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PloS one2024; 19(2); e0297303; doi: 10.1371/journal.pone.0297303

microRNAs are differentially expressed in equine plasma of horses with osteoarthritis and osteochondritis dissecans versus control horses.

Abstract: Osteoarthritis (OA) is a leading cause of lameness in horses with no effective disease-modifying treatment and challenging early diagnosis. OA is considered a disease of the joint involving the articular cartilage, subchondral bone, synovial membrane, and ligaments. Osteochondritis dissecans (OCD) is a joint disease consisting of focal defects in the osteochondral unit which may progress to OA later in life. MicroRNAs (miRNAs) have been recognized as small non-coding RNAs that regulate a variety of biological processes and have been detected in biological fluids. MiRNAs are currently investigated for their utility as biomarkers and druggable targets for a variety of diseases. The current study hypothesizes that miRNA profiles can be used to actively monitor joint health and differences in miRNA profiles will be found in healthy vs diseased joints and that differences will be detectable in blood plasma of tested horses. Five horses with OA, OCD, and 4 controls (C) had blood plasma and synovial fluid collected. Total RNA, including miRNA was isolated before generating miRNA libraries from the plasma of the horses. Libraries were sequenced at the Schroeder Arthritis Institute (Toronto). Differential expression analysis was done using DESeq2 and validated using ddPCR. KEGG pathway analysis was done using mirPath v.3 (Diana Tools). 57 differentially expressed miRNAs were identified in OA vs C plasma, 45 differentially expressed miRNAs in OC vs C plasma, and 21 differentially expressed miRNAs in OA vs OCD plasma. Notably, miR-140-5p expression was observed to be elevated in OA synovial fluid suggesting that miR-140-5p may serve as a protective marker early on to attenuate OA progression. KEGG pathway analysis of differentially expressed plasma miRNAs showed relationships with glycan degradation, glycosaminoglycan degradation, and hippo signaling pathway. Interestingly, ddPCR was unable to validate the NGS data suggesting that isomiRs may play an integral role in miRNA expression when assessed using NGS technologies.
Copyright: © 2024 Antunes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Publication Date: 2024-02-23 PubMed ID: 38394252PubMed Central: PMC10890772DOI: 10.1371/journal.pone.0297303Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research explores the presence and variance in microRNAs in the blood plasma and synovial fluid of horses suffering from osteoarthritis and osteochondritis dissecans compared to healthy ones. The research attempts to use this as a marker for detecting and monitoring these joint health issues.

Introduction and Hypothesis

  • The study is situated in the context of osteoarthritis and osteochondritis dissecans (OCD), two common joint diseases in horses which lack effective treatments and present difficulties in early diagnosis.
  • The researchers hypothesize that profiling of microRNAs (miRNAs) – small non-coding RNAs found in biological fluids – could be used to monitor the state of joint health in the animals.
  • They expect to find differences in miRNA profiles between healthy and diseased joints, and believe that such differences would be detectable in the blood plasma of the horses under study.

Methods

  • They collected blood plasma and synovial fluid from five horses diagnosed with osteoarthritis or OCD, and four healthy control horses.
  • Total RNA, inclusive of miRNA, was isolated from the plasma of these horses to create miRNA libraries.
  • These libraries were then sequenced at the Schroeder Arthritis Institute and differentially expressed miRNAs were identified using a data analysis technique known as DESeq2.
  • The sequencing data was validated by droplet digital PCR (ddPCR) and a pathway analysis was conducted using mirPath v.3.

Results

  • Differential expression analysis revealed 57, 45, and 21 differently expressed miRNAs in the plasma of OA vs control, OCD vs control, and OA vs OCD horses, respectively.
  • The microRNA miR-140-5p was found to be over expressed in osteoarthritic synovial fluid, suggesting its potential as a protective marker that could attenuate OA progression.
  • Pathway analysis showed correlations between the differently expressed plasma miRNAs and pathways involved in glycan degradation, glycosaminoglycan degradation, and the Hippo signaling pathway.
  • The researchers noted that ddPCR could not validate the next-generation sequencing (NGS) data, highlighting the critical role of isomiRs (miRNA variants) in miRNA expression determined by NGS.

