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Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein

A pressing need for new chronic kidney disease (CKD) biomarkers persists. MicroRNAs (miRNAs) are emerging as a novel class of disease biomarkers in body fluids, but mechanisms conferring their stability in urine have not been fully elucidated. Here we investigated stabilization in human urine of ubi...

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Autores principales: Beltrami, Cristina, Clayton, Aled, Newbury, Lucy J., Corish, Peter, Jenkins, Robert H., Phillips, Aled O., Fraser, Donald J., Bowen, Timothy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932545/
https://www.ncbi.nlm.nih.gov/pubmed/29861421
http://dx.doi.org/10.3390/ncrna1020151
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author Beltrami, Cristina
Clayton, Aled
Newbury, Lucy J.
Corish, Peter
Jenkins, Robert H.
Phillips, Aled O.
Fraser, Donald J.
Bowen, Timothy
author_facet Beltrami, Cristina
Clayton, Aled
Newbury, Lucy J.
Corish, Peter
Jenkins, Robert H.
Phillips, Aled O.
Fraser, Donald J.
Bowen, Timothy
author_sort Beltrami, Cristina
collection PubMed
description A pressing need for new chronic kidney disease (CKD) biomarkers persists. MicroRNAs (miRNAs) are emerging as a novel class of disease biomarkers in body fluids, but mechanisms conferring their stability in urine have not been fully elucidated. Here we investigated stabilization in human urine of ubiquitously expressed miR-16, and miR-192, which we have shown previously to be downregulated in renal fibrosis, by association with extracellular vesicles and with argonaute protein (AGO) 2. Endogenous urinary miR-16 was significantly more resistant to RNase-mediated degradation than exogenous, spiked-in, Caenorhabditis elegans cel-miR-39. We used our previously optimized high-resolution exosome isolation protocol with sucrose gradient ultracentrifugation to sub-fractionate the primary extracellular vesicle-rich urinary pellet. MiR-16 and miR-192 were enriched in exosomal sucrose gradient fractions, but were also detected in all other fractions. This suggested association of urinary miRNAs with other urinary extracellular vesicles and/or pellet components, complicating previous estimates of miRNA:exosome stoichiometry. Proteinase K digestion destabilized urinary miR-16 and we showed, for the first time, RNA-immunoprecipitation of urinary miR-16:AGO2 and miR-192:AGO2 complexes. Association with exosomes and AGO2 stabilized urinary miR-16 and miR-192, suggesting quantitative urinary miRNA analysis has the potential to identify novel, non-invasive CKD biomarkers.
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spelling pubmed-59325452018-05-14 Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein Beltrami, Cristina Clayton, Aled Newbury, Lucy J. Corish, Peter Jenkins, Robert H. Phillips, Aled O. Fraser, Donald J. Bowen, Timothy Noncoding RNA Article A pressing need for new chronic kidney disease (CKD) biomarkers persists. MicroRNAs (miRNAs) are emerging as a novel class of disease biomarkers in body fluids, but mechanisms conferring their stability in urine have not been fully elucidated. Here we investigated stabilization in human urine of ubiquitously expressed miR-16, and miR-192, which we have shown previously to be downregulated in renal fibrosis, by association with extracellular vesicles and with argonaute protein (AGO) 2. Endogenous urinary miR-16 was significantly more resistant to RNase-mediated degradation than exogenous, spiked-in, Caenorhabditis elegans cel-miR-39. We used our previously optimized high-resolution exosome isolation protocol with sucrose gradient ultracentrifugation to sub-fractionate the primary extracellular vesicle-rich urinary pellet. MiR-16 and miR-192 were enriched in exosomal sucrose gradient fractions, but were also detected in all other fractions. This suggested association of urinary miRNAs with other urinary extracellular vesicles and/or pellet components, complicating previous estimates of miRNA:exosome stoichiometry. Proteinase K digestion destabilized urinary miR-16 and we showed, for the first time, RNA-immunoprecipitation of urinary miR-16:AGO2 and miR-192:AGO2 complexes. Association with exosomes and AGO2 stabilized urinary miR-16 and miR-192, suggesting quantitative urinary miRNA analysis has the potential to identify novel, non-invasive CKD biomarkers. MDPI 2015-09-14 /pmc/articles/PMC5932545/ /pubmed/29861421 http://dx.doi.org/10.3390/ncrna1020151 Text en © 2015 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 license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Beltrami, Cristina
Clayton, Aled
Newbury, Lucy J.
Corish, Peter
Jenkins, Robert H.
Phillips, Aled O.
Fraser, Donald J.
Bowen, Timothy
Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
title Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
title_full Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
title_fullStr Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
title_full_unstemmed Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
title_short Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
title_sort stabilization of urinary micrornas by association with exosomes and argonaute 2 protein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932545/
https://www.ncbi.nlm.nih.gov/pubmed/29861421
http://dx.doi.org/10.3390/ncrna1020151
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