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Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing
MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3’untranslated region (3’UTR). The identification of bona fide targets is challenging for researchers working on the functional as...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268774/ https://www.ncbi.nlm.nih.gov/pubmed/28125734 http://dx.doi.org/10.1371/journal.pone.0170999 |
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author | De Santi, Chiara Vencken, Sebastian Blake, Jonathon Haase, Bettina Benes, Vladimir Gemignani, Federica Landi, Stefano Greene, Catherine M. |
author_facet | De Santi, Chiara Vencken, Sebastian Blake, Jonathon Haase, Bettina Benes, Vladimir Gemignani, Federica Landi, Stefano Greene, Catherine M. |
author_sort | De Santi, Chiara |
collection | PubMed |
description | MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3’untranslated region (3’UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth. |
format | Online Article Text |
id | pubmed-5268774 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-52687742017-02-06 Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing De Santi, Chiara Vencken, Sebastian Blake, Jonathon Haase, Bettina Benes, Vladimir Gemignani, Federica Landi, Stefano Greene, Catherine M. PLoS One Research Article MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3’untranslated region (3’UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth. Public Library of Science 2017-01-26 /pmc/articles/PMC5268774/ /pubmed/28125734 http://dx.doi.org/10.1371/journal.pone.0170999 Text en © 2017 De Santi et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article De Santi, Chiara Vencken, Sebastian Blake, Jonathon Haase, Bettina Benes, Vladimir Gemignani, Federica Landi, Stefano Greene, Catherine M. Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing |
title | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing |
title_full | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing |
title_fullStr | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing |
title_full_unstemmed | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing |
title_short | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing |
title_sort | identification of mir-21-5p as a functional regulator of mesothelin expression using microrna capture affinity coupled with next generation sequencing |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268774/ https://www.ncbi.nlm.nih.gov/pubmed/28125734 http://dx.doi.org/10.1371/journal.pone.0170999 |
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