China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (3): 1025-1036.doi: 10.16431/j.cnki.1671-7236.2023.03.018

• Genetics and Breeding • Previous Articles     Next Articles

Effect of RNA Methylation Transfer Enzyme METTL3 on the Proliferation and Myogenic Differentiation of Bovine Skeletal Muscle Satellite Cells

ZHEN Zhen, WANG Mei, WANG Yimin, HU Debao, ZHANG Linlin, LI Xin, GUO Yiwen, GUO Hong, DING Xiangbin   

  1. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China
  • Received:2022-08-24 Online:2023-03-05 Published:2023-03-02

Abstract: 【Objective】 The aim of this study was to investigate the effects of RNA methyltransferase like 3 (METTL3) on the proliferation and differentiation of bovine skeletal muscle satellite cells (BSMSCs).【Method】 In this study,the growth and development of bovine skeletal muscle was simulated by the BSMSCs model induced differentiation in vitro.The difference of expression level of mRNA and proteins of METTL3 before and after differentiation of BSMSCs were detected by Real-time quantitative PCR (qRT-PCR) and Western blotting methods.The METTL3 interfering RNA (si-METTL3) was designed and synthesized,and the overexpression vector pcDNA3.1-METTL3 of METTL3 was constructed and transfected into BSMSCs respectively.The transfection effect and the expression of paired box 7 (Pax7) in BSMSCs were detected by qRT-PCR and Western blotting,and the cell proliferation was detected by EdU method.The BSMSCs transfected with si-METTL3 and pcDNA3.1-METTL3 were induced to differentiate in vitro,and the cell morphology of BSMSCs in the differentiation stage was observed by light microscope.The expression of myosin heavy chain (MyHC) and myogenin (MyoG),the differentiation marker of BSMSCs was detected by qRT-PCR and Western blotting.The m6A RNA methylation levels of BSMSCs at different periods after interference or overexpression of METTL3 were detected by colorimetry.【Result】 The results showed that after the differentiation of BSMSCs,the expression level of METTL3 protein was extremely significantly higher than that in the proliferation period (P<0.01).After METTL3 was interfered or overexpressed,there was no significant difference in the mRNA and protein levels of Pax7 (P>0.05),and neither of the EdU positive cell rate had no significant difference compared with the control group (P>0.05).After interfering with the expression of METTL3,the number and diameter of cellular myotubes increased significantly,the levels of mRNA and protein expression of MyHC were extremely significantly higher than that of the control group (P<0.01),the level of protein expression of MyoG was extremely significantly higher than that of the control group (P<0.01),and the m6 A RNA methylation levels during BSMSCs proliferation and differentiation phase were extremely significantly downregulated(P<0.01).After overexpressing of METTL3,the number and diameter of cellular myotubes were significantly reduced,the levels of mRNA and protein expression of MyHC were extremely significantly lower than that of the control group (P<0.01),the level of protein expression of MyoG was extremely significantly lower than that of the control group (P<0.01),and the m6 A RNA methylation levels during BSMSCs proliferation and differentiation phase were increased extremely significantly (P<0.01).【Conclusion】 The METTL3 had no significant effect on the proliferation of the BSMSCs,but it could regulate the myogenic differentiation process of BSMSCs,and affect the m6 A mRNA methylation level of BSMSCs.Interfering with METTL3 could promote myogenic differentiation progression,and reduce the m6 A RNA methylation levels of BSMSCs. While overexpressing METTL3 could inhibit the myogenic differentiation progression,and increase the m6A RNA methylation levels of BSMSCs.The results provided the foundation for further exploring the regulatory mechanism of METTL3 in bovine muscle development.

Key words: bovine; METTL3; skeletal muscle satellite cells(BSMSCs); cell proliferation; muscle differentiation

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