肺癌细胞中调控ezrin基因基本转录活性的顺式作用元件及转录因子的鉴定

Identification of Elements and Transcription Factors for ezrin Basal Transcriptional Activity in Lung Cancer Cells

研究发现,质膜-细胞骨架连接蛋白Ezrin在多种肿瘤细胞中异常表达,而且Ezrin的表达上调与肿瘤细胞的移动侵袭相关,但是调控ezrin基因转录的分子机制却不清楚.为了探明ezrin基因的转录调控机制,以肺癌细胞A549为材料,首先采用双荧光素酶报告基因分析系统检测ezrin基因5′侧翼嵌套缺失序列和位点突变序列的转录活性,鉴定肺癌细胞中ezrin基因的基本启动子区以及关键的顺式作用元件Sp1结合位点(-75/-69)和AP-1结合位点(-64/-58).其次,利用凝胶电泳迁移率变动分析证明,肺癌细胞核蛋白提取物能够与ezrin基因含有关键顺式作用元件的DNA序列结合,形成DNA-核蛋白复合物,而且Sp1结合位点和AP-1结合位点与重组蛋白rhSp1和rhAP-1的结合具有位点特异性.最后,利用瞬时转染实验证实,转录因子Sp1和AP-1(由c-Jun和c-Fos组成的异源二聚体)分别通过Sp1结合位点和AP-1结合位点,增强ezrin基因基本转录活性,而且,过表达转录因子Sp1、c-Jun或c-Fos上调了Ezrin蛋白表达.研究确定,肺癌细胞中调控ezrin基因基本转录活性的关键顺式作用元件是Sp1结合位点(-75/-69)和AP-1结合位点(-64/-58),与之作用的转录因子Sp1和AP-1对于ezrin基因的转录激活作用至关重要.

It has been found that Ezrin is aberrantly expressed in some carcinomas and there is a relationship between the high level of Ezrin expression and metastatic potential of carcinomas. However, the molecular mechanisms underlying the regulation of the human ezrin gene transcription are not well understood. Transcriptional regulation regions of the human ezrin gene were examined by linking 5＇ -deletion mutants and site-directed mutagenesis of the 5＇-flanking region to a luciferase reporter gene, and the basal promoter region and key DNA sequence elements （an Spl binding site, -75/-69; and an AP-1 binding site, -64/-58） involved in the expression of the human ezrin gene in lung cancer A549 cells were explored. Furthermore, electrophoretic mobility shift assay （EMSA） showed that DIG-ddUTP labeled DNA sequences containing ezrin key elements could bind nuclear extracts from lung cancer cells to form DNA-protein complex, and the bindings to Spl and AP-1 sites by rhSpl and rhAP-l, respectively, were specific. Finally, the analysis of transient transfection of A549 cells by transcription factor expression vectors indicated that Spl and AP-1 （c-Jun/c-Fos heterodimer） could increase the human ezrin basal transcriptional activity significantly through the Spl and AP-1 binding sites, respectively. In addition, over-expression of Spl, c-Jun or c-Fos could up-regulate Ezrin expression. The data suggested that the Spl and AP-1 binding sites were two key elements regulating ezrin gene basal transcriptional activity in lung cancer cells, and the corresponding transcription factors Sp 1 and AP-1 were crucial for the transactivation.