Stabilization of urinary microRNAs by association with exosomes and argonaute 2 protein

Beltrami, Cristina, Clayton, Aled ORCID: https://orcid.org/0000-0002-3087-9226, Newbury, Lucy, Corish, Peter, Jenkins, Robert Hywel ORCID: https://orcid.org/0000-0001-8500-9044, Phillips, Aled Owain ORCID: https://orcid.org/0000-0001-9744-7113, Fraser, Donald James ORCID: https://orcid.org/0000-0003-0102-9342 and Bowen, Timothy ORCID: https://orcid.org/0000-0001-6050-0435 2015. Stabilization of urinary microRNAs by association with exosomes and argonaute 2 protein. Non-Coding RNA 1 (2) , pp. 151-166. 10.3390/ncrna1020151

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Abstract

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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine Systems Immunity Research Institute (SIURI)
Subjects:	R Medicine > R Medicine (General)
Publisher:	MDPI
ISSN:	2311-553X
Date of First Compliant Deposit:	6 December 2018
Date of Acceptance:	15 July 2015
Last Modified:	05 Jan 2024 06:44
URI:	https://orca.cardiff.ac.uk/id/eprint/77256

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