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International Journal of Genomics
Volume 2017 (2017), Article ID 5214806, 7 pages
Research Article

The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer

1Non-coding RNA Laboratory, Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, Italy
2Tuscan Tumor Institute (ITT), Via Alderotti 26/N, 50139 Firenze, Italy
3Instute of Life Science, Scuola Superiore Sant’Anna, Piazza Martiri delle Libertà 33, 56127 Pisa, Italy
4Laboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT) and Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, Italy
5Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark

Correspondence should be addressed to Milena Rizzo; ti.rnc.cfi@ozzir.anelim

Received 30 May 2017; Accepted 1 August 2017; Published 20 September 2017

Academic Editor: Davide Barbagallo

Copyright © 2017 Milena Rizzo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


miR-28-5p is an intragenic miRNA which is underexpressed in several tumor types showing a tumor suppressor (TS) activity. Routinely, the known miR-28-5p targets are validated in specific tumor contexts but it is unclear whether these targets are also being regulated in other tumor types. To this end, we adopted the miRNA pull out assay to capture the miR-28-5p targets in DU-145 prostate cancer (PCa) cells. Firstly, we demonstrated that miR-28-5p acts as a TS-miRNA in PCa, affecting cell proliferation, survival, and apoptosis. Secondly, we evaluated the enrichment of the 10 validated miR-28-5p targets in the pull out sample. We showed that E2F6, TEX-261, MAPK1, MPL, N4BP1, and RAP1B but not BAG1, OTUB1, MAD2L1, and p21 were significantly enriched, suggesting that not all the miR-28-5p targets are regulated by this miRNA in PCa. We then verified whether the miR-28-5p-interacting targets were regulated by this miRNA. We selected E2F6, the most enriched target in the pull out sample, and demonstrated that miR-28-5p downregulated E2F6 at the protein level suggesting that our approach was effective. In general terms, these findings support the miRNA pull out assay as a useful method to identify context-specific miRNA targets.