MicroRNAs (miRNAs) play a crucial role in various physiological and pathological conditions in a broad range of organisms from viruses to animals [1]–[2]. The short non-coding RNAs regulate the expression of thousands of genes by partial or complementary binding to target mRNAs, resulting in translational inhibition and/or degradation of the target mRNAs [3]–[5]. The mature miRNAs are generated from serial stepwise processing [6]. The enzyme Drosha cleaves the single stranded primary miRNA transcripts(pri-miRNAs) to produce a precursor stem-loop secondary structure(pre-miRNAs) [7]. In turn, the pre-miRNAs are cleaved by the enzyme Dicer to produce mature miRNA duplexes [8]. Only one of the two strands acts as a regulatory RNA associated with RNA-induced silencing complex (RISC) [9].

The importance of miRNAs has been recently recognized in veterinary medicine. There is increasing interest in canine miRNAs because of their clinical relevance for human breast cancers [10]–[11]. Down-regulation of miR-130 and miR-30 in canine heart plays an important role in the pathogenesis of chronic atrial fibrillation [12]. Distinct sets of miRNAs including miR-122, miR-193b, and miR-483 were reported to be involved in the development of type-2 diabetes mellitus in cats [13]. A large number of miRNAs have been identified in normal tissues of swine and bovine species [14]–[17]. However, information about equine miRNAs and their role in clinical conditions is scarce. Recently, in silico analytical methodology was applied to identify and characterize mature 354 equine miRNAs [18]. A study revealed 82 new miRNAs in equine sperms, suggesting the role of miRNAs in the regulation of sperm function, fertility and reproduction [19]. Barrey et al. [20] described the expression profile of muscular miRNAs in healthy and myopathic horses using a direct cloning technology.

Forward genetics, bioinformatics and direct cloning have been utilized to study miRNAs [21]. Recently, next generation sequencing (NGS) technology has emerged as a major tool to scrutinize small RNAs including miRNAs [22]. An important feature of the NGS is parallel sequencing of clonally amplified or single DNA molecules that are spatially separated in a flow cell [23]. Due to its ability to generate millions of reads with determined lengths, NGS greatly improves the capacity to identify a large number of novel miRNAs on a genomic scale [24].

Domestic horse, Equus caballus, has been a crucial part of human civilization [25]–[26]. It is economically important throughout the world and has been used for transportation and entertainment. Moreover, horse is a medically valuable animal model because the species shares over 90 hereditary conditions with human disorders [27]–[28] as well as many medical conditions such as allergies and osteoarthritis [29]–[30]. Furthermore, horse can be a valuable model organism for studying biomechanics and exercise physiology [31]. The present study was performed to characterize miRNAs in normal horse tissues by using Illumina high-throughput NGS technology. Our study identifies a list of known miRNAs as well as candidates for novel miRNAs in horse tissues. Additionally, characteristics of horse microRNAs including expression patterns in major tissues, sequence composition and chromosomal distribution were determined. Our study provides background data and information to facilitate research on the role of miRNAs in the pathogenesis of various conditions in horses.

Using Illumina short read sequencing technology, a total of 292 known and 329 novel miRNAs were identified in equine tissues including muscle, liver, and colon. Furthermore, it was found that subsets of miRNAs were differentially expressed in horse tissues, suggesting tissue-specific behavior of equine miRNAs. The identification and characterization of equine miRNAs was carried out using NGS technology to obtain millions of small RNA sequences and to construct small RNA differential expression profile in target organs [23]. Although NGS has the advantage of cloning small RNAs exclusively, it has several disadvantages compared to computational approaches [41]. The limitations include the difficulty of finding miRNAs that are expressed at a low level and a cloning inability due to physical properties such as sequence composition and/or post-transcriptional modifications [42]. Further studies are warranted to elucidate a comprehensive list of equine miRNAs that are biologically important.

Although horses are one of the most important domestic animals, research on equine miRNAs has been limited [36]. Attempts have recently been made to investigate the role of equine miRNAs in various conditions. A study using direct sequencing verified two miRNAs, miR-433 and -127, in horse, thus proving the conservation of the miRNAs throughout mammalian species [37]. Zhou et al.[18] discovered a total of 354 mature miRNAs through an integrated computational analysis of an equine genome, providing a baseline for horse miRNA research. Additionally, they presented valuable references for all major sequence characteristics of horse miRNAs, such as the contents of each nucleotide, A+U, C+G, and base composition at each position of pre-miRNA and mature miRNA sequences. Recently, expression profiles of 82 miRNAs were characterized in equine sperms, indicating their role in the regulation of sperm function, fertility, and reproduction [19]. A study revealed that 10 miRNAs were significantly expressed in equine muscles, some of which were also expressed in blood samples [20]. The present study provides the first experimental data on expression profile of global equine miRNAs using NGS technology, some of which were previously reported in an in silico study [18].

