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The role of CREM2 gene in the expression of CYP1A1 gene

CREM2基因在CYP1A1基因表达中的作用

基本信息

批准号：
15310032
负责人：
KIKUCHI Hideaki
金额：
$ 9.92万
依托单位：
Hirosaki University (2004-2006)Tohoku University (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15310032/
关键词：
CREM Ah receptor dioxin transcripton factor HepG2 CYP1A1 EMSA Hepa1c1c7

项目摘要

Our group identified CERM gene as a one of the components in a new signal transduction pathway that mediates CYP1A1 induction. In order to clarify the function of CREM gene product, we analyzed the cis-element in the regulatory sequence of Cyp1a1 gene using reporter gene assay. We successfully narrowed down the responsive sequence of CREM and found BTE (basic transcription element) in this sequence. Then, several mutations in the consensus sequence were introduced to test the initiation ability of transcription by the method of reporter gene assay. The mutated cis-element of BTE decreased the transcription of Cyp1a1.Furthermore, a possible protein factor(s) that can bind to this sequence was investigated by EMSA (electromobility shift assay) method. The nuclear extract was prepared from HepG2 cells and was mixed with 32P-labeled cis-element. After 20 min incubation at 25℃, the nuclear protein and oligonucleotide mixture was separated on non-denaturation acrylamide gel and the dried gel was exposed to imaging plate to visualize the retarded band that was a complex of proteins with DNA. There were several specific bands that were abolished by the addition of vast excess of non-labeled oligonucleotide. Although these bands were observed in the nuclear extract from non-treated sample, the band intensity increased remarkably by the treatment of omeprazole. Therefore, it was suggested that CREM might make a complex with Sp1 and might stimulate the transcription of Cyp1a1. To verify the possible involvement of CREM in the process of Cyp1a1 induction, gene-silencing experiment of CREM in HepG2 cells is performing with siRNA of CREM.

本课题组将CERM基因鉴定为介导CYP1A1诱导的新的信号转导通路的组成部分之一。为了阐明CREM基因产物的功能，我们采用报告基因分析法对Cyp1a1基因调控序列中的顺式元件进行了分析。我们成功地缩小了CREM的响应序列，并在该序列中发现了BTE（基本转录元件）。然后，在共有序列中引入几个突变，通过报告基因分析的方法测试转录起始能力。突变的BTE顺式元件使Cyp1a1的转录水平降低，并通过EMSA（electromobility shift assay）方法研究了与该顺式元件结合的蛋白质因子。从HepG2细胞制备核提取物，并与32P标记的顺式元件混合。在25℃孵育20 min后，将核蛋白和寡核苷酸混合物在非变性丙烯酰胺凝胶上分离，并将干燥的凝胶暴露于成像板，以观察蛋白质与DNA的复合物的延迟条带。有几条特异性条带被添加大量过量的未标记寡核苷酸消除。尽管在来自未处理样品的核提取物中观察到这些条带，但通过奥美拉唑处理，条带强度显著增加。因此，CREM可能与Sp1形成复合物，并可能刺激Cyp1a1的转录。为了验证CREM在Cyp1a1诱导过程中的可能作用，我们在HepG2细胞中进行了CREM的siRNA沉默实验。