MicroRNAs (miRNAs) are small, non-coding RNAs that bind to mRNAs with complementary sequences and inhibit their translation. The miRNAs are released extracellularly into the blood in a stable state and circulating miRNAs are potential biomarkers for various physiological states. In humans, research on miRNA for anti-doping purposes is ongoing and several miRNAs have been reported to be altered by the administration of drugs. Circulating miRNAs may also have potential applications in detecting endogenous substances such as hydrocortisone (HYD) and erythropoietin (EPO) which are difficult to distinguish as exogenous or endogenous using direct drug testing. However, there are few reports on circulating miRNA after drug administration in horses. Therefore, this study aimed to identify circulating miRNAs that could be used for drug testing, and to evaluate their potential as biomarkers for doping control in horses.
Plasma samples were collected from six HYD-treated horses. Small RNA sequencing (small RNA-seq) and reverse transcription quantitative PCR (RT-qPCR) were performed to identify miRNAs altered by HYD administration. An additional six horses were used in experiments involving endogenous HYD elevation through exercise and ACTH administration. As a result, three miRNAs (two novel ones and miR-133a) showed potential for distinguishing between exogenous and endogenous HYD. Plasma samples were also collected from 14 pregnant mares in studies investigating biomarkers associated with increased endogenous EPO. Same approach was conducted to identify miRNAs that fluctuated during the increase in endogenous EPO levels. Three miRNAs (one novel miRNA, miR-182 and miR-9180) were found to vary in response to elevated endogenous EPO.
In conclusion, we identified circulating miRNAs that could serve as potential biomarkers for the detection of HYD administration and elevated endogenous EPO levels in horses. This study provides foundational knowledge of circulating miRNAs in horses and suggests that circulating miRNAs could be useful biomarkers for doping control in horses.