miR-374b及其靶基因Myf6调控成肌细胞增殖分化的研究

Proliferation and Differentiation Regulation of miR-374b on Ovine Myoblasts by Targeting Myf6 Gene

本实验旨在研究miR-374b及其靶基因Myf6调控肌细胞增殖分化的作用机制。首先采集70日龄胎羊,培养成肌细胞并诱导分化成肌管,通过荧光定量PCR测定诱导分化时期(0、24、72、120 h)miR-374b、Myf6基因以及成肌细胞增殖标志基因MyoD和分化标志基因MyoG、MyHC在mRNA水平的表达量;通过转染miR-374b过表达载体(mimics)及抑制载体(inhibitor),研究各基因在细胞中mRNA水平的表达规律;采用蛋白免疫印迹技术检测Myf6基因在细胞中蛋白水平表达变化规律。结果表明:细胞在分化第72、120小时出现大量肌管,诱导分化成功;miR-374b的表达趋势为先上升后下降,细胞增殖标志基因MyoD在细胞增殖期(0 h)表达量较高;分化标志基因MyHC、MyoG及Myf6基因在细胞增殖期表达量较低,进入分化阶段后表达量极显著上调;转染miR-374b过表达载体(mimics)后,miR-374b表达量极显著高于阴性对照组,而Myf6基因、MyHC基因表达量极显著低于阴性对照组;Myf6基因蛋白表达量极显著低于阴性对照组;转染miR-374b抑制载体(inhibitor)后,miR-374b表达量极显著低于阴性对照组,而Myf6、MyHC、MyoD基因表达量极显著高于阴性对照组;Myf6基因蛋白表达量极显著高于阴性对照组;MyoG基因表达量显著低于阴性对照组。上述结果表明,miR-374b可能通过靶向Myf6基因抑制绵羊成肌细胞分化。

In order to explore the mechanism of miR-374b and its target gene Myf6 regulating muscle cell proliferation and differentiation. First of all, the muscles of 70-day-old fetal sheep were collected for myoblast cultivation and induce differentiation into myotubes. Real-time quantitative PCR was used to study the mRNA expression levels of miR-374b, Myf6 and myoblast proliferation marker gene MyoD and cell differentiation marker genes MyoG and MyHC during the induced differentiation period(0, 24, 72, 120 h). By transfecting miR-374b overexpression vectors(mimics) and inhibitor vectors(inhibitors), the mRNA expression of each gene in cells was studied;Protein expression of Myf6 in cells was detected by western blotting methods. The results showed that a large number of myotubes appeared at 72 h and 120 h after cell differentiation, indicating that differentiation was successful. The results of real-time quantitative PCR showed that the expression of miR-374b increased first and then decreased, the cell proliferation marker gene MyoD was highly expressed in cell proliferation phase(0 h), the expression of differentiation marker genes MyHC, MyoG and Myf6 genes were low in the cell proliferative phase and up-regulated significantly while entering the cell differentiation stage. After cells transfection with miR-374b mimics, the expression of miR-374b mRNA was significantly higher than that of the negative control group, while the expression of Myf6 and MyHC gene was significantly lower than that of the negative control group, Myf6 protein expression was significantly lower than the negative control group. After transfection with miR-374b inhibitor, the expression of miR-374b was significantly lower than that of the negative control group, while the expression of Myf6, MyHC, MyoD gene was significantly higher than that of the negative control group, the expression of MyoG gene was significantly lower than that of the negative control group.Myf6 protein expression was significantly higher than the negative control group. The above results indicate that miR-374b may inhibit sheep myoblast differentiation by targeting Myf6.