Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse

Abstract

The adaptive response to extreme endurance exercise might involve transcriptional and translational regulation by microRNAs (miRNAs). Therefore, the objective of the present study was to perform an integrated analysis of the blood transcriptome and miRNome (using microarrays) in the horse before and after a 160 km endurance competition. A total of 2,453 differentially expressed genes and 167 differentially expressed microRNAs were identified when comparing pre-and post-ride samples. We used a hypergeometric test and its generalization to gain a better understanding of the biological functions regulated by the differentially expressed microRNA. In particular, 44 differentially expressed microRNAs putatively regulated a total of 351 depleted differentially expressed genes involved variously in glucose metabolism, fatty acid oxidation, mitochondrion biogenesis, and immune response pathways. In an independent validation set of animals, graphical Gaussian models confirmed that miR-21-5p, miR-181b-5p and miR-505-5p are candidate regulatory molecules for the adaptation to endurance exercise in the horse. To the best of our knowledge, the present study is the first to provide a comprehensive, integrated overview of the microRNA-mRNA co-regulation networks that may have a key role in controlling post-transcriptomic regulation during endurance exercise.The physiological and biochemical demands of endurance exercise elicit both muscle-based and systemic responses. The main adaptations to endurance exercise include improvement of mechanical, metabolic, neuromuscular and contractile functions in muscle 1 , correction of electrolyte imbalance 2 , a decrease in glycogen storage 3 and an increase in mitochondrial biogenesis in muscle tissue 4 , and the modulation of oxidative stress 5 , intestinal permeability, muscle damage, systemic inflammation and immune responses 5 . Consequently, adaptations to endurance exercise are influenced by the transcriptional and translational regulation of genes that encode the proteins controlling these processes 5 . Over the past decade, microRNAs (miRNAs) have emerged as novel elements in the rapid, reversible regulation of transcription and translation 6 . MiRNAs are small non-coding RNAs molecules (~19-24 bp in length) that are synthesized from short hairpin precursors and that reportedly degrade or inhibit the translation of their target genes by binding to the 3′ untranslated region (UTR) of coding mRNAs 7 . In fact, miRNAs molecules may regulate up to one-third of the mammalian transcriptome 8 and appear to be stable outside the cell (e.g. when incorporated into exosomes 9 , microvesicles 10 , lipoproteins 11 or Argonaute2 protein complexes 12 ).

Novel equine tissue miRNAs and breed-related miRNA expressed in serum

“…From the 329 novel miRNAs reported by Kim et al [9] only 11 had exactly the same mature sequence as novel miRNAs reported here. From the 896 putative novel miRNAs included in the microarrays used by Mach et al [32] only two had exactly the same mature sequence and further 24 had few nucleotides shorter mature sequence compared to the miRNAs identified here (Additional file 4: Table S3). One of the reasons of such discrepancy is the different method used for putative novel miRNA identification in all of those studies.…”

“…A future EquCab3 reference genome is believed to decrease the number of unplaced scaffolds and improve the quality of the reference genome. Interestingly, we identified a putative novel miRNA encoded by the mitochondrial genome that has been previously described in horse [32], however, the mitomiR was identified only in one heart sample and was excluded from further analysis.…”

“…All samples had an RNA integrity number (RIN) > 8. For further details on SOLiD samples, preparation and sequencing, see [10, 32]. …”

“…The PSSM-affected horses were diagnosed based on GYS2 genotype and histological examination of glycogen digestion as described in [52, 53]. Within each library, three different horses were pooled to decrease the effect of the individual variability, except for the blood samples where each library comes from the same animal (Additional file 1: Table S1) but the samples come from different physiological moments (before and after exercise [32]. Approximately 200 ng of mRNA extracted from the different tissues and blood were used for library construction following the protocol for the SOLiD Total RNA-Seq Kit (Applied Biosystems, Life Technologies).…”

Identification of long non-coding RNA in the horse transcriptome

“…Most efforts towards identifying noncoding RNAs has gone to miRNA identification [12–14]. There are, however, over 4,000 lncRNA transcripts predicted by ENSEMBL and NCBI represented in our transcriptome that we have considered as input for our annotation pipeline.…”