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Potential of circulating microRNAs as biomarkers in veterinary medicine
The Veterinary Journal 212 (2016) 78–79
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Guest Editorial
Potential of circulating microRNAs as biomarkers in veterinary medicine
MicroRNAs (miRNAs) are small non-coding RNA molecules that are conserved across species (Wang et al., 2016). They play an essential role in the post-transcriptional control of gene expression by binding complimentary messenger RNA. They can either repress or up-regulate gene expression (Szabo and Bala, 2013). There are over 1400 mammalian miRNAs and each of these can alter the expression of many genes. Additionally, the expression of a particular gene can be influenced by more than one miRNA (Szabo and Bala, 2013). As well as being involved in normal physiological processes, miRNAs are also involved in the pathogenesis of many diseases, including various tumours, inflammatory diseases, metabolic diseases, autoimmune diseases and viral infections (Wang et al., 2016). miRNAs also have the potential to be used as biomarkers and even as therapeutic agents/targets. In a recent issue of The Veterinary Journal, there is an interesting and important original study by Dirksen et al. (2016) that evaluates the use of circulating hepatocyte-derived miRNAs as markers of hepatocellular injury in Labrador retrievers. It is possible for miRNAs to be used as biomarkers because of their release from tissues into the circulation and their extracellular stability. Dirksen et al. (2016) quantified serum hepatocytederived miR-122 and miR-148a in 66 Labrador retrievers. Of these dogs, 11 had normal livers, 11 had increased (>400 mg/kg dry weight) hepatic copper concentrations, but no histological evidence of liver injury, and 44 had histological evidence of liver injury. The authors not only found that expression of these hepatocyte-derived miRNAs was increased in the serum of dogs with liver injury, but they also found that the sensitivity of miRNA-122 for the detection of liver injury in these dogs (84%) was superior to that of ALT activity (55%; Dirksen et al., 2016). The results of this study suggest that miR122 could be a useful and more sensitive marker of canine hepatocellular injury than traditional liver enzyme activity assays, but also, perhaps more importantly, they serve as proof of principle that circulating miRNAs can be used as biomarkers in dogs. Researchers are working to define the miRNA profiles of many diseases principally in human beings and laboratory animals. In liver diseases, in addition to using miRNAs as non-aetiology specific markers of hepatocellular injury, investigators are hoping to characterise miRNA signatures that can distinguish between different liver disease processes. In human beings with liver fibrosis/ cirrhosis, expression of miR-29 and miR-652 are decreased, while expression of miR-571 is increased (Roderburg et al., 2011, 2012). The ability to detect a serum miRNA profile that is specific to a certain disease/process offers tremendous scope for the development of novel biomarkers in veterinary species. miRNA profiles have also http://dx.doi.org/10.1016/j.tvjl.2016.04.005 1090-0233/© 2016 Elsevier Ltd. All rights reserved.
been described in human patients with various forms of cancer, viral infections, nervous system disorders, metabolic disorders and cardiovascular disorders (Wang et al., 2016). In veterinary science, miRNAs have been studied in a variety of diseases, including feline hypertrophic cardiomyopathy (Weber et al., 2015), feline diabetes (Fleischhacker et al., 2013), canine myxomatous mitral valve disease (Li et al., 2015), canine lymphosarcoma/lymphoma (Fujiwara-Igarashi et al., 2015), canine osteosarcomas (Pazzaglia et al., 2015), Golden retriever muscular dystrophy (Jeanson-Leh et al., 2014) and Doberman pinscher dilated cardiomyopathy (Steudemann et al., 2013). The study by Dirksen et al (2016) is one of the first to take the next step of directly comparing the diagnostic utility of miRNA biomarkers to that of an established diagnostic test. Two main experimental designs are used to evaluate miRNAs. The first is an untargeted approach where multiple miRNAs are quantified in parallel by microarray analysis, real-time PCR array or next generation sequencing. The advantage of this approach is that the diagnostic utility of multiple molecules can be screened simultaneously and that a complete miRNA expression profile can be produced. The disadvantages are the relatively high cost, the need for advanced equipment and the complexity of