Small RNA sequencing of red blood cell specific miRNAs reveals expression changes after 6 weeks of recombinant EPO (rEPO) administration in healthy recreational athletes

Recombinant erythropoietins (rEPOs) as well as other erythropoiesis-stimulating agents (ESAs) are used for medical treatment of anemia, but likewise ESAs can be misused for performance enhancing purposes in sports. Using small RNAseq, we investigated the effects of low dose administration of rEPO for 6 weeks on the miRNA profile of purified red blood cells (RBCs), in 7 male healthy recreational athletes. Despite lacking most of long RNAs, RBCs contain diverse and abundant miRNAs. For each of the 14 samples 100 ng total RNA, of highly purified RBCs, were used for sequencing with Illumina NextSeq500 system. Following bowtie alignment and mapping of the raw reads, edgeR was used for normalization and differentially expression analysis of mature miRNA raw reads. In addition to the miRNA analysis, typical blood parameters were determined using Sysmex XT-2000i. Out of 1202 discovered mature miRNAs, 13 miRNAs were differentially expressed at a P ≤ 0.05 and FDR ≤ 0.05 with fold changes between 1.680 and -2.176. Out of the 13 miRNAs, 3 miRNAs are well described in literature to be of pivotal importance during the process of erythropoiesis. In silico analysis for the most up-regulated miRNA (MIMAT0017990) revealed a putative binding site at the 3´-untranslated region of GATA2, which is down-regulated during erythropoiesis. MiRNA transfection experiments will be prepared to validate miRNA:mRNA interactions in vitro. In addition to the physiological value of discovering novel miRNAs, which are relevant during the process of erythropoiesis, the results might be of high value for indirect doping detection purposes. Recently, indirect blood doping detection approaches are dependent on changes in traditional blood parameters such as percentage of reticulocytes. The changes in RBC miRNA expression after microdoses rEPO administration, occurring without significant changes of reticulocyte percentage, showed that RBC miRNA can potentially be more sensitive than reticulocyte percentage in detecting microdoses of rEPO.