A quantitative shRNA screen identifies ATP1A1 as a gene that regulates cytotoxicity by aurilide B

Autor: Shohei Takase, Rumi Kurokawa, Daisuke Arai, Kind Kanemoto Kanto, Tatsufumi Okino, Yoichi Nakao, Tetsuo Kushiro, Minoru Yoshida, Ken Matsumoto
Jazyk: angličtina
Rok vydání: 2017
Předmět:
Zdroj: Scientific Reports, Vol 7, Iss 1, Pp 1-9 (2017)
Druh dokumentu: article
ISSN: 2045-2322
DOI: 10.1038/s41598-017-02016-4
Popis: Abstract 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.
Databáze: Directory of Open Access Journals
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