应用抑制消减杂交技术克隆人大肠癌转移相关新基因

Differential screening of novel human metastasis-associated genes of colorectal carcinoma

目的 克隆新的大肠癌转移相关基因,为进一步阐述大肠癌转移的分子机制提供线索。方法 利用抑制消减杂交技术,以1对来自同一亲本、转移能力不同的大肠癌细胞株SW620和SW480为研究对象,构建2种大肠癌转移促进基因和转移抑制基因的cDNA消减文库。随机挑取文库克隆进行差异筛选,对所得的差异片段进行测序和同源性分析,利用Northern Blot对新基因的表达情况进行验证。结果 两种消减文库分别含有235和232个白色克隆,90％以上的白色克隆含有插入片段。随机挑取约200个阳性克隆进行差异筛选。共获得29个差异片段。测序及同源分析发现15个为未知基因,GenBank登陆号为CD485499-CD485513。其中与5号染色体序列同源的基因有6个。结论 抑制消减杂交技术是筛选、克隆大肠癌转移相关新基因的有效手段;5号染色体上可能存在多个与大肠癌转移相关的新基因位点;筛选到的新基因片段为进一步克隆其全长、研究基因功能提供了实验基础。

Objective To clone novel metastasis-associated genes of colorectal carcinoma and provide clues for the molecular mechanisms of metastasis of colorectal carcinoma. Methods By suppression subtractive hybridization (SSH), a pair of colorectal carcinoma cell lines with different metastatic potentials derived from the same parental cell line was used to construct two subtractive cDNA library specific for metastasis-accelerating genes or metastasis-suppressor genes. Partial positive clones in the two libraries were selected randomly and differentially screened, and the differentially expressed gene fragments obtained were sequenced and analyzed with BLAST software. The mRNA expressions of several new genes were confirmed by Northern blot analysis. Results Two subtractive cDNA libraries had 235 and 232 white clones respectively and more than 90% of the white clones had the inserts. About 200 positive clones selected randomly were differentially screened, and 29 differentially expressed genes were obtained. Fifteen unknown genes were found by sequence and BLAST analysis and had been collected by the GenBank dbEST database with the entry number of CIM85499 to CD485513, among which 6 novel genes were located in the fifth chromosome. Conclusion SSH is a reliable strategy for screening novel genes differentially expressed in metastasis of colorectal carcinoma. The fifth chromosome may harbor many new genes related with metastasis of colorectal carcinoma, and identified new genes can be cloned for their full length for further study of their functions.