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该文采用Western blot技术检测人食管癌EC109细胞、鼻咽癌CNE2细胞和宫颈癌HeLa细胞中Ezrin蛋白的表达;采用DNA片段定向克隆技术构建一系列携带ezrin基因增强子区–1541/–706序列的报告基因表达载体,将载体瞬时转染EC109、CNE2和HeLa细胞,检测荧光素酶活性;研究肿瘤细胞中ezrin基因增强子区的转录调控特性。实验结果显示,在被检测的三种肿瘤细胞中,Ezrin蛋白的表达水平没有明显不同。EC109细胞中,当ezrin基因–1541/–706片段正向位于无启动子的报告基因上游时,表现出类似启动子的转录激活作用;当这一片段反向连接时转录激活作用几乎消失。当–1541/–706片段正向位于ezrin启动子或SV40启动子上游时,显著增强荧光素酶表达;然而,当这一片段反向位于启动子上游以及正向或反向位于启动子控制的报告基因下游时,转录增强作用消失。ezrin基因–1541/–706片段在CNE2和HeLa细胞中的转录调控作用,与其在EC109细胞中的转录调控作用部分相似,但不完全相同。结果表明,ezrin基因增强子区具有转录激活和转录增强双重作用,这种作用具有DNA序列位置和方向依赖性以及细胞特异性。
Western blot was used to detect the expression of Ezrin protein in human esophageal carcinoma EC109 cells, nasopharyngeal carcinoma CNE2 cells and cervical cancer HeLa cells. A series of ezrin gene enhancer region -1541 / -706 sequences were constructed by DNA fragment directed cloning technique The reporter vector was transiently transfected into EC109, CNE2 and HeLa cells to detect the luciferase activity. The transcriptional regulation of ezrin gene enhancer region in tumor cells was investigated. The experimental results show that in the three tumor cells tested, Ezrin protein expression levels did not differ significantly. EC109 cells, promoter-like transcriptional activation was observed when the ezrin gene -1541 / -706 fragment was located upstream of the promoterless reporter gene; transcriptional activation almost disappeared when the fragment was reversely ligated. When the -1541 / -706 fragment was located directly upstream of the ezrin promoter or the SV40 promoter, luciferase expression was significantly enhanced; however, when this fragment was located upstream of the promoter and in the forward or reverse direction under promoter control Downstream of the reporter gene, the transcriptional enhancement disappears. The transcriptional regulation of ezrin gene -1541 / -706 in CNE2 and HeLa cells was partly similar to that of ezrin gene in EC109 cells, but not completely the same. The results show that the ezrin gene enhancer region has the dual role of transcriptional activation and transcriptional enhancement, which has the DNA sequence location and direction-dependent and cell-specific.