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

doi:10.1016/j.jia.2023.05.025

Journal of Integrative Agriculture

2024, Vol. 23

Issue (8): 2764-2777 DOI: 10.1016/j.jia.2023.05.025

Animal Science · Veterinary Medicine

Advanced Online Publication | Current Issue | Archive | Adv Search

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

Xiaoxu Shen^{1, 2*}, Yongtong Tian^{1, 2*}, Wentao He^{1, 2}, Can He^{1, 2}, Shunshun Han^{1, 2}, Yao Han^{1, 2}, Lu Xia^{1, 2}, Bo Tan3, Menggen Ma⁴, Houyang Kang⁵, Jie Yu⁶, Qing Zhu^{1, 2#}, Huadong Yin^{1, 2#}

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
²Farm 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
⁴College 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

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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mRNA的3’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-5p和miR-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。TGFBR1和TGF-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

Keywords: MiRNA-181-5p family SMSCs differentiation TGFBR1 TGF-β signaling

Received: 16 January 2023 Accepted: 19 March 2023

Fund:

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).

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.

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Xiaoxu Shen, Yongtong Tian, Wentao He, Can He, Shunshun Han, Yao Han, Lu Xia, Bo Tan, Menggen Ma, Houyang Kang, Jie Yu, Qing Zhu, Huadong Yin. 2024. Gga-miRNA-181-5p family facilitates chicken myogenesis via targeting TGFBR1 to block TGF-β signaling. Journal of Integrative Agriculture, 23(8): 2764-2777.

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