Molecular profiling of hepatocellular carcinoma

人类肝癌在美国的发病率越来越高，是世界上第五大最常见的恶性肿瘤。它是癌症死亡的第四大原因，估计每年有100万人死于癌症。肝细胞癌是原发性肝癌的一种主要类型。肝细胞癌被认为是一种晚期疾病，目前，在发现导致消退的有效治疗方法方面进展甚微。这在很大程度上是由于缺乏早期诊断的方法，以及缺乏与肝细胞癌发展相关的表型变化的信息。我们的目标是确定共同的基因簇，负责肝癌的发生，并发现新的对病毒性肝炎介导的肝癌至关重要的基因以及转移所必需的基因。这些研究将有助于建立具有潜在诊断和预后价值的新标记物，对这些基因的分析将进一步了解肝癌的发生机制，并为设计肝癌导向的分子治疗策略提供进一步的见解。 我们采用了两种方法，即基因表达序列分析(SAGE)和基因芯片技术，探索在感染HBx或HC-core这两种病毒性肝炎癌蛋白的原代人肝细胞以及不同状态的慢性活动性肝炎患者或肝细胞癌患者的肝组织中异常表达的潜在细胞基因。此外，我们还比较了原发性和转移性肝癌之间的基因表达谱。HBx和HC-core感染正常肝细胞是通过复制缺陷型腺病毒载体实现的。利用SAGE，我们构建了一个来源于感染HBx的原代人肝细胞的文库。对19,000多个转录本(代表1443个独特基因)的分析提供了正常肝细胞特有的转录本的分布。在表达HBx的肝细胞中，共诱导了57个转录本，46个转录本被抑制了5倍以上。有趣的是，大多数受HBx表达上调的转录本主要聚集在三大类中，包括编码核糖体蛋白的基因、带有锌指基序的转录因子和与蛋白质降解途径相关的蛋白质。这些结果表明，HBx可能在一个共同的细胞途径中发挥主要调节作用，该途径反过来调节蛋白质合成、基因转录和蛋白质降解。此外，我们还比较了表达HBx的原代肝细胞以及慢性肝病(包括乙肝病毒或丙型肝炎病毒感染)和肝癌患者的肝脏样本中的基因表达谱。聚类算法被用来识别这些样本中不同基因表达谱的放松调控。采用Northern印迹分析对芯片数据进行验证。我们已经在这些样本中发现了多个上调或下调的基因。对这些基因的进一步分析将有助于理解乙肝病毒和丙型肝炎病毒介导的肿瘤发生的机制。

Human liver cancer, with increasing occurrence in the United States, is the 5th most prevalent malignant disease in the world. It is the fourth leading cause of cancer mortality, which accounts for an estimated 1 million deaths annually. Hepatocellular carcinoma (HCC) is a major type of primary liver cancer. HCC is considered to be a terminally ill disease and currently, there is little progress toward the discovery of efficient therapies leading to regression. This is due largely to the lack of a method for early diagnosis and the lack of information on the phenotypic changes associated with the development of HCC. Our goals are to identify common gene clusters that are responsible for the genesis of HCC and to discover new genes critical for viral hepatitis-mediated HCC as well as genes necessary for metastasis. These studies will contribute to the establishment of novel markers with potential diagnostic and prognostic value, and analysis of these genes would provide further understanding of the genesis of liver cancer and provide further insights into designing strategies for HCC-directed molecular therapy. We have taken two approaches, namely, Serial Analysis of Gene Expression (SAGE) and cDNA microarray, to explore potential cellular genes that are expressed abnormally in primary human hepatocytes infected with the two viral hepatitis oncoproteins, HBx or HC-core, and in liver samples from chronic active hepatitis patients or HCC patients that differ in the status of HBV or HCV. In addition, we are comparing gene expression profiles between primary HCC and metastatic HCC. Infection of normal hepatocytes with HBx and HC-core is achieved by a replication-defective adenoviral vector. Using SAGE, we have constructed a library derived from primary human hepatocytes infected with HBx. Analysis of over 19,000 transcripts (representing 1443 unique genes) provides a distribution of transcriptomes characteristic of normal hepatocytes. In the HBx-expressing hepatocytes, a total of 57 transcripts were induced and 46 transcripts were repressed with greater than 5-fold. Interestingly, most of transcripts that are upregulated by HBx expression are clustering in three major classes including genes that encode ribosomal proteins, transcription factors with zinc finger motifs and proteins associated with protein degradation pathway. These results suggest that HBx may function as a major regulator in a common cellular pathway that in turns regulating protein synthesis, gene transcription and protein degradation. In addition, we have compared gene expression profiles in primary hepatocytes expressing HBx as well as liver samples from chronic liver diseases including HBV or HCV infection and in HCC. Clustering algorithms were used to identify deregulation of distinctive gene expression profiles in these samples. Northern blotting analysis was used to verify the microarray data. We have identified multiple genes that are either up- or down-regulated in these samples. Further analysis of these genes will be useful for understanding the mechanism of HBV- and HCV-mediated oncogenesis.