Why the hype — What are microRNAs and why do they provide unique investigative, diagnostic, and therapeutic opportunities in veterinary medicine?

Abstract

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by inhibiting translation or inducing transcript degradation. MiRNAs act as fine-tuning factors that affect the expression of up to 60% of all mammalian protein coding genes. In contrast to proteins, there is widespread conservation of miRNA sequences across species. This conservation strongly suggests that miRNAs appeared early in evolution and have retained their functional importance. Cross-species conservation provides advantages when compiling candidate markers for health and disease compared to protein-based discoveries. This broad utility is accompanied by the emergence of inexpensive sequencing protocols for the identification of all RNAs in a sample (including miRNAs). With the use of miRNA mimics and antagonists, unique research questions can be answered in biological systems with ‘cause and effect’ methodology. MiRNAs are readily detectable in blood making them attractive candidates as biomarkers for disease. Here, we review their utility as biomarkers and their potential as therapeutic agents or targets to combat disease.

Introduction

MicroRNAs (miRNAs) are potent regulators that have been widely implicated in normal cellular function and in pathogenesis of disease. They affect cellular function primarily by decreasing protein expression through the inhibition of mRNA translation or by inducing destruction of the mRNA template (1). Basic discovery is aided by the cell and tissue specificity of some miRNAs and the availability of miRNA mimic and antagonist molecules to overexpress or suppress specific miRNAs and thereby prove cause and effect and biological functionality (2–5). Another miRNA area of focus is their utility as novel biomarkers due to their stability and presence in circulating and excreted fluids. The phylogenetic conserved nature of many miRNA molecules makes them ideal candidates for cross-species discoveries and translation.

Materials and methods

This is a qualitative narrative review based on keyword searches in search engines common to the field, combined with articles sourced from literature references, conference proceedings, abstracts, and personal knowledge of publications. PubMed, Google Scholar Medline, and the University of Guelph Scholar Portal were used to identify references. Keywords used in the search engines were: miRNA, miRNA biogenesis, role of miRNA, miRNA expression control, miRNA veterinary, biomarkers, osteoarthritis (OA) biomarkers, miRNA biomarkers, miRNA veterinary medicine, miRNA therapy, tissue, stem cell, mesenchymal stem/stromal cell, cartilage, and potency. The reference lists of selected articles revealed additional relevant studies.

MicroRNAs in veterinary medicine

MicroRNA research is an emerging area in veterinary medicine as demonstrated by the increasing number of articles published and indexed in PubMed and the variety of topics investigated. MicroRNAs are of interest to veterinarians for 3 key reasons: their utility as biomarkers, their ability to perform cause and effect biological research, and their utility as therapeutic agents (Figure 1). As of July 2019, 89 papers have been published regarding canine miRNA research since 2007. Six of these papers were published from 2007 to 2010, 11 were published from 2011 to 2013, and 72 were published from 2014 to the present. With respect to miRNA research in horses, only 36 papers have been published since 2010. Thirty-one of these have been published since 2014. Bovine publications are also increasing, with 145 papers since 2008. Only 4 of these papers were published from 2008 to 2010. From 2011 to 2014, 43 papers were published, and from 2015 to the present 98 papers were published. Other domestic species show similar growing interest of miRNA research with 489 papers in total published from 2005 to the present in canine, bovine, equine, porcine, ovine, and caprine miRNA. First we outline the evolution, mechanisms of action, biogenesis, and regulation of miRNAs. Secondly, we review and exemplify implications of miRNA during development, as biomarkers, and their potential use as therapeutic agents.

Figure 1.

miRNA key pillars of interest to veterinarians: Biomarkers, Cause and Effect Biology, and Therapeutic Potential.

Definition of microRNAs

MicroRNAs are small, non-coding RNAs that regulate gene expression by either inhibiting translation or inducing transcript decay. Mature miRNAs are 19 to 23 nucleotides long, single-stranded, and are prominent in the control of post-transcriptional regulation of gene expression (1). Significant evidence has accumulated in the last few years, demonstrating that miRNAs play key roles in regulating cellular processes, including cell proliferation, development, differentiation, and apoptosis (6).

Evolution and microRNAs

In contrast to proteins, there is significant conservation of miRNA sequences across species, which suggests that miRNAs emerged early in the course of evolution and their persistence throughout the animal kingdom reflects their functional importance (7). Positive correlations have been made between the number of miRNAs and the morphological complexity of an organism, further emphasizing their importance in evolution (8).

