m6A甲基化酶相关基因在牛骨骼肌生成中的表达

【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METTL3,METTL14,WTAP,FTO和ALKBH5，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m^6...

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

【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.