analysing the huge datasets that these experiments produce. The other approach is to evaluate targeted miRNAs by reverse transcribing them into complementary DNA (cDNA) and then quantifying them using real time PCR (qPCR). This was the approach used by Dirksen et al (2016). Since many miRNAs are conserved across species, if a particular miRNA or set of miRNAs has previously been shown to have diagnostic utility in human beings or laboratory animals, they are good candidates to be studied in veterinary species. The advantages of this targeted approach are the lower costs and the reduced complexity of the resulting dataset. There are several obstacles that need to be overcome before miRNAs can be used as biomarkers in a clinical setting. More research needs to be done to fully define the miRNA signatures of different diseases states to ensure their specificity. Large scale clinical studies need to be performed to rigorously assess the diagnostic utility of miRNAs. Additionally, sample handling, RNA extraction, method of quantification and normalisation of samples can have a profound effect on experimental results; therefore, more work needs to be performed to optimise and standardise these steps (Arrese et al., 2015). The technology involved must be repeatable, reproducible, reliable and cost effective before miRNA biomarkers can be implemented clinically and commercialised. If these challenges can be overcome, miRNA analysis has the potential to revolutionise the diagnosis of diseases in veterinary species. The work
Guest Editorial / The Veterinary Journal 212 (2016) 78–79
described in the paper is an important early step towards this goal in dogs. Jonathan Lidbury Gastrointestinal Laboratory Texas A&M University College Station, TX 77843, USA E-mail address: [email protected] References Arrese, M., Eguchi, A., Feldstein, A.E., 2015. Circulating microRNAs: Emerging biomarkers of liver disease. Seminars in Liver Disease 35, 43–54. Dirksen, K., Verzijl, T., van den Ingh, T.S., Vernooij, J.C., van der Laan, L.J., Burgener, I.A., Spee, B., Fieten, H., 2016. Hepatocyte-derived microRNAs as sensitive serum biomarkers of hepatocellular injury in Labrador retrievers. The Veterinary Journal doi:10.1016/j.tvjl.2016.01.010. Fleischhacker, S.N., Bauersachs, S., Wehner, A., Hartmann, K., Weber, K., 2013. Differential expression of circulating microRNAs in diabetic and healthy lean cats. The Veterinary Journal 197, 688–693. Fujiwara-Igarashi, A., Igarashi, H., Mizutani, N., Goto-Koshino, Y., Takahashi, M., Ohno, K., Tsujimoto, H., 2015. Expression profile of circulating serum microRNAs in dogs with lymphoma. The Veterinary Journal 205, 317–321. Jeanson-Leh, L., Lameth, J., Krimi, S., Buisset, J., Amor, F., Le Guiner, C., Barthelemy, I., Servais, L., Blot, S., Voit, T., et al., 2014. Serum profiling identifies novel muscle
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miRNA and cardiomyopathy-related miRNA biomarkers in Golden Retriever muscular dystrophy dogs and Duchenne muscular dystrophy patients. American Journal of Pathology 184, 2885–2898. Li, Q., Freeman, L.M., Rush, J.E., Laflamme, D.P., 2015. Expression profiling of circulating microRNAs in canine myxomatous mitral valve disease. International Journal of Molecular Science 16, 14098–14108. Pazzaglia, L., Leonardi, L., Conti, A., Novello, C., Quattrini, I., Montanini, L., Roperto, F., Del Piero, F., Di Guardo, G., Piro, F., et al., 2015. miR-196a expression in human and canine osteosarcomas: A comparative study. Research in Veterinary Science 99, 112–119. Roderburg, C., Urban, G.W., Bettermann, K., Vucur, M., Zimmermann, H., Schmidt, S., Janssen, J., Koppe, C., Knolle, P., Castoldi, M., et al., 2011. Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis. Hepatology (Baltimore, Md.) 53, 209–218. Roderburg, C., Mollnow, T., Bongaerts, B., Elfimova, N., Vargas Cardenas, D., Berger, K., Zimmermann, H., Koch, A., Vucur, M., Luedde, M., et al., 2012. Micro-RNA profiling in human serum reveals compartment-specific roles of miR-571 and miR-652 in liver cirrhosis. PLoS ONE 7, e32999. Steudemann, C., Bauersachs, S., Weber, K., Wess, G., 2013. Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls. BMC Veterinary Research 9, 12. Szabo, G., Bala, S., 2013. MicroRNAs in liver disease. Nature Reviews. Gastroenterology and Hepatology 10, 542–552. Wang, J., Chen, J., Sen, S., 2016. MicroRNA as biomarkers and diagnostics. Journal of Cellular Physiology 231, 25–30. Weber, K., Rostert, N., Bauersachs, S., Wess, G., 2015. Serum microRNA profiles in cats with hypertrophic cardiomyopathy. Molecular and Cellular Biochemistry 402, 171–180.
