食管癌细胞NGAL5′侧翼区存在TPA应答元件  

Identification of TPA Response Elements in NGAL 5′ Flanking Region of the Esophageal Cancer Cells

作　　者：许丽艳[1] 李恩民[2] 王子良[2] 牛永东[2] 蔡唯佳[3] 袁华敏[2] 常静霞[2] 沈忠英[3] 曾毅[1] 

机构地区：[1]北京工业大学生命科学与生物工程学院病毒与药理研究室,北京100022 [2]汕头大学医学院生物化学与分子生物学教研室,汕头515041 [3]汕头大学医学院肿瘤病理研究室,汕头515041

出　　处：《中国生物化学与分子生物学报》2006年第2期135-141,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金面上项目(No.39900069;No.30170428;No.30370641;No.30570849);广东省自然科学基金重点项目(No.37788;No.05104541);广东省自然科学基金面上项目(No.010431)~~

摘　　要：在TPA诱导永生化食管上皮细胞癌变中,NGAL(neutrophil gelatinase-associated lipocalin)过表达,提示食管癌细胞NGAL转录调控区可能存在TPA应答元件.为了对食管癌细胞NGAL的这一TPA应答元件进行分段定位鉴定,首先将NGAL5′侧翼区不同长度片段-1 431^+84-、1 137^+84、-945^+84、-657^+84、-416^+84和-152^+84等依次插入质粒pGLP,构建NGAL/pGLP系列报告基因荧光素酶表达载体pGLP-1431、pGLP-1137、pGLP-945、pGLP-657、pGLP-416和pGLP-152;然后将上述质粒分别同pRL-TK共转染食管癌细胞EC109,最后用5 ng/ml的TPA刺激转染的EC109,检测报告基因荧光素酶活力,综合判定报告基因荧光素酶活力变化趋势,并以此对食管癌细胞NGAL5′侧翼区TPA应答元件进行分段定位.实验结果表明,食管癌细胞NGAL5′侧翼-657^-417区段内存在着较强的TPA应答元件.生物信息学分析显示,NGAL5′侧翼-657^-417区段至少存在3个潜在的TPA应答元件,表明有应答TPA刺激的结构基础.这些研究有助于从分子水平揭示TPA诱导永生化食管上皮细胞癌变中NGAL基因过表达机制.Overexpression of the neutrophil gelatinase-associated lipocalin gene （NGAL） is related to malignant transformation of human immortalized esophageal epithelial cell induced by 12-O-tetradecanoyl phorbol 13-acetate （TPA）. This suggested that there might be TPA response elements in the 5＇ transcription regulation region of NGAL. To locate the position of TPA response elements, the fragments - 1 431 - ＋ 84, -1 137- ＋84, -945- ＋84, -657- ＋84, -416- ＋84 and - 152- ＋84 of NGAL 5＇ flanking region were cloned into pGL3-promoter （pGLP）, respectively, and followed by constructing NGAL/pGLP report gene expression vectors pGLP- 1431, pGLP- 1137, pGLP-945, pGLP-657, pGLP-416 and pGLP-152. And then they were individually cotransfected with pRL-TK vector into EC109 esophageal cancer cells stimulated with TPA （5 ng/ml）. Relative luciferase activity of whole cell extracts from ceils was measured with Dual Luciferase Report Gene Assay System （DLR） to verify whether there is a TPA response element in above fragments. Results indicated that a strong TPA response element might be located on the position -657 -417 of NGAL in the esophageal cancer cells. The analysis by bioinformatics showed that there is the structural character responding to TPA induction in the gene and at least 3 putative TPA response elements in -657 - -417 position. These results will help to clarify the molecular mechanisms of overexpression of NGAL gene in malignant transformation of human immortalized esophageal epithelial cell induced by TPA.

关 键 词：NGAL基因 食管癌细胞 基因表达调控 TPA应答元件 

分 类 号：Q784[生物学—分子生物学] R735.1[医药卫生—肿瘤]