Circulating microRNAs as biomarkers for recombinant human erythropoietin (r-HuEPO) doping detection

Administration of recombinant human erythropoietin (rHuEpo) improves performance and hence is subject to abuse by athletes. The detection of rHuEpo doping remains a major challenge at present. The aim of the current study was to investigate whether circulating microRNA (miRNA) can be used for detecting r-HuEpo doping. Twenty trained males received rHuEpo injections of 50 IU∙kg -1 body mass every two days for 4 weeks. Blood was obtained 2 weeks before, during and 4 weeks after administration. For this pilot analysis, plasma miRNA expression was assessed at selected time points using the Affymetrix GeneChip 3.0 and the miScript 384 HC PCR Array (Qiagen). For the Affymetrix microarray data, GC content adjustment was carried out prior to background correction and quantile normalization. For the Qiagen PCR Array data, raw CT values exported from the RQ manager were firstly normalized to the spike-in control (i.e. cel-miR-39-3p), then to the mean of commonly expressed miRNAs with a CT < 35 prior to formal analysis. 1733 human miRNAs are present on the GeneChip 3.0. Eighty-two of the 1733 miRNAs were commonly expressed across all samples and 773 miRNAs were found in at least one sample. Six miRNAs showed significant fold changes following r-HuEpo, with 5 miRNAs up-regulated (fold change ranging from 2.03-3.07, P < 0.05) and 1 miRNA down-regulated (fold change: 2.56, P = 0.042). Out of 372 human miRNAs present (Qiagen), 205 human miRNAs were detected in all samples and 164 miRNAs were found in at least one sample. Two miRNAs were differentially expressed (fold changes: 3.4 - 3.84, P ≤ 0.03) following r-HuEpo. For significantly expressed miRNAs identified by both methods, 6 common miRNAs were found to be either predicted or validated miRNAs potentially involved in Epo signalling pathways. Analysis of identified miRNAs in the remaining samples will be required in order to determine whether circulating miRNAs can be effectively used for r-HuEpo doping detection. miRNAs isolated from red blood cells before and after r-HuEpo are also currently being analysed using next generation sequencing.