Abstract
BackgroundCirculating miRNAs (ci-miRNAs) are endogenous, non-coding RNAs emerging as potential diagnostic biomarkers. Equine miRNAs have been previously identified including subsets of tissue-specific miRNAs. In order to investigate ci-miRNAs as diagnostic tools, normal patterns of expression for different scenarios including responses to exercise need to be identified. Human studies have demonstrated that many ci-miRNAs are up-regulated following exercise with changes in expression patterns in skeletal muscle. However, technical challenges such as haemolysis impact on accurate plasma ci-miRNA quantification, with haemolysis often occurring naturally in horses following moderate-to-intense exercise. The objectives of this study were to identify plasma ci-miRNA profiles and skeletal muscle miRNAs before and after exercise in Thoroughbreds (Tb), and to evaluate for the presence and effect of haemolysis on plasma ci-miRNA determination. Resting and post-exercise plasma ci-miRNA profiles and haemolysis were evaluated in twenty 3 year-old Tbs in sprint training. Resting and post-exercise skeletal muscle miRNA abundance was evaluated in a second cohort of eleven 2 year-old Tbs just entering sprint training. Haemolysis was further quantified in resting blood samples from twelve Tbs in sprint training. A human plasma panel containing 179 miRNAs was used for profiling, with haemolysis assessed spectrophotometrically. Data was analysed using a paired Student’s t-test and Pearson’s rank correlation.ResultsPlasma ci-miRNA data for 13/20 horses and all skeletal muscle miRNA data passed quality control. From plasma, 52/179 miRNAs were detected at both time-points. Haemolysis levels were greater than the threshold for accurate quantification of ci-miRNAs in 18/25 resting and all post-exercise plasma samples. Positive correlations (P < 0.05) between haemolysis and miRNA abundance were detected for all but 4 miRNAs, so exercise-induced changes in plasma ci-miRNA expression could not be quantified. In skeletal muscle samples, 97/179 miRNAs were detected with 5 miRNAs (miR-21-5p, let-7d-3p, let-7d-5p, miR-30b-5p, miR-30e-5p) differentially expressed (DE, P < 0.05) between time-points.ConclusionsThe degree of haemolysis needs to be determined prior to quantifying plasma ci-miRNA expression from horses in high-intensity exercise training. Identification of DE miRNAs in skeletal muscle indicates modification of miRNA expression may contribute to adaptive training responses in Tbs. Using a human plasma panel likely limited detection of equine-specific miRNAs.
Highlights
Circulating miRNAs are endogenous, non-coding RNAs emerging as potential diagnostic biomarkers
Cohort A: Plasma ci-miRNA In the equine plasma samples, 29% (52/179) of the miRNAs on the human plasma panel were detected at both timepoints, all of which had increased expression (3.23 ± 1.5 fold mean increase, 1.1–10.3 range increase) at 5 mins after exercise (T5min) when compared to before exercise (T0)
Absorbance >0.2 absorption units (AU) at 414 nm were detected in 11/13 T0 and all T5min plasma samples for horses in cohort A, with absorbance peaks significantly higher in the T5min samples (P = 1.2−05; Fig. 1)
Summary
Circulating miRNAs (ci-miRNAs) are endogenous, non-coding RNAs emerging as potential diagnostic biomarkers. MicroRNAs (miRNAs) are endogenous, non-coding RNAs with complex roles causing translational repression/degradation of bound mRNA [1], regulating many cellular processes [2] They have intracellular effects, are extremely stable when secreted into the bloodstream [3] and have been identified in multiple body fluids [4]. MiRNAs are often tissue-specific and can be secreted from their tissue of origin into the bloodstream bound to lipoproteins [5] or packaged into extracellular microparticles [6] When in blood they are termed circulating miRNAs (ci-miRNAs) [7] and are emerging as potential diagnostic biomarkers due to their stability [8] and ease of access for sample collection. It has been hypothesised that skeletal muscle may contribute to changes in differentially expressed (DE) ci-miRNAs in response to exercise training by releasing exosomes into plasma [15, 16], supporting the possibility that DE plasma ci-miRNAs could be used to assess adaptive training responses of skeletal muscle
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