MicroRNA expression profile in the process of cyclic mechanical stretch promoting C2C12 myogenesis

doi:10.3969/j.issn.2095-4344.2017.28.013

Abstract

Abstract:

BACKGROUND: In recent years, the incidence of skeletal muscle injury becomes higher and higher, but the skeletal muscle repair ability is limited; therefore, studies on the molecular mechanism of skeletal muscle repair play a positive role in the treatment of skeletal muscle injury.
OBJECTIVE: To explore the role of microRNA in skeletal muscle regeneration.
METHODS: C2C12 myoblasts were cyclic stretched in vitro by the Flexercell-5000 flexible device, and the appropriate stretch condition which could induce myogenesis was selected. The microRNA expression alteration during mechanical stretch-induced myoblast myogenesis was explored using high-throughout sequencing, and the differentially expressed microRNAs were further studied by the bioinformatics analysis.
RESULTS AND CONCLUSION: 10% deformation, 0.125 Hz cyclic mechanical stretch could promote myoblast proliferation and increase MyoD and Myogenin expressions in C2C12 myoblasts. MicroRNA expression profile alteration, including the downregulated miR-500-3p/1934-5p/31-3p/378a-5p/3473b/331-3p/5097 and upregulated miR-340-5p/449c-5p/1941-3p, were all involved in the stretch-mediated myoblast myogenesis, and the MAPK signal pathway seemed to participate in this process. These results suggest that the low frequency of the cyclic mechanical stretch can upregulate the expression levels of myogenic regulatory factors through the alteration of MicroRNA expression, further inducing myogenesis, which the MAPK signal pathway may be involved in.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: MicroRNAs, Myoblasts, Tissue Engineering

CLC Number:

R318

He Yu-tong, Zhang Ma-hui, Song Chen, Ye Gen-lan, Yu Lei, Qiu Xiao-zhong, Wang Le-yu. MicroRNA expression profile in the process of cyclic mechanical stretch promoting C2C12 myogenesis[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(28): 4505-4511.

Figures/Tables 4

2.2 10%形变、0.125 Hz拉伸频率可促进C2C12细胞myoD和myogenin基因表达升高由图2所示，0.125 Hz拉伸频率的C2C12细胞myoD和myogenin基因的表达与对照组比较显著升高(P < 0.05)；0.25 Hz拉伸频率的C2C12细胞中成肌因子的表达与对照组比较差异无显著性意义；0.5 Hz拉伸频率的C2C12细胞中myoD和myogenin基因的表达与对照组比较显著降低(P < 0.05)。 2.3 10%形变、0.125 Hz拉伸C2C12成肌细胞后microRNA表达谱的变化通过高通量测序检测，0.125 Hz的拉伸组对照组C2C12成肌细胞比较其表达有明显差异且P < 0.05的共有10个microRNA。其中拉伸组有7个下调的microRNA，分别是miR-331-3p， miR-5097，miR-500-3p，miR-3473b，miR-1934-5p，miR-31-3p，miR-378a-5p。而有3个是拉伸组上调的microRNA，分别是miR-1941- 3p，miR-340-5p，miR-449c-5p(表3)。 2.4 生物信息学结果分析(GO分析，KEGG分析) 从GO富集分析的结果显示，拉伸组与对照组表达有差异的microRNA预测靶点的分子，其功能在金属离子结合，锌离子结合，蛋白结合，离子结合等方面富集较为显著。因此预测靶点所在的位置应在细胞内，细胞内部分，胞内有界膜细胞器，有界膜细胞器，细胞器等条目富集显著。预测靶点参与的生命活动在刺激反应调节，信号调节，细胞新陈代谢过程调节，信号传导调节，细胞正分组成等方面富集显著，而P值越小越有理由说明在该条目富集越显著(图3)。 KEGG通路分析的结果显示，显著富集的通路共有29条，而其中与骨骼肌的损伤修复有关的通路有：T细胞受体信号通路，磷脂酰肌醇信号通路，肌萎缩侧索硬化，肌动蛋白细胞骨架调节通路，B细胞受体信号通路，MAPK信号通路，转化生长因子β信号通路，趋化因子信号通路，黏附连接，磷酸盐和亚磷酸盐代谢通路，统计学分析显示P < 0.05为显著富集，且P值越小，说明在该条目越富集(图4)。"

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