Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1 St., 32-083 Balice, Poland.
Institute of Veterinary Medicine, University Centre of Veterinary Medicine JU-AU, Al. Mickiewicza 24/28, 30-059 Cracow, Poland.
Cells. 2022 Jun 14;11(12):1917. doi: 10.3390/cells11121917.
Horses are of great importance in recreation, livestock production, as working animals in poorly developed countries, and for equine-assisted therapy. Equine sarcoids belong to the most commonly diagnosed tumors in this species. They may cause discomfort, pain, and can lead to the permanent impairment of motor function. The molecular bases of their formation are still under investigation. Our previous studies revealed altered microRNA (miRNA) expression and DNA methylation levels in sarcoid tumors. Abnormal patterns of methylation may be responsible for changes in gene expression levels, including microRNAs. Recently, the DNA methylation of gene bodies has also been shown to have an impact on gene expression. Thus, the aim of the study was to investigate the methylation pattern of gene bodies of chosen miRNAs identified in sarcoid tissue (miR-101, miR-10b, miR-200a, and miR-338-3p), which have also been established to play roles in neoplastic transformation. To this end, we applied qRT-PCR, Bisulfite Sequencing PCR (BSP), and Mquant methods. As a result, we identified the statistically significant downregulation of pri-mir-101-1, pri-mir-10b, and pri-mir-200a in the sarcoid samples in comparison to the control. The DNA methylation analysis revealed their hypermethylation. This suggests that DNA methylation may be one mechanism responsible for the downregulation of theses miRNAs. However, the identified differences in the methylation levels are not very high, which implies that other mechanisms may also underlie the downregulation of the expression of these miRNAs in equine sarcoids. For the first time, the results obtained shed light on microRNA expression regulation by gene body methylation in equine sarcoids and provide bases for further deeper studies on other mechanisms influencing the miRNA repertoire.
马在娱乐、畜牧业生产、欠发达国家的役用动物以及马辅助治疗中都具有重要作用。马的肉瘤是该物种中最常见的诊断肿瘤之一。它们可能引起不适和疼痛，并导致运动功能永久受损。其形成的分子基础仍在研究中。我们之前的研究揭示了肉瘤肿瘤中 miRNA（微小 RNA）表达和 DNA 甲基化水平的改变。异常的甲基化模式可能是导致基因表达水平变化的原因，包括 microRNAs。最近，基因体的 DNA 甲基化也被证明会影响基因表达。因此，本研究旨在探讨在肉瘤组织中已确定的选定 miRNA（miR-101、miR-10b、miR-200a 和 miR-338-3p）的基因体甲基化模式，这些 miRNA 也被证明在肿瘤转化中发挥作用。为此，我们应用了 qRT-PCR、亚硫酸氢盐测序 PCR（BSP）和 Mquant 方法。结果表明，与对照相比，在肉瘤样本中 pri-mir-101-1、pri-mir-10b 和 pri-mir-200a 的前体 miRNA 明显下调。DNA 甲基化分析显示其高度甲基化。这表明 DNA 甲基化可能是这些 miRNA 下调的一种机制。然而，鉴定出的甲基化水平差异并不高，这意味着其他机制也可能导致这些 miRNA 在马肉瘤中的表达下调。研究结果首次揭示了马肉瘤中基因体甲基化对 miRNA 表达调控的作用，并为进一步研究影响 miRNA 谱的其他机制提供了基础。
