Identification of novel gastric cancer-related genes with transforming activity

本研究的目的是确定在导致胃癌形成的转化过程中涉及的新因素。利用逆转录病毒载体构建了人胃癌细胞cDNA文库。通过筛选转化NIH3T3细胞的转化活性进行功能克隆。共分离到6个cDNA克隆，其中一个编码延伸因子1α亚基，该亚基已被认为在肿瘤发生中起作用。在筛选过程中反复分离的一个cDNA（克隆56.2）编码了一个与g蛋白偶联受体蛋白相同的蛋白质GPR35。此外，另一个cDNA克隆（72.3）被发现是GPR35基因的选择性剪接产物，其中GPR35的n端增加了31个氨基酸。因此，克隆56.2和72.3编码的蛋白分别被命名为GPR35a和GPR35b。RT-PCR实验显示，GPR35基因在周围非癌区表达低或不存在，而这两种mrna在所有检测的胃癌中都存在。72.3编码mRNA水平始终显著高于56.2编码mRNA水平。在正常肠黏膜中检测到与胃癌相似的表达模式。基于两个GPR35克隆在NIH3T3细胞中的明显转化活性，以及在癌组织中表达水平的显著上调，推测这两个GPR35新亚型在胃癌形成过程中发挥了重要作用。

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.