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Int J Mol Sci. 2019 Mar 5;20(5). pii: E1108. doi: 10.3390/ijms20051108.

Identification of miRNA Reference Genes in Extracellular Vesicles from Adipose Derived Mesenchymal Stem Cells for Studying Osteoarthritis.

Author information

1
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, 20161 Milan, Italy. enrico.ragni@grupposandonato.it.
2
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, 20161 Milan, Italy. carlotta.perucca@grupposandonato.it.
3
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, 20161 Milan, Italy. deluca.paola@grupposandonato.it.
4
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, 20161 Milan, Italy. alessandra.colombini@grupposandonato.it.
5
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, 20161 Milan, Italy. marco.vigano@grupposandonato.it.
6
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, 20161 Milan, Italy. gaia.lugano@grupposandonato.it.
7
Università degli Studi di Milano, EPIGET-Epidemiology, Epigenetics and Toxicology Lab, Department of Clinical Sciences and Community Health, 20122 Milan, Italy. valentina.bollati@unimi.it.
8
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, 20161 Milan, Italy. laura.degirolamo@grupposandonato.it.

Abstract

Osteoarthritis (OA) leads to chronic pain and disability, and traditional conservative treatments are not effective in the long term. The intra-articular injection of mesenchymal stem cells (MSCs) is considered a novel therapy for OA whose efficacy mainly relies on the adaptive release of paracrine molecules which are either soluble or extracellular vesicles (EVs) embedded. The correct quantification of EV-miRNAs using reliable reference genes (RGs) is a crucial step in optimizing this future therapeutic cell-free approach. The purpose of this study is to rate the stabilities of literature-selected proposed RGs for EV-miRNAs in adipose derived-MSCs (ASCs). EVs were isolated by ultracentrifugation from ASCs cultured with or without inflammatory priming mimicking OA synovial fluid condition. Expression of putative RGs (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA) was scored by using the algorithms geNorm, NormFinder, BestKeeper and ΔCt method. miR-16a-5p/miR-23a-3p yielded the most stable RGs, whereas let-7a-5p/miR-425-5p performed poorly. Outcomes were validated by qRT-PCR on miR-146a-5p, reported to be ASC-EVs enriched and involved in OA. Incorrect RG selection affected the evaluation of miR-146a-5p abundance and modulation by inflammation, with both values resulting strongly donor-dependent. Our findings demonstrated that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for ASC-EVs miRNAs evaluation. A correct approach would increase the accuracy of embedded molecule assessments aimed to develop therapeutic strategies for the treatment of OA based on EVs.

KEYWORDS:

adipose-derived mesenchymal stem cells; extracellular vesicles; miRNA, reference genes; osteoarthritis

PMID:
30841483
DOI:
10.3390/ijms20051108
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