RNA甲基化转移酶METTL3对牛骨骼肌卫星细胞增殖与成肌分化的影响

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

【目的】探究RNA甲基化转移酶样3(methyltransferase-like 3,METTL3)对牛骨骼肌卫星细胞(bovine skeletal muscle satellite cells,BSMSCs)增殖和成肌分化的影响。【方法】利用体外诱导BSMSCs分化模型模拟牛骨骼肌生长发育过程,采用实时荧光定量PCR技术和蛋白质免疫印迹技术(Western blotting)检测METTL3在BSMSCs分化前后mRNA和蛋白表达水平的差异。设计合成METTL3干扰RNA(si-METTL3),且构建METTL3的过表达载体pcDNA3.1-METTL3,分别转染至BSMSCs中,通过实时荧光定量PCR和Western blotting检测转染效果和BSMSCs增殖标志因子盒转录家族成员7(paired box 7,Pax7)的表达情况,EdU检测细胞增殖情况。将转染si-METTL3和pcDNA3.1-METTL3的BSMSCs进行体外诱导分化,通过光学显微镜观察BSMSCs进入分化期的细胞形态,利用实时荧光定量PCR和Western blotting检测BSMSCs分化标志因子肌球蛋白重链(myosin heavy chain,MyHC)、肌细胞生成素(myogenin,MyoG)的表达情况。通过比色法检测干扰或过表达METTL3后,BSMSCs不同时期的m 6 A RNA甲基化水平。【结果】BSMSCs分化后METTL3蛋白表达水平极显著高于增殖期(P<0.01);干扰或过表达METTL3后,Pax7的mRNA水平以及蛋白水平均无显著变化(P>0.05),EdU阳性细胞率与对照组相比均无显著差异(P>0.05);干扰METTL3后,细胞肌管数量明显增多,肌管直径明显增大,MyHC的mRNA和蛋白表达水平极显著升高(P<0.01),MyoG的蛋白表达水平极显著升高(P<0.01),并且BSMSCs增殖和分化期的m 6 A RNA甲基化水平均极显著下调(P<0.01);过表达METTL3后,细胞肌管数量明显减少,肌管直径明显变小,MyHC的mRNA和蛋白表达水平极显著降低(P<0.01),MyoG的蛋白表达水平极显著降低(P<0.01),并且BSMSCs增殖和分化期的m 6 A RNA甲基化水平均极显著上升(P<0.01)。【结论】METTL3对BSMSCs的增殖过程没有显著影响,但对BSMSCs的成肌分化过程具有调控作用,并影响BSMSCs的m 6 A RNA甲基化水平。干扰METTL3可以促进BSMSCs的成肌分化进程,降低BSMSCs的m 6 A RNA甲基化水平;而过表达METTL3会抑制BSMSCs的分化,提高BSMSCs的m 6 A RNA甲基化水平。试验结果为进一步探明METTL3在牛肌肉发育中的调控机制奠定了基础。

【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 m 6 A 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 m 6 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 m 6 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 m 6 A mRNA methylation level of BSMSCs.Interfering with METTL3 could promote myogenic differentiation progression,and reduce the m 6 A RNA methylation levels of BSMSCs.While overexpressing METTL3 could inhibit the myogenic differentiation progression,and increase the m 6 A RNA methylation levels of BSMSCs.The results provided the foundation for further exploring the regulatory mechanism of METTL3 in bovine muscle development.