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

Scientific Reports
Shohei TakaseKen Matsumoto

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.

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Citations

Oct 23, 2018·Bioscience, Biotechnology, and Biochemistry·Minoru Yoshida
Nov 18, 2018·Cancer Science·Masataka AmisakiGoshi Shiota
Jul 29, 2019·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Xiao LiangHendrik Luesch
Jan 10, 2020·The Journal of Biological Chemistry·Amita GuptaChaitan Khosla
May 1, 2021·International Journal of Molecular Sciences·Jia-Nan ZhangHong-Jie Zhang
Jun 28, 2021·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Hong-Wei ZhangXin Luan

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Methods Mentioned

BETA
PCR
transfection
column chromatography
electrophoresis

Software Mentioned

Cellecta
R
Decipher

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