High-thoughput siRNA screen to mechanistically dissect Vascular Endothelial Growth Factor (VEGF) Internal Ribosomal Entry Site (IRES)-mediated translation in tumorigenesis

高通量 siRNA 筛选可机械剖析血管内皮生长因子 (VEGF) 内部核糖体进入位点 (IRES) 介导的肿瘤发生翻译

• 批准号: 70817203
• 负责人: Dr. Christian Thoma
• 金额: --
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/70817203?language=en
• 关键词: thoughput; siRNA; screen; mechanistically; dissect

VEGF is critical for angiogenesis in solid tumors and important for tumor survival under hypoxic conditions1. Although hypoxia leads to global repression of translation by the canonical cap-dependent pathway, the VEGF mRNA remains efficiently translated. This is achieved by IRES-driven translation of the VEGF mRNA2 3. However, the molecular mechanism responsible for VEGF IRES translation is poorly understood. In particular, it has not been addressed whether specific functional modulators are necessary to promote VEGF IRES-mediated translation. We will apply previously established functional genomic, biochemical and cell culture approaches to mechanistically dissect VEGF IRES-driven translation. We have previously developed an in vivo assay based on RNA transfections4 that allows to study the function of cellular IRESs in vivo. Using this system we will perform a highthroughput RNAi screen to identify positive and negative modulators of VEGF IRES translation. Subsequently, we will validate the identified factors in a secondary RNAi screen and functionally characterize these factors using in vivo and in vitro approaches. The proposed research will contribute to our understanding of the molecular and cellular mechanisms that enable selective translation of VEGF mRNA in cancer cells and could ultimately provide novel in vivo-validated targets for therapeutic intervention.

VEGF对于实体瘤中的血管生成至关重要，并且对于缺氧条件下的肿瘤存活很重要1。尽管缺氧会导致典型的帽依赖性途径对翻译的全面抑制，但VEGF mRNA仍然有效翻译。这是通过IRES驱动的VEGF mRNA 2 3的翻译实现的。然而，负责VEGF IRES翻译的分子机制知之甚少。特别是，尚未解决是否需要特定的功能调节剂来促进VEGF IRES介导的翻译。我们将应用先前建立的功能基因组学，生物化学和细胞培养方法来机械解剖VEGF IRES驱动的翻译。我们先前已经开发了一种基于RNA转染的体内测定法4，其允许在体内研究细胞IRES的功能。使用这个系统，我们将进行高通量RNAi筛选，以确定VEGF IRES翻译的阳性和阴性调节剂。随后，我们将在二次RNAi筛选中验证所鉴定的因子，并使用体内和体外方法对这些因子进行功能表征。拟议的研究将有助于我们理解能够在癌细胞中选择性翻译VEGF mRNA的分子和细胞机制，并最终为治疗干预提供新的体内验证靶点。