Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (8): 2764-2777.DOI: 10.1016/j.jia.2023.05.025

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miRNA-181-5p家族通过靶向TGFBR1基因抑制TGF-β信号通路从而促进鸡骨骼肌的肌生成

  

  • 收稿日期:2023-01-16 接受日期:2023-03-19 出版日期:2024-08-20 发布日期:2024-07-29

Gga-miRNA-181-5p family facilitates chicken myogenesis via targeting TGFBR1 to block TGF-β signaling

Xiaoxu Shen1, 2*, Yongtong Tian1, 2*, Wentao He1, 2, Can He1, 2, Shunshun Han1, 2, Yao Han1, 2, Lu Xia1, 2, Bo Tan3, Menggen Ma4, Houyang Kang5, Jie Yu6, Qing Zhu1, 2#, Huadong Yin1, 2#   

  1. 1 Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
    2 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China 
    3 College of Forestry, Sichuan Agricultural University, Chengdu 611130, China
    4 College of Resources, Sichuan Agricultural University, Chengdu 611130, China
    5 Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, China
    6 Key Laboratory for Animal Disease Resistance Nutrition of China, Institute of Animal Nutrition, Ministry of Education/Sichuan Agricultural University, Chengdu 611130, China
  • Received:2023-01-16 Accepted:2023-03-19 Online:2024-08-20 Published:2024-07-29
  • About author:Xiaoxu Shen, E-mail: shenxiaoxu@stu.sicau.edu.cn; Yongtong Tian, E-mail: tianyongtong@stu.sicau.edu.cn; #Correspondence Qing Zhu, E-mail: zhuqing@sicau.edu.cn; Huadong Yin, E-mail: yinhuadong@sicau.edu.cn * These authors contributed equally to this study.
  • Supported by:

    This study was economically supported by the National Key Research and Development Program of China (2022YFF10002020) and Sichuan Science and Technology Program, China (2021YFYZ0007 and 2021YFYZ0031).

摘要:

鸡是一种重要的农业动物,其提供的骨骼肌是人类社会重要的蛋白质食物来源之一。骨骼肌的产量主要取决于肌纤维的发育,而该生物学过程受到许多基因,信号通路和非编码RNA的调控。microRNAs (miRNAs)是一类22个碱基左右的小非编码RNA,通常可以结合靶基因mRNA3’端非翻译区3’UTR)从而抑制靶基因的表达。有研究表明miR-181-5p家族(主要是miR-181a-5pmiR-181b-5p)可以调控哺乳动物骨骼肌的发育,然而miR-181-5p家族在鸡骨骼肌发育过程中的功能和调控机制仍然不清楚等。在本研究中,我们利用肌肉发育速度显著不同的蛋鸡与肉鸡这对极端模型,发现miR-181-5p家族在肌肉快速发育的肉鸡骨骼肌中高表达。然后我们采集鸡的骨骼肌卫星细胞来研究miR-181-5p家族在鸡骨骼肌体外肌生成中的作用,结果发现miR-181-5p家族显著促进成肌分化相关基因的RNA和蛋白表达,并且促进骨骼肌卫星细胞分化成多核的肌管,说明miR-181-5p家族在鸡骨骼肌发育过程中起正向的作用。随后靶基因预测发现miR-181-5p家族可能靶向TGF-β信号通路的重要受体TGFBR1,我们通过双荧光素酶报告等试验技术验证了miR-181-5p家族可以通过结合TGFBR1基因的3’UTR从而抑制TGFBR1基因的表达。TGFBR1TGF-β信号通路都是已知的肌肉发育限制因子,我们利用鸡骨骼肌卫星细胞验证发现TGFBR1基因负调控鸡骨骼肌体外肌生成过程。在TGF-β信号通路中,TGF-β蛋白通过结合细胞膜上的TGFBR1/2蛋白复合体将细胞外的TGF-β信号转导进入细胞质中,促进下游SMAD2/3蛋白的磷酸化并促使其进入细胞增强肌肉发育抑制基因的转录。我们通过蛋白质免疫印迹实验发现干扰TGFBR1会导致SMAD2/3蛋白的磷酸化程度降低,TGF-β信号通路转导受阻,此外miR-181-5p家族可以同时抑制TGFBR1的表达和SMAD2/3蛋白的磷酸化程度总之,我们的研究结果发现miR-181-5p家族可以通过靶向TGFBR1基因抑制TGF-β信号通路的转导,从而促进鸡骨骼肌的体外肌生成,表明miR-181-5p家族是鸡骨骼肌发育的促进因子,可能是导致肉鸡肌肉快速发育的重要调控因子之一,并可能成为分子标记辅助育种提高地方鸡肌肉产能的有效分子靶点

Abstract:

MicroRNAs (miRNAs) have been demonstrated to control chicken skeletal muscle growth, however, the potential function of the miR-181-5p family in chicken myogenesis remains largely unknown.  Here, our study identified the two chicken (Gallus gallus; Gga) miR-181-5p family members widely expressed in various tissues, specifically miR-181a-5p and miR-181b-5p.  Besides, the breast muscles of fast-growing broilers expressed higher levels of miR-181a-5p and miR-181b-5p than those of slow-growing layers.  Functionally, miR-181a-5p and miR-181b-5p both promote the expression level of myogenic factors including myogenin (MyoG), myogenic differentiation 1 (MyoD1), and myosin heavy chain (MyHC), meanwhile accelerating the myotube formation of skeletal muscle satellite cells (SMSCs).  Mechanistically, miR-181a-5p and miR-181b-5p directly bind to the 3´ untranslated region (UTR) of the transforming growth factor beta receptor 1 (TGFBR1) mRNA, further reducing the expression of TGFBR1.  TGFBR1 is a key Transforming growth factor beta (TGF-β) signaling transduction receptor and had a negative function in muscle cell differentiation.  Furthermore, knockdown of TGFBR1 facilitated the expression of chicken myogenic factors, boosted myotube formation, and decreased the SMAD family member 2/3 (SMAD2/3) phosphorylation in chicken SMSCs.  SMAD2/3 are downstream of TGF-β signaling, and miR-181a-5p and miR-181b-5p could reduce the expression of TGFBR1 to further diminish the SMAD2/3 phosphorylation.  Our findings revealed that the miR-181-5p family targets TGFBR1 to break the TGF-β signaling transduction, which resulted in promoting chicken skeletal muscle development

Key words: MiRNA-181-5p family ,  SMSCs ,  differentiation ,  TGFBR1 ,  TGF-β signaling