MicroRNA structure and mechanism of action

MicroRNAs consist of 2 functionally distinct parts: the seed sequences and the seed-distal portion. The seed sequence is shared among miRNAs of the same family and it is located at position 2–7 from the miRNAs’ 5′ end. The unique seed sequence is 7 to 8 nucleotides long and determines miRNAs target specificity (9). Since only the seed distal differs between family members, more than 1 member of the miRNA’s family has the potential to bind to the same mRNA and influence expression downstream. Simultaneously, most mRNAs that contain a binding site for 1 miRNA also harbor sites for additional miRNAs. Therefore, miRNAs exhibit redundancy in function and compensation.

MicroRNAs regulate gene expression though RNA-dependent post-transcriptional silencing mechanisms of mRNA (Figure 2). MiRNAs exert their function as part of a multiprotein complex with RNase activity known as the miRNA-induced silencing complex (miRISC) that forms once a specific miRNA binds with 1 of 4 Argonaute (AGO) proteins and various accessory factors including TRBP1/2 and Dicer (1). MiRNAs interact with the 3′ untranslated region (3′UTR) of the target mRNAs through complementary sequences known as the miRNA response elements (MREs). Once a miRNA binds, the mRNA target is either degraded or blocked from downstream translation depending on the similarity (fidelity) of the miRNA to the target mRNA sequence (10). If the sequence of the miRNA guide strand does not perfectly match the target mRNA, translation of that mRNA is inhibited, but the mRNA remains structurally intact. In contrast, high fidelity interactions occur when the sequence of the miRNA guide strand matches the target mRNA perfectly, which leads to mRNA cleavage by AGO2 and degradation (11). In situations in which multiple miRNAs imperfectly bind to the 3′UTR of a target gene, either of these processes may occur.

Figure 2.

Biogenesis and miRNA functions. Multiple processing steps are present in the biogenesis of mature, single-stranded miRNAs. The miRNAs are processed into pre-miRNAs using either the canonical or non-canonical pathways as shown. Exportin-5 then transports the pre-miRNA to the cytoplasm where the stem-region is cleaved by Dicer, forming an miRNA duplex. One strand of mature miRNA is unwound from a double-stranded miRNA and incorporates RISC and anneals to the 3′UTR of target genes to either repress mRNA translation or induce mRNA degradation. The mature miRNA can also be exported through extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies, or protein bound to lipoproteins (HDL and LDL) or AGO. Figure by Lee and Koch modified from (1).

Biogenesis of microRNAs

The biogenesis of mature, single-stranded miRNAs that form functional miRISCs with AGO proteins, involves multiple processing steps in both the nucleus and the cytoplasm (1). There are 2 miRNA biogenesis pathways; canonical and non-canonical (Figure 2). In the dominant canonical pathway, the transcription of miRNAs begins in the nucleus. Here they are encoded by genes, polycistronic clusters, or intronic regions and transcribed by RNA polymerase II as long primary transcripts known as pri-miRNAs (Figure 2). Pri-miRNAs form unique structures that distinguish miRNAs from other types of RNAs — a hairpin loop with 3 spiral turns with single-stranded RNA attached to the terminus. Pri-miRNAs are processed by the microprocessor complex containing the RNase III enzyme Drosha and DiGeorge Syndrome Critical Region 8 (DGCR8) to form a pre-miRNA, a 70-nucleotide precursor miRNA. Pre-miRNAs are subsequently transported to the cytoplasm by exportin-5, where another RNase III enzyme — Dicer — cleaves the stem region. The resulting miRNA duplex is cleaved with 1 of the strands destined to become the mature miRNA (1,9,12). The non-canonical pathway is less elucidated. Two classes of non-canonical pathways — the Drosha/DGCR8-independent and Dicer-independent pathways (Figure 2) — utilize different combinations of proteins involved in the canonical pathways, including Drosha, Dicer, exportin-5, and AGO2, but ultimately the mature miRNA associates with an AGO protein to form a miRISC as in the canonical pathway (1).

Regulation of microRNA expression

MicroRNAs are encoded in the genome and the locations of genes encoding miRNAs are diverse. Approximately 40% of miRNAs are encoded by the introns of protein coding genes (intragenic miRNAs), about another 50% are the products of non-coding transcripts (intergenic miRNAs), and the remaining miRNAs are encoded by exons (exonic) (13). Like mRNAs, most miRNAs are transcribed by RNA polymerase II although a select few are transcribed by RNA polymerase III (9). The expression of miRNAs is regulated at both the transcriptional and posttranscriptional levels in the biogenesis pathway.

