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Analysis of liver diesease related gene expression using subtraction cloning

消减克隆技术分析肝脏疾病相关基因表达

基本信息

批准号：
12670467
负责人：
ENOMOTO Nobuyuki
金额：
$ 2.5万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670467/
关键词：
subtraction hepatoma chronic hepatitis autoimmune hepatitis primary biliary cirrhosis

项目摘要

The genetic basis of hepatocellular carcinoma (HCC) has not yet been fully understood. Although various methods have been developed to detect differentially expressed genes in malignant diseases, efficient analysis from clinical specimens is generally difficult to perform due to the requirement of a large amount of samples. In the present study, we analyzed differentially expressed genes with a small amount of human HCC samples using suppression subtractive hybridization (SSH). Total RNA were obtained from the hepatitis C virus-associated HCC and adjacent to non-HCC liver tissues. cDNA was synthesized using modified RT-PCR, and then tester cDNA was ligated with two different kinds of adaptors and hybridized with an excess amount of driver cDNA. Tester specific cDNA was obtained by suppression PCR and the final PCR product was subcloned and sequenced. Seven known genes (focal adhesion kinase, deleted in colon cancer, guanine binding inhibitory protein a, glutamine synthetase, ornithine aminotransferase, M130, and pepsinogen C) and two previously unknown genes as being overexpressed in HCC, and 1 gene (decorin) as suppressed in HCC. Quantitative analysis of gene expression using quantitative RT-PCR demonstrated the differential expression of these genes in the original and other HCC samples. These findings demonstrated that it is possible to identify the previously unknown, differential gene expression from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in HCC pathogenesis, developing the new diagnosic markers, or determining novel therapeutic targets. Using similar technique, we found overexpression of IP10 in autoimmune hepatitis and chronic hepatitis C. In PBC, several mitochondrial genes are up-regulated.

肝细胞癌（HCC）的遗传基础尚未完全了解。尽管已经开发了各种方法来检测恶性疾病中的差异表达基因，但是由于需要大量样品，通常难以从临床标本进行有效的分析。在本研究中，我们用抑制性消减杂交（SSH）技术分析了少量人肝癌标本中差异表达的基因。从丙型肝炎病毒相关的HCC和邻近的非HCC肝组织中获得总RNA。用改良的RT-PCR合成cDNA，然后将测试者cDNA与两种不同的接头连接，并与过量的驱动者cDNA杂交。通过抑制PCR获得测试者特异性cDNA，并将最终PCR产物亚克隆并测序。7个已知基因（在结肠癌中缺失的粘着斑激酶、鸟嘌呤结合抑制蛋白a、谷氨酰胺合成酶、鸟氨酸氨基转移酶、M130和胃蛋白酶原C）和2个先前未知的基因在HCC中过表达，1个基因（核心蛋白聚糖）在HCC中被抑制。使用定量RT-PCR对基因表达进行定量分析，证实了这些基因在原始和其他HCC样品中的差异表达。这些发现表明，从少量临床样本中鉴定以前未知的差异基因表达是可能的。这些基因表达改变的信息可能有助于阐明HCC发病机制中的遗传事件，开发新的诊断标志物或确定新的治疗靶点。使用类似的技术，我们发现IP 10在自身免疫性肝炎和慢性丙型肝炎中过表达。在PBC中，几个线粒体基因被上调。