Functional characterization of the human translocator protein (18 kDa) gene promoter in human breast cancer cell lines
Functional characterization of the human translocator protein (18 kDa) gene promoter in human breast cancer cell lines
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DOI：
10.1016/j.bbagrm.2011.09.001
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发表时间：
2012-01-01
影响因子：
4.7
通讯作者：
Papadopoulos, Vassilios
中科院分区：
文献类型：
--
作者：
Batarseh, Amani;Barlow, Keith D.;Papadopoulos, Vassilios
转运蛋白（18kda； TSPO）是一种线粒体药物和胆固醇结合蛋白，参与多种过程，包括类固醇生成、细胞增殖和细胞凋亡。人类TSPO基因的表达在几种癌症中升高。为了了解人类乳腺癌细胞中调控TSPO表达的分子机制，我们在TSPO激素依赖性低、非侵袭性的MCF-7细胞和TSPO激素依赖性高、侵袭性强、转移性强的MDA-MB-231乳腺癌细胞中鉴定、克隆了TSPO启动子，并对其进行了功能表征。RNA连接酶介导的cDNA末端5'快速扩增分析表明，转录始于富含gc的启动子下游的多个位点，该启动子缺乏功能性的TATA和CCAAT盒。缺失分析表明，从-121到+66的区域，包含5个被认为是GC盒的调控位点，足以诱导MCF-7和MDA-MB-231细胞中高达24倍和近120倍的报告基因活性。电泳迁移率转移和染色质免疫沉淀实验表明，Sp1、Sp3和Sp4在体外与这些GC盒和内源性TSPO启动子结合。沉默Sp1、Sp3和Sp4基因表达可降低TSPO水平。此外。TSPO的表达在一个或多个鉴定的GC盒中受到表观遗传调控。在MCF-7和MDA-MB-231细胞中，TSPO主要起始位点下游序列的破坏可差异调节TSPO启动子活性，表明必需元件在这些细胞中促进其差异表达。综上所述，这些实验首次对人类TSPO基因启动子及其转录调控进行了深入的功能分析。(C) 2011 Elsevier B.V.版权所有
The translocator protein (18 kDa; TSPO) is a mitochondrial drug- and cholesterol-binding protein that has been implicated in several processes, including steroidogenesis, cell proliferation, and apoptosis. Expression of the human TSPO gene is elevated in several cancers. To understand the molecular mechanisms that regulate TSPO expression in human breast cancer cells, the TSPO promoter was identified, cloned, and functionally characterized in poor-in-TSPO hormone-dependent, non-aggressive MCF-7 cells and rich-in-TSPO hormone-independent, aggressive, and metastatic MDA-MB-231 breast cancer cells. RNA ligase-mediated 5'-rapid amplification of cDNA ends analysis indicated transcription initiated at multiple sites downstream of a GC-rich promoter that lacks functional TATA and CCAAT boxes. Deletion analysis indicated that the region from -121 to +66, which contains five putative regulatory sites known as GC boxes, was sufficient to induce reporter activity up to 24-fold in MCF-7 and nearly 120-fold in MDA-MB-231 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1, Sp3 and Sp4 bind to these GC boxes in vitro and to the endogenous TSPO promoter. Silencing of Sp1, Sp3 and Sp4 gene expression reduced TSPO levels. In addition. TSPO expression was epigenetically regulated at one or more of the identified GC boxes. Disruption of the sequence downstream of the main start site of TSPO differentially regulated TSPO promoter activity in MCF-7 and MDA-MB-231 cells, indicating that essential elements contribute to its differential expression in these cells. Taken together, these experiments constitute the first in-depth functional analysis of the human TSPO gene promoter and its transcriptional regulation. (C) 2011 Elsevier B.V. All rights reserved.