Cite This Article

APA
Antunes J, Salcedo-Jiménez R, Lively S, Potla P, Coté N, Dubois MS, Koenig J, Kapoor M, LaMarre J, Koch TG. (2024). microRNAs are differentially expressed in equine plasma of horses with osteoarthritis and osteochondritis dissecans versus control horses. PLoS One, 19(2), e0297303. https://doi.org/10.1371/journal.pone.0297303

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 19
Issue: 2
Pages: e0297303
PII: e0297303

Researcher Affiliations

Antunes, Joshua
  • Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Salcedo-Jiménez, Ramés
  • Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Lively, Starlee
  • Osteoarthritis Research Program, Division of Orthopedics, Schroeder Arthritis Institute, University Health Network, Toronto, Ontario, Canada.
Potla, Pratibha
  • Osteoarthritis Research Program, Division of Orthopedics, Schroeder Arthritis Institute, University Health Network, Toronto, Ontario, Canada.
Coté, Nathalie
  • Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Dubois, Marie-Soleil
  • Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Koenig, Judith
  • Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Kapoor, Mohit
  • Osteoarthritis Research Program, Division of Orthopedics, Schroeder Arthritis Institute, University Health Network, Toronto, Ontario, Canada.
LaMarre, Jonathan
  • Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Koch, Thomas Gadegaard
  • Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.

MeSH Terms

  • Animals
  • Horses / genetics
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Osteochondritis Dissecans / genetics
  • Osteochondritis Dissecans / veterinary
  • Osteoarthritis / genetics
  • Osteoarthritis / veterinary
  • Osteoarthritis / diagnosis
  • Joint Diseases
  • Synovial Membrane / metabolism

Conflict of Interest Statement

I have read the journal’s policy and the authors of the manuscript have the following competing interests: Mohit Kapoor has a patent on microRNA biomarkers for cartilage degeneration (Patent Number: 10888577). There are no other competing interests.

