Modulation of epithelial mesenchymal transition by miRNA125b in cholestatic bile duct ligated (BDL) mice

Cholangiocytes are the target of cholestatic liver diseases such as primary sclerosing cholangitis that are characterized by biliary proliferation/damage and the progression of fibrosis. Biliary damage and liver fibrosis are coordinately regulated by a number of neuroendocrine factors including secretin. We have previously shown that: (i) secretin stimulates biliary growth by downregulation of miR125b; and (ii) miRNA125b is downregulated in cholestatic bile duct ligated (BDL) and mdr2-/- mice. miRNA125b has been shown to play a key role in the regulation of epithelial mesenchymal transition (EMT). Moreover, a number of studies have shown that the progression of liver fibrosis is associated with enhanced EMT. The aim of this study was to evaluate the role of miRNA125b in the modulation of liver fibrosis by secretin through changes in EMT. METHODS: Liver tissue was collected from wild-type (WT) mice underwent sham surgery or BDL for 7 days. The mice were treated by tailvein injections with Vivo Morpholino sequence against miRNA125b (to reduce the hepatic expression of miRNA125b) or mismatched Morpholino at days 3 and 7 after surgery. Studies were also performed in normal WT and mdr2-/- mice treated with secretin by osmotic minipumps (10 ng/kg BW/day) during 7 days. Thereafter, we measured: (i) liver fibrosis by Sirius red staining in liver sections and by qPCR for transforming growth factor-beta1 (TGF-beta1), collagen type 1 (Col1A1) and fibronectin-1 (Fn-1) in total liver tissue; and (ii) EMT by qPCR for N-cadherin and Vimentin in total liver tissue. RESULTS: We observed an increase in the % of connective tissue in liver sections and enhanced expression of TGFbeta1, Col1A1 and Fn-1 in normal and BDL mice treated with Vivo miRNA125b Morpholino, and normal WT and mdr2-/- mice chronically treated with secretin (where miRNA125b levels are decreased) compared to control mice. In BDL mice treated with Vivo miRNA125b Morpholino and normal WT and mdr2-/- mice treated with secretin, there was enhanced expression of N-Cadherin and Vimentin compared to respective control mice. CONCLUSION: We have demonstrated that downregulation of miR125b by secretin induces liver fibrosis by selective upregulation of EMT. Delivery of miRNA125b mimics may be an important therapeutic approach to ameliorate biliary damage and liver fibrosis by modulation of EMT