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Cloning of suppressor gene of metastasis using subtraction hybridization method

消减杂交法克隆转移抑制基因

基本信息

批准号：
05671026
负责人：
KUBOTA Tetsuro
金额：
$ 1.41万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

A gene (HFREP-1) that is overexpressed in hepatocellular carcinomas (HCCs) has been identified using a subtractive and differential cDNA cloning approach. We isolated the gene from a lambdagt10 cDNA library that was constructed from the poly(A)+-enriched RNA of a HCC specimen. The expression patterns observed in HCCs from 20 patients were classified into three types ; HFREP-1 was overexpressed in HCCs compared with non-tumorous regions (NTRs) in 55%, expressed weakly in both regions (15%) and strongly in both (30%). The largest cDNA insert contained 1231 base pairs coding 312 amino acids. The deduced protein sequence contained a hydrophobic leader peptide with a site for cleavage. The putative protein sequence showed strong homology with fibrinogen beta- and gamma- subunites and other fibrinogen-related proteins (FRPs), including four cysteine conserved residues. However, the HFREP-1 protein lacked a platelet-binding site, cross-linking region and thrombin-sensitive site, which are crucial for fibrin clot formation. The gene expression was studied in various organs in the rat and was specific to the liver, but little or none was detected in the fetal rat liver. Of the human carcinoma cell lines investigated, HFREP-1 was detected only in the HCC cell lines. We suggest that the HFREP-1 gene is a new member of the family of fibrinogens and has different features from other known FRPs and its expression is regulated oncodevelopmentally in hepatocytes.

采用消减和差异表达的基因克隆方法，鉴定了一个在肝细胞癌中过表达的基因(HFREP-1)。我们从从肝癌标本的Poly(A)+丰富的RNA中构建的lambdagt10 cDNA文库中分离到该基因。20例患者肝细胞癌组织中HFREP-1的表达可分为三种类型：HFREP-1在肝细胞癌中高表达，非肿瘤区高表达(55%)，两区弱表达(15%)，强表达(30%)。最大的插入片段包含1231个碱基对，编码312个氨基酸。推导出的蛋白质序列含有一个疏水的前导肽，带有一个切割位点。推测的蛋白质序列与纤维蛋白原β和γ亚基以及其他纤维蛋白原相关蛋白(FRP)有很强的同源性，包括四个半胱氨酸保守残基。然而，HFREP-1蛋白缺乏对纤维蛋白凝块形成至关重要的血小板结合部位、交联区和凝血酶敏感部位。该基因在大鼠的不同器官中都有表达，并且只在肝脏中特异表达，但在胎鼠肝脏中很少或没有检测到。在所研究的人癌细胞系中，仅在肝癌细胞系中检测到HFREP-1。我们认为HFREP-1基因是纤维蛋白原家族的一个新成员，具有不同于其他已知FRP的特征，其表达在肝细胞中受到共同发育的调节。