Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation
Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation
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DOI：
10.1371/journal.pone.0045943
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发表时间：
2012-09-27
期刊：
影响因子：
3.7
通讯作者：
Thoma, Christian
中科院分区：
文献类型：
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作者：
Casanova, Claudia M.;Sehr, Peter;Thoma, Christian
促进血管生成的蛋白质，如血管内皮生长因子(VEGF)，是癌症治疗的主要靶点。因此，特异性激活血管内皮生长因子等因子表达的蛋白质是潜在的替代治疗靶点，可能有助于对抗血管生成抑制剂的逃避耐药。血管内皮生长因子mRNA含有两个内部核糖体进入位点(IRESS)，能够在低氧条件下选择性地激活血管生成过程中合成的血管内皮生长因子蛋白。为了确定人类细胞中VEGFIRES驱动的翻译的新调节因子，我们开发了一种高通量筛选方法，将siRNA处理和VEGFIRES报告mRNA的转染相结合。我们确定MAPK3是一种新的VEGFIRES驱动翻译的正向调节因子，并验证了其对内源性VEGF3的调节作用。我们的自动化方法是可扩展的，并且很容易与其他mRNA调控元件一起使用。因此，它应该是一种普遍有用的方法来高通量地鉴定新的mRNA翻译调控因子。
Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.