References

This article includes 43 references
  1. Bailey CJ, Reid SWJ, Hodgson DR, Rose RJ. Impact of injuries and disease on a cohort of two- and three-year-old thoroughbreds in training.. Veterinary Record 1999;145: 487–493.
    doi: 10.1136/vr.145.17.487pubmed: 10596871google scholar: lookup
  2. Cokelaere S, Malda J, van Weeren R. Cartilage defect repair in horses: Current strategies and recent developments in regenerative medicine of the equine joint with emphasis on the surgical approach.. Veterinary Journal Bailliere Tindall Ltd; 2016. pp. 61–71.
    doi: 10.1016/j.tvjl.2016.02.005pubmed: 27387728google scholar: lookup
  3. Man GS, Mologhianu G. Osteoarthritis pathogenesis—a complex process that involves the entire joint.. Journal of medicine and life J Med Life; 2014. pp. 37–41.
    pmc: PMC3956093pubmed: 24653755
  4. Olstad K, Ekman S, Carlson CS. An Update on the Pathogenesis of Osteochondrosis.. Vet Pathol 2015;52: 785–802.
    doi: 10.1177/0300985815588778pubmed: 26080832google scholar: lookup
  5. Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity: microRNA biogenesis pathways and their regulation.. Nat Cell Biol 2009;11: 228–234.
    doi: 10.1038/ncb0309-228pubmed: 19255566google scholar: lookup
  6. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL. Circulating microRNAs as stable blood-based markers for cancer detection.. Proceedings of the National Academy of Sciences 2008;105: 10513–10518.
    doi: 10.1073/pnas.0804549105pmc: PMC2492472pubmed: 18663219google scholar: lookup
  7. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.. Nature 2000;408: 86–9.
    doi: 10.1038/35040556pubmed: 11081512google scholar: lookup
  8. Lewis BP, Shih I, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets.. Cell 2003;115: 787–98.
    doi: 10.1016/s0092-8674(03)01018-3pubmed: 14697198google scholar: lookup
  9. Murata K, Yoshitomi H, Tanida S, Ishikawa M, Nishitani K, Ito H. Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis.. Arthritis Res Ther 2010;12: R86.
    doi: 10.1186/ar3013pmc: PMC2911870pubmed: 20470394google scholar: lookup
  10. Yamasaki K, Nakasa T, Miyaki S, Ishikawa M, Deie M, Adachi N. Expression of MicroRNA-146a in osteoarthritis cartilage.. Arthritis Rheum 2009;60: 1035–1041.
    doi: 10.1002/art.24404pmc: PMC2670476pubmed: 19333945google scholar: lookup
  11. Si HB, Zeng Y, Liu SY, Zhou ZK, Chen YN, Cheng JQ. Intra-articular injection of microRNA-140 (miRNA-140) alleviates osteoarthritis (OA) progression by modulating extracellular matrix (ECM) homeostasis in rats.. Osteoarthritis Cartilage 2017;25: 1698–1707.
    doi: 10.1016/j.joca.2017.06.002pubmed: 28647469google scholar: lookup
  12. Nakamura A, Rampersaud YR, Sharma A, Lewis SJ, Wu B, Datta P. Identification of microRNA-181a-5p and microRNA-4454 as mediators of facet cartilage degeneration.. JCI Insight 2016;1.
    doi: 10.1172/jci.insight.86820pmc: PMC5033882pubmed: 27699225google scholar: lookup
  13. Nakamura A, Rampersaud YR, Nakamura S, Sharma A, Zeng F, Rossomacha E. microRNA-181a-5p antisense oligonucleotides attenuate osteoarthritis in facet and knee joints.. Ann Rheum Dis 2019;78: 111–121.
    doi: 10.1136/annrheumdis-2018-213629pubmed: 30287418google scholar: lookup
  14. Castanheira C, James V, Taylor S, Skiöldebrand E, Clegg PD, Peffers MJ. Synovial fluid and serum small non-coding RNA signatures in equine osteoarthritis.. Osteoarthritis Cartilage 2021;29: S162.
    doi: 10.1016/j.joca.2021.02.226google scholar: lookup
  15. Castanheira C, Balaskas P, Falls C, Ashraf-Kharaz Y, Clegg P, Burke K. Equine synovial fluid small non-coding RNA signatures in early osteoarthritis.. BMC Vet Res 2021;17: 1–12.
    doi: 10.1186/S12917-020-02707-7pmc: PMC7796526pubmed: 33422071google scholar: lookup
  16. Potla P, Ali SA, Kapoor M. A bioinformatics approach to microRNA-sequencing analysis.. Osteoarthr Cartil Open 2021;3: 100131.
    doi: 10.1016/j.ocarto.2020.100131pmc: PMC9718162pubmed: 36475076google scholar: lookup
  17. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.. Genome Biol 2014;15: 1–21.
    doi: 10.1186/s13059-014-0550-8pmc: PMC4302049pubmed: 25516281google scholar: lookup
  18. Ding PN, Becker T, Bray V, Chua W, Ma Y, Xu B. Plasma next generation sequencing and droplet digital PCR‐based detection of epidermal growth factor receptor (EGFR) mutations in patients with advanced lung cancer treated with subsequent‐line osimertinib.. Thorac Cancer 2019;10: 1879.
    doi: 10.1111/1759-7714.13154pmc: PMC6775001pubmed: 31414729google scholar: lookup
  19. Mattox AK, D’Souza G, Khan Z, Allen H, Henson S, Siewert TY. Comparison of next generation sequencing, droplet digital PCR, and quantitative real-time PCR for the earlier detection and quantification of HPV in HPV-positive oropharyngeal cancer.. Oral Oncol 2022;128.
    doi: 10.1016/j.oraloncology.2022.105805pmc: PMC9058207pubmed: 35334415google scholar: lookup
  20. Ye P, Cai P, Xie J, Wei Y. The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: A systematic review and meta-analysis.. PLoS One 2021;16.
    doi: 10.1371/journal.pone.0248775pmc: PMC7997033pubmed: 33770081google scholar: lookup
  21. Murata K, Furu M, Yoshitomi H, Ishikawa M, Shibuya H, Hashimoto M. Comprehensive microRNA Analysis Identifies miR-24 and miR-125a-5p as Plasma Biomarkers for Rheumatoid Arthritis.. PLoS One 2013.
    doi: 10.1371/journal.pone.0069118pmc: PMC3715465pubmed: 23874885google scholar: lookup
  22. Nakamura A, Rampersaud YR, Sharma A, Lewis SJ, Wu B, Datta P. Identification of microRNA-181a-5p and microRNA-4454 as mediators of facet cartilage degeneration.. JCI Insight 2016;1: e86820.
