MiR-106a-5p靶向E2F3和SP-1抑制C2C12细胞成肌分化的研究

MiR-106a-5p Inhibits C2C12 Cells Myogenic Differentiation by Targeting E2F3 and SP1

为探究miR-106a-5p对成肌分化的作用及潜在调控靶点,在线预测miR-106a-5p潜在靶基因,通过RT-qPCR及Westernblot检测miR-106a-5p对靶基因的调控作用,利用双荧光素酶报告系统验证miR-106a-5p与靶基因之间的互作,并以C2C12细胞系为实验模型,采用过表达技术,在细胞形态学水平初步探索其对成肌分化的作用。在线预测发现E2F3和SP-1可能是miR-106a-5p的潜在靶基因;RT-qPCR分析表明,miR-106a-5p与E2F3和SP-1的表达模式相反;Westernblots结果发现,miR-106a-5p抑制E2F3和SP-1蛋白翻译;双荧光素酶报告载体系统表明,E2F3和SP-1是miR-106a-5p调控成肌分化的靶基因;此外,超表达miR-106a-5p抑制C2C12细胞分化,肌管数目显著减少。结果表明,E2F3和SP-1是miR-106a-5p抑制C2C12细胞成肌分化的靶基因。

To explore the effect of miR-106a-5p on myoblast differentiation and its potential targets, the potential target genes of miR-106a-5p were predicted by online software, the regulatory effects of miR-106a-5p on target genes were detected by RT-qPCR and Western blot, and the interaction between miR-106a-5p and target genes was verified by using dual-luciferase reporter assay. The C2C12 mouse skeletal muscle cell line was used as a model system, overexpression technology was adopted to analyze its effect on myoblast differentiation by cells morphology. Bioinformatics analysis software found that E2F3 and SP-1 may be the potential target genes of miR-106a-5p. The expression patterns of miR-106a-5p was opposite with both E2F3 and SP-1 during differentiation, and miR-106a-5p significantly down-regulated the expression of E2F3 and SP-1 protein. The dual-luciferase reporter assay showed that E2F3 and SP-1 are the target genes of miR-106a-5p in myoblast differentiation. In addition, overexpression miR-106a-5p inhibits the myoblasts differentiation, significantly reduces the number of myofiber. In conclusion, E2F3 and SP-1 are targets of miR-106a-5p during C2C12 cells myogenic differentiation.