曲古霉素A诱导人乳腺癌细胞MCF-7凋亡反义cDNA文库的构建和效应基因的初步筛选

Construction of anti-sense cDNA library of human breast cancer cells during apoptosis induced by trichostatin A and preliminary screening of essential genes

目的构建曲占霉素A诱导人乳腺癌细胞凋亡反义cDNA文库，以筛选曲古霉素A抗肿瘤的效应基因。方法收集经曲古霉素A处理不同时间点的人乳腺癌细胞MCF-7，提取poly（A）^＋RNA，将逆转录合成的cDNA反向插入载体PCEP4中以构建反义cDNA文库。文库DNA随机转入人宫颈癌细胞HeLa中，以转染PCEP4空载体细胞为对照组，用200nmol／L曲古霉素A和200μg／ml潮霉素同时筛选，至对照组细胞全部死亡，文库组仍有存活细胞时停止曲古霉素A筛选。存活克隆扩增后提取Hirt DNA并转化感受态细胞，获得的转化克隆扩增后进行酶切鉴定并测序，得到多个EST片段，经生物信息学分析后选择感兴趣的EST片段进行初步功能验证。结果构建的反义cDNA文库含有2×10^6重组子，重组效率〉90％；经DNA测序和生物信息学分析提示第27号存活克隆是锌转运蛋白LIV1，该克隆在功能验证时表现出对曲古霉素A非常显著的抵抗效应。结论构建的反cDNA文库容量较大，质量较高；基因LIV1可能是曲古霉素A抗肿瘤的效应基因之一。

Objective To construct an anti-sense cDNA library of human breast cancer cells to screen essential genes with anti-tumor effects on apoptosis of human breast cancer cells induced by trichostatin A. Methods Poly （A） ^＋ RNA was extracted from human breast cancer ceils of the line MCF-7 treated by trichostatin A for 0, 12, 24, 36, 48, 60, or 72 h. cDNA were synthesized and inserted reversely into PCEP 4 vector to construct an anti-sense eDNA library. HeLa cells were tansfected with the library DNA or blank PCEP 4 vector as control group. All the transfected cells were screened by 200 nmol/L trichostatin A and 200 μg/ml hygromycin B. Screening was stopped when the control cells died. Then the surviving cell clones were amplified and Hirt DNA was extracted. Several expressed sequence tags were thus obtained. The data were analyzed by bioinformatics and interested EST fragment was chosen for preliminary functional screening. Results An anti-sense cDNA library was constructed containing 2 × 10^6 independent clones with an insert efficiency of more than 90% ;DNA sequencing and bioinfonnatic analysis suggested that the No. 27 survival clone was zinc transporter LIV1 showing a strong resistance against trichostatin A-induced apoptosis during functional screening. Conclusion An anti-sense cDNA library with high quantity and quality has been successfully constructed; LIV1 gene may be one of the essential genes with anti-tumor effects on apoptosis induced by trichostatin A.