A Quantitative ShRNA Screen Identifies ATP1A1 As a Gene That Regulates Cytotoxicity by Aurilide B

Overview

Journal Sci Rep

Specialty Science

Date 2017 May 19

PMID 28515454

Citations 15

Authors

Shohei Takase

Rumi Kurokawa

Daisuke Arai

Kind Kanemoto Kanto

Tatsufumi Okino

Yoichi Nakao

Tetsuo Kushiro

Minoru Yoshida

Ken Matsumoto

Affiliations

Soon will be listed here.

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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