人卵巢癌相关候选基因HE4(WFDC2)的转录调控主要由Sp1与位于-71和-48的Egr-1位点结合所介导  

The interaction of Sp1 transcription factor with two Erg-1 sites at-71/-48 upstream contributes significantly the promoter activity of the HE-4 gene in tumor cells

作　　者：赵莹珺[1] 杨剑峰[1] 朱景德[2] 

机构地区：[1]复旦大学医学院,上海200032 [2]上海市肿瘤研究所癌基因及相关基因国家重点实验室

出　　处：《肿瘤》2004年第6期517-525,共9页Tumor

摘　　要：目的　阐明HE4基因转录调控机制的细节。方法　 1.用半定量RT PCR的方法评估HE4基因在肿瘤细胞株中的表达情况 ;2 .以荧光素酶报告基因载体为基础 ,对HE4基因的上游顺序构建了 3′、5′缺失突变和一系列连接体扫描突变 ;3.通过瞬时转染 /体外双荧光素酶报告系统对这些重组质粒中插入片段进行启动子活性的分析 ;4 .针对蛋白和关键顺式区域的结合的特异性和能力 ,开展泳动滞后和抗体介导的超迁移分析。结果　通过对 ( 186 0 /+ 2 9)的HE4基因上游片段及其剪切体的分析 ,将HE4基因最小启动子确定为 10 7/+ 15的DNA片段。通过对连接体扫描突变体的分析 ,将关键的顺式作用元件确定在W4 5 ( 71/ 4 8)片段 ,生物信息学提示该区存在两个Egr 1位点。通过用已知的反式作用因子与报告基因质粒分别进行共转染 ,提示Sp1是最为有效的反式作用因子 ,而不是Egr 1。通过泳动滞后和抗体介导的超迁移实验 ,证实Sp1确实是参与作用的转录因子。结论　HE4基因的转录活性主要是由转录因子Sp1与位于 ( 71/ 4 8)区域的两个Egr 1位点结合所介导的。Objective To uncover the molecular details of the mechanisms underlying the transcriptional regulation of human HE4 gene in tumor cells Methods 1, Using a semi-quantitative PCR method to determine the expression profile of HE4 gene in a panel of tumor cell lines; 2, Using molecular dissection to construct, based upon the luciferase reporter gene, a set of the 5', or 3' deletion mutants from the full-length upstream region(-1860/+29), and 10 linker-scanning mutants of (-107/+15) of the HE4 minimal promoter; 3, Via the transient transfection/dual reporter assay, the promoter activities of the above constructs were vigorously tested; 4, Via the analyses combining bioinformatics, co-transfection and electrophoresis mobility shift assay (EMSA), the proteins binding specific to the cis-elements within the key region ( -71/-48) with significant functional consequence was determined. Results It has been found that the minimal promoter of the HE4 genes is mapped at the region from -107 to +15. The key cis-elements are within the region from -71 to -48. The bioinformatics analysis revealed two Egr-1 sites as the candidate cis-elements. Co-transfection with each of a panel of the expression plasmids of the known transcription factors with the relevant reporter construct indicates Sp1, but not Egr-1, is the most potent transcription factor for enhancement of the promoter activity. Via the EMSA assay, Sp1 indeed binds to the Egr-1 site with the comparable specificity and affinity as to Sp1 site. Conclusion The interaction of Sp1 protein to two Egr-1 sites at -71/-48 region contributes significantly to the promoter activity of the HE4 gene in tumor cells.

关 键 词：HE4基因 转录调控 最小启动子 转染 顺式作用元件 转录因子SP1 肿瘤细胞 培养的 卵巢癌 

分 类 号：R737.31[医药卫生—肿瘤]