonstrated that miR-30and miR-101could negatively regulate the expression of Snail1and ZEB1respectively by direct targeting the predicted3’UTR binding site of Snail1and ZEB1. More importantly, tansfection of miR-30or miR-101mimics significantly inhibited the the expression of Snail1and ZEB1during TGF-β1-induced EMT in AML12cells, Thereby increasing the content of E-cadherin and effectively suppress the occurrence of the EMT. Finally, we demonstrated that TGF-β1-induced hepatocyte migration was greatly suppressed in cells transfected with miR-30or miR-101mimics. Our results initially revealed the mechanism of miR-101and miR-30in regulating EMT by inhibiting the expression of Snail1and ZEB1and provide a basis for understanding the effect of miR-101and miR-30in liver fibrosis/cirrhosis and liver cancer/transfer process.