S. De Minicis

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

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.

36 PDX-1/HES-1 INTERACTIONS DETERMINE CHOLANGIOCYTE PROLIFERATIVE RESPONSE TO INJURY: THE SIGNIFICANCE FOR SCLEROSING CHOLANGITIS

in PLINK v1.07. For selected SNPs, previously published summary statistics (Melum et al., Nat. Gen. 2011) were used to perform a meta-analysis. Results: Significant association (P < 8.5×10−4) corrected for multiple testing (Bonferroni method) was observed for three SNPs at 10p15 and one SNP at 4q27 (Table 1). In addition, nominal significance (P < 0.05) was seen for 10/28 SNPs at 10p15 and 9/27 SNPs at 4q27. Genome-wide significance (P < 5×10−8) was observed for rs4147359 (10p15) in the combined analysis.

365 ROLE OF BACTERIAL TRANSLOCATION IN LIVER FIBROSIS AND HCC DEVELOPMENT

Results: mRNA for Collagen and aSMA were increased in HFD-BDL-mice versus CTRL-BDL-mice. Similarly, colllagen depostion, aSMA immunohistochemistry and hydroxyproline content were higher in the HFD-BDL-mice, while no differences were observed between CTRL-CCl4-mice and HFDCCl4-mice. Culture-positivity of mesenteric lymphnodes showed higher density of infection in HFD-BDL-mice respect to CTRL-BDL-mice, suggesting higher bacterial translocation rate in the first group. Due to the peritoneum toxic effect of CCl4, no evidence of bacterial translocation was observed in the group CCl4-treated-mice. Moreover, HFD-DEN-mice showed an increase in number and size of tumors in comparison to CTRL-DEN-mice. Conclusions: HFD enhances liver fibrosis in BDL-mice but not in CCl4-mice, and this is associated to potential effect of bacterial translocation. Moreover, HFD is also able to enhance the process of cancerogenesis: HFD-mice develop more liver cancer then CTRL-mice. Thus, the different habit in dietary feeding, through the mechanisms of bacterial translocation, may enhance, in the course of chronic hepatic liver damage, the development of fibrosis and cancer in the liver.

29 FROM NAFLD TO HCC: THE ROLE OF FIBROSIS AS A COFACTOR IN A NEW MOUSE MODEL

Materials and Methods: Seventy OLT recipients were enrolled. The aetiology was HCV infection in 25 patients, HBV infection in 9, alcoholic disease in 30 and other in 6. Two per protocol liver biopsies (BIO1, BIO2) were performed: BIO1 within the first 60 postoperative days, BIO2 at one year. Significant ACR episodes were recorded during the first year; Ishak staging was assessed in BIO2. A fragment of BIO1 was stored for RT-PCR quantification of the following genes: Retinoic-acid-Inducible Gene I (RIG-I), CARD adaptor inducing IFN-b (CARDIF), Interferon Regulatory Factor 3 (IRF-3), Ubiquitin Specific Peptidase 18 (USP-18), Interferon-Stimulated Gene 15 (ISG-15), Suppressor Of Cytokine Signaling 1 and 3 (SOCS-1, SOCS-3), 2′-5′ OligoAdenylate Synthase 2 (OAS-2). Results: Seven patients (6 HCV positives) reached an Ishak staging >2 on BIO2. In 19 HCV+ patients with Ishak staging 2 on BIO2, higher mRNA expression of CARDIF (19.7±32.8 Vs 0.1±0.1; p 2. Eighteen patients experienced ACR. In these patients a lower mRNA expression of USP-18 (30.4±78.3 Vs 871.4±2631.2; p = 0.02) and RIG-I (0.3±0.5 Vs 13.5±38.5; p = 0.05) was found in comparison to the 52 patients who did not show significant ACR. The latter association was confirmed in HCV positive patients (0.4±0.6 Vs 16.6±45.3; p< 0.05). Conclusions: Early activation of genes involved in the II response may reduce liver fibrosis progression in HCV positive recipients. The activation of USP-18 and RIG-I may prevent the occurrence of ACR.