Transcriptional regulation of miRNA expression is a major level of control for tissue and developmentally specific miRNAs. Similar to protein-coding genes, several mechanisms govern the expression of miRNAs at the transcriptional level. The expression of both intragenic and intergenic miRNAs can vary due to mutations in genes encoding proteins involved in miRNA biogenesis or can be regulated by hypermethylation of the promoters, which suppresses miRNA expression (9). Furthermore, many transcription factors and proteins, including Myc, P53 and REST, activate or inhibit specific miRNA expression.

Post-transcriptional regulations include interference with transportation of pre-miRNA from the nucleus to the cytoplasm, decreased expression of enzymes such as Dicer and Drosha needed for biogenesis, chemical compounds of either endogenous origin (hormones, cytokines), or exogenous origin (xenobiotics) (9,12). The miRNA transport factor, exportin-5, is a major regulator of pre-miRNA transport from the nucleus to the cytoplasm (14).

Intracellular and extracellular microRNAs

MicroRNAs are important intracellular regulators of mRNA expression, but miRNAs are also secreted from the cells. Once secreted, miRNAs are often contained in extracellular vesicles (EVs) such as exosomes, microvesicles, and apoptotic bodies (15). These vesicles differ in both cargo and production. Whereas microvesicles are produced by an outward budding of the plasma membrane, exosomes are the result of exocytosis of multivesicular bodies (MVBs). Microvesicles are also much larger (100 to 1000 nm) compared to exosomes (50 to 90 nm). The combination of these differences produces significantly different cargos in each. Microvesicle contents tend to be more heterogeneous when compared to exosomes. We also now know that miRNAs can be preferentially packaged into exosomes through a special EXOmotif. It is still not understood how microvesicles select miRNAs for export. Apoptotic bodies are a third class of EV and the largest of those discussed (1 to 5 μm). They are produced in the late stages of cell apoptosis and can also contain miRNAs. It is unknown whether the packaging of miRNAs within apoptotic bodies is a completely random process during cell death or if they are loaded in response to a stimulus (16,17).

In addition to being packaged in EVs, extracellular miRNAs can be bound to high-density lipoproteins (HDL), low-density lipoproteins (LDL), and AGO-protein complexes. In comparison to miRNAs contained within EVs which can contain both miRNAs and mRNAs, protein bound miRNAs are only rich in small non-coding RNAs. HDL-bound miRNAs have been shown to exhibit specific signatures in pathologic conditions indicating their potential as biomarkers. AGO2-bound miRNAs within EVs have been demonstrated to be more resistant to RNAses and make up potentially 90% of miRNAs within circulation (16,17). Protein-bound miRNAs are present in synovial fluid, blood and urine, which has made them attractive biomarker candidates for diagnostic and prognostic purposes (18) (Figure 1).

MicroRNAs and development

The importance of miRNAs on organ and organism development has been investigated through Dicer knock-out experiments in Drospohila and mouse models. Following Dicer knock-out, spatial-temporal analysis of miRNA expression revealed tissue-specific miRNA expression during development, suggesting miRNA involvement in defining and retaining tissue identity (19,20). In addition, the conservation and expression patterns of miRNAs in the central nervous system (e.g., miRNA-124), muscles (e.g., miR-1) and during anterior-posterior patterning (e.g., miR-10), clearly support conserved function and an important role of miRNAs in development, propagation, and overall survival of animals (21,22). Mouse fetuses deficient in Dicer die between days 12.5 and 14.5 of gestation (23). In order to bypass the embryonic lethality of Dicer-deficient mutants and characterize tissue-specific roles, numerous conditional approaches have been utilized in the mouse model. These studies have revealed essential roles for Dicer and miRNAs in the morphogenesis of the lung epithelium (24), skin (25), and vertebrate limb (26) in mouse models. A reduction in cellular proliferation and differentiation has also been observed in Dicer-deficient embryonic stem cell lines (27). In addition, mutant germline stem cells of Drosophila lacking Dicer-1 suggest the role miRNAs have in stem cell division by bypassing the G1/S checkpoint of the cell cycle (28).

