miR-34c通过YY1调控骨骼肌细胞增殖及分化的机制研究

发布时间：2018-03-30 10:47

本文选题：miR-34c　切入点：YY1　出处：《中国农业大学》2017年博士论文

【摘要】：骨骼肌的发育是一个漫长而复杂的过程,其中成肌细胞的增殖和分化在这个过程中起到了关键的作用。成肌细胞的增殖与分化受到一些信号通路、转录因子以及microRNAs(miRNAs)的精确调控。miRNAs是一类非编码的单链小RNA,可以通过与靶基因mRNA的3'端非编码区(3'UTR)结合来抑制靶基因的表达,从而调控多种生物学进程。很多研究证明miRNAs在骨骼肌发育与再生、卫星细胞的激活以及成肌细胞的增殖与分化方面起着重要的作用。miR-34c作为抑癌基因成为近些年研究的热点,它可以通过抑制细胞的增殖来抑制多种肿瘤的生长、迁移和入侵,但是对miR-34c在骨骼肌中功能的研究却很少。因此本研究主要以小鼠C2C12成肌细胞系为模型,来研究miR-34c在成肌细胞增殖和分化中所起到的作用,同时探究miR-34c发挥功能所涉及的靶基因,并进一步阐明其在骨骼肌发育过程中的功能及其调控机制。首先,本研究发现miR-34c在处于增殖状态的C2C12成肌细胞中低表达,并随着细胞的成肌分化进程,表达量逐渐升高。其次,在C2C12成肌细胞以及从小鼠骨骼肌中分离出的原代成肌细胞中过表达miR-34c,可以抑制细胞的增殖,使细胞周期阻滞在G0/G1期,细胞周期相关基因的表达量下调;相反,抑制miR-34c的表达可以促进成肌细胞的增殖,使更多的细胞通过G0/G1期,细胞周期相关基因的表达量上调。再次,在C2C12成肌细胞中过表达miR-34c可以促进细胞的分化,表现为MyoG和MyHC的表达量上调,多核肌管的数量增加;而抑制miR-34c的表达会抑制C2C12成肌细胞的分化,MyoG和MyHC的表达量下调,多核肌管的数量减少。最后,发现YY1是miR-34c的靶基因,miR-34c可以抑制YY1的表达。与过表达miR-34c的表型相似,在C2C12成肌细胞中抑制YY1的表达可以抑制细胞的增殖,使细胞周期阻滞在G0/G1期,细胞周期相关基因的表达量下调,并且可以促进细胞的分化,MyoG和MyHC的表达量上调,多核肌管的数量增加。在对卫星细胞的研究中发现,miR-34c对卫星细胞的激活也有着重要的调控作用。与静息态的卫星细胞相比,在激活态的卫星细胞中miR-34c表达量上调。在卫星细胞中抑制miR-34c的表达,可导致Pax7和MyoD的表达量下调,分化后MyoG和MyHC的表达量下调,多核肌管数量减少,说明抑制了 miR-34c在卫星细胞中的表达会抑制卫星细胞的激活以及分化。除此之外,本研究还发现miR-34c对横纹肌肉瘤的增长也具有抑制作用。在横纹肌肉瘤细胞RD细胞系中过表达miR-34c,可以抑制RD细胞的增殖,使细胞周期阻滞在G0/G1期,细胞周期相关基因的表达量下调。综上所述,本研究发现miR-34c可以通过其靶基因YY1来调控成肌细胞的增殖与分化,主要是使细胞周期阻滞在G0/G1期,抑制成肌细胞的增殖,促进成肌细胞的分化。同时,miR-34c对卫星细胞的激活与分化也有重要的调控作用,而且可以抑制横纹肌肉瘤细胞的增殖。因此,本研究为探究miR-34c在骨骼肌发育及再生过程的功能提供了新的观点,为治疗横纹肌肉瘤提供了新的靶点。
[Abstract]:Skeletal muscle development is a long and complex process, including myoblast proliferation and differentiation in this process plays a key role. Proliferation and differentiation of muscle cells by some signaling pathways, transcription factor microRNAs (miRNAs) and the precise control of.MiRNAs is a kind of non encoding single stranded small RNA the target gene mRNA, the 3'non encoding region (3'UTR) to inhibit the expression of target genes, which regulate a variety of biological processes. Many studies have shown that miRNAs in skeletal muscle development and regeneration, the activation of satellite cells and the proliferation and differentiation of muscle cells plays an important role for.MiR-34c as a tumor suppressor gene become a research hot spot in recent years, it can inhibit cancer cell proliferation by inhibiting the growth, migration and invasion, but few studies on miR-34c in skeletal muscle function. Therefore this study Mainly in the C2C12 mouse myoblast cell line as a model to study miR-34c in play muscle cell proliferation and differentiation in the role, and explore the miR-34c play the function of the target genes involved, and further clarify the function of skeletal muscle development and regulation mechanism. Firstly, this study found that miR-34c in proliferation the C2C12 low expression of muscle cells and muscle cells with differentiation, the expression level increased gradually. Secondly, myoblasts and isolated from mouse skeletal muscle in primary muscle cells overexpressing miR-34c in C2C12 can inhibit cell proliferation, induce cell cycle arrest in G0/G1 phase. The expression of cell cycle related genes down regulated; on the contrary, the inhibition of miR-34c expression can promote the proliferation of skeletal muscle cells, so that more cells through G0/G1, up-regulated expression of cell cycle related genes. Again, in C2C12 MiR-34c can promote cell differentiation and expression of myogenic cells, showed expression of MyoG and MyHC, the number of multinucleated myotubes increase; and inhibit the expression of miR-34c can inhibit the differentiation of C2C12 myoblasts, the expression of MyoG and downregulation of MyHC, the number of multinucleated myotubes decreased. The YY1 is found, the target gene of miR-34c, miR-34c can inhibit the expression of YY1. Overexpression of miR-34c and phenotypic similarity into the inhibition of YY1 expression in muscle cells in C2C12 can inhibit cell proliferation, induce cell cycle arrest in G0/G1 phase, the expression of cell cycle related genes under the regulation, and can promote cell differentiation, expression MyoG and MyHC, the number of multinucleated myotubes increase. In a study of satellite cells, miR-34c activation of satellite cells also plays an important role. Compared with the resting state of the satellite cells in activated satellite cells in M Expression of iR-34c. The inhibition of miR-34c expression in satellite cells, can lead to the expression of Pax7 and downregulation of MyoD, expression of MyoG and downregulation of MyHC after differentiation, the number of multinucleated myotubes reduced that inhibited the expression of miR-34c in satellite cells can inhibit satellite cell activation and differentiation. In addition, the the study also found that miR-34c on rhabdomyosarcoma growth also has inhibitory effect. The overexpression of miR-34c in rhabdomyosarcoma cell line RD, can inhibit the proliferation of RD cells, induce cell cycle arrest in G0/G1 phase, the expression of cell cycle related genes down regulated. In summary, this study found that miR-34c can through its target gene YY1 in regulating proliferation with the differentiation of muscle cells, mainly to cell cycle arrest in G0/G1 phase, inhibit the proliferation of skeletal muscle cells, promote myogenic differentiation. At the same time, the activation of miR-34c and satellite cells Differentiation also plays an important regulatory role, and can inhibit the proliferation of rhabdomyosarcoma cells. Therefore, this study provides a new perspective for exploring the function of miR-34c in skeletal muscle development and regeneration, and provides a new target for the treatment of rhabdomyosarcoma.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q445

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