中国农业科学 ›› 2023, Vol. 56 ›› Issue (1): 165-178.doi: 10.3864/j.issn.0578-1752.2023.01.013

• 畜牧·兽医 • 上一篇    下一篇

m6A甲基化酶相关基因在牛骨骼肌生成中的表达

杨昕冉1(),马鑫浩1,杜嘉伟1,昝林森1,2()   

  1. 1.西北农林科技大学动物科技学院,陕西杨凌 712100
    2.国家肉牛改良中心,陕西杨凌 712100
  • 收稿日期:2021-09-30 接受日期:2022-04-12 出版日期:2023-01-01 发布日期:2023-01-17
  • 通讯作者: 昝林森
  • 作者简介:杨昕冉,E-mail:yangxinran93@nwafu.edu.cn
  • 基金资助:
    国家重点研发计划(2018YFD0501700);国家自然科学基金(31972994);国家肉牛牦牛产业技术体系(CARS-37)

Expression Pattern of m6A Methylase-Related Genes in Bovine Skeletal Muscle Myogenesis

YANG XinRan1(),MA XinHao1,DU JiaWei1,ZAN LinSen1,2()   

  1. 1. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi
    2. National Beef Cattle Improvement Center, Yangling 712100, Shaanxi
  • Received:2021-09-30 Accepted:2022-04-12 Online:2023-01-01 Published:2023-01-17
  • Contact: LinSen ZAN

摘要:

【目的】近年来,RNA m6A甲基化修饰在肌肉发育中的作用不断被发现,通过探究m6A甲基化酶相关基因,包括METTL3METTL14WTAPFTOALKBH5,在牛肌肉组织以及骨骼肌卫星细胞(skeletal muscle satellite cells, SMSCs)增殖和分化过程中的表达,同时分析体外成肌分化过程中m6A甲基化水平的变化,为阐明m6A修饰在骨骼肌发育中的作用及机制提供参考。【方法】使用实时荧光定量PCR(RT-qPCR)技术检测m6A甲基化酶相关基因在新生和成年牛不同部位骨骼肌中的表达。随后在秦川牛肉用新品系背最长肌中分离SMSCs,通过生长曲线、免疫荧光和RT-qPCR技术验证SMSCs的增殖和成肌分化功能,使用RT-qPCR检测m6A甲基化酶相关基因在SMSCs增殖期24、36、48、60、72 h和分化第0、2、4、6、8天中的时序表达谱。最后利用LC-MS/MS和Dot blot技术检测SMSCs分化过程中m6A甲基化水平的变化。【结果】METTL3METTL14WTAP等m6A甲基化转移酶基因在成年牛背最长肌、前腿肌和后腿肌中的表达量均显著低于新生牛(P<0.01)。FTOALKBH5等m6A去甲基化酶基因在成年牛后腿肌中的表达更高(P<0.01),ALKBH5在成年牛背最长肌中的表达也较高(P<0.01)。分离的SMSCs具有良好的生长状态且可正常成肌分化。在SMSCs增殖期,METTL3表达逐渐下降,但在增殖后期时明显提高。METTL14在增殖期的表达变化差异不明显,WTAP则在增殖48 h后表达逐渐降低。而FTOALKBH5在增殖期的时序表达类似,60 h之前变化不显著,但在72 h时显著提高。在SMSCs成肌分化过程中,METTL3METTL14WTAP的表达模式基本一致,分化前期上升,随后下降,在分化末期表达增加。而FTO的表达随分化进行逐渐增加,ALKBH5则在分化前4天表达上升,随后不断下降。此外,在SMSCs分化过程中,mRNA的整体m6A水平下降(P<0.01)。【结论】m6A甲基转移酶和去甲基化酶在新生牛和成年牛骨骼肌中的表达变化存在较为明显的差异,表明m6A修饰可能对秦川牛骨骼肌的发育具有重要作用。同时,这些m6A相关甲基化酶可能参与调控牛骨骼肌卫星细胞的增殖和分化。这一发现为研究m6A甲基化修饰调控骨骼肌生成的作用及机制提供理论依据。

关键词: m6A, m6A甲基化酶, 牛, 骨骼肌卫星细胞, 细胞增殖, 成肌分化

Abstract:

【Objective】 The role of RNA m6A methylation modification in muscle development has been continuously discovered in recent years. The aim of this study was to explore the mRNA expression of m6A methylases, including METTL3, METTL14, WTAP, FTO, and ALKBH5, in bovine muscle tissue and in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). Meanwhile, the changes of m6A level during SMSCs myogenic differentiation in vitro were analyzed. This study could provide a reference for clarifying the role and mechanism of m6A modification in skeletal muscle development. 【Method】 The expression of m6A methylases in skeletal muscle of newborn and adult cattle was detected by real-time quantitative PCR (RT-qPCR). Then, SMSCs were isolated from the Longissimus dorsi muscle of Qinchuan beef cattle. The proliferation and myogenic differentiation of SMSCs were verified by cell growth curve, immunofluorescence and RT-qPCR, and the temporal expression profiles of m6A methylases in proliferation (24 h, 36 h, 48 h, 60 h, and 72 h) and differentiation (Day 0, 2, 4, 6, and 8) of SMSCs were detected by RT-qPCR. Finally, the m6A levels during SMSCs differentiation were detected using LC-MS/MS and dot blot assays. 【Result】 The mRNA expression levels of m6A methyltransferases, including METTL3, METTL14, and WTAP, in the Longissimus dorsi muscle, forelegs muscle and hind legs muscle of adult cattle were significantly lower than those of newborn cattle (P<0.01). The mRNA expression of demethylases such as FTO and ALKBH5 in the hind leg muscle of adult cattle was higher (P<0.01), and ALKBH5 was higher in the Longissimus dorsi muscle of adult cattle (P<0.01). The isolated SMSCs had the functions of normal growth, proliferation and myogenic differentiation. The expression of METTL3 decreased gradually in SMSCs proliferation, but increased significantly at 72 h. The expression of METTL14 did not change significantly, while WTAP reached the highest level at 48 h, and then decreased gradually. The temporal expression profiles of FTO and ALKBH5 were similar in the proliferative phase. They did not change obviously before 60 h and increased significantly at 72 h. The expression patterns of METTL3, METTL14 and WTAP were consistent during SMSCs differentiation, with an increase in the early stage of differentiation, followed by a decrease, and an increase in the late stage of differentiation. The expression of FTO increased gradually with differentiation. The expression of ALKBH5 increased during the first 4 days of differentiation and then continuously decreased. Furthermore, the overall m6A level of mRNA declined during the myogenic differentiation in SMSCs (P<0.01). 【Conclusion】 The expression changes of m6A methyltransferases and demethylases in skeletal muscle of newborn and adult cattle were significantly different, suggesting that m6A modification might have an important role in the development of skeletal muscle in Qinchuan cattle. Meanwhile, these m6A methylases might regulate the proliferation and differentiation of bovine SMSCs. These results provide a theoretical basis for the study of the role and molecular mechanism of m6A methylation modifications in regulating skeletal myogenesis.

Key words: N6-methyladenosine (m6A), m6A methylase, cattle, skeletal muscle satellite cells, cell proliferation, myogenic differentiation