Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts.
Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts.
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DOI：
10.7150/ijbs.4729
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发表时间：
2013
影响因子：
9.2
通讯作者：
Terruzzi I
中科院分区：
文献类型：
--
作者：
Montesano A;Luzi L;Senesi P;Terruzzi I
肌发生是一个多步骤的过程，其中成肌细胞退出细胞周期，停止分裂，伸长和融合形成多核肌管。细胞周期转换由细胞周期蛋白依赖性蛋白激酶（CDKs）家族控制，该家族通过与细胞周期蛋白、负调节亚基和磷酸化的结合来调节。肌肉分化是由生肌调节因子（MRF），如MyoD和Myf-5协调的。DNA甲基化在参与肌生成的基因的转录控制中是至关重要的。以前的工作表明，用DNA去甲基化剂5-氮杂胞苷（AZA）处理成纤维细胞可促进MyoD表达。我们研究了AZA对C2 C12成肌细胞增殖和早期成肌阶段细胞周期调控和MRFs合成的影响。在增殖阶段，将细胞在含有5μM AZA（GMAZA）或不含AZA（GM）的生长培养基中孵育24小时。在70%汇合时，将细胞保持在生长培养基中以自发实现分化，或转移到含有5μM AZA（DMAZA）或不含AZA（DM）的分化培养基中12小时和24小时。用作对照的细胞未受刺激。在增殖阶段，AZA处理的细胞似乎失去了其特征性的圆形形状，并变得细长。AZA的存在导致Cyclin-D（FC：1.23 GMAZA vs GM p≤0.05）、p21（FC：1.23 GMAZA vs GM p≤0.05）、Myf-5（FC：1.21 GMAZA vs GM p≤0.05）和MyoD（FC：1.20 GMAZA vs GM p≤0.05）的蛋白含量显著增加。这些结果表明，AZA可以抑制细胞增殖。在分化的12小时内，AZA降低了参与细胞周期阻滞和限制点（G1和G1/S期）的基因的下调，以及几种细胞周期蛋白、E2 F转录因子、细胞周期蛋白依赖性激酶抑制剂、负责细胞周期负调控的特异性基因的表达。在24小时的分化过程中，AZA诱导Myf-5蛋白表达增加（FC：1.57 GMAZA vs GM p≤0.05），MyoD（FC：1.14 DM vs GM p≤0.05; FC：1.47 DMAZA vs GM p≤0.05），p21（FC：1.36 GMAZA vs GM p≤0.01; FC：1.49 DM vs GM p≤0.05; FC：1.82 DMAZA vs GM p≤0.01）和MyHC（FC：1.40 GMAZA vs GM p≤0.01; FC：2.39 DM vs GM p≤0.05; FC：3.51 DMAZA vs GM p≤0.01）。我们的研究结果表明，AZA诱导的DNA去甲基化可以调节细胞周期进程，增强肌生成。AZA的作用可能在肌肉损伤研究和治疗领域开辟新的临床用途。
Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein expression of Myf-5 (FC: 1.57 GMAZA vs GM p≤0.05), MyoD (FC: 1.14 DM vs GM p≤0.05; FC: 1.47 DMAZA vs GM p≤0.05), p21 (FC: 1.36 GMAZA vs GM p≤0.01; FC: 1.49 DM vs GM p≤0.05; FC: 1.82 DMAZA vs GM p≤0.01) and MyHC (FC: 1.40 GMAZA vs GM p≤0.01; FC: 2.39 DM vs GM p≤0.05; FC: 3.51 DMAZA vs GM p≤0.01). Our results suggest that AZA-induced DNA demethylation can modulate cell cycle progression and enhance myogenesis. The effects of AZA may open novel clinical uses in the field of muscle injury research and treatment.
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