食管癌细胞NGAL基因-152～-60区段存在TPA反应元件  

There are TPA Response Elements in -152～-60 Position of NGAL Gene 5′Flanking Region in The Esophageal Cancer Cells EC109

作　　者：许丽艳[1] 李恩民[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：《生物化学与生物物理进展》2006年第2期140-148,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金面上资助项目(39900069;30170428;30370641;30570849);广东省自然科学基金重点项目(37788;05104541);广东省自然科学基金面上项目(010431).~~

摘　　要：以往研究发现,在TPA诱导永生化食管上皮细胞癌变中中性粒细胞明胶酶相关载脂蛋白(neutrophilgelatinase-associatedlipocalin,NGAL)基因过表达,但过表达机制不明.最近研究提示,食管癌细胞NGAL启动子及其邻近区域可能存在着TPA反应元件.为了对NGAL的这一TPA反应元件进行更准确定位,采用PCR法结合嵌套缺失实验从食管癌细胞中克隆了NGAL5′侧翼区-152～+84、-140～+84、-78～+84、-59～+84、-50～+84、-41～+84、-37～+84、-29～+84和-10～+84等片段,并定向插入pGLB、pGLP或pGLE等萤火虫荧光素酶报告基因表达载体中,构建了pGLB-152、pGLP-152、pGLE-152、pGLB-140、pGLB-78、pGLB-59、pGLB-50、pGLB-41、pGLB-37、pGLB-29和pGLB-10等系列报告基因表达载体.将上述报告基因表达载体分别同pRL-TK共转染食管癌细胞EC109,并用TPA刺激,检测TPA刺激转染EC109的相对荧光素酶活力,综合判定NGAL-152～+84区不同长度片段的TPA反应性,对NGAL启动子区的TPA反应元件给予进一步分段定位.结果表明,NGAL启动子区的TPA反应元件位于-152～-60区段,而且应答TPA刺激的反应能力很强.生物信息学分析结果显示,NGAL启动子区所存在的TPA反应元件很可能是一种新结构类型.研究说明,NGAL在DNA序列上有应答TPA刺激的结构基础,将有助于深入到分子水平揭示TPA诱导永生化食管上皮细胞癌变中NGAL过表达机制,也有助于进一步认识TPA信号细胞内传递途径网络在肿瘤发生发展中的作用.Overexpression of the neutrophil gelatinase-associated lipocalin （NGAL） gene is related to malignant transformation of human immortalized esophageal epithelial cell induced by 12-O-tetradecanoyl phorbol 13-acetate （TPA）. However, the mechanisms of the transcriptional activation of this gene remain unclear. The recent study indicated that there might be TPA response elements in the 5＇ flanking promoter region and the sequences surrounding it. In order to locate the position of TPA response elements, the NGAL fragments for - 152～＋84, - 140 ～＋84, -78 ～＋84, -59 ～＋84, -50～＋84, -41～＋84, -37～＋84, -29～＋84 and - 10～＋84 were obtained from esophageal cancer cells by PCR and nested deletion and cloned into pGL3-Basic （pGLB）, pGL3-Enhancer （pGLE） and pGL3-promoter （pGLP） vector which are luciferase reportors, respectively. The constructed recombinant expression plasmids pGLB- 152, pGLP- 152, pGLE- 152, pGLB-140, pGLB-78, pGLB-59, pGLB-50, pGLB-41, pGLB-37, pGLB-29 and pGLB-10 were adopted to cotransfect with pRL-TK into EC109 esophageal cancer cells stimulated with TPA （5 p^g/L） respectively or not. Relative luciferase activity of whole cell extracts from cells was measured with Dual Luciferase Report Gene Assay System （DLR） to verify the position of TPA response elements. Results showed that the TPA response elements of NGAL are located on -152 ^-60 region and have a strong response to TPA stimulation in the esophageal cancer cells. The analysis by bioinformatics showed that the potential TPA responsive element in -152～-60 position of NGAL is a new sequence. This suggested that there is the structural character responding to TPA induction in the gene. These results will help to clarify the molecular mechanisms of NGAL overexpression in malignant transformation of human immortalized esophageal epithelial cell induced by TPA.

关 键 词：中性粒细胞明胶酶相关载脂蛋白基因 食管癌细胞 基因表达调控 TPA反应元件 

分 类 号：Q784[生物学—分子生物学] R735.1[医药卫生—肿瘤]