Identification of novel gastric cancer-related genes with transforming activity

本研究的目的是确定新的因素参与的转化过程中导致胃癌的形成。用逆转录病毒载体构建人胃癌细胞cDNA文库。通过在转导的NIH 3 T3细胞中筛选转化活性来进行功能性克隆。共分离出6个cDNA克隆，其中一个克隆编码已知在肿瘤发生中起作用的延伸因子1α亚基。在筛选过程中反复分离的一个cDNA（克隆56.2）编码与G蛋白偶联受体蛋白GPR 35相同的蛋白质。此外，发现另一个cDNA克隆（72.3）是GPR 35基因的可变剪接产物，其中在GPR 35的N末端添加了额外的31个氨基酸。因此，由克隆56.2和72.3编码的蛋白质分别命名为GPR 35 a和GPR 35 b。RT-PCR实验显示，GPR 35基因表达低或不存在于周围的非癌区域，而这两种mRNA存在于所有的胃癌检查。72.3编码的mRNA的水平始终显著高于56.2编码的mRNA的水平。在正常肠粘膜中检测到类似于在胃癌中观察到的表达模式。基于两个GPR 35克隆在NIH 3 T3细胞中的明显转化活性，以及它们在癌组织中的表达水平的显著上调，推测这两个新的GPR 35异构体在胃癌形成过程中起重要作用。

The present study was directed towards the identification of novel factors involved in the transformation process leading to the formation of gastric cancer. A cDNA library from human gastric cancer cells was constructed using a retrovirus vector. Functional cloning was performed by screening for transformation activity in transduced NIH3T3 cells. A total of 6 cDNA clones were isolated, including one encoding the elongation factor 1α subunit which has already been known to play a role in tumorigenesis. One cDNA (clone 56.2), which was repeatedly isolated during the course of screening, encoded a protein identical to a G-protein-coupled receptor protein, GPR35. In addition, another cDNA clone (72.3) was found to be an alternatively spliced product of the GPR35 gene, whereby an additional 31 amino acids were added to the N-terminus of GPR35. Hence, the proteins encoded by clones 56.2 and 72.3 were designated GPR35a and GPR35b, respectively. RT-PCR experiments revealed that GPR35 gene expression is low or absent in the surrounding non-cancerous regions, while both mRNAs were present in all of the gastric cancers examined. The level of 72.3 encoded mRNA was consistently significantly higher than that of 56.2 encoded mRNA. An expression pattern similar to that observed in gastric cancers was detected in normal intestinal mucosa. Based on the apparent transformation activities of the two GPR35 clones in NIH3T3 cells, and the marked up-regulation of their expression levels in cancer tissues, it is speculated that these two novel isoforms of GPR35 play significant roles during the course of gastric cancer formation.