Circulating miRNome detection analysis reveals 537 miRNAS in plasma, 625 in extracellular vesicles and a discriminant plasma signature of 19 miRNAs in children with retinoblastoma from which 14 are also detected in corresponding primary tumors.

Abstract

miRNAs regulate post-transcriptional gene expression in metazoans, and thus are involved in many fundamental cellular biological processes. Extracellular miRNAs are also found in most human biofluids including plasma. These circulating miRNAs constitute a long distance inter cellular communication system and are potentially useful biomarkers. High throughput technologies like microarrays are able to scan a complete miRNome providing useful detection scores that are underexplored. We proposed to answer how many and which miRNAs are detectable in plasma or extracellular vesicles as these questions have not yet been answered. We set out to address this knowledge gap by analyzing the mirRNome in plasma and corresponding extracellular vesicles (EVs) from 12 children affected by retinoblastoma (Rb) a childhood intraocular malignant tumor, as well as from 12 healthy similarly aged controls. We calculated an average of 537 detectable miRNAs in plasma and 625 in EVs. The most miRNA enriched compartment were EVs from Rb cases with an average of 656 detectable elements. Using hierarchical clustering with the detection scores, we generated broad detection mirnome maps and identified a plasma signature of 19 miRNAs present in all Rb cases that is able to discriminate cases from controls. An additional 9 miRNAs were detected in all the samples; within this group, miRNA-5787 and miRNA-6732-5p were highly abundant and displayed very low variance across all the samples, suggesting both are good candidates to serve as plasma references or normalizers. Further exploration considering participant's sex, allowed discovering 5 miRNAs which corresponded only to females and 4 miRNAs corresponding only to males. Target and pathway analysis of these miRNAs revealed hormonal function including estrogen, thyroid signaling pathways and testosterone biosynthesis. This approach allows a comprehensive unbiased survey of a circulating miRNome landscape, creating the possibility to define normality in mirnomic profiles, and to locate where in these miRNome profiles promising and potentially useful circulating miRNA signatures can be found.