Hunecke Danele, Spanel Reinhard, Länger Florian, Nam Suk Woo, Borlak Juergen
Centre for Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany; Department of Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, 30625 Hannover, Germany.
J Pathol. 2012 Dec;228(4):520-33. doi: 10.1002/path.4059. Epub 2012 Nov 6.
Foci of liver cell dysplasia (LCD) are distinct morphological entities and may evolve into hepatocellular carcinomas (HCCs). While most HCCs overexpress c-Myc, its role in LCD remains uncertain. Therefore, a c-Myc transgenic model of HCC was investigated to understand the genetic events forcing liver cells into dysplasia and subsequent malignant transformation. Specifically, whole genome scans enabled fingerprinting of genes at different stages of disease, ie LCD and HCC, while laser microdissected LCD lesions were used to validate regulation of candidate genes by quantitative real-time RT-PCR, ie Mybbp1a, Rps7, Rps19, Rpl10a, Skp1a, Tfdp1, Nhp2, and Bola2. EMSA band shift assays confirmed c-Myc DNA binding at regulatory sequences of candidate gene-specific promoters. Additionally, published ChIP-seq data helped to define the candidate genes as c-Myc bona fide targets. Treatment of the human hepatoma cell line HepG2 with hepatic growth factor (Hgf) caused c-Myc protein induction and transcriptional up-regulation of candidate genes, albeit at different levels when individual genes were compared. A significant increase of HepG2 entering the G1-phase was associated with up-regulation of the candidate genes in an Hgf concentration-dependent matter. Finally, we confirmed regulation of candidate genes in patients' samples with low- and high-grade dysplasia and HCC staged T1 to T3, while their expression was unchanged in focal nodular hyperplasia and hepatic adenoma, therefore asserting the diagnostic value and clinical significance of these candidate genes. Overall, novel c-Myc targeted genes were identified and may contribute to hepatocyte transformation by altering cell cycle control, thereby contributing to c-Myc's oncogenic activity.
肝细胞发育异常灶（LCD）是独特的形态学实体，可能演变为肝细胞癌（HCC）。虽然大多数HCC中c-Myc过表达，但其在LCD中的作用仍不确定。因此，研究了一种HCC的c-Myc转基因模型，以了解促使肝细胞发育异常及随后发生恶性转化的遗传事件。具体而言，全基因组扫描能够对疾病不同阶段（即LCD和HCC）的基因进行指纹识别，而激光显微切割的LCD病变用于通过定量实时RT-PCR验证候选基因（即Mybbp1a、Rps7、Rps19、Rpl10a、Skp1a、Tfdp1、Nhp2和Bola2）的调控。电泳迁移率变动分析（EMSA）证实c-Myc与候选基因特异性启动子的调控序列结合。此外，已发表的染色质免疫沉淀测序（ChIP-seq）数据有助于将候选基因定义为c-Myc的真正靶点。用肝生长因子（Hgf）处理人肝癌细胞系HepG2会导致c-Myc蛋白诱导和候选基因的转录上调，尽管在比较单个基因时上调水平不同。HepG2进入G1期的显著增加与候选基因在Hgf浓度依赖性方面的上调相关。最后，我们在低级别和高级别发育异常以及T1至T3期HCC患者样本中证实了候选基因的调控，而它们在局灶性结节性增生和肝腺瘤中的表达未改变，因此确定了这些候选基因的诊断价值和临床意义。总体而言，鉴定出了新的c-Myc靶向基因，它们可能通过改变细胞周期控制来促进肝细胞转化，从而有助于c-Myc的致癌活性。
