整合蛋白b1亚基对Skp2转录调控的机制研究

项目名称： 整合蛋白b1亚基对Skp2转录调控的机制研究

项目编号： No.30800182

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 傅奕

作者单位： 扬州大学

项目金额： 20万元

中文摘要： 整合蛋白在细胞的增殖分化、凋亡及肿瘤细胞的迁移、浸润等过程中发挥重要作用。我们已发现，整合蛋白b1亚基过表达可抑制肝癌细胞的增殖，下调Skp2的表达，进而抑制p27蛋白的降解。因此本课题研究了整合蛋白b1亚基对Skp2表达的调控机制。结果发现，整合蛋白b1亚基过表达没有影响Skp2 mRNA的稳定性，表明整合蛋白b1亚基在转录水平调节Skp2的表达，因此从转录因子以组蛋白乙酰化系统两方面展开研究。研究发现，Skp2启动子（-439~+51）的片段是其核心启动子区域，该区域中Sp1结合位点突变可增加Skp2启动子活性，而Sp1过表达可抑制其转录活性，表明Sp1作为负性转录因子调节Skp2表达。整合蛋白b1亚基主要通过Skp2核心启动子（-100~ -95）区域的Sp1结合位点发挥转录抑制作用。PI3K信号通路也可在转录水平抑制Skp2表达，但不依赖于PKB。整合蛋白b1亚基过表达还可增加组蛋白乙酰化水平，但却使Skp2转录水平下调，PI3K通路没有参与该机制的调节。综上所述，整合蛋白b1亚基可以通过Sp1转录因子和组蛋白乙酰化调节Skp2的转录，PI3K通路只涉及转录因子的调节。

中文关键词： 整合蛋白;Skp2;转录;Sp1;乙酰化

英文摘要： As heterodimeric transmembrane receptors, integrins recognize and bind ECM (extracellular matrix) ligands, participating in regulation of cell proliferation, differentiation, apoptosis, migration and invasion. In our previous studies, it has been demonstrated that overexpression of integrin beta1（?1） subunit could inhibit SMMC-7721 proliferation and decreased the level of Skp2, which inhibited the degradation of p27 protein. Therefore, the aim of this study is to illuminate the machanism of integrin ?1 on Skp2 expression. It was found that integrin βsubunit overexpression did not affect the stability of Skp2 mRNA, which indicated that integrin ?1 regulated the Skp2 expression in transcriptional level. Then we investigated the transcriptional mechanism from the key transcription factors and histone acetylation/deacetylation system. Aftrer 5' serial deletion of Skp2 promoter analysis, we found that the region from -439~+51 of Skp2 promoter is the core region of Skp2 promoter. In this region, there were four Sp1 binding sites. Mutation these Sp1 binding sites could increased the Skp2 promoter activity. Overexpression of Sp1 reduced the activity of Skp2 promoter, which suggested that Sp1 might be a suppressor for Skp2 transcription. Overexpression of integrin βsubunit inhibited the Skp2 transcription mainly through the second Sp1 binding site, which located at the region from -100~ -95 contained in the core region of Skp2 promoter. PI3K signaling pathway also inhibited Skp2 expression transcriptionally, but PKB was independent. By western blot, we found that overexpression of integrin βsubunit could increase the activity of histone acetylation. However, activated acetylation repressed the transcription of Skp2. In this regulation, PI3K pathway had not obvious effect. In summary, overexpression of integrin ?1 prevented the Skp2 transcription through Sp1 and histone acetylation, PI3K signaling pathway was only involved in the former mechanism.

英文关键词： integrin; Skp2; transcription; Sp1; acetylation