MicroRNA Profiling of Exosomes Derived from Red Blood Cell Units: Implications in Transfusion-Related Immunomodulation

Abstract

Purpose. To elucidate the microRNAs existent in exosomes derived from stored red blood cell (RBC) unit and their potential function. Materials and Methods. Exosomes were isolated from the supernatant derived from stored RBC units by sequential centrifugation. Isolated exosomes were characterized by TEM (transmission electron microscopy), western blotting, and DLS (dynamic light scattering). MicroRNA (miRNA) microarray was performed to detect the expression of miRNAs in 3 exosome samples. Results revealed miRNAs that were simultaneously expressed in the 3 exosome samples and were previously reported to exist in mature RBCs. Functions and potential pathways of some detected miRNAs were illustrated by bioinformatic analysis. Validation of the top 3 abundant miRNAs was carried out by qRT-PCR (quantitative reverse transcription‐polymerase chain reaction). Results. TEM and DLS revealed the mean size of the exosomes (RBC-derived) as 64.08 nm. These exosomes exhibited higher abundance of short RNA than the long RNA. 78 miRNAs were simultaneously detected in 3 exosome samples and mature RBCs. Several biological processes might be impacted by these miRNAs, through their target gene(s) enriched in a particular signalling pathway. The top 3 (abundant) miRNAs detected were as follows: miR-125b-5p, miR-4454, and miR-451a. qRT-PCR revealed higher abundance of miR-451a than others. Only miR-4454 and miR-451a abundance tended to increase with increasing storage time. Conclusion. Exosomes derived from stored RBC units possessed multiple miRNAs and, hence, could serve various functions. The function of exosomes (RBC-derived) might be implemented partly by the predominantly enriched miR-451a.

Extracellular Vesicles from Red Blood Cells and Their Evolving Roles in Health, Coagulopathy and Therapy

“…Approximately 78 miRNAs are reported within EVs isolated from stored RBCs (3 separate donor units) with a mean size of 64.08 nm. Three miRs—mir-125-b-5p, 4454, and 451a—were most abundant and present in all three donor unit isolated exosomes and miR-4454 and miR-451a levels were observed to increase with the duration of refrigerator storage time [ 93 ]. RNA sequencing [ 93 ] predicted target genes for the top ten most abundant RBC exosomal miRNAs: miR29a-3p, 101-3p, 125b-5p, 22-3p, 30b-5p, 451a, 30c-5p, 4454, 1260b, and 96-5P [ 93 ].…”

“…Three miRs—mir-125-b-5p, 4454, and 451a—were most abundant and present in all three donor unit isolated exosomes and miR-4454 and miR-451a levels were observed to increase with the duration of refrigerator storage time [ 93 ]. RNA sequencing [ 93 ] predicted target genes for the top ten most abundant RBC exosomal miRNAs: miR29a-3p, 101-3p, 125b-5p, 22-3p, 30b-5p, 451a, 30c-5p, 4454, 1260b, and 96-5P [ 93 ]. Among those identified, MiR-125b-5p acts as a negative regulator of inflammatory genes through the TRAF6/MAPKs/NF-κB pathway in human osteoarthritic chondrocytes [ 94 ] and modulates the inflammatory state of macrophages by targeting B7-H4 [ 95 ].…”

“…MiRNA-144-5p and miR-30b-5p demonstrate increased expression levels in high altitude dweller’s RBCs based on RNA sequencing data and both miRNAs may be involved in erythroid and nitric oxide (NO) related signaling pathways during hypoxia. Some of the miRNAs reported in RBCEVs [ 93 ] were either upregulated (miR-30b-5p [ 101 ], miR-125b-5p [ 102 ], and miR-451a [ 102 , 103 ]) or downregulated (miR-101 [ 104 ]) during hypoxia. A comprehensive cataloging [ 105 ] of cell specific miRNAs derived from human peripheral blood identifies 271 RBC miRNAs, 90 serum miRNAs, and 5 miRNAs compartmentalized within exosomes.…”

MiRNAs Regulating Oxidative Stress: A Correlation with Doppler Sonography of Uteroplacental Complex and Clinical State Assessments of Newborns in Fetal Growth Restriction

“…Moreover, miRNAs can suppress or enhance angiogenesis, thus realizing the fetal-maternal signal transmission mechanism leading to vascular maladaptation of the fetus [ 40 ]. According to the data of Huang H. et al, miR-125b-5p and miR-451a are the most abundant in exosomes [ 41 ]. In connection with the above, we suggest that a change in miR-125b-5p and miR-451a expression in FGR can contribute to reducing the vascularization of placental villi through inhibition of angiogenic and proliferative factors.…”

Red Blood Cells as Potential Repositories of MicroRNAs in the Circulatory System

“…Their results showed that 38 miRNA species are shared by serum, exosomes, and RBCs, suggesting that blood may contain exosomes derived from RBC and serum may contain miRNAs released by RBC (Juzenas et al, 2017). Researchers have detected that 78 miRNAs are present in exosomes of stored RBCs, and miR-125b-5p, miR-4454, and miR-451a are the most abundant miRNAs with potential functions (Huang et al, 2019). The above studies suggest the possibility that RBCs as repositories of miRNA function by releasing EVs that are loaded with miRNAs.…”