人NPCEDRG基因启动子的克隆及CCAAT/NFY结合位点初步分析

Cloning of Human NPCEDRG Core Promoter and Preliminary Identification of Its CCAAT/NFY Binding Site

NPCEDRG基因是采用基因定位候选克隆策略获得的一个鼻咽癌候选抑瘤基因.NPCEDRG在鼻咽癌细胞和组织中表达下调,重新恢复NPCEDRG基因在CNE2细胞系的表达,可部分逆转CNE2的恶性表型.为揭示NPCEDRG基因在鼻咽癌细胞和组织中表达下调的分子机制,联合应用生物信息学和报告基因载体系统分析方法对NPCEDRG基因启动子区进行克隆及功能分析,系统发育进化足迹分析结果表明,NPCEDRG基因5′端调控区-180～+235 bp区间在脊椎动物中高度保守,该保守区域中存在包括CCAAT/NFY、STAT1和SP1等转录因子结合位点.构建Luc和/或EGFP报告基因表达载体并检测其启动子活性,-146～-8 bp区域有较强的启动子活性,电泳迁移阻滞分析实验(EMSA)提示,CCAAT/NFY转录因子结合位点是NPCEDRG基因的转录调控元件.因此,研究确定-146～-8 bp区域是NPCEDRG基因核心启动子区域且启动子核心元件CCAAT/NFY可能参与NPCEDRG基因的转录调控.

NPCEDRG is an NPC associated suppressive gene cloned by positional candidate cloning strategy.Its transcriptional down-expression has been shown in the cell lines and primary tumor tissues of NPC.Reintroduction of NPCEDRG into CNE2,a cell line derived from NPC,was effective to induce cell differentiation,control cell growth,and regulate the cell cycle.To uncover the molecular mechanisms underlying down-expression of NPCEDRG in NPC cells,bioinformatics approaches and functional assays in different tumor cell lines were used to identify and characterize the NPCEDRG core promoter and cis-acting elements.The conserved region from-180 to ＋235 bp was found in the potential promoter among 6 vertebrate species by the ECR browser,and there have several potential binding sites for transcription factors,such as CCAAT/NFY,STAT1 and SP1.To characterize the NPCEDRG core promoter,transient luciferase and/or EGFP reporter assay were carried out with the construct pGL3-en138.The results demonstrated that the core promoter is located at the conserved region from-146 to-8 nucleotides.Gel shift assay revealed the specific binding of some nuclear proteins to probes containing a putative CCAAT/NFY site,suggesting that the CCAAT/NFY site contributes to the regulation of NPCEDRG gene expression.