Apoptosis is a central cellular mechanism during development and miRNAs play an essential role in the regulation of this process. In Drosophila, miR-14 targets inositol 1,4,5-trisphosphate kinase 2 (ip3k2) and thereby suppresses autophagy during developmentally regulated cell death (29). Likewise, bantam miRNA promotes proliferation and prevents apoptosis by regulating the proapoptotic gene hid (30). P53 acts as a transcription factor that facilitates apoptosis during development and stress responses. MiR-30a/b/d, miR-125b, miR-138, miR-214, and miR-504 have been reported as direct negative regulators of human p53 (31).

Organogenesis is another developmental process influenced by miRNAs. For example, myogenic differentiation of cardiac myocytes as well as striated skeletal myocytes is modulated by miR-1. MiR-1 is a primary regulator of muscle differentiation in flies, mice, and humans (22). MiR-1 is associated with the expression of transcription factors such as Mef2, MyoD, Hand2, and SRF, which are involved in muscle-differentiation in mice (22). In the developing lung mesenchyme, miR-142-3p modulates mesenchymal cell differentiation and proliferation in the embryonic mouse via the regulation of Wnt signalling (32). Wnt signalling is negatively regulated in part by adenomatous polyposis coli (APC). Following miR-142-3p knockdown, Apc was highly expressed at 12 and 24 h and Wnt signalling was decreased. MiR-142-3p downregulation led to the differentiation of parabronchial smooth muscle cells through the dysregulation of the WNT-FGF signalling positive feedback loop in the developing tissue (32).

MicroRNAs are important regulators during the chondrogenic differentiation of mesenchymal stromal cells (MSCs). It was recently shown that miR-140 is present in normal equine cartilage and that levels increase during chondrogenic differentiation of equine cord blood derived MSCs in vitro (3). The chondrogenic differentiation pathway consists of 6 phases (chondroprogenitors, condensed mesenchymal cells, chondrocytes, proliferating chondrocytes, pre-hypertrophic chondrocytes, and hypertrophic chondrocytes), which are regulated by several transcription factors and cytokines such as the SOX family and bone morphogenetic proteins (BMPs) (4). Numerous miRNAs participate in the regulation of chondrogenesis by targeting these transcription factors and cytokines including miR-145 targeting Sox9 (33).

Extracellular/paracrine functions of microRNAs

Maintaining homeostasis is a key challenge for organisms and evidence suggests miRNAs are key regulators of this process. Once miRNAs are released into the extracellular environment, they can be delivered to neighboring or distant cells through different mechanisms including apoptotic bodies, microvesicles, exosomes, and bound proteins which have been previously discussed (Figure 2). One of the more widely studied interactions occurs via microvesicle fusion and discharge of miRNAs directly into the cytoplasm (34). In vertebrates, expression of miR-375 in pancreatic island cells led to a reduction of insulin secretion by targeting the myotrophin (Mtpn) gene (35). Human miR-33 was shown to regulate high-density lipoprotein (HDL) levels by directly suppressing ATP-binding cassette transporter A1 (ABCA1) (36). Delivering miR-33 antagonists in an atherosclerosis mouse model reversed cholesterol transport and improved the condition of the disease, which supports the potential therapeutic value of miR-33. Likewise, repression of miR-33 in green monkeys led to a decrease in HDL metabolism (36).

Biomarkers

Biomarkers are defined as any substance that can be measured within an organism and the presence of which is indicative of a disease of interest. Traditionally, proteins and metabolites have been used (37). More recently, prognostic biomarkers have emerged, which are used to predict disease progression, treatment response, and even drug resistance (38). For biomarkers to be of clinical utility, they must be highly specific and their method of detection reproducible. RNA biomarkers can include long noncoding RNAs and miRNAs. This review is focused on the use of miRNAs.

MicroRNAs share several features with protein biomarkers. Like proteins, miRNA expression profiles differ in various disease states compared to healthy controls (39). They have also been detected in a variety of biofluids including synovial fluid and blood plasma, which provides a promising means of minimally invasive sample collection for analysis when compared to the traditional tissue biopsy. MiRNAs in collected biofluid samples are uniquely stable when compared to other RNA molecules, even at room temperature for up to 48 h (40,41).

MicroRNAs offer several key benefits as biomarkers compared to proteins. First, miRNAs are highly conserved among different species compared to proteins (7). The conservation of miRNAs allows biomarkers of interest to be applied across species, making them attractive biomarker candidates in veterinary medicine. Second, tissue specific miRNAs exist which, combined with ease of detection in blood, make miRNAs suitable in the identification of novel biomarkers of tissue-specific diseases.

