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MiRAR—miRNA Activity Reporter for Living Cells

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe...

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Autores principales: Turk, Matthew A., Chung, Christina Z., Manni, Emad, Zukowski, Stephanie A., Engineer, Anish, Badakhshi, Yasaman, Bi, Yumin, Heinemann, Ilka U.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027049/
https://www.ncbi.nlm.nih.gov/pubmed/29921790
http://dx.doi.org/10.3390/genes9060305
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author Turk, Matthew A.
Chung, Christina Z.
Manni, Emad
Zukowski, Stephanie A.
Engineer, Anish
Badakhshi, Yasaman
Bi, Yumin
Heinemann, Ilka U.
author_facet Turk, Matthew A.
Chung, Christina Z.
Manni, Emad
Zukowski, Stephanie A.
Engineer, Anish
Badakhshi, Yasaman
Bi, Yumin
Heinemann, Ilka U.
author_sort Turk, Matthew A.
collection PubMed
description microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.
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spelling pubmed-60270492018-07-13 MiRAR—miRNA Activity Reporter for Living Cells Turk, Matthew A. Chung, Christina Z. Manni, Emad Zukowski, Stephanie A. Engineer, Anish Badakhshi, Yasaman Bi, Yumin Heinemann, Ilka U. Genes (Basel) Article microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models. MDPI 2018-06-19 /pmc/articles/PMC6027049/ /pubmed/29921790 http://dx.doi.org/10.3390/genes9060305 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Turk, Matthew A.
Chung, Christina Z.
Manni, Emad
Zukowski, Stephanie A.
Engineer, Anish
Badakhshi, Yasaman
Bi, Yumin
Heinemann, Ilka U.
MiRAR—miRNA Activity Reporter for Living Cells
title MiRAR—miRNA Activity Reporter for Living Cells
title_full MiRAR—miRNA Activity Reporter for Living Cells
title_fullStr MiRAR—miRNA Activity Reporter for Living Cells
title_full_unstemmed MiRAR—miRNA Activity Reporter for Living Cells
title_short MiRAR—miRNA Activity Reporter for Living Cells
title_sort mirar—mirna activity reporter for living cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027049/
https://www.ncbi.nlm.nih.gov/pubmed/29921790
http://dx.doi.org/10.3390/genes9060305
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