Molecular mechanisms of liver carcinogenesis in the Mdr2-knockout mice

Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-KO mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling demonstrated 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 datasets. 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 RT-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We demonstrate 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 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. Keywords: Hepatocellular carcinoma, Mouse model, Mdr2-knockout. The liver RNA samples from six Mdr2-KO tumors, six non-tumorous liver tissues (four matched and two unmatched), as well as from three control heterozygous mice at 16 months of age were subjected to gene expression profiling using the genome scale Affymetrix Mouse Genome 430 2.0 Array.

肝细胞癌（Hepatocellular carcinoma, HCC）小鼠模型可模拟人类肝细胞癌的特定亚型。本研究以炎症相关肝细胞癌模型Mdr2基因敲除（Mdr2-KO）小鼠为对象，采用基因表达谱分析与免疫组织化学分析手段，探究其肝细胞癌变过程。基因表达谱分析结果显示，尽管Mdr2-KO小鼠与已发表的其他小鼠肝癌模型存在差异，但二者存在若干关键失调通路，且与人类肝细胞癌数据集存在大量协同差异表达基因。对肝脏肿瘤中差异表达基因的基因组位置分析显示，在6个分析的肿瘤样本中的3个样本里，小鼠8号染色体上存在一段持续下调的基因区域。该区域与人类肝细胞癌中常见缺失且携带有多个肿瘤抑制基因的人类染色体区域具有同线性。对16份肿瘤样本的实时荧光定量逆转录聚合酶链反应（real-time RT-PCR）分析证实，多数肿瘤中存在多个肿瘤抑制基因的下调表达。本研究证实，在老年Mdr2-KO小鼠中，未形成结节的异型增生肝细胞内细胞核周期蛋白D1（cyclin D1）水平升高；而在异型增生结节与肝脏肿瘤中，该蛋白水平则出现降低。我们发现，该水平降低主要发生在蛋白层面，而非信使RNA（mRNA）层面。上述发现引发了关于周期蛋白D1在Mdr2-KO肝细胞癌模型的肝细胞癌变早期阶段所发挥作用的相关疑问。此外，本研究发现Mdr2-KO小鼠的多数肝脏肿瘤以β-连环蛋白（β-catenin）激活缺失为特征。综上，Mdr2-KO小鼠可作为人类肝细胞癌β-连环蛋白阴性亚型的模型，该亚型的特征为肿瘤细胞核内周期蛋白D1水平较低，且存在多个肿瘤抑制基因的下调表达。关键词：肝细胞癌、小鼠模型、Mdr2基因敲除。本研究采用全基因组规模的Affymetrix小鼠基因组430 2.0阵列（Affymetrix Mouse Genome 430 2.0 Array），对6份Mdr2-KO小鼠肝脏肿瘤样本、6份非肿瘤性肝脏组织（4份配对样本与2份非配对样本）以及3只16月龄的杂合子对照小鼠的肝脏RNA样本进行基因表达谱分析。