Zhi Yunxia, Hu Boxin, Tian Shujun, Bai Ying, Chen Xiaoyong
College of Animal Science and Technology, Hebei Agricultural University, Baoding, China.
School of Life Science and Food Engineering, Hebei University of Engineering, Handan, China.
PLoS One. 2025 Jun 12;20(6):e0325054. doi: 10.1371/journal.pone.0325054. eCollection 2025.
BACKGROUND/OBJECTIVES: In sheep farming, the economic significance of meat characteristics is substantial, and advancing the genetic quality of livestock relies heavily on understanding the cellular mechanisms behind muscle growth and its regulation. This study examined miRNA expression patterns in the longissimus dorsi muscle tissue of Hanper sheep of various ages, with the goal of determining their biological functions and identifying miRNAs and their target mRNAs that influence muscle development and meat quality.
Using the Image-Pro Plus 6.0 program and HE and fluorescent staining procedures, we measured the diameter of muscle fibers in the longissimus dorsi of Hanper sheep at three distinct ages (1, 7, and 13 months) in order to calculate the average fiber size. For the analysis of muscle fiber area, one-way Analysis of Variance was conducted using Statistical Package for the Social Sciences 25.0, with Least Significant Difference tests applied afterward to compare the different groups. Transcriptome sequencing was conducted to identify miRNAs, and bioinformatics tools were applied to predict their target genes. GO and KEGG functional annotations were used to analyze the biological functions of these target genes. RT-qPCR was performed to validate the expression levels of differential expressed miRNAs.
Muscle fiber diameter and area increased progressively with age, as indicated by HE and fluorescence staining. Four novel miRNAs identified for the first time in sheep were among the 116 differential expressed miRNAs that were found. These miRNAs were found to be involved in key pathways such as TGF-β, mTOR, Wnt, and MAPK, which regulate muscle growth and development. It was determined that three new miRNA-mRNA pairs included oar-miR-133/MSC, oar-miR-148a/FST, and oar-miR-410-3p/NIN may be essential for muscle growth. RT-qPCR results confirmed the expression trends observed in the transcriptome sequencing data.
Our knowledge of the fundamental molecular mechanisms underpinning muscle growth and development is improved by the discovery of new miRNAs and the target genes that correspond to them. These findings may serve as new breeding targets for improving meat quality in sheep.
背景/目的：在养羊业中，肉品质的经济意义重大，提高家畜的遗传质量在很大程度上依赖于了解肌肉生长及其调控背后的细胞机制。本研究检测了不同年龄汉普夏羊背最长肌组织中的miRNA表达模式，旨在确定其生物学功能，并鉴定影响肌肉发育和肉质的miRNA及其靶mRNA。
使用Image-Pro Plus 6.0软件以及苏木精-伊红（HE）和荧光染色程序，我们测量了三个不同年龄（1、7和13个月）的汉普夏羊背最长肌的肌纤维直径，以计算平均纤维大小。对于肌纤维面积分析，使用社会科学统计软件包25.0进行单因素方差分析，随后应用最小显著差异检验来比较不同组。进行转录组测序以鉴定miRNA，并应用生物信息学工具预测其靶基因。使用基因本体论（GO）和京都基因与基因组百科全书（KEGG）功能注释来分析这些靶基因的生物学功能。进行逆转录定量聚合酶链反应（RT-qPCR）以验证差异表达miRNA的表达水平。
HE和荧光染色表明，肌纤维直径和面积随年龄增长而逐渐增加。在所发现的116个差异表达的miRNA中，有4个是首次在绵羊中鉴定出的新miRNA。这些miRNA参与了诸如转化生长因子-β（TGF-β）、哺乳动物雷帕霉素靶蛋白（mTOR）、Wnt和丝裂原活化蛋白激酶（MAPK）等调节肌肉生长和发育的关键途径。确定了三个新的miRNA- mRNA对，即oar-miR-133/间充质干细胞（MSC）、oar-miR-148a/卵泡抑素（FST）和oar-miR-410-3p/核仁蛋白（NIN）可能对肌肉生长至关重要。RT-qPCR结果证实了转录组测序数据中观察到的表达趋势。
新miRNA及其相应靶基因的发现增进了我们对肌肉生长和发育基本分子机制的了解。这些发现可能成为改善绵羊肉质的新育种目标。
