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.
Emerging evidence indicates that extracellular vesicles, particularly exosomes, play a role in several biological processes and actively contribute to cancer development and progression, by carrying and delivering proteins, transcripts and small RNAs (sRNAs). There is high interest in studying exosomes of cancer patients both to develop non-invasive liquid biopsy tests for risk stratification and to elucidate their possible involvement in disease mechanisms. We profiled by RNA-seq the sRNA content of circulating exosomes of 20 pediatric patients with Anaplastic Large Cell Lymphoma (ALCL) and five healthy controls. Our analysis disclosed that non-miRNA derived sRNAs constitute the prominent fraction of sRNA loaded in exosomes and identified 180 sRNAs significantly more abundant in exosomes of ALCL patients compared to controls. YRNA fragments, accounting for most of exosomal content and being significantly increased in ALCL patients, were prioritized for further investigation by qRT-PCR. Quantification of RNY4 fragments and full-length sequences disclosed that the latter are massively loaded into exosomes of ALCL patients with more advanced and aggressive disease. These results are discussed in light of recent findings on the role of RNY4 in the modulation of tumor microenvironment.
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