    doi: 10.1172/jci.insight.86820pmc: PMC5033882pubmed: 27699225google scholar: lookup
  23. Xue J, Min Z, Xia Z, Cheng B, Lan B, Zhang F. The hsa-miR-181a-5p reduces oxidation resistance by controlling SECISBP2 in osteoarthritis.. BMC Musculoskelet Disord 2018;19.
    doi: 10.1186/S12891-018-2273-6pmc: PMC6172777pubmed: 30286747google scholar: lookup
  24. Zhai X, Meng R, Li H, Li J, Jing L, Qin L. miR-181a Modulates Chondrocyte Apoptosis by Targeting Glycerol-3-Phosphate Dehydrogenase 1-Like Protein (GPD1L) in Osteoarthritis.. Med Sci Monit 2017;23: 1224–1231.
    doi: 10.12659/msm.899228pmc: PMC5360418pubmed: 28280258google scholar: lookup
  25. Xie Z, Shen P, Qu Y, Xu J, Zheng C, Gao Y. MiR-20a inhibits the progression of human arthritis fibroblast-like synoviocytes and inflammatory factor expression by targeting ADAM10.. Environ Toxicol 2020;35: 867–878.
    doi: 10.1002/tox.22923pubmed: 32198911google scholar: lookup
  26. Xu R, Wei Y, Yin X, Shi B, Li J. miR-20a suppresses chondrogenic differentiation of ATDC5 cells by regulating Atg7.. Sci Rep 2019;9.
    doi: 10.1038/S41598-019-45502-7pmc: PMC6592888pubmed: 31239522google scholar: lookup
  27. Liu J, Tang G, Liu W, Zhou Y, Fan C, Zhang W. MiR-20a-5p facilitates cartilage repair in osteoarthritis via suppressing mitogen-activated protein kinase kinase kinase 2.. Bioengineered 2022;13: 13801.
    doi: 10.1080/21655979.2022.2084270pmc: PMC9276018pubmed: 35707845google scholar: lookup
  28. Zhao H, Gong N. miR-20a regulates inflammatory in osteoarthritis by targeting the IκBβ and regulates NK-κB signaling pathway activation.. Biochem Biophys Res Commun 2019;518: 632–637.
    doi: 10.1016/J.BBRC.2019.08.109pubmed: 31451219google scholar: lookup
  29. Ntoumou E, Tzetis M, Braoudaki M, Lambrou G, Poulou M, Malizos K. Serum microRNA array analysis identifies miR-140-3p, miR-33b-3p and miR-671-3p as potential osteoarthritis biomarkers involved in metabolic processes.. Clin Epigenetics 2017;9.
    doi: 10.1186/s13148-017-0428-1pmc: PMC5728069pubmed: 29255496google scholar: lookup
  30. Deng Y, Lu J, Li W, Wu A, Zhang X, Tong W. Reciprocal inhibition of YAP/TAZ and NF-κB regulates osteoarthritic cartilage degradation.. Nat Commun 2018;9.
    doi: 10.1038/s41467-018-07022-2pmc: PMC6212432pubmed: 30385786google scholar: lookup
  31. Wang C, Shen J, Ying J, Xiao D, O’Keefe RJ. FoxO1 is a crucial mediator of TGF-β/TAK1 signaling and protects against osteoarthritis by maintaining articular cartilage homeostasis.. Proc Natl Acad Sci U S A 2020;117: 30488–30497.
    doi: 10.1073/PNAS.2017056117pmc: PMC7720227pubmed: 33199631google scholar: lookup
  32. Matsuzaki T, Alvarez-Garcia O, Mokuda S, Nagira K, Olmer M, Gamini R. FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis.. Sci Transl Med 2018;10.
    doi: 10.1126/scitranslmed.aan0746pmc: PMC6204214pubmed: 29444976google scholar: lookup
  33. Guo L, Chen F. A challenge for miRNA: multiple isomiRs in miRNAomics.. Gene 2014;544: 1–7.
    doi: 10.1016/j.gene.2014.04.039pubmed: 24768184google scholar: lookup
  34. Schamberger A, Orbán TI. 3′ IsomiR Species and DNA Contamination Influence Reliable Quantification of MicroRNAs by Stem-Loop Quantitative PCR.. PLoS One 2014;9: e106315.
    doi: 10.1371/journal.pone.0106315pmc: PMC4149544pubmed: 25170848google scholar: lookup
  35. Tan GC, Chan E, Molnar A, Sarkar R, Alexieva D, Isa IM. 5′ isomiR variation is of functional and evolutionary importance.. Nucleic Acids Res 2014;42: 9424–9435.
    doi: 10.1093/nar/gku656pmc: PMC4132760pubmed: 25056318google scholar: lookup
  36. Aiso T, Ueda M. 5’-isomiR is the most abundant sequence of miR-1246, a candidate biomarker of lung cancer, in serum.. Mol Med Rep 2023;27.
    doi: 10.3892/MMR.2023.12979,pmc: PMC10073804pubmed: 36960865google scholar: lookup
  37. Lunn M-L, Mouritzen P, Faber K, Jacobsen N. MicroRNA quantitation from a single cell by PCR using SYBR® Green detection and LNA-based primers.. Nature Methods 2008;5: iii–iv.
    doi: 10.1038/nmeth.f.205google scholar: lookup
  38. Noren Hooten N, Fitzpatrick M, Wood WH, De S, Ejiogu N, Zhang Y. Age-related changes in microRNA levels in serum.. Aging (Albany NY) 2013;5: 725.
    doi: 10.18632/aging.100603pmc: PMC3838776pubmed: 24088671google scholar: lookup
  39. Sharma S, Eghbali M. Influence of sex differences on microRNA gene regulation in disease.. Biol Sex Differ 2014;5: 3.
    doi: 10.1186/2042-6410-5-3pmc: PMC3912347pubmed: 24484532google scholar: lookup
  40. Sadkowski T, Ciecierska A, Oprzadek J, Balcerek E. Breed-dependent microRNA expression in the primary culture of skeletal muscle cells subjected to myogenic differentiation.. BMC Genomics 2018;19.
    doi: 10.1186/S12864-018-4492-5pmc: PMC5793348pubmed: 29390965google scholar: lookup
  41. Pacholewska A, Mach N, Mata X, Vaiman A, Schibler L, Barrey E. Novel equine tissue miRNAs and breed-related miRNA expressed in serum.. BMC Genomics 2016;17: 1–15.
    doi: 10.1186/S12864-016-3168-2pmc: PMC5080802pubmed: 27782799google scholar: lookup
  42. Kirschner MB, Edelman JJB, Kao SCH, Vallely MP, Van Zandwijk N, Reid G. The Impact of Hemolysis on Cell-Free microRNA Biomarkers.. Front Genet 2013;4.
    doi: 10.3389/FGENE.2013.00094pmc: PMC3663194pubmed: 23745127google scholar: lookup
  43. Unger L, Gerber V, Pacholewska A, Leeb T, Jagannathan V. MicroRNA fingerprints in serum and whole blood of sarcoid-affected horses as potential non-invasive diagnostic biomarkers.. Vet Comp Oncol 2019;17: 107–117.
    doi: 10.1111/vco.12451pubmed: 30430738google scholar: lookup

