Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (1): 165-178.doi: 10.3864/j.issn.0578-1752.2023.01.013

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

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;


【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

Table 1

Primers for RT-qPCR"

基因名称 Gene name 引物序列 Primer sequence (5′-3′) 产物长度 Product length (bp)

Fig. 1

Expression of m6A methylase genes in different parts of skeletal muscle in Qinchuan beef cattle A: Relative mRNA expression of METTL3 in different parts of skeletal muscle in newborn and adult Qinchuan beef cattle; B: METTL14; C: WTAP; D: FTO; E: ALKBH5. *means significant difference (P<0.05), **means extremely significant difference (P<0.01), no makers mean no significant difference (P>0.05). The same as below"

Fig. 2

Detection of proliferation phase of skeletal muscle satellite cells in Qinchuan beef cattle A: Phenotypic observation of bovine SMSCs proliferation (scale bar: 200 μm); B: Growth curve of bovine SMSCs; C: Identification of SMSCs based on PAX7 and MYOD1 expression in growth medium for 48 h (scale bar: 50 μm)"

Fig. 3

Detection of bovine SMSCs myogenic differentiation A: Observation of myotube formation on days 0, 2, 4, 6 and 8 of culture in differentiation medium after the induction of myogenic differentiation (scale bar: 100 μm); B: Identification of differentiated myotubes based on MYHC expression after culture in differentiation medium for 2, 4 and 6 days (scale bar: 200 μm); C: Relative mRNA expression of myogenic genes (MYOD1, MYOG, MYF6 and MYH3) during bovine SMSCs differentiation. Different capital letters indicate very significant differences (P<0.01), different lowercase letters indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05). The same as below"

Fig. 4

Temporal expression profile of m6A methylation-related genes during SMSCs proliferation A: Temporal mRNA expression profile of METTL3 during SMSCs proliferation; B: METTL14; C: WTAP; D: FTO; E: ALKBH5"

Fig. 5

Temporal expression profile of m6A methylation-related genes during SMSCs myogenic differentiation A: Temporal mRNA expression profile of METTL3 during SMSCs myogenic differentiation; B: METTL14; C: WTAP; D: FTO; E: ALKBH5"

Fig. 6

Changes of m6A level during SMSCs myogenic differentiation A: LC-MS/MS assay showing the amount of mRNA m6A during myogenic differentiation; **means extremely significant difference (P<0.01). B: Dot blotting was used to detect the m6A modification of mRNA at D0, D2 and D4 during SMSCs myogenic differentiation. Methylene blue staining was used as a loading control"

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