FAK突变体的亚细胞定位及生物学特性  

Subcellular location and biological characteristics of a FAK splicing mutant

作　　者：方旭前[1] 刘湘帆[2] 姚玲[3] 顾志冬[1] 陈长强[1] 倪培华[2] 郑新民 樊绮诗[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院北院检验科,201801 [2]上海交通大学医学院检验系 [3]上海交通大学基础医学院生化与分子生物学教研室

出　　处：《中华临床实验室管理电子杂志》2014年第1期32-36,共5页Chinese Journal of Clinical Laboratory Management(Electronic Edition）

基　　金：国家自然科学基金资助项目(81071745)

摘　　要：目的探讨外显子33缺失型黏着斑激酶(focal adhesion kinase,FAK)突变体(FAK-Del33)的亚细胞定位及生物学特性。方法采用基础实验研究设计。将野生型FAK以及突变型FAK-Del33分别构建到pEGFP表达载体,并转染乳腺癌细胞株,以研究分析其细胞表达和定位情况。另构建过表达FAK或FAK-Del33的MDA-MB-468乳腺癌稳转细胞株,研究评价其细胞表达和体外克隆形成能力。结果构建FAK-GFP融合蛋白表达载体转染MDA-MB-468后的细胞定位显示,野生型FAK在胞浆中弥散分布,而FAK突变体蛋白在胞浆中呈点状不均匀分布,并聚集成簇。经限制性内切酶酶切分析与测序分析,成功构建出过表达FAK的慢病毒载体,以293T细胞包装的重组慢病毒能够高效感染乳腺癌细胞;经抗生素puromycin筛选和荧光定量PCR鉴定,成功筛选到稳定表达FAK的细胞株。在软琼脂克隆形成实验中,FAK野生型的克隆形成数为(335±48)/1000个,FAK突变体为(735±91)/1000个,后者对MDA-MB-468乳腺癌稳转细胞株的增殖能力高于前者(t=9.437,P<0.01)。结论初步研究表明外显子33缺失可改变FAK的亚细胞定位;并能增强肿瘤细胞株的体外克隆形成能力,这种FAK突变体可能参与肿瘤的发生发展。Objective To investigate the subcellular localization and the biological characteristics of the novel FAK splicing mutant （FAK-Del33）. Methods Basic experimental research applied. FAK wide-type and FAK-Del33 cDNA were cloned into pEGFP expression vector, and then transfected into breast cancer cell lines. The expression and subcellular localization of FAK wild-type and FAK-De133 cDNA were analyzed. FAK or FAK-De133 overexpressing MDA-MB-468 were constructed. Its expression and in vitro colony forming ability was evaluated. Results FAK-GFP fusion protein expression plasmids were constructed and then transfected into MDA-MB-468. FAK-WT dispersed evenly in the cytoplasma while FAK-De133 signal enriched into strong signal points at different subcellular sites; restriction enzyme analysis and sequencing confirmed the correct construction of lentivirus plasmids. The stable cell lines with expression of FAK were generated by lentivirus infection, puromycin screening and fluorescence quantitative PCR identification. Soft agar colony formation test showed that clone number from mutant FAK had significantly higher levels than FAK wide-type [（735±91）/1000 vs. （335±48）/1000, t=-9.437, P 〈 0.01）. Conclusion This work clearly indicates that the exon 33 deletion change the subcellular localization of FAK and can enhance tumor cell line＇ s in vitro colony- forming ability and the FAK mutant may participate in the occurrence and development of tumor.

关 键 词：剪接突变体 酪氨酸激酶 黏着斑激酶 肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]