摘要
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。
To investigate transcriptional regulatory properties of ezrin gene enhancer in carcinoma cells, Ezrin expression in human esophageal carcinoma EC 109, nasopharyngeal carcinoma CNE2 and cervical carcinoma HeLa cells was detected by Western blot. A series of reporter gene expression vectors carrying ezrin enhancer -1541/-706 sequence were constructed using DNA fragments orientating clone method and then transfected into EC109, CNE2 and HeLa cells for luciferase assay. It was found that the expression levels of Ezrin in the detected three cell lines were not obviously different. In EC109 cells, when the ezrin -1541/-706 segment was located upstream of luc reporter gene without promoter in the forward orientation, it exhibited transcriptional activation like a promoter; while this transactivation was nearly abolished when this segment was reversed. When this segment was located upstream of the ezrin promoter or SV40 promoter in the forward orientation, it dramatically increased luciferase expression. However, the transcriptional enhancement disappeared when this segment was located upstream of promoters in the reverse orientation, or downstream of reporter genes in the forward or reverse orientation. The transcriptional regulation of ezrin -1541/-706 segment in CNE2 and HeLa cells was partly similar, but not completely identical to that in EC109 cells. These data suggested that the ezrin enhancer could exhibit transcriptional activation and enhancement, in a position/orientation-dependent and cell-type-specific manner.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2014年第5期610-616,共7页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:31360212)
深圳市科技研发资金基础研究计划(批准号:JC201005260209A
JC201105201028A)资助的课题~~
