Identification of miRNA Reference Genes in Extracellular Vesicles from Adipose Derived Mesenchymal Stem Cells for Studying Osteoarthritis.
Int J Mol Sci
; 20(5)2019 Mar 05.
Article
en En
| MEDLINE
| ID: mdl-30841483
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.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Osteoartritis
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MicroARNs
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Trasplante de Células Madre Mesenquimatosas
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Células Madre Mesenquimatosas
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Vesículas Extracelulares
Tipo de estudio:
Diagnostic_studies
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Prognostic_studies
Límite:
Female
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Humans
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Middle aged
Idioma:
En
Revista:
Int J Mol Sci
Año:
2019
Tipo del documento:
Article
País de afiliación:
Italia