中国农业科学 ›› 2013, Vol. 46 ›› Issue (14): 3010-3021.doi: 10.3864/j.issn.0578-1752.2013.14.017

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

成肌细胞成脂过程中PPARγ、C/EBPα和Myogenin 基因启动子的甲基化变化

 姜美华, 巨婷婷, 刘洋, 许玲, 孙文娟, 赵泮峰, 尹靖东   

  1. 中国农业大学动物科技学院/动物营养学国家重点实验室,北京 100193
  • 收稿日期:2013-01-22 出版日期:2013-07-15 发布日期:2013-04-11
  • 通讯作者: 通信作者尹靖东,Tel:010-62733590-1404;E-mail:yinjd@cau.edu.cn
  • 作者简介:姜美华,Tel:010-62733691;E-mail:Jiangmh@mafic.ac.cn
  • 基金资助:

    国家自然科学基金项目(31272452,30972113)、国家“973”计划项目(2012CB124700)

Dynamics of PPARγ, C/EBPα and Myogenin Gene Promoter Methylation During Myoblasts Intracellular Lipid Accumulation

 JIANG  Mei-Hua, JU  Ting-Ting, LIU  Yang, XU  Ling, SUN  Wen-Juan, ZHAO  Pan-Feng, YIN  Jing-Dong   

  1. College of Animal Science and Technology, China Agricultural University/State Key Laboratory of Animal Nutrition, Beijing 100193
  • Received:2013-01-22 Online:2013-07-15 Published:2013-04-11

摘要: 【目的】利用C2C12成肌细胞探讨肌源性干细胞成脂过程与调控成脂和成肌分化的关键转录因子PPARγ(peroxisome proliferator-activated receptor gamma)、C/EBPα(CCAAT/enhancer binding protein alpha)和Myogenin启动子区甲基化的关系。【方法】分别用2%马血清和三联诱导剂诱导C2C12细胞成肌和成脂分化,在马血清促进的成肌分化第0、1、3和5天收集细胞进行姬姆萨染色观察肌管形成情况;在三联诱导的成脂分化第0、2、4、6和10天收集细胞进行油红O染色观察脂滴形成情况;提取成肌诱导第0、1、3、5天和成脂诱导第0、2、4、6天的RNA和DNA,分别采用qRT-PCR检测成肌和成脂分化相关基因的表达,采用重亚硫酸盐测序的方法检测PPARγ、C/EBPα和Myogenin启动子区甲基化的变化,并分析基因表达与甲基化状态的相关关系。【结果】①C2C12细胞经马血清诱导形成了多核肌管,表达成肌相关基因,但不表达脂肪特异性基因;三联诱导使C2C12细胞自主分化的肌管中沉积了脂滴,同时表达成脂和成肌相关基因;②重亚硫酸盐测序结果表明,在未分化的成肌细胞中,PPARγ基因启动子的甲基化水平是61%,在三联诱导第2、4和6天,其甲基化程度依次为49%、39%和42%,呈逐渐去甲基化趋势,与PPARγ基因转录负相关;在马血清诱导第3和5天,甲基化水平为56%和48%,与未分化的成肌细胞相比差异不显著,同时PPARγ基因的表达水平也没有显著变化;③Myogenin基因在马血清促进的成肌过程中甲基化水平不断降低(49%、42%、35%和34%),转录水平急剧增加;在三联诱导过程中,Myogenin启动子的甲基化水平由0天的49%下降为第1天的37%、第3天的41%和第5天的38%,但下降幅度弱于马血清诱导的成肌过程,这一结果与降低的Myogenin转录上调相吻合;④C/EBPα基因在未分化的C2C12细胞中呈低甲基化状态,甲基化程度仅为1.6%,在成肌分化和脂肪沉积过程中均未发生显著变化,与基因表达无显著关联。【结论】成脂和成肌关键转录因子PPARγ和Myogenin启动子区的DNA甲基化变化参与了成肌细胞的分化和脂肪沉积的调控。

关键词: C2C12细胞 , DNA甲基化 , 成肌分化 , 脂肪沉积

Abstract: 【Objective】This experiment was designed to investigate whether DNA methylation of adipogenic promoters (peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα)) and myogenic promoter (myogenin) was involved in C2C12 myoblasts intracellular lipid accumulation.【Method】C2C12 cells were treated, respectively, by 2% horse serum and an adipogenesis cocktail which typically contains 3-isobutyl-methylxanthine, dexamethasone and insulin (MDI). In order to observe the formation of myotubes, cells were incubated for 0 d, 1 d, 3 d, and 5 d by 2% horse serum and then were collected to perform Giemsa staining. To confirm the lipid accumulation, cells treated by adipogenesis program for 0 d, 2 d, 4 d, 6 d and 10 d were fixed to conduct Oil Red O staining. Genome DNA and total RNA were extracted from C2C12 cells during the myogenesis differentiation and intracellular lipid accumulation. Real-time quantitative reverse transcription PCR was used to detect the mRNA levels of myogenic and adipogenic related genes. The bisulfite sequencing was adopted to explore the sequential DNA methylation changes in PPARγ, C/EBPα and myogenin promoter regions, and the relationship between DNA methylation and gene expression was analyzed.【Result】After horse serum treatment, the morphology of multinucleated myotubes and up-regulation of myogenic genes were displayed in C2C12 cells, but the adipocyte-specific genes showed no significant changes. The adipogenesis cocktail induced the intracellular lipid accumulation during automatical differentiation into myotubes of C2C12 cells, and both the myogenic and adipigenic genes were up-regulated. The bisulfite sequencing showed that the methylation level of PPARγ gene in undifferentiated myoblasts was 61%, and progressively demethylated upon adipogenic differentiation with methylation levels of 49%, 39% and 42% on d 2, 4 and 6, which was accompanied by an increase of mRNA level of PPARγ. But during the horse serum treatment, there were no significant changes in PPARγ methylation levels (56% and 48% on d 3 and 5) and mRNA levels. In the case of myogenin, it showed decreased methylation levels after horse serum inducement with methylation levels of 49%, 42%, 35% and 34% on d 0, 1, 3 and 5, respectively, while the expression of myogenin increased rapidly. During the adipogenic inducement, myogenin gene also demethylated (49%, 37%, 41% and 38% on d 0, 2, 4 and 6), but the degree was weaker than that in horse serum treatment, which agreed with the reduced up-regulation of myogenin mRNA level. C/EBPα showed hypomethylated status with only 1.6% in undifferentiated C2C12 cells and maintained the hypomethylated status regardless the myogenesis or lipid accumulation, so the methylation of C/EBPα promoter seemed unrelated to its transcription.【Conclusion】The dynamics of DNA methylation of PPARγ and myogenin promoter are closely correlated with their gene expression, which indicates that DNA methylation may play a role in regulating the myogenesis and lipid accumulation in C2C12 cells.

Key words: C2C12 cells , DNA methylation , myogenesis differentiation , lipid accumulation