Y‐RNA subtype ratios in plasma extracellular vesicles are cell type‐ specific and are candidate biomarkers for inflammatory diseases

Abstract

Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV-and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant noncoding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EVassociated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific "Y-RNA signatures" in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases.

Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery

“…HDLs were reported to transport miRNA 54 , 55 , but the results are not yet conclusive 56 . Still, when miRNA characterization is the end goal of the study, we propose separate quantification of HDLs in EV-enriched sample to evaluate their potential contribution to miRNA expression or treatment of EV-enriched sample with protease/RNase to release and degrade lipoprotein associated miRNAs, as proposed by Driedonks et al 57 . Alternatively, HDLs can be removed from plasma by antibody-mediated clearing of lipoproteins 58 .…”

Immunosuppressive Cell Subsets and Factors in Myeloid Leukemias

“…Other crucial cargo contained and shuttled via EVs is different types of non-coding RNAs, such as microRNAs (miRNAs), lncRNAs (long non-coding RNAs) or Y-RNAs [ 149 ]. These can be shaped by immune stimuli in the microenvironment and thus constitute immune mediators [ 150 ].…”

Distinct Extracellular RNA Profiles in Different Plasma Components

“…In addition, plasma treated by thrombin contains a high abundance of fibrin, which can be precipitated along with its binding RNAs by simple centrifugation. Although RNA profiles from EVs and whole plasma samples have been reported ( Arroyo et al, 2011 ; Turchinovich et al, 2011 ; Allen et al, 2018 ; Driedonks et al, 2020 ), the comprehensive analysis of RNA profiles among different plasma fractions is needed. Furthermore, due to low yield and significant degradation of exRNAs, plasma-derived RNA profiling analysis is technically challenging.…”