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

VEGF对实体瘤血管生成至关重要，对低氧条件下的肿瘤存活至关重要1。尽管缺氧通过典型的帽依赖途径导致翻译的全局抑制，但VEGF mRNA仍然有效翻译。这是通过ires驱动的VEGF mrna23翻译实现的。然而，对VEGF IRES翻译的分子机制了解甚少。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，生化和细胞培养方法来机械解剖VEGF ires驱动的翻译。我们之前已经开发了一种基于RNA转染的体内实验，可以在体内研究细胞IRESs的功能。使用该系统，我们将进行高通量RNAi筛选，以鉴定VEGF IRES翻译的阳性和阴性调节因子。随后，我们将在二次RNAi筛选中验证鉴定的因子，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了解癌细胞中VEGF mRNA选择性翻译的分子和细胞机制，并最终为治疗干预提供新的体内验证靶点。

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.