Abstract
BackgroundTransfusion-related immunomodulation (TRIM) can be caused by exosomes, in which case, microRNAs (miRNAs) are one critical factor impacting exosome behavior. This study aims to investigate and analyze the expression profiles of exosomal miRNA in red blood cell (RBC) suspensions during storage and to identify potential TRIM-related miRNAs as well as their potential functions.MethodsA total of 25 packs of RBC suspensions were randomly collected. Exosome were extracted by ultracentrifugation and then identified and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot (WB). Exosomal miRNA profiles were acquired using gene chips in five packs on week 1 and week 5. The expression data were compared from the two time points identifying accumulated miRNAs with statistical significance and their predicted targeting genes were analyzed. Based on the gene chip results, quantitative reverse transcription-polymerase chain reactions (qRT-PCR) were performed to verify miRNA accumulation in the rest 20 packs sampling on week 1, 3 and 5.ResultsGene chip analysis revealed that most exosomal miRNAs were enriched as the storage period progressed. Compared to samples from week 1, week 5 samples exhibited a total of 539 differential miRNA expressions, among which, 159 were statistically significant (P < 0.05) and 148 (93.08%) were accumulated. In the bioinformatics functional analysis, significant immunoregulatory annotations related to the thyroid hormone, mitogen-activated protein kinase (MAPK), focal adhesion and RAS signaling pathways were identified. The top 17 differential expression miRNAs were validated by qRT-PCR. The results confirmed that all the 17 miRNAs were accumulated with increasing storage time. In particular, miRNA-1246 and miRNA-150-3p were the most enriched strands by more than 150-folds in the 5-week storage period.ConclusionsAs storage progressed, numerous exosomal miRNAs accumulated in the RBC suspensions, which are informatically connected to multiple immuno-signaling pathways. MiRNA-1246 and miRNA-150-3p may be essential mediators impacting the immunoregulation functions of exosomes in RBC suspensions, considering their significant accumulating scales. Further research should therefore focus on the relationship between these miRNAs and TRIM.
Highlights
Transfusion-related immunomodulation (TRIM) can be caused by exosomes, in which case, microRNAs are one critical factor impacting exosome behavior
This study focuses on the miRNA contained in exosomes and intends to analyze the dynamic alterations in the expression of exosomal miRNA in Red blood cell (RBC) suspensions during the storage period
Exosomes extracted from five packs underwent gene chip analysis while the rest exosome samples were collected for quantitative reverse transcription-polymerase chain reaction validation based on gene chip analysis results
Summary
Transfusion-related immunomodulation (TRIM) can be caused by exosomes, in which case, microRNAs (miRNAs) are one critical factor impacting exosome behavior. Red blood cell (RBC) suspension is commonly transfused in clinic for patients suffering from anemia. It can be stored for 35 days at 4 °C in an additive solution with A-form of acid–citrate–dextrose (ACD-A). Studies have reported that prolonged RBC storage may increase the occurrence of complications and adverse clinical outcomes. For trauma patients, prolonged RBC storage may increase the risk of deep vein thrombosis and bacterial infections [5, 6]. These complications are considered to be related to the changes in both RBCs and bioactive substances accumulated during storage [7]
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