A quantitative shRNA screen identifies ATP1A1 as a gene that regulates cytotoxicity by aurilide B.
A quantitative shRNA screen identifies ATP1A1 as a gene that regulates cytotoxicity by aurilide B.
复制标题
DOI：
10.1038/s41598-017-02016-4
复制
发表时间：
2017-05-17
影响因子：
4.6
通讯作者：
Matsumoto K
中科院分区：
文献类型：
--
作者：
Takase S;Kurokawa R;Arai D;Kanemoto Kanto K;Okino T;Nakao Y;Kushiro T;Yoshida M;Matsumoto K
基因组范围的RNA干扰(RNAi)与汇集和条形码的短发夹状RNA(ShRNA)文库一起提供了一个强大的工具来识别与生物活性化合物的作用模式/机制(MOA)相关的细胞成分。影响细胞对给定化合物敏感性的shRNA可以通过对shRNA特定条形码的深度测序来识别。在序列文库制备过程中，我们使用了多重条形码测序技术，通过在聚合酶链式反应中添加特定于样本的索引标签，实现了对多个样本的并行分析。利用该系统进行的shRNA文库筛选表明，Na+/K+ATPase的α亚基ATP1A1的下调使其对诱导线粒体介导的细胞凋亡的天然海洋产物银杏内酯B具有显著的敏感性。通过靶向α亚基抑制Na+/K+ATPase的哇巴因联合作用增强了对金莲内酯B的敏感性，提示ATP1A1调节线粒体介导的细胞凋亡。我们的结果表明，多重测序有助于使用汇集的shRNA文库筛选来识别联合药物治疗靶点。
Genome-wide RNA interference (RNAi) with pooled and barcoded short-hairpin RNA (shRNA) libraries provides a powerful tool for identifying cellular components that are relevant to the modes/mechanisms of action (MoA) of bioactive compounds. shRNAs that affect cellular sensitivity to a given compound can be identified by deep sequencing of shRNA-specific barcodes. We used multiplex barcode sequencing technology by adding sample-specific index tags to PCR primers during sequence library preparation, enabling parallel analysis of multiple samples. An shRNA library screen with this system revealed that downregulation of ATP1A1, an α-subunit of Na+/K+ ATPase, conferred significant sensitivity to aurilide B, a natural marine product that induces mitochondria-mediated apoptosis. Combined treatment with ouabain which inhibits Na+/K+ ATPase by targeting α-subunits potentiated sensitivity to aurilide B, suggesting that ATP1A1 regulates mitochondria-mediated apoptosis. Our results indicate that multiplex sequencing facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets.