McGivney B A, Griffin M E, Gough K F, McGivney C L, Browne J A, Hill E W, Katz L M
UCD School of Agriculture and Food Science, Section of Animal & Crop Sciences, University College Dublin, Belfield, Dublin 4, Ireland.
UCD School of Veterinary Medicine, Section of Veterinary Biomedical Sciences, University College Dublin, Belfield, Dublin 4, Ireland.
BMC Vet Res. 2017 Nov 22;13(1):347. doi: 10.1186/s12917-017-1277-z.
Circulating 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.
Plasma 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.
The 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.
循环微小RNA（ci-miRNAs）是内源性非编码RNA，正成为潜在的诊断生物标志物。先前已鉴定出马的微小RNA，包括组织特异性微小RNA的子集。为了研究ci-miRNAs作为诊断工具，需要确定不同情况下（包括运动反应）的正常表达模式。人体研究表明，许多ci-miRNAs在运动后会随着骨骼肌表达模式的变化而上调。然而，诸如溶血等技术挑战会影响血浆ci-miRNA定量的准确性，而在中高强度运动后，马体内经常会自然发生溶血。本研究的目的是确定纯种马（Tb）运动前后的血浆ci-miRNA谱和骨骼肌微小RNA，并评估溶血对血浆ci-miRNA测定的影响。对20匹进行短跑训练的3岁Tb马的静息和运动后血浆ci-miRNA谱及溶血情况进行了评估。对另一组11匹刚开始短跑训练的2岁Tb马的静息和运动后骨骼肌微小RNA丰度进行了评估。对12匹进行短跑训练的Tb马的静息血样中的溶血情况进行了进一步定量。使用包含179种微小RNA的人血浆检测板进行分析，通过分光光度法评估溶血情况。数据采用配对学生t检验和皮尔逊等级相关性分析。
20匹马中的13匹马的血浆ci-miRNA数据以及所有骨骼肌微小RNA数据均通过质量控制。在血浆中，两个时间点均检测到179种微小RNA中的52种。在18/25个静息血浆样本和所有运动后血浆样本中，溶血水平高于准确定量ci-miRNAs的阈值。除4种微小RNA外，其余所有微小RNA的溶血与微小RNA丰度之间均存在正相关（P<0.05），因此无法量化运动诱导的血浆ci-miRNA表达变化。在骨骼肌样本中，检测到179种微小RNA中的97种，其中5种微小RNA（miR-21-5p、let-7d-3p、let-7d-5p、miR-30b-5p、miR-30e-5p）在不同时间点差异表达（DE，P<0.05）。
在对高强度运动训练的马进行血浆ci-miRNA表达定量之前，需要确定溶血程度。骨骼肌中差异表达微小RNA的鉴定表明，微小RNA表达的改变可能有助于纯种马的适应性训练反应。使用人血浆检测板可能会限制对马特异性微小RNA的检测。
