学术
人PRL-3基因启动子的克隆及转录因子Snail结合位点的初步鉴定
周军[1,2,3,4] 李建明[1,2,3,4] 杨发达[1,2,3,4] 柳玉红[1,2,3,4] 丁彦青[1,2,3,4]
[1]南方医科大学南方医院病理科,广州510515 [2]南方医科大学基础医学院病理学教研室,广州510515 [3]广东省分子肿瘤病理重点实验室,广州510515 [4]教育部广东省共建人类重大疾病转录组学和蛋白质组学重点实验室,广州510515
摘 要:
促肝细胞再生磷酸酶-3（PRL-3）是重要的肿瘤转移相关基因，其转录调控机制-直未被阐明．应用TRED在线分析系统共获得3种可能的人PRL-3基因启动子区域．通过与人基因组序列进行比对，发现其中3号启动子序列距离人PRL-3基因距离最近，位于该基因上游约1kb的DNA区域，与5′端非翻译区域邻接．在线Consite分析系统发现，-500bp至-451bp之间存在Snail结合的核心寡核苷酸序列CACCTG．运用分子克隆的方法获得PRL-3基因启动子2段区域-699bp至299bp及-642bp至-383bp区域，后者具有Snail结合位点核心寡核苷酸序列CACCTG．构建具有荧光素酶报告基因的pGL3载体并检测其启动子活性．-699-299bp区域与-642～-383bp区域的DNA片段在SW480、SW620、CNE2、293A细胞中均具有启动子活性，其中含有Snail结合位点核心寡核苷酸序列CACCTG的短片段活性强于较完整的序列．染色质免疫沉淀结合PCR扩增技术及凝胶迁移阻滞实验确定PRL-3基因启动子区域具有Snail结合位点．研究确定，PRL-3基因的启动子位于转录起始位点上游700bp与下游300bp的DNA区域，PRL-3基因启动子存在转录因子Snail结合元件．[著者文摘]
文章出处:
《生物化学与生物物理进展》-2008年35卷2期 -187-194页
栏目信息:
Cloning of Human PRL-3 Gene Promoter and Preliminary Identification of Its Snail Binding Site
ZHOU Jun, LI Jian-Ming, YANG Fa-Da, LIU Yu-Hong, DING Yan-Qing（Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China）
Abstract:
PRL-3 is a key gene related to metastasis of colorectal carcinoma. However, it is known little about the possible regulatory mechanisms of PRL-3 gene expression. There were three possible promoter regions predicted by TRED, a promoter prediction software, which were all located in the upstream regions of PRL-3 gene. One of PRL-3 gene candidate promoters was located in the region of about -1kb upstream proximal to 5＇ UTR of PRL-3 gene. Many possible transcription factor binding sites such as Snail, n-MYC, ARNT, E74A, NF-kappaB, NRF-2 and AML-1 were predicted in the region by Consite, a promoter analysis web system. Interestingly, a 5′ CACCTG 3′core sequence and other related sequences of snail binding sites were found in promoter region of PRL-3 genes. Two PRL-3 gene promoters between -699 to 299 nt and between -642 to -383 nt were cloned into pGL3 vector with luciferase report gene. Both of them had promoter activities in four different cell lines including colorectal carcinoma cell lines SW480 and SW620, nasopharyngeal carcinoma cell line CNE2 and human embryo kidney cell line 293A. Interestingly, the luciferase activities of the short DNA fragmentations with Snail binding site＇s core sequence 5′ CACCTG 3′ were higher than that of the longer one. PRL-3 promoter obtaining the 5′ CACCTG 3′ core sequence of Snail binding sites, was validated to bind to snail by chromatin immunoprecipitation （CHIP） analysis and electrophoretic mobility shift assay （EMSA） in SW480 cells. The data suggested that Snail was involved in regulation of PRL-3.[著者文摘]
Key words:
PRL-3, PRL-3 promoter, promoter activity, Snail, regulatory elements
基金资助:
国家自然科学基金资助项目（30500241,30670968和30700286）.
更多>>免费文章
cqvip.com