RNA-sequencing of equine whole blood identified biomarkers linked to recombinant human erythropoietin (rhEPO)-induced erythropoiesis in thoroughbred horses.

ABSTRACT

Erythropoiesis-stimulating agents (ESAs) continue to be a significant threat to the integrity of human and equine sports. Besides conventional direct testing, monitoring the biomarkers associated with the effects of ESAs may provide a complementary approach via indirect detection to enhance doping control. In this study, we applied RNA-sequencing (RNA-seq) to discover blood RNA biomarkers in Thoroughbred horses after administration with a long-acting form of recombinant human erythropoietin (rhEPO), methoxy polyethylene glycol epoetin beta, Mircera®. A single subcutaneous administration of Mircera® at ~ 4.2 μg/kg was effective in elevating haematocrit, haemoglobin and erythrocyte levels to varying extents in as early as 4 days post-administration in all three horses, which persisted for 40 days post-administration (the last sample collected). RNA-seq was applied to analyse blood transcriptomic changes. Differential gene expression analysis has allowed the identification of 46 genes that showed dramatic and temporary upregulation at 4–11 days after Mircera® administration. STRING analysis has identified the functional enrichment of 15 genes important for erythropoiesis and erythrocyte function, supporting the idea of an increased release into the peripheral circulation of residual RNA-containing reticulocytes after rhEPO exposure, which would otherwise mature normally inside the bone marrow in horses. Machine learning of blood transcriptomes has enabled the discrimination of samples with or without Mircera administration. Therefore, our study has provided new insights into the biological mechanism of erythropoiesis caused by rhEPO administration in horses and has provided evidence supporting the control of misuse of ESAs by monitoring the equine blood transcriptome.