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Guest Editorial
Potential of circulating microRNAs as biomarkers in veterinary medicine
MicroRNAs (miRNAs) are small non-coding RNA molecules that are conserved across species (Wang et al., 2016). They play an essential role in the post-transcriptional control of gene expression by binding complimentary messenger RNA. They can either repress or up-regulate gene expression (Szabo and Bala, 2013). There are over 1400 mammalian miRNAs and each of these can alter the expression of many genes. Additionally, the expression of a particular gene can be influenced by more than one miRNA (Szabo and Bala, 2013). As well as being involved in normal physiological processes, miRNAs are also involved in the pathogenesis of many diseases, including various tumours, inflammatory diseases, metabolic diseases, autoimmune diseases and viral infections (Wang et al., 2016). miRNAs also have the potential to be used as biomarkers and even as therapeutic agents/targets. In a recent issue of The Veterinary Journal, there is an interesting and important original study by Dirksen et al. (2016) that evaluates the use of circulating hepatocyte-derived miRNAs as markers of hepatocellular injury in Labrador retrievers. It is possible for miRNAs to be used as biomarkers because of their release from tissues into the circulation and their extracellular stability. Dirksen et al. (2016) quantified serum hepatocytederived miR-122 and miR-148a in 66 Labrador retrievers. Of these dogs, 11 had normal livers, 11 had increased (>400 mg/kg dry weight) hepatic copper concentrations, but no histological evidence of liver injury, and 44 had histological evidence of liver injury. The authors not only found that expression of these hepatocyte-derived miRNAs was increased in the serum of dogs with liver injury, but they also found that the sensitivity of miRNA-122 for the detection of liver injury in these dogs (84%) was superior to that of ALT activity (55%; Dirksen et al., 2016). The results of this study suggest that miR122 could be a useful and more sensitive marker of canine hepatocellular injury than traditional liver enzyme activity assays, but also, perhaps more importantly, they serve as proof of principle that circulating miRNAs can be used as biomarkers in dogs. Researchers are working to define the miRNA profiles of many diseases principally in human beings and laboratory animals. In liver diseases, in addition to using miRNAs as non-aetiology specific markers of hepatocellular injury, investigators are hoping to characterise miRNA signatures that can distinguish between different liver disease processes. In human beings with liver fibrosis/ cirrhosis, expression of miR-29 and miR-652 are decreased, while expression of miR-571 is increased (Roderburg et al., 2011, 2012). The ability to detect a serum miRNA profile that is specific to a certain disease/process offers tremendous scope for the development of novel biomarkers in veterinary species. miRNA profiles have also http://dx.doi.org/10.1016/j.tvjl.2016.04.005 1090-0233/© 2016 Elsevier Ltd. All rights reserved.
been described in human patients with various forms of cancer, viral infections, nervous system disorders, metabolic disorders and cardiovascular disorders (Wang et al., 2016). In veterinary science, miRNAs have been studied in a variety of diseases, including feline hypertrophic cardiomyopathy (Weber et al., 2015), feline diabetes (Fleischhacker et al., 2013), canine myxomatous mitral valve disease (Li et al., 2015), canine lymphosarcoma/lymphoma (Fujiwara-Igarashi et al., 2015), canine osteosarcomas (Pazzaglia et al., 2015), Golden retriever muscular dystrophy (Jeanson-Leh et al., 2014) and Doberman pinscher dilated cardiomyopathy (Steudemann et al., 2013). The study by Dirksen et al (2016) is one of the first to take the next step of directly comparing the diagnostic utility of miRNA biomarkers to that of an established diagnostic test. Two main experimental designs are used to evaluate miRNAs. The first is an untargeted approach where multiple miRNAs are quantified in parallel by microarray analysis, real-time PCR array or next generation sequencing. The advantage of this approach is that the diagnostic utility of multiple molecules can be screened simultaneously and that a complete miRNA expression profile can be produced. The disadvantages are the relatively high cost, the need for advanced equipment and the complexity of analysing the huge datasets that these experiments