NGAL基因新型TPA反应元件结合蛋白的研究  

Study of New TPA Response Element Binding Proteins of NGAL Gene

作　　者：孟令英[1] 缪成贵[2] 杜则澎[2] 蔡唯佳[1] 许丽艳[1] 李恩民[2] 

机构地区：[1]汕头大学医学院肿瘤病理研究室,汕头515041 [2]汕头大学医学院生物化学与分子生物学教研室,汕头515041

出　　处：《生物化学与生物物理进展》2008年第6期661-670,共10页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划(863)(2006AA02A403);国家自然科学基金资助项目(30370641,30570829,30672376);广东省自然科学基金重点项目(37788,5104541,7118419);教育部高等学校博士点重点学科专项基金项目(20050560002,20050560003).~~

摘　　要：以往研究发现, 食管癌细胞的中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)基因的过表达具有明显的 12-O- 十四烷酰佛波醇 -13- 醋酸酯(12-O-tetradecanoyl phorbol-13-acetate, TPA)诱导性, 在启动子- 152～- 60 区段存在着一种新型的 TPA 反应元件(TPA response element, TRE), 但是该元件的结合蛋白尚未被鉴定出来. 采用寡核苷酸 DNA 亲和层析法(oligonucleotide trapping)从食管癌细胞中分离纯化了 NGAL 基因 TRE 结合蛋白; 经 SDS-PAGE进一步分离, 银染显示目标蛋白带. 人工切取各目标蛋白带进行 MALDI-TOF-MS 分析, 通过 Mascot 软件搜索 NCBI 数据库, 根据蛋白质的功能、细胞定位和分子质量大小等指标确定 8 个核蛋白因子: C19、KIAA1949、TDRD1、RXR β、FAM54A、KLF15、KLF10 和 YY-1. 最后, 利用 RT-PCR 验证了这些核蛋白因子表达的 TPA 反应性, 结果表明 C19、KIAA1949、TDRD1、RXR β和 KLF15 等编码基因的转录表现出了明显的 TPA 反应性, 提示可能是食管癌细胞中应答 TPA刺激, 参与 NGAL 基因过表达的转录激活因子.The previous studies have showed that neutrophil gelatinase-associated lipocalin （NGAL） was overexpressed in the progress of human esophageal epithelial cells and novel 12-O-tetradecanoyl phorbol-13- acetate （TPA） response elements （TRE） might be located on the -152 ～-60 position of the 5′ flanking region of the promoter. However, the nucleic proteins bound to the elements have not been identified. In present study, the TRE binding-proteins from EC109 cells following TPA induction were purified by oligonucleotide trapping DNA affinity chromatograph, further separated by SDS-PAGE and stained with silver staining. And then the protein bands were subsequently identified by MALDI-TOF-MS and the resulting spectra were searched to NCBI protein data bank through Mascot. C19, KIAA1949, TDRD1, RXRβ, FAM54A, KLF15, KLF10 and YY-1 eight proteins were identified according to their function, localization and molecular mass. Finally, RT-PCR was performed to determine the TPA responsiveness of these nucleic proteins. Results showed that C 19, KIAA1949, TDRD 1, RXRβ and KLF15 proteins have the obvious TPA responsiveness in the transcription level. The results suggest these proteins might respond the stimulation of TPA and regulate the expression of NGA L in EC 109 cells.

关 键 词：NGAL基因 寡核苷酸DNA亲和层析 MALDI-TOF-MS TPA反应元件 TPA反应元件结合蛋白 TPA反应性 

分 类 号：Q781[生物学—分子生物学] R735.1[医药卫生—肿瘤]