Cloning of suppressor gene of metastasis using subtraction hybridization method

一个基因（HFREP-1）是在肝细胞癌（HCC）中过表达已被确定使用消减和差异cDNA克隆方法。我们从肝癌标本富含poly（A）+的RNA构建的cDNA文库中分离出该基因。在20例患者的HCC中观察到的表达模式被分为三种类型：与非肿瘤区域（NTR）相比，HFREP-1在HCC中过表达（55%），在两个区域中弱表达（15%），在两个区域中强表达（30%）。最大的cDNA插入片段含有1231个碱基对，编码312个氨基酸。推导的蛋白质序列含有一个疏水性的前导肽与切割位点。推测的蛋白质序列显示出与纤维蛋白原β-和γ-亚基以及其他纤维蛋白原相关蛋白（FRPs）的高度同源性，包括四个半胱氨酸保守残基。然而，HFREP-1蛋白缺乏血小板结合位点、交联区域和凝血酶敏感位点，这些对于纤维蛋白凝块形成至关重要。在大鼠的各种器官中研究了该基因的表达，并对肝脏具有特异性，但在胎鼠肝脏中检测到很少或没有。在研究的人癌细胞系中，仅在HCC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新成员，具有与其他已知的纤维蛋白原不同的特征，其表达在肝细胞中受到肿瘤发育的调控。

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.