The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer
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...
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doaj-27541680fe694539a26070cbfa0bd0f52020-11-24T22:02:27ZengHindawi LimitedInternational Journal of Genomics2314-436X2314-43782017-01-01201710.1155/2017/52148065214806The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate CancerMilena Rizzo0Gabriele Berti1Francesco Russo2Monica Evangelista3Marco Pellegrini4Giuseppe Rainaldi5Non-coding RNA Laboratory, Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, ItalyNon-coding RNA Laboratory, Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, ItalyLaboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT) and Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, ItalyNon-coding RNA Laboratory, Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, ItalyLaboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT) and Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, ItalyNon-coding RNA Laboratory, Institute of Clinical Physiology (IFC), CNR, Via Moruzzi 1, 56124 Pisa, ItalymiR-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.http://dx.doi.org/10.1155/2017/5214806 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Milena Rizzo Gabriele Berti Francesco Russo Monica Evangelista Marco Pellegrini Giuseppe Rainaldi |
spellingShingle |
Milena Rizzo Gabriele Berti Francesco Russo Monica Evangelista Marco Pellegrini Giuseppe Rainaldi The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer International Journal of Genomics |
author_facet |
Milena Rizzo Gabriele Berti Francesco Russo Monica Evangelista Marco Pellegrini Giuseppe Rainaldi |
author_sort |
Milena Rizzo |
title |
The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer |
title_short |
The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer |
title_full |
The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer |
title_fullStr |
The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer |
title_full_unstemmed |
The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer |
title_sort |
mirna pull out assay as a method to validate the mir-28-5p targets identified in other tumor contexts in prostate cancer |
publisher |
Hindawi Limited |
series |
International Journal of Genomics |
issn |
2314-436X 2314-4378 |
publishDate |
2017-01-01 |
description |
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. |
url |
http://dx.doi.org/10.1155/2017/5214806 |
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