P353 ACTIVATION OF THE DEVELOPMENTAL PATHWAY Ngn-3/miR-7a REGULATES CHOLANGIOCYTES PROLIFERATION IN RESPONSE TO INJURY

Abstract

Background and Aims: The activation of the biliary stemcell signalling pathway Hes-1/PDX-1 in mature cholangiocytes determines cell proliferation. Neurogenin-3 (Ngn-3) is required for pancreas development and for ductal cell neogenesis. PDX-1dependent activation of Ngn-3 initiates the differentiation program, by inducting microRNA (miR)-7 expression. We aimed to verify whether Ngn-3 regulates cholangiocyte proliferation. Methods: Expression levels of Ngn-3 and miR-7 isoforms were tested in cholangiocytes from normal and cholestatic livers. Ngn-3 was knocked down in vitro by siRNA. In vivo, wild type (WT) and Ngn-3-heterozygous (+/−) mice were subjected to Bile Duct Ligation (BDL) for 2 weeks. Results: In the liver, Ngn-3 is expressed in cholangiocytes of mice subjected to BDL and of patients affected by PSC, but not in normal conditions. Expression of miR-7a-1 and miR-7a-2 isoforms, but not miR-7b, was increased in BDL cholangiocytes as compared to normal ones. In vitro, Ngn-3 siRNA neutralized the increases in cell proliferation and in the expression of IGF-1 (a pro-proliferative effector) and miR-7a, but not of PDX-1 or VEGF, observed after exposure to FBS or exendin-4. Anti-sense miR-7 neutralized the FBS or exendin-4 induced increases in cell proliferation but not in PDX-1 and Ngn-3 synthesis. In vivo, increases in bile duct mass and collagen deposition induced by BDL were significantly reduced in Ngn-3 mice. Conclusions: Ngn-3-dependent activation of miR-7a is a determinant of cholangiocyte proliferation. These findings indicate that the re-acquisition of a molecular profile typical of organ development is essential for the biological response to injury by mature cholangiocytes.