siRNA沉默Myh3基因抑制C2C12细胞糖酵解  

Silencing of Myh3 Gene by siRNA Inhibits Glycolysis in C2C12 Cells

作　　者：温作晨 楚菡 代宇星 罗云燕 张建斌 李淑英 洪亮 蒲蕾 张颖锋 WEN Zuo-Chen;CHU Han;DAI Yu-Xing;LUO Yun-Yan;ZHANG Jian-Bin;LI Shu-Ying;HONG Liang;PU Lei;ZHANG Ying-Feng(Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Breeding,College of Animal Science and Animal Medicine,Tianjin 300384,China;Surgery Department of Xian Jiren Hospital,Xian 710300,China)

机构地区：[1]天津农学院动物科学与动物医学学院,天津市农业动物繁育与健康养殖重点实验室,天津300384 [2]西安济仁医院手术部,西安710300

出　　处：《中国生物化学与分子生物学报》2022年第11期1511-1519,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：天津市自然科学基金(No.20JCQNJC00650);天津市教委科研计划项目(No.2018KJ188);青海省重点研发与转化计划—科技援青合作专项(No.2021-QY-204);广西首牧种猪育种有限公司委托项目(No.TNHXKJ2020039);泾阳百川合汇农业开发有限公司委托项目(No.TNHXKJ2020040);天津生猪产业技术体系创新团队(No.ITTPRS2021006)资助。

摘　　要：肌球蛋白重链3(myosin heavy chain 3,Myh3)基因为肌肉细胞分化的标志基因,调节肌肉细胞能量的利用,但其是否会影响肌肉细胞不同状态下的糖酵解过程尚鲜有报道。本文以成肌和成脂分化不同阶段的小鼠C2C12细胞为模型,利用qRT-PCR方法研究Myh3与糖酵解相关基因Pkm(M-type pyruvate kinse)、Prkag3(protein kinase adenosine monophosphate-activatedγ3-subunit)和Gsk3β(glycogen synthase kinase-3β)的表达模式。发现在C2C12细胞成肌分化过程中,Myh3与糖酵解基因Prkag3和Pkm的相对表达趋势基本一致,都呈现相对表达水平先上升,分化第2 d达到峰值,之后下降的趋势;糖原合酶抑制基因Gsk3β的表达趋势相对平稳。而在C2C12细胞成脂分化过程中,Myh3依然与糖酵解基因Prkag3和Pkm的相对表达趋势基本一致,相对表达量逐渐上升,在分化第8 d达到最高值;糖原合酶抑制基因Gsk3β的表达保持稳定状态。在C2C12细胞成肌分化状态下,qRT-PCR和Western印迹检测干扰Myh3对细胞糖酵解相关基因Pkm、Prkag3和Gsk3βmRNA和蛋白质表达的影响。结果显示,干扰Myh3后,糖酵解基因Pkm和Prkag3的mRNA表达量极显著降低(P<0.01),糖原合酶抑制基因Gsk3β的mRNA表达无明显变化(P>0.05);Myh3干扰组中Myh3和Pkm的蛋白质水平显著低于空白组和NC组细胞。在C2C12细胞成脂分化状态下,干扰Myh3,糖原合酶抑制基因Gsk3β和糖酵解基因Prkag3的mRNA表达量极显著升高(P<0.01),糖酵解基因Pkm的mRNA表达下降;Myh3干扰组中Myh3和Pkm的蛋白质水平也低于空白组和NC组细胞。综合以上研究,C2C12细胞成肌和成脂状态下糖酵解水平存在明显差异,Myh3与酵解基因的表达模式相似,进一步研究发现,干扰Myh3可以抑制C2C12细胞成肌状态下的糖酵解,不影响糖原合成。与成肌状态不同,在C2C12细胞成脂状态下干扰Myh3,抑制了糖原合成和糖酵解。The Myh3(myosin heavy chain 3)gene is a marker gene of muscle cell differentiation and regulates the utilization of energy in muscle cells,but whether it affects the glycolysis process of muscle cells in different states is rarely reported.In this paper,the expression patterns of Myh3 and glycolysis-related genes Pkm(M-type pyruvate kinse),Prkag3(protein kinase adenosine monophosphate-activatedγ3-subunit),and Gsk3β(glycogen synthase kinase-3β)were studied by the qRT-PCR(quantitative-Real-Time-PCR)method using C2 C12 cells at different stages of myoblast and adipogenic differentiation as models.It was found that in the process of myoblast differentiation of C2 C12 cells,the relative expression trends of Myh3 and glycolysis genes Prkag3 and Pkm were basically the same,and the relative expression levels first increased,reached the peak on the second day of differentiation,and then decreased;glycogen synthase the expression trend of the inhibitory gene Gsk3βwas relatively stable.In the process of adipogenic differentiation of C2 C12 cells,the relative expression trend of Myh3 and glycolysis genes Prkag3 and Pkm remained basically the same,and the relative expression gradually increased,reaching the highest value on the 8 th day of differentiation;glycogen synthase inhibitory gene Gsk3βexpression remained stable.In the myogenic differentiation state of C2 C12 cells,qRT-PCR and Western blotting were used to detect the effects of interfering Myh3 on the mRNA and protein expressions of glycolysis-related genes Pkm,Prkag3,and Gsk3β.The results showed that after interfering with Myh3,the mRNA expressions of glycolysis genes Pkm and Prkag3 were significantly decreased(P<0.01),while the mRNA expression of glycogen synthase inhibitory gene Gsk3βhad no significant change(P>0.05).The protein levels of Myh3 and Pkm were significantly lower than those in the blank group and NC group.Under the adipogenic differentiation state of C2 C12 cells,after interfering with Myh3,the mRNA levels of glycogen synthase inhibitor Gsk3βan

关 键 词：肌球蛋白重链3 SIRNA干扰 C2C12细胞 酵解基因 

分 类 号：Q254[生物学—细胞生物学]