Plasma miRNAs have been identified as possible biomarkers of chronic degenerative valvular disease in dogs (42). Dachshunds suffering this disease were compared to healthy controls. Afflicted patients were further divided into groups according to their American College of Veterinary Internal Medicine (ACVIM) classification. The study found that miR-30b and miR-133b both had the potential to be biomarkers of ACVIM stage B and stage C heart failure, respectively (42).

MicroRNAs have been examined as potential biomarkers of cancer. One study profiled miRNAs of dogs with transitional cell carcinoma (TCC). MiRNA profiles of blood and urine in dogs with TCC, healthy controls, and dogs with non-neoplastic urinary diseases were assessed. No differences in miRNA profiling were detected in blood however changes were observed in urine (43). Both miR-103b and miR-16 expression levels were decreased in dogs with TCC compared to the 2 control groups (43).

Serum and synovial fluid miRNAs have been evaluated in human patients with joint disease (44). Synovial fluid concentrations of miR-16, miR-146a, miR-155, and miR-223 in rheumatoid arthritis (RA) were significantly higher compared to those in OA patients. Plasma miR-132 was significantly higher in healthy control patients and could differentiate healthy controls from patients with either RA or OA with high sensitivity and selectivity (45). MiR-146a has also been assessed in patients with OA (46). Human patients suffering OA were divided into mild OA, moderate OA, and severe OA according to their Mankin score. Cartilage samples from patients with mild OA had significantly higher levels of miR-146a than those from moderate and severe OA patients.

Serum miRNAs have been explored as makers of pregnancy; in buffalo cows following artificial insemination (AI) (47), miR-451, miR-452, miR-375, miR423-5p, miR-301a, miR103, and miR-200b were all evaluated based on their involvement in pregnancy and oocyte maturation at 0, 25, and 40 days post AI. Interestingly, all the examined miRNAs were differentially expressed at 40 days post AI in pregnant compared to non-pregnant cows. On day 25 miR-452, miR-453-5p, and miR-200b were all differentially expressed compared to non-pregnant cows (47).

Next generation sequencing as a tool for identifying novel transcripts, microRNAs, and gene functions

MicroRNA discoveries in cells, tissues, or species are facilitated by 1 of 2 strategies: i) candidate strategy where a limited number of candidate miRNAs is chosen based on past discoveries and then experimentally interrogated; or ii) non-biased global screening approach using microarrays or next generation sequencing (NGS). Candidates identified in the screen are then subjected to the candidate approach to verify robust and consistent expression.

Next generation sequencing permits non-biased global screening and identification of all RNAs contained within a sample, including miRNAs. The resulting data are analyzed using bio-informatics methods. A unique benefit of NGS is the ability to identify novel miRNAs. These are previously undescribed miRNAs and can be predicted from sequencing data. Novel miRNAs are identified based on their pre-miRNA stem-loop structure and several computational algorithms are available to predict these (48).

MicroRNAs as powerful investigative tools of basic biology — cause and effect

Functional interrogation of a given miRNAs is possible through the use of miRNA mimetics and inhibitors. The use of miRNA mimetics is associated with reduced expression of mRNA if the mRNA is in fact a target of the miRNA. Conversely, the use of miRNA inhibitors will effectively suppress specific miRNAs of interest leading to increased expression of target mRNAs.

Our laboratory has previously investigated miR-140 in equine MSCs undergoing chondrogenic differentiation (3). We identified correlations between miR-140 and the expression of CXCL12, and ADAMTS5 mRNAs. As MSCs underwent chondrogenic differentiation, miR-140 expression rose to similar levels to that observed in native chondrocytes. Interestingly, CXCL12 and ADAMTS5 expression decreased as miR-140 expression increased suggesting that miR-140 may target CXCL12 and ADAMTS5. The use of miRNA mimetics and inhibitors in the culture system will be important to reveal whether the predicted functional associations occur.