Citations

This article has been cited 3 times.
  1. Li L, Xu Z, Gao F, Xu J. Current status and future prospects of nanocarrier-mediated miRNA delivery for osteoarthritis therapy. Front Med (Lausanne) 2025;12:1728944.
    doi: 10.3389/fmed.2025.1728944pubmed: 41625748google scholar: lookup
  2. Cullen JN, Cieslak J, Petersen JL, Bellone RR, Finno CJ, Kalbfleisch TS, Calloe K, Capomaccio S, Cappelli K, Coleman SJ, Distl O, Durward-Akhurst SA, Giulotto E, Hamilton NA, Hill EW, Katz LM, Klaerke DA, Lindgren G, MacHugh DE, Mackowski M, MacLeod JN, Metzger J, Murphy BA, Orlando L, Raudsepp T, Silvestrelli M, Strand E, Tozaki T, Trachsel DS, Valderrama Figueroa LS, Velie BD, Wade CM, Waud B, Mickelson JR, McCue ME. Charting the equine miRNA landscape: An integrated pipeline and browser for annotating, quantifying, and visualizing expression. PLoS Genet 2025 Sep;21(9):e1011835.
    doi: 10.1371/journal.pgen.1011835pubmed: 40911641google scholar: lookup
  3. Azhar G, Verma A, Patyal P, Zhang W, Sharma S, Savary PE, Riklon S, Kabua PM, McElfish PA, Wei JY. Differential microRNA profiling of the Marshallese population in Arkansas reveals a higher association with chronic diseases. PLoS One 2025;20(8):e0329321.
    doi: 10.1371/journal.pone.0329321pubmed: 40788916google scholar: lookup
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