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      Diagnostic and prognostic potential of eight whole blood microRNAs for equine sarcoid disease

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          Abstract

          MicroRNAs have been proposed as biomarkers for equine sarcoids, the most prevalent equine skin tumors globally. This study served to validate the diagnostic and prognostic potential of whole blood microRNAs identified in a previous study for long-term equine sarcoid diagnosis and outcome prediction. Based on findings of a clinical examination at the age of 3 years and a follow-up following a further 5–12 years, 32 Franches-Montagnes and 45 Swiss Warmblood horses were assigned to four groups: horses with regression (n = 19), progression (n = 9), new occurrences of sarcoid lesions (n = 19) and tumor-free control horses (n = 30). The expression levels for eight microRNAs (eca-miR-127, eca-miR-432, eca-miR-24, eca-miR-125a-5p, eca-miR-134, eca-miR-379, eca-miR-381, eca-miR-382) were analyzed through reverse transcription quantitative polymerase chain reaction in whole blood samples collected on initial examination. Associations of sex, breed, diagnosis, and prognosis with microRNA expression levels were examined using multivariable analysis of variance. Sex and breed influenced the expression level of five and two microRNAs, respectively. Eca-miR-127 allowed discrimination between sarcoid-affected and tumor-free horses. No variation in microRNA expression was found when comparing horses with sarcoid regression and progression. Expression levels of eca-miR-125a-5p and eca-miR-432 varied in male horses that developed sarcoids throughout the study period in comparison to male control horses. While none of the investigated miRNAs was validated for predicting the prognosis of sarcoid regression / progression within young horses with this condition, two miRNAs demonstrated potential to predict if young male (though not female) tumor-free horse can develop sarcoids within the following years. Sex- and breed- biased miRNAs exist within the equine species and have an impact on biomarker discovery.

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          Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation

          MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3′ untranslated region (3′ UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interaction of miRNAs with other regions, including the 5′ UTR, coding sequence, and gene promoters, have also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription. The interaction of miRNAs with their target genes is dynamic and dependent on many factors, such as subcellular location of miRNAs, the abundancy of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. miRNAs can be secreted into extracellular fluids and transported to target cells via vesicles, such as exosomes, or by binding to proteins, including Argonautes. Extracellular miRNAs function as chemical messengers to mediate cell-cell communication. In this review, we provide an update on canonical and non-canonical miRNA biogenesis pathways and various mechanisms underlying miRNA-mediated gene regulations. We also summarize the current knowledge of the dynamics of miRNA action and of the secretion, transfer, and uptake of extracellular miRNAs.
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            Accessories to the crime: functions of cells recruited to the tumor microenvironment.

            Mutationally corrupted cancer (stem) cells are the driving force of tumor development and progression. Yet, these transformed cells cannot do it alone. Assemblages of ostensibly normal tissue and bone marrow-derived (stromal) cells are recruited to constitute tumorigenic microenvironments. Most of the hallmarks of cancer are enabled and sustained to varying degrees through contributions from repertoires of stromal cell types and distinctive subcell types. Their contributory functions to hallmark capabilities are increasingly well understood, as are the reciprocal communications with neoplastic cancer cells that mediate their recruitment, activation, programming, and persistence. This enhanced understanding presents interesting new targets for anticancer therapy. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

              Introduction microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases. Background Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. Methods Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA. Conclusion This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: SupervisionRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: MethodologyRole: Writing – review & editing
                Role: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 December 2021
                2021
                : 16
                : 12
                : e0261076
                Affiliations
                [1 ] Swiss Institute of Equine Medicine, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern and Agroscope, Bern, Switzerland
                [2 ] Institute of Genetics, Vetsuisse Faculty, University of Bern, Bern, Switzerland
                University of Bologna, ITALY
                Author notes

                Competing Interests: The authors have declared that no competing interests exists.

                Author information
                https://orcid.org/0000-0003-2300-5237
                https://orcid.org/0000-0001-9798-842X
                https://orcid.org/0000-0003-0553-4880
                Article
                PONE-D-21-19643
                10.1371/journal.pone.0261076
                8699634
                34941894
                f31fdc86-ecf8-4892-a67f-2bb16b29ee80
                © 2021 Cosandey 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.

                History
                : 15 June 2021
                : 23 November 2021
                Page count
                Figures: 5, Tables: 2, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100013348, Innosuisse - Schweizerische Agentur für Innovationsförderung;
                Award ID: 37900.1
                Award Recipient :
                L.U. Grant number: 37900.1 IP-LS Innosuisse ( https://www.innosuisse.ch) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Natural antisense transcripts
                MicroRNAs
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Mammals
                Equines
                Horses
                Biology and Life Sciences
                Zoology
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                Amniotes
                Mammals
                Equines
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                Biology and Life Sciences
                Biochemistry
                Biomarkers
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                Anatomy
                Body Fluids
                Blood
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
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                Physiology
                Body Fluids
                Blood
                Biology and Life Sciences
                Organisms
                Eukaryota
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                Mammals
                Equines
                Biology and Life Sciences
                Zoology
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                Amniotes
                Mammals
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                Medicine and Health Sciences
                Clinical Medicine
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                All relevant data are within the paper and its Supporting information files.

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