MicroRNAs have been implicated in the pathogenesis of OA. When OA synovial explants were stimulated with IL-1β; miR-23a, miR-24, miR-27a, miR-27b, miR-29c, miR-186 and miR-378a all had significantly higher levels of expression. MiR-23a and miR-27b were excreted into the explant medium following IL-1β treatment suggesting that it may be involved in the release of miRNAs into the synovial fluid from the synovium (44). MiR-181a and miR-4454 were elevated in patients with facet joint OA. Following treatment with miR-181a and miR-4454 mimics, markers of inflammation, catabolism, and cell death in facet joint chondrocytes were elevated while COL2A1 was decreased. The same experiment was then repeated using inhibitor and the opposite effect was observed. Taken together, these results suggest that miR-181a and miR-4454 may play a pivotal role in cartilage degradation (49).

MicroRNAs as potential therapeutic agents

Unmodified miRNAs have limited stability clinically in vivo. Two popular technologies involve miRNA agomirs and antagomirs. These are single-stranded RNA molecules with chemical modifications at their 3′ and 5′ ends to enhance their stability and cellular uptake. Even with these modifications delivery systems are needed to be effective in vivo. These can be broadly grouped into viral vectors and nonviral carriers. Although the specific delivery methods are beyond the scope of this review, a recent paper by Fu et al (50) highlights the current and upcoming delivery systems of miRNA therapeutics.

Clinical trials investigating the therapeutic utility of miRNA mimetics and antagonists are now being conducted. Intravenous delivery of tumor-targeted anti-miR-21 has been shown to increase both the mRNA and protein levels of RhoB, a miR-21 target (51). The antisense miR-21 nanoparticle constructs were shown to select tumor cells almost exclusively within the brain and no systemic immune response was detected when compared to saline controls. When combined with the tyrosine kinase inhibitor sunitinib, the nanoparticles decreased tumor cell proliferation, increased apoptosis, decreased tumor size, and increased animal survival in glioblastoma mice compared to sunitinib alone (51).

One study examining chronic dilated cardiomyopathy (DCM) in mice found that miR-669a therapy increased survival (52). MiR-699a AAV vectors were administered to Sgcb-null dystrophic mice intraventricularly. The miR-699a AAV vector induced miR-699a overexpression for at least 18 months. Compared to controls, mice undergoing miR-699a AAV vector treatment had decreased cardiomyocyte remodeling and fibrosis while sarcomere organization increased. Genes and miRNAs associated with DCM were generally lower (52).

Recently, several miRNAs have shown therapeutic value in osteoarthritis. MiR-140 actively targets MMP-13 and ADAMTS-5 in human chondrocytes. Furthermore, intra-articular injections of miR-140 in rats with surgically induced OA showed delayed disease progression compared to controls (53). Rats that received the miR-140 injections also had cartilage of higher thickness and chondrocyte number. Previously we discussed miR-181a and miR-4454 in FJ OA. A follow-up study examined the use of miR-181a-5p antisense oligonucleotides (ASO) in induced rat FJ OA and mouse knee OA models. Intra-articular injections of the ASO attenuated cartilage destruction, and the expression of catabolic, hypertrophic, apoptotic, and markers of type II collagen breakdown (54).

A potential concern, particularly in vivo, is the occurrence of “off-target” effects, which can be considered as either direct or indirect. Direct off target effects occur when a targeted miRNA is implicated in a variety of biological processes. While targeting an miRNA expressed in a disease state may prove efficacious in vitro, they may also interact in other important processes in vivo. Indirect off target effects occur when anti-miRs execute their therapeutic effect by binding to and recognizing miRNA-RISCs and therefore prevent the further binding and inhibition of mRNAs. Many miRNAs within the same family feature almost identical seed sequences. It is therefore possible that an anti-miR could bind to an unintended miRNA within the same family causing unintended effects in vivo (55). In-vivo miRNA therapies would need to be designed to closely monitor adverse events related to such off-target effects. More effective delivery techniques that preferentially target the tissue of interest will reduce the risk of off-target effects. Tissue-specific anti-miR and miRNAs would also overcome the risk of off-target effects.

In conclusion, from a veterinary and comparative One-Health perspective, the high degree of cross-species conservation of miRNAs provides real advantages when compiling candidate markers from published knowledge compared to protein-based discoveries. Combined with the emergence of relatively affordable NGS for the identification of all RNAs contained within a sample, including miRNAs, a powerful research strategy emerges compared to traditional qPCR screening. Through these strategies we can elucidate cell and tissue functionality, identify novel biomarkers, and druggable therapeutic targets. Together, these factors suggest that miRNAs will ultimately be of significant clinical utility in veterinary medicine as diagnostic, prognostic and therapeutic tools.