MiR-26a-5p as a Reference to Normalize MicroRNA qRT-PCR Levels in Plasma Exosomes of Pediatric Hematological Malignancies

Carlotta C Damanti,Lara Mussolin,Marta Pillon,Stefania Bortoluzzi,Enrico Gaffo,Piero Di Battista,Anna Tosato,Elisa Carraro,Anna Garbin,Caterina Elia,Alessandra Biffi,Maurizio Mascarin,Ilaria Gallingani,Federica Lovisa

doi:10.3390/cells10010101

Abstract

Plasma exosomal microRNAs (miRNAs) are considered as valid circulating biomarkers for cancer diagnosis and prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR), the most commonly used technique to assess circulating miRNA levels, requires a normalization step involving uniformly expressed endogenous miRNAs. However, there is still no consensus on reference miRNAs for plasma exosomal miRNA abundance normalization. In this study, we identified a panel of miRNAs with stable abundance by analyzing public plasma exosome RNA-seq data and selected miR-486-5p, miR-26a-5p, miR-423-5p and miR191-5p as candidate normalizers. Next, we tested the abundance variation of these miRNAs by qRT-PCR in plasma exosomes of healthy donors and pediatric patients with anaplastic large cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia. MiR-486-5p and miR-26a-5p showed the most stable levels, both between healthy controls and patients and among the malignancies analyzed. In light of previous reports on miRNA stability in different exosome isolation methods, our data indicated that miR-26a-5p is a bona fide reference miRNA for qRT-PCR normalization to evaluate miRNA abundance from circulating plasma exosomes in studies of hematological malignancies.

Highlights

The use of liquid biopsy is of high interest in cancer research as a valuable noninvasive source of biomarkers [1]
We evaluate the expression stability of different miRNAs in plasma exosomes derived from healthy donors and pediatric patients with different hematological malignancies and identify a reliable reference miRNA for this clinical context
Vesicle-enclosed circulating miRNA studies using Quantitative real-time polymerase chain reaction (qRT-PCR) aim to disclose noninvasive biomarkers depending on the choice of an endogenous reference miRNA to normalize the measured abundances

Summary

Introduction

The use of liquid biopsy is of high interest in cancer research as a valuable noninvasive source of biomarkers [1]. Among different biological materials that circulate in the bloodstream, such as circulating tumor cells, cell-free DNA and RNA, proteins and metabolites, extracellular vesicles (EVs) are considered as the most promising carriers of circulating biomarkers. An important category of EVs is represented by exosomes, 40–150 nm endosome-derived vesicles originating from multivesicular bodies [4]. Exosomes are involved in cell–cell communications as they transfer proteins, lipids, DNAs, messenger RNAs (mRNAs) and noncoding RNAs originating from the source cell to different recipient cells [5,6]. MicroRNAs (miRNAs) enriched in serum and plasma exosomes have been suggested as promising diagnostic and prognostic biomarkers for cancer, in view of their relatively high stability [8] and their well-known role as post-transcriptional regulators of gene expression [9]. In hematological malignancies [10], several studies have demonstrated the pathogenetic importance of exosomal miRNAs and suggested their use for diagnostic and prognostic purposes [11]

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Results

Conclusion