m6A甲基化酶相关基因在牛骨骼肌生成中的表达

doi:10.3864/j.issn.0578-1752.2023.01.013

摘要/Abstract

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m⁶A甲基化水平的变化。【结果】METTL3、METTL14和WTAP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）。FTO和ALKBH5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FTO和ALKBH5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，METTL3、METTL14和WTAP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

杨昕冉,马鑫浩,杜嘉伟,昝林森. m⁶A甲基化酶相关基因在牛骨骼肌生成中的表达[J]. 中国农业科学, 2023, 56(1): 165-178.

YANG XinRan,MA XinHao,DU JiaWei,ZAN LinSen. Expression Pattern of m⁶A Methylase-Related Genes in Bovine Skeletal Muscle Myogenesis[J]. Scientia Agricultura Sinica, 2023, 56(1): 165-178.

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基因名称 Gene name	引物序列 Primer sequence (5′-3′)	产物长度 Product length (bp)
METTL3	F: TCGAAAGCTGCACTTCAGAC	199
METTL3	R: TCCAACGCTCTGTGTAAGGG	199
METTL14	F: TGACATCAGAGAACTGACACCC	198
METTL14	R: AGGTCCAATCCTTCCCCAGA	198
WTAP	F: GCCTGGAAGTTTACGCCTGA	176
WTAP	R: TCCTGACTGCTTTTAAGCTCCT	176
FTO	F: AGCAGCGTACAACGTCACTT	193
FTO	R: AGGGTCGTCCTCACTTTCCT	193
ALKBH5	F: TACTTCTTCGGCGAGGGCTA	191
ALKBH5	R: TGGTAGTCGTTGATGACGGC	191
MYOD1	F: AACCCCAACCCGATTTACC	196
MYOD1	R: CACAACAGTTCCTTCGCCTCT	196
MYOG	F: GGCGTGTAAGGTGTGTAAG	85
MYOG	R: CTTCTTGAGTCTGCGCTTCT	85
MYF6	F: GTGATAACTGCCAAGGAAGGAG	93
MYF6	R: CGAGGAAATGCTGTCCACGA	93
MYH3	F: TGAACGCCCTCTCCAAATCC	101
MYH3	R: AATGAAGTGCTGTCTCGGCA	101
GAPDH	F: AGTTCAACGGCACAGTCAAGG	124
GAPDH	R: ACCACATACTCAGCACCAGCA	124

m⁶A甲基化酶相关基因在牛骨骼肌生成中的表达

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

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