干扰素调节因子3多个启动子及其剪接异构体的特性

项目名称： 干扰素调节因子3多个启动子及其剪接异构体的特性

项目编号： No.30872804

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 周国平

作者单位： 南京医科大学

项目金额： 34万元

中文摘要： 干扰素调节因子3（IRF-3）在肿瘤和抗感染免疫中起重要作用，本研究寻找其新的剪接异构体，探索这些RNA转录调控的机制。用5' 快速cDNA 末端扩增法，在IRF-3的第二内含子内发现了两个新的转录起始位点，分别位于第二内含子内、第三外显子前的第718bp和162bp位。启动子功能研究和蛋白-DNA结合实验发现，野生型IRF-3的核心启动子受Sp1和Sp3正调控、E2F1负调控,呼吸道合胞病毒可诱导IRF3的RNA增加，IRF3可诱导microRNA-221表达下调。剪接异构体IntV1的核心启动子受Sp1正调控。EB病毒患儿的外周血中IntV1的RNA较正常人低，病毒类似物可增强该剪接异构体的启动子活性和RNA表达。CREB位点是调控IntV2启动子的关键位点，病毒类似物可提高IntV2启动子活性和RNA水平。IntV1在人肺癌细胞表达量增高，在肝癌细胞没有表达；IntV2在肺癌细胞表达量较高，在宫颈癌和卵巢癌细胞中没有表达。由于EB病毒成分可通过Sp和E2F1调控基因转录，这些数据为研究EB病毒调控IRF-3的免疫逃避机制开辟了新领域，为研究IRF-3在肿瘤发病中的作用打下了基础。

中文关键词： 干扰素调节因子；剪接异构体；启动子；转录调控；基因表达

英文摘要： Interferon regulatory factor 3 (IRF-3) plays a key role in cancers and anti-infectious immunity. The aims of this study were to search for new spliced isoforms of IRF-3 and to explore the mechanism of the transcription regulation of these isoforms. Two novel transcription start sites were found in the second intron of the wild type of IRF-3 by the method of 5' rapid amplification of cDNA ends, located at 718 bp and 162 bp before the 3rd exon of IRF-3. The core promoter of the wild type of IRF-3 was positively regulated by Sp1 and Sp3, and negatively regulated by E2F1, by methods of luciferase promoter functional analysis, electrophoretic mobitity shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Respiratory syncytial virus induced the increased RNA level of IRF-3. IRF-3 decreased the expression of microRNA-221. The core promoter of the spliced isoform of IRF-3, IntV1, was positively regulated by Sp1. The RNA of IntV1 from peripheral blood of patients with EB virus infection was lower than that of the healthy controls. Viral analogues increased the promoter activity and RNA level of IntV1. The transcription factor binding site of CREB was the critical site for the promoter function of IntV2. Viral analogues also increased the promoter activity and RNA level of IntV2. IntV1 has a higher RNA expression in A549 cells. RNA of IntV1 was not found in HepG2. Higher RNA of IntV2 was also detected in A549 cell, but not in Hela and Skov3 cells. It was reported that the components of EB virus regulated transcriptions by transcription factors of Sp and E2F1. These data may open a new area for the research of the immune escape molecular mechanism of transcription regulation of IRF-3 by the components of EB virus and set a basis for the role of IRF-3 in the pathogenesis of cancers.

英文关键词： interferon regulatory factor; spliced isoform; promoter; transcription regulation; gene expression