Profiling of plasma extracellular vesicle transcriptome reveals that circRNAs are prevalent and differ between multiple sclerosis patients and healthy controls

Abstract

Background Extracellular vesicles (EVs) are released by almost all cell types and are implicated in a number of biological and pathological processes including autoimmune diseases such as multiple sclerosis (MS). Differences in the number and cargo of plasma derived EVs have been described in MS. In this work, we attempt to characterise the EV RNA cargo of MS patients with particular attention to a recently discovered non coding RNA type, circular RNAs (circRNAs), which have been shown to play important roles in physiology and disease and hold a great biomarker potential. Methods Plasma was collected from 20 MS patients and 8 healthy controls (HC) and total RNA was isolated from plasma-derived extracellular vesicles isolated by differential centrifugation. Samples were pooled in disease status, sex and age paired groups and RNA-Sequenced with Illumina HiSeq X Ten after rRNA depletion. CircRNAs were detected by both find_circ and CIRI2 and their quantification was based on BSJ-spanning reads. Linear transcripts were quantified by HTSEq. Differential expression analysis was performed using DESeq2. RNA type distribution was analyzed based on biomart classification. MiRNA binding site number and density for circRNAs was calculated based on the TargetScan prediction performed by Circinteractome. CircRNA secondary structure prediction was calculated by their length normalized Gibbs free energy. All the statistical analysis were performed in Rstudio. Results The EV linear and circular transcriptome of MS patients and controls is characterized and compared to the transcriptome previously described in leucocytes. Results reveal differences in the RNA type distribution, showing that circRNAs are enriched in EVs compared to leucocytes. Nevertheless, highly structured circRNAs are preferentially retained in leukocytes. Additionally, differential expression analysis reports significant differences in circRNA and linear RNA expression between MS patients and controls as well as between different MS types. Conclusions The plasma derived EV RNA cargo is not a representation of leukocytes’ cytoplasm but a message that must be studied. Moreover, our results reveal the interest of circRNAs as part of this message highlighting the importance to further understand the RNA regulation in MS.