Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Cells. 2023 Mar 20;12(6):943. doi: 10.3390/cells12060943.
Muscle development is a complex biological process involving an intricate network of multiple factor interactions. Through the analysis of transcriptome data and molecular biology confirmation, this study aims to reveal the molecular mechanism underlying sheep embryonic skeletal muscle development. The RNA sequencing of embryos was conducted, and microRNA (miRNA)-mediated competitive endogenous RNA (ceRNA) networks were constructed. qRT-PCR, siRNA knockdown, CCK-8 assay, scratch assay, and dual luciferase assay were used to carry out gene function identification. Through the analysis of the ceRNA networks, three miRNAs (miR-493-3p, miR-3959-3p, and miR-410-5p) and three genes (, and ) were identified. The qRT-PCR of the DE-miRNAs and genes in the muscle tissues of sheep showed that the expression levels of the gene and miR-410-5p were correlated with the growth rate. The knockdown of the gene by siRNA could significantly inhibit the proliferation of sheep primary embryonic myoblasts, and the expression levels of , and were significantly downregulated. The targeting relationship between miR-410-5p and the gene was validated by a dual luciferase assay, and miR-410-5p can significantly downregulate the expression of in sheep primary embryonic myoblasts. We proved the regulatory relationship between miR-410-5p and the gene, which was related to the proliferation of sheep embryonic myoblasts. The results provide a reference and molecular basis for understanding the molecular mechanism of embryonic muscle development.
肌肉发育是一个复杂的生物学过程,涉及到多个因素相互作用的复杂网络。本研究通过分析转录组数据和分子生物学验证,旨在揭示绵羊胚胎骨骼肌肉发育的分子机制。对胚胎进行 RNA 测序,并构建 microRNA(miRNA)介导的竞争性内源 RNA(ceRNA)网络。采用 qRT-PCR、siRNA 敲低、CCK-8 检测、划痕实验和双荧光素酶报告基因实验进行基因功能鉴定。通过 ceRNA 网络分析,鉴定出三个 miRNA(miR-493-3p、miR-3959-3p 和 miR-410-5p)和三个基因(、和)。绵羊肌肉组织中 DE-miRNAs 和基因的 qRT-PCR 结果显示,基因和 miR-410-5p 的表达水平与生长速度相关。siRNA 敲低基因可显著抑制绵羊原代胚胎成肌细胞的增殖,和的表达水平显著下调。双荧光素酶报告基因实验验证了 miR-410-5p 与基因的靶向关系,miR-410-5p 可显著下调绵羊原代胚胎成肌细胞中基因的表达。我们证明了 miR-410-5p 与基因的调控关系,该关系与绵羊胚胎成肌细胞的增殖有关。这些结果为理解胚胎肌肉发育的分子机制提供了参考和分子基础。