produce. The other approach is to evaluate targeted miRNAs by reverse transcribing them into complementary DNA (cDNA) and then quantifying them using real time PCR (qPCR). This was the approach used by Dirksen et al (2016). Since many miRNAs are conserved across species, if a particular miRNA or set of miRNAs has previously been shown to have diagnostic utility in human beings or laboratory animals, they are good candidates to be studied in veterinary species. The advantages of this targeted approach are the lower costs and the reduced complexity of the resulting dataset. There are several obstacles that need to be overcome before miRNAs can be used as biomarkers in a clinical setting. More research needs to be done to fully define the miRNA signatures of different diseases states to ensure their specificity. Large scale clinical studies need to be performed to rigorously assess the diagnostic utility of miRNAs. Additionally, sample handling, RNA extraction, method of quantification and normalisation of samples can have a profound effect on experimental results; therefore, more work needs to be performed to optimise and standardise these steps (Arrese et al., 2015). The technology involved must be repeatable, reproducible, reliable and cost effective before miRNA biomarkers can be implemented clinically and commercialised. If these challenges can be overcome, miRNA analysis has the potential to revolutionise the diagnosis of diseases in veterinary species. The work
Guest Editorial / The Veterinary Journal 212 (2016) 78–79
described in the paper is an important early step towards this goal in dogs. Jonathan Lidbury Gastrointestinal Laboratory Texas A&M University College Station, TX 77843, USA E-mail address: [email protected] References Arrese, M., Eguchi, A., Feldstein, A.E., 2015. Circulating microRNAs: Emerging biomarkers of liver disease. Seminars in Liver Disease 35, 43–54. Dirksen, K., Verzijl, T., van den Ingh, T.S., Vernooij, J.C., van der Laan, L.J., Burgener, I.A., Spee, B., Fieten, H., 2016. Hepatocyte-derived microRNAs as sensitive serum biomarkers of hepatocellular injury in Labrador retrievers. The Veterinary Journal doi:10.1016/j.tvjl.2016.01.010. Fleischhacker, S.N., Bauersachs, S., Wehner, A., Hartmann, K., Weber, K., 2013. Differential expression of circulating microRNAs in diabetic and healthy lean cats. The Veterinary Journal 197, 688–693. Fujiwara-Igarashi, A., Igarashi, H., Mizutani, N., Goto-Koshino, Y., Takahashi, M., Ohno, K., Tsujimoto, H., 2015. Expression profile of circulating serum microRNAs in dogs with lymphoma. The Veterinary Journal 205, 317–321. Jeanson-Leh, L., Lameth, J., Krimi, S., Buisset, J., Amor, F., Le Guiner, C., Barthelemy, I., Servais, L., Blot, S., Voit, T., et al., 2014. Serum profiling identifies novel muscle
79
miRNA and cardiomyopathy-related miRNA biomarkers in Golden Retriever muscular dystrophy dogs and Duchenne muscular dystrophy patients. American Journal of Pathology 184, 2885–2898. Li, Q., Freeman, L.M., Rush, J.E., Laflamme, D.P., 2015. Expression profiling of circulating microRNAs in canine myxomatous mitral valve disease. International Journal of Molecular Science 16, 14098–14108. Pazzaglia, L., Leonardi, L., Conti, A., Novello, C., Quattrini, I., Montanini, L., Roperto, F., Del Piero, F., Di Guardo, G., Piro, F., et al., 2015. miR-196a expression in human and canine osteosarcomas: A comparative study. Research in Veterinary Science 99, 112–119. Roderburg, C., Urban, G.W., Bettermann, K., Vucur, M., Zimmermann, H., Schmidt, S., Janssen, J., Koppe, C., Knolle, P., Castoldi, M., et al., 2011. Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis. Hepatology (Baltimore, Md.) 53, 209–218. Roderburg, C., Mollnow, T., Bongaerts, B., Elfimova, N., Vargas Cardenas, D., Berger, K., Zimmermann, H., Koch, A., Vucur, M., Luedde, M., et al., 2012. Micro-RNA profiling in human serum reveals compartment-specific roles of miR-571 and miR-652 in liver cirrhosis. PLoS ONE 7, e32999. Steudemann, C., Bauersachs, S., Weber, K., Wess, G., 2013. Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls. BMC Veterinary Research 9, 12. Szabo, G., Bala, S., 2013. MicroRNAs in liver disease. Nature Reviews. Gastroenterology and Hepatology 10, 542–552. Wang, J., Chen, J., Sen, S., 2016. MicroRNA as biomarkers and diagnostics. Journal of Cellular Physiology 231, 25–30. Weber, K., Rostert, N., Bauersachs, S., Wess, G., 2015. Serum microRNA profiles in cats with hypertrophic cardiomyopathy. Molecular and Cellular Biochemistry 402, 171–180.