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目的:克隆食管癌侵袭转移相关基因Ezrin的转录调控区序列,进行启动子活性鉴定及初步的生物信息学分析。方法:利用在线程序对Ezrin基因可能的转录调控区进行GC含量和CpG岛分析以及转录因子结合位点预测;采用PCR法从食管癌细胞基因组DNA中克隆Ezrin基因-1759/+134和-726/+134区段,构建萤火虫荧光素酶报告基因表达质粒pGLB-hE(-1759/+134)和pGLB-hE(-726/+134),检测所克隆片段的启动子活性。结果:Ezrin基因5′侧翼区为高GC含量区,存在CpG岛,无典型的TATA盒,然而转录因子Sp1结合位点却无处不在。与对照质粒pGLB相比,重组质粒pGLB-hE(-726/+134)具有较强的荧光素酶活性,pGLB-hE(-1759/+134)的荧光素酶活性约是pGLB-hE(-726/+134)的2倍。结论:Ezrin基因的-726/+134区段具有启动子活性,含有Ezrin基因的核心启动子区和调节性启动子区;-1759/-726区段具有增强启动子活性的作用,含有Ezrin基因增强子元件区。
OBJECTIVE: To clone the sequence of transcriptional regulatory region of Ezrin, a gene involved in invasion and metastasis of esophageal cancer, and to identify the promoter activity and preliminary bioinformatics analysis. Methods: GC and CpG island analysis and transcription factor binding site prediction were performed on potential transcriptional regulatory regions of Ezrin gene by using online programs. Ezrin genes -1759 / +134 and -726 / -726 were cloned from genomic DNA of esophageal cancer cells by PCR. +134, the firefly luciferase reporter gene expression plasmids pGLB-hE (-1759 / + 134) and pGLB-hE (-726 / + 134) were constructed and the promoter activity of the cloned fragment was tested. RESULTS: The 5 ’flanking region of Ezrin gene was a high GC content region with CpG island and no typical TATA box. However, the Sp1 binding site of transcription factor was ubiquitous. Compared with the control plasmid pGLB, the recombinant plasmid pGLB-hE (-726 / + 134) had a strong luciferase activity and the pGLB-hE (-1759 / + 134) 726 / + 134). CONCLUSION: The -726 / + 134 segment of Ezrin gene has promoter activity and contains the core promoter region and regulatory promoter region of Ezrin gene. The -1759 / -726 segment has the effect of enhancing promoter activity. The Ezrin gene Enhancer element area.