食管癌低表达cDNA片段C6-2A的克隆及表达分析

目的　克隆人食管癌发生相关的基因。方法　用ｍ Ｒ Ｎ Ａ 差异显示技术分离、克隆食管癌组织中不表达或低表达的ｃ Ｄ Ｎ Ａ 片段，再通过 Ｎｏｒｔｈｅｒｎ ｂｌｏｔ 、ｄｏｔ ｂｌｏｔ 和 Ｒ Ｔ Ｐ Ｃ Ｒ 证实。结果　获得２８０ｂｐ 的ｃ Ｄ Ｎ Ａ 片段，命名为 Ｃ６２ Ａ。与 Ｇｅｎ Ｂａｎｋ 基因数据库比较，未发现 Ｃ６２ Ａ 与任何已知基因有同源性。查询 Ｅ Ｓ Ｔ 数据库，发现 Ｃ６２ Ａ 与ｎｅ２７ｂ０３ ，ｓ１ Ｎ Ｃ Ｉ Ｃ Ｇ Ａ Ｐ Ｃ０３ 人ｃ Ｄ Ｎ Ａ 克隆８９８５４１ 及人卵巢肿瘤ｃ Ｄ Ｎ Ａ 克隆７５５１９６ 等高度同源。 Ｎｏｒｔｈｅｒｎ ｂｌｏｔ 结果显示６／６ 例食管癌组织表达丧失或低表达，ｄｏｔ ｂｌｏｔ 分析表明７／８例食管癌组织低表达， Ｒ Ｔ Ｐ Ｃ Ｒ 显示食管癌细胞系 Ｅ Ｃ１０９ 、 Ｅ Ｃ８７１２ 、 Ｅ Ｃ９７０６ 和肺腺癌细胞系 Ｇ Ｌ Ｃ８２ 极弱表达，１７／２０ 例食管癌组织表达丧失或低表达，胎儿食管、皮肤、大脑、胎盘较高表达，胎儿胃、肝较弱表达，胎儿心、小肠、肾不表达。结论　在食管癌细胞系和食管癌组织高频率的不表达或低表达提示， Ｃ６２ Ａ 很可能与食管癌的发生、发展有关。

Cloning and expression analyses of down regulated cDNA C6 2A in human esophageal cancer

OBJECTIVE: To clone genes associated with the genesis of human esophageal cancer. METHODS: Identifying missing or low expressing cDNAs in human esophageal cancer tissues by mRNA differential display and examining its mRNA expression in 4 human cancer cell lines, 9 fetal tissues and other matched esophageal cancer tissues by Northern blot, dot blot and RT-PCR. RESULTS: One cDNA fragment named C6-2A, was cloned and sequenced. There was no identical sequence with C6-2A in BLASTN database; but in querying Genbank EST, the authors found that C6-2A was identical with ne27b03.s1NCI-CGAP-C03 humans sapiens cDNA clone IMAGE:898541 3' and zv30g07.rl Soares ovary tumor NbHOT homo sapiens cDNA clone 755196. 6/6 esophageal cancer tissues in Northern blot and 7/8 in dot blot did not or slightly express C6-2A. RT-PCR analysis showed that C6-2A was expressed much lower in 17/20 esophageal cancer tissues than adjacent microscopically normal mucosa, highly expressed in fetal esophageal mucosa, skin, cerebrum, placenta; moderately expressed in fetal stomach and liver, but not detected in fetal heart, small intestine and kidney. CONCLUSION: The high frequency of deletion of decreased expression of C6-2A in esophageal cell lines and human esophageal cancer tissues suggested that C6-2A might be involved in the carcinogenesis of esophagus.