荧光差异显示PCR克隆参与胃腺癌转移的基因wcl1  

Cloning of a Novel Gastric Adenocarcinoma Metastasis Related Gene wcl1 by Fluorescent Differential Display-PCR

作　　者：王建华[1] 陈诗书[1] 

机构地区：[1]上海第二医科大学分子生物学实验室,人类基因治疗研究中心上海200025

出　　处：《中国生物化学与分子生物学报》2002年第2期134-138,共5页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：使用来源于同一个胃腺癌病人的原发灶RF 1(ATCC编号CRL 186 4 )和转移灶RF 4 8(ATCC编号CRL 186 3)作为研究肿瘤转移的模型 .通过荧光差异显示PCR(FDD PCR)技术 ,克隆了 4 5个涉及胃腺癌转移相关基因 .和原发灶CRL 186 4相比 ,发现在转移灶CRL 186 3细胞中有 38个基因被显著上调 ,7个基因被显著下调 ,包括未被发现的基因 3个 .利用生物信息学技术对其中 1个在RF 4 8中高度上调的wcl1进行克隆和鉴定 ,发现wcl1的cDNA全长为 6 6 4bp ,含有 1个 2 4 0bp的完整阅读框 .用RT PCR和Northern印迹证实 ,结果与克隆拼接的大小完全一致 (NCBI数据库收录号AF36 4 86 3) .wcl1编码肽位于胞浆内含 79个氨基酸 ,理论上的pI Mr:5 2 8 896 1 72 .氨基酸序列分析发现 ,其N端 4～ 5 5氨基酸处为未知功能区UPF0 0 16蛋白 ,5 7～ 78处有一段亮氨酸拉链结构域 ,未发现与已知的蛋白质有高度的同源性 .该基因定位于 11q14.组织分布表明 ,wcl1除在分化差的胃腺癌中的表达要高于相应的正常胃组织 ,在微血管内皮中表达也明显高于乳腺管癌、脑纤维状星形细胞瘤 ,未检测到在其它组织有分布 .研究提示 ,Wcl1可能为胃腺癌转移过程中参与核内基因转录的相关蛋白质 .The differential expression of mRNA between RF-1 cell line (primary tumor of gastric adenocarcinoma patient) and RF-48 cell line (its metastatic counterpart) was cloned by fluorescent differential display-PCR(FDD-PCR).45 differential fragments were finally obtained. Comparison of expression of mRNA between RF-1 and RF-48 cells enabled us to identify 38 genes, including 3 novel genes, that were highly up-regulated, and 7 genes that were apparently down-regulated. One gene named wcl1, highly up-regulated in RF-48 cells, was cloned and identified using bio-informatics techniques. Full-length of approximately 664 bp wcl1 cDNA containing a largest opening-reading frame of 240 bp was obtained, which located at human chromosome 11q14. The stretched result from electron-clone by internet was confirmed by RT-PCR and Northern-blot(Accession Number AF364863). wcl1 encodes a polypeptide containing 79 amino acid residues with a predicted pI/M r of 5.28/8 961.72, located to cytoplasm. Analyzing its amino acid sequence, it was found that Wcl1 contained a unknown functional region similar to UPF0016 protein and a leucine zipper motif at amino acids 57-78, no highly homologous with known protein. Compared to brain polocytic astrocytoma, invasive breast ductal tumor and the normal gastric tissue, wcl1 mRNA expression is apparently increased in microvascular endothelial cells and the poorly differential gastric cancer, respectively. These data indicated that Wcl1 might involve in gene transcription and regulation during gastric adenocarcinoma metastasis process.

关 键 词：荧光差异显示PCR 克隆 胃腺癌转移 wcl1 转移相关基因 

分 类 号：R735.202[医药卫生—肿瘤] R394.8[医药卫生—临床医学]