Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year.

Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-knockout (Mdr2-KO) mice, a model of inflammationassociated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling showed that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC data sets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time reverse transcription-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We show that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in most dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA, level.These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of B-catenin activation.In conclusion, the Mdr2-KO mouse may serve as a model for B-catenin -negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes.

Background/AimsmicroRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. Human embryonic stem cells (hESC), which exhibit the characteristics of pluripotency and self-renewal, may serve as a model to study the role of miRNAs in early human development. We aimed to determine whether endodermally-differentiated hESC demonstrate a unique miRNA expression pattern, and whether overexpression of endoderm-specific miRNA may affect hESC differentiation.MethodsmiRNA expression was profiled in undifferentiated and NaButyrate-induced differentiated hESC of two lines, using microarray and quantitative RT-PCR. Then, the effect of lentiviral-based overexpression of liver-specific miR-122 on hESC differentiation was analyzed, using genomewide gene microarrays.ResultsThe miRNA profiling revealed expression of three novel miRNAs in undifferentiated and differentiated hESC. Upon NaButyrate induction, two of the most upregulated miRNAs common to both cell lines were miR-24 and miR-10a, whose target genes have been shown to inhibit endodermal differentiation. Furthermore, induction of several liver-enriched miRNAs, including miR-122 and miR-192, was observed in parallel to induction of endodermal gene expression. Stable overexpression of miR-122 in hESC was unable to direct spontaneous differentiation towards a clear endodermal fate, but rather, delayed general differentiation of these cells.ConclusionsOur results demonstrate that expression of specific miRNAs correlates with that of specific genes upon differentiation, and highlight the potential role of miRNAs in endodermal differentiation of hESC.

Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)-knockout (KO) mouse (adenosine triphosphate-binding cassette b4 2/2 ), a model of inflammation-mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2-KO strains: Friend virus B-type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2-KO/B6 mice versus the Mdr2-KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2-KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age-matched Mdr2-KO/FVB males. A comparative genome-scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain-specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin-1 (Gal-1), possesses both anti-inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal-1-KO mutation (lectin galactoside-binding soluble 1 2/2 ) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal-1 protected the liver against concanavalin A-induced hepatitis with the B6 genetic background but not the FVB genetic background. Conclusion: Decreased chronic hepatitis in Mdr2-KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2-KO/FVB strain. We found candidate factors that may determine strain-specific differences in the course of chronic hepatitis and HCC development in the Mdr2-KO model, including inefficient anti-inflammatory activity of the endogenous lectin Gal-1 in the FVB strain. (HEPATOLOGY 2013;58:192-204) Abbreviations: Abcb4, adenosine triphosphate-binding cassette b4; Aif1, allograft inflammatory factor 1; ALP, alkaline phosphatase; ALT, alanine aminotransferase; Angptl4, angiopoietin-like 4; B6, C57 black 6; BrdU, bromodeoxyuridine; ConA, concanavalin A; CoxIV, cytochrome c oxidase subunit IV; Cxcl2, chemokine (C-X-C motif) ligand 2; Defb1, defensin beta 1; FVB, Friend virus B-type/N; Gal-1, galectin-1; GCRMA, GeneChip robust multiarray analysis; GO, Gene Ontology; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; Hexim1, hexamethylene bisacetamide inducible 1; Hprt, hypoxanthine-guanine phosphoribosyltransferase; Ifn, interferon; Il, interleukin; Inhbe, inhibin beta E; KO, knockout; LBI, liver-to-body weight index; Lgals1, lectin galactoside-binding soluble 1; Lpin, lipin; Mat, methionine adenosyltransferase; Mbd1, methyl-CpG binding domain protein 1; Mdr2, multidrug resistance 2; mRNA, messenger RNA; Mrps21, mitochondrial ribosomal protein S21;...

Dietary antioxidants and selenium compounds were shown to have a therapeutic effect against hepatocellular carcinoma in several mouse models. We tested the effects of tannic acid and selenomethionine on hepatocellular carcinoma development in Mdr2 knockout (Mdr2-KO) mice. Mdr2-KO and age-matched Mdr2 heterozygous control mice were fed with tannic acid or selenomethionine during the first 3 months of life. Then, several mice from each group were sacrificed, and liver tissue samples were removed for analysis. The remaining mice were fed a regular diet until the age of 16 months, at which time the number and size of liver tumors were determined. Liver tissue samples of 3-month-old mice were subjected to gene expression profiling analysis using cDNA macroarrays containing probes for 240 genes that regulate responses to oxidative stress and inflammation or lipid metabolism. Both tannic acid and selenomethionine had partial chemopreventive effect on development of hepatocellular carcinoma in Mdr2-KO mice: they reduced the incidence of large tumor nodules (diameter >1 cm) at age 16 months. Both agents inhibited gene expression and reversed up-regulation of many genes that control inflammation or response to oxidative stress in Mdr2-KO livers at age 3 months. This inhibitory effect on gene expression correlated with the ability of agents to reduce incidence of large tumors: selenomethionine was more active than tannic acid in both aspects. Understanding the molecular mechanism of chemoprevention effect could improve our therapeutic modalities while using these agents. [Mol Cancer Ther 2007;6(4):1283 -91]