Abstract 2810: Loss of microRNA-122 in mice causes hepatic inflammation and hepatocarcinogenesis

Background: miR-122 is the most abundant liver-specific microRNA that modulates hepatic lipid metabolism, hepatitis C viral replication and is downregulated in hepatocellular carcinoma (HCC) of both rodent and human origin. However, it is not clear whether loss of miR-122 directly promotes or induces hepatocarcinogenesis. To address this question we have generated liver specific (LKO) and systemic (KO) miR-122 knockout mice and have studied the effect of the miR-122 deficiency on the liver function, hepatocyte proliferation and hepatocarcinogenesis with or without administration of a chemical carcinogen, diethylnitrosamine (DEN). Materials and methods: LKO and KO mice were generated by crossing miR-122 conditional KO (floxed) mice to Albumin-Cre and E2A-Cre mice, respectively. Southern blot/PCR and Northern blot/RT-PCR analysis were performed to confirm deletion of the gene and depletion of miR122 in the mutants compared to the controls (floxed). Phenotypic changes in the mutants were identified by histopathological and biochemical analysis of the liver and serum, respectively. Microarray analysis of hepatic RNA was performed to identify molecular pathways altered in LKO mice. Identify novel miR-122 targets among the upregulated genes in LKO mice by RT-PCR, luciferase assay, immunoblot and immunohistochemical analysis. Development of HCC in LKO, KO and control mice with or without DEN injection was monitored. Results and conclusions: Both LKO and KO mice were born alive and fertile but developed inflammation, steatosis and fibrosis with age. Hepatic miR-122 level was depleted by ∼83% and 98% on postnatal day 0 in LKO and KO mice, respectively and by 99% at adult stage in both mice. Serology showed reduced total, HDL- and LDL-associated cholesterol. Increased CK19- and A6-postitve cells as well as elevated serum alkaline phosphatase indicate abnormality in the biliary system in knockout mice. These mice developed hepatic inflammation in early adult life, which progressed to bridging inflammation and fibrosis with age. T cells and macrophages predominantly constituted the inflammatory cells in the mutant mice. Upregulation of Ccng1, Ccnd1 and Igf2 correlated with increased proliferation (as determined by Ki-67 positivity) and dedifferentiation (increased expression of Afp, Igf2 and H19) of miR-122 deficient hepatocytes. Eight months after DEN injection, LKO mice exhibited pronounced liver damage, higher incidence of HCC compared to the control mice. After one year, both LKO and KO mice developed spontaneous liver cancers, including HCC and cholangiocarcinoma, and exhibited liver damage as demonstrated by increased serum ALT, ALP, GGT and bilirubin levels. Taken together, these observations suggest that miR-122 deficiency causes inflammation that leads to hepatocarcinogenesis. Data will be presented on the underlying molecular mechanism of the development of liver damage and cancer in the knockout mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2011-2810