In horse, the characterization of miRNAs in major organs including liver, skeletal muscle, and large intestine has significant clinical relevance to important equine diseases. Horses are more prone to develop colic, a fatal disease complex resulting from numerous factors including strangulation, obstruction and volvulus [38]. Approximately 34% of horses undergoing an exploratory laparotomy had displacement or volvulus of the large colon due to its free mobility in the abdominal cavity [38]–[39]. Unfortunately, despite the magnitude of the problem of equine colic, relatively little has been known about exact causes. Because of their nature and human usage, horses are affected by various types of muscle diseases including exercise-associated myopathy, post-exhaustion syndrome, and nutritional myopathy [40]. Horses frequently develop liver disease because of their grazing habits, causing significant economic losses [41]. Given that the expression profile of miRNA is specific for organs and/or tissues [3], it is clinically important to discover subsets of organ-specific miRNAs. Our study revealed that both known and novel miRNAs in colon have a larger proportion of total miRNAs than other tissues. In contrast to other mammals, the equine large intestine has relatively complicated anatomical structures and physiological functions to facilitate a steady flow of nutrient [42]. Our data may indicate that the colon-specific miRNAs have more sophisticated roles in the colon's regulatory system [17].

Barrey et al. [20] demonstrated that muscle-specific miRNAs were also detected in blood samples of horses with heritable muscular pathology. The study suggested the potential value of the miRNAs for the development of novel and minimally invasive methodology for diagnosing muscular pathology in horses [20]. Similarly, further studies are warranted to investigate expression profile of tissue-specific miRNAs in the blood and may provide valuable biomarker to identify organ damage and various disease conditions.

Characterization of 5′-end sequences of the miRNA is important because seed sequence of a miRNA is critical in miRNA-target mRNA binding [4]–[43]. Analysis of nucleotide sequences in eukaryotic miRNAs showed a clear bias for U or A at the 5′ position [44]. Our study revealed that U was the most frequent nucleotide at the 5′-end of tissue-specific miRNAs, followed by A. This finding is consistent with results from the published studies on miRNAs of other organisms [18–32–33–45]. In plant, U at the end of 5′ was proposed as being critical for the biogenesis of miRNAs through the recognition of targeted miRNA precursor by RISC [46]. The C+G contents in the 6^th position of the seed region were known to induce or enhance the function of a miRNA [45]. In our study, all tissue miRNAs had high percentages of C+G from the third to sixth positions in the sequence. The miRNAs in colon had more distinct sequence bias showing high percentage of C+G in all of the seed nucleotides such a bias may be responsible for distinct functions in colon compared to different organs [45]. The base bias in seed sequence may provide a valuable reference for further study on the identification of regulatory cellular function of miRNAs.

In our study, the equine miRNAs were located on diverse chromosomes. An intriguing feature was that the distribution of miRNAs along the chromosomes was uneven; some chromosomes were relatively miRNA-rich while others were miRNA-poor. Based on chromosomal mapping, more than half of the known miRNAs that exists within 3 kb threshold of each other were considered as a polycistronic transcript. It is interesting to note that our study revealed a number of putative miRNA clusters in horse genome. Consistent with a published study on horse miRNAs [18], a miR-17-92 cluster was also detected on chromosome 17 in our study. This cluster is reportedly harboring miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a, and is a well-preserved cluster in human and all vertebrates [11]–[18]. In mammals, the miR-17-92 cluster is divided into two cluster paralogs: miR-106b-25 cluster and the miR-106a-363 cluster [47]. In our study, the former cluster was located on chromosome 13 and the latter cluster on chromosome X. Further studies addressing the precise role of polycistronic transcripts in horse may enhance our understanding about the mechanisms of miRNA generation.

The present study revealed that approximately 53% of the known miRNAs were observed as part of a polycistronic unit. The finding suggests that the clustering property of miRNAs is preserved in horse, although there is partial discrepancy in chromosomal location compared with human data [47]. The proportion of miRNAs in polycistronic units is similar to that of zebrafish miRNAs (50%) [48]. It is noteworthy that approximately 72% of clustered equine miRNAs in our study correspond to those detected by the computational analysis of horse [18]. However, 45 of the 160 miRNAs that are considered as a polycistron could not be identified at orthologous positions in the in silico study [18]. We speculated that difference in analysis methodologies is responsible for this discrepancy in miRNA clustering. In our study the miRNA clustering was made based on sequence data from NGS technology, whereas the in silico study utilized a limited number of miRNAs identified by BLAST search. Zhou et al. [18] reported polycistronic miRNAs such as miR-302a, -302b, -302c, -302d, and -367 on chromosome 2 and miR-1912 and -1264 on chromosome X, these were not found in our result. In contrast, newly found polycistronic miRNAs in our study were not reported by Zhou et. al [16]. A number of questions for the clustering property for equine miRNAs remain to be clarified in further studies.

In the present study, we identified 292 known and 329 novel miRNAs in normal equine tissues by using NGS technology. This study significantly enriches the horse miRNA database and presents a valuable reference for equine miRNAs. In addition, the global miRNA expression profile in equine tissues revealed that distinct sets of miRNAs were expressed in a tissue-specific pattern. Clustering in chromosomes and sequence characteristics of miRNA may be useful for dissecting their biological functions in horse. Taken together, the present study suggests that equine miRNAs may play an important role in development and function of specific tissues, and therefore, they can be developed as a valuable molecular marker for various pathophysiological conditions.