Characterizing the factors that regulate the growth and development of muscle is central to animal production. Skeletal muscle satellite cells (SMSCs) provide an important material for simulating the proliferation and differentiation of muscle cells. YAP1, which can promote muscle growth, is closely related to the proliferation of SMSCs in Hu sheep (Ovis aries). In addition, some miRNAs, such as miR-541-3p, miR-142-5p, and miR-29a, can play critical roles in muscle growth by specifically binding with their target mRNAs. Meanwhile, lncRNA can competitively bind these miRNAs and reduce the regulatory effect of miRNAs on their target genes and thus play critical roles themselves in muscle growth. However, the regulatory molecular mechanism of miRNA and lncRNA on SMSC proliferation through YAP1 remains unclear. Here, we characterized the regulatory network among YAP1 and its targeted miRNAs and lncRNAs in Hu sheep SMSCs. The potential ncRNAs that regulate YAP1 (miR-29a and CTTN-IT1) were predicted through multilevel bioinformatics analysis. Dual-luciferase assays, RT-qPCR, and western blots revealed that miR-29a can significantly reduce the mRNA and protein expression level by binding to a specific 3′-UTR of YAP1 (P < 0.05), while CTTN-IT1 can restore the expression of YAP1 through competitive binding to miR-29a. Furthermore, the mRNA and protein expression levels of MyoG, MyoD, and MyHC showed that miR-29a can inhibit the expression of genes related to the differentiation of SMSCs, and CTTN-IT1 can increase the expression of these same genes. Thus, miR-29a may inhibit the differentiation of SMSCs and CTTN-IT1 can restore this inhibition. The EdU staining assay indicated that excessive miR-29a can significantly reduce the proliferation ability of SMSCs (P < 0.05), while overexpression of CTTN-IT1 can significantly increase the proliferation of SMSCs (P < 0.01). CTTN-IT1 is a novel lncRNA that is a competing endogenous RNA (ceRNA) of miR-29a and can promote SMSC proliferation and differentiation by restoring the expression of YAP1 when it is inhibited by miR-29a in Hu sheep. Overall, our findings construct a CTTN-IT1-miR-29a-YAP1 regulatory network that will help contribute new insight into improving the muscle development of Hu sheep.

表征调节肌肉生长和发育的因素对动物生产至关重要。骨骼肌卫星细胞（SMSC）为模拟肌肉细胞的增殖和分化提供了重要的材料。YAP1可以促进肌肉生长，与胡羊中的SMSC增殖密切相关（卵巢）。此外，某些miRNA，例如miR-541-3p，miR-142-5p和miR-29a，可以通过与目标mRNA特异性结合而在肌肉生长中发挥关键作用。同时，lncRNA可以竞争性地结合这些miRNA，并降低miRNA对其靶基因的调节作用，从而在肌肉生长中发挥关键作用。然而，miRNA和lncRNA的调控分子机制通过YAP1还不清楚。在这里，我们描述了监管网络之间YAP1及其在胡羊SMSC中的靶向miRNA和lncRNA。潜在的调控ncRNAYAP1（miR-29a和CTTN-IT1）通过多级生物信息学分析进行预测。双重荧光素酶检测，RT-qPCR和Western印迹显示，miR-29a可通过与特定的3'-UTR结合而显着降低mRNA和蛋白质表达水平。YAP1 （P <0.05），而CTTN-IT1可以恢复 YAP1通过竞争结合miR-29a。此外，MyoG，MyoD和MyHC的mRNA和蛋白质表达水平表明miR-29a可以抑制与SMSC分化相关的基因的表达，而CTTN-IT1可以增加这些相同基因的表达。因此，miR-29a可以抑制SMSC的分化，而CTTN-IT1可以恢复这种抑制。EdU染色试验表明，过量的miR-29a会显着降低SMSC的增殖能力（P <0.05），而CTTN-IT1的过表达可显着增加SMSC的增殖（P<0.01）。CTTN-IT1是一种新型的lncRNA，它是miR-29a的竞争性内源RNA（ceRNA），可以通过恢复mRNA的表达来促进SMSC增殖和分化。YAP1在胡羊中被miR-29a抑制时。总体而言，我们的发现构成了CTTN-IT1-miR-29a-YAP1 监管网络，将有助于改善湖羊肌肉的发育。