Identification of targets of tumor suppressor microRNA-34a using a reporter library system
Creators
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1.
National Center For Child Health and Development
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2.
Imperial College London
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3.
Nagoya City University
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4.
California Institute of Technology
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5.
Japan Science and Technology Agency
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6.
Scripps Research Institute
Abstract
miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3′ UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks.
Additional Information
© 2017 National Academy of Sciences. Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, University Health Network, Toronto, Canada, and approved March 9, 2017 (received for review December 8, 2016). Published online before print March 29, 2017. We thank Drs. Akio Yamashita, Masato Yano, Tomoaki Tanaka, and Kazuhiko Kuwahara for helpful discussions and Mayuko Yoda, Yukinari Osaka, Nobuhiro Yamamoto, and Ayaka Ichise for technical assistance. This work was supported by Core Research for the Evolutionary Science and Technology (CREST) funding from the Japan Science and Technology Agency; CREST funding from the Japan Agency for Medical Research and Development; Grants-in-Aid for Scientific Research (KAKENHI) Grants 26113008, 15H02560, 15K15544, 15K15026, and 25860139 from the Japan Society for the Promotion of Science; NIH Grants AR050631 and AR065379; the Naito Foundation; and a Bristol-Myers K.K. RA Clinical Investigation Grant. Author contributions: Y.I., A. Inoue, and H.A. designed research; Y.I., A. Inoue, T.S., Y.H., A. Igarashi, and T.T. performed research; Y.I., A. Inoue, and H.A. contributed new reagents/analytic tools; Y.I., A. Inoue, T.S., Y.H., T.T., K.D.T., M.P.B., and H.A. analyzed data; and Y.I. and H.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1620019114/-/DCSupplemental.Attached Files
Published - PNAS-2017-Ito-3927-32.pdf
Supplemental Material - pnas.1620019114.sd01.xlsx
Supplemental Material - pnas.201620019SI.pdf
Files
PNAS-2017-Ito-3927-32.pdf
Additional details
Identifiers
- PMCID
- PMC5393199
- Eprint ID
- 75522
- DOI
- 10.1073/pnas.1620019114
- Resolver ID
- CaltechAUTHORS:20170329-152704022
Funding
- Japan Science and Technology Agency (JST)
- Japan Agency for Medical Research and Development
- Japan Society for the Promotion of Science (JSPS)
- 26113008
- Japan Society for the Promotion of Science (JSPS)
- 15H02560
- Japan Society for the Promotion of Science (JSPS)
- 15K15544
- Japan Society for the Promotion of Science (JSPS)
- 15K15026
- Japan Society for the Promotion of Science (JSPS)
- 25860139
- NIH
- AR050631
- NIH
- AR065379
- Naito Foundation
- Bristol-Myers
Dates
- Created
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2017-03-29Created from EPrint's datestamp field
- Updated
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2022-03-28Created from EPrint's last_modified field