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目的:通过沉默微小RNA-19b(miR-19b)研究miR-19b对P19细胞分化及线粒体功能的影响。方法:转染miR-19b沉默质粒与空载质粒进入P19细胞建立起稳定的细胞系;线粒体功能分析用以检测细胞凋亡;诱导分化P19细胞,实时定量荧光PCR检测miR-19b沉默后细胞分化关键基因表达水平。结果:Target Scan 5.1软件及双荧光素酶报告基因系统共同证实,Wnt1为miR-19b在P19细胞中发挥作用的靶点(P<0.05);测定第0、4、6、8、10、12天c Tn T、NKX2.5、GATA4等基因的核酸表达量显示,心肌分化相关标志基因逐渐增高,miR-19b沉默组明显低于对照组(P<0.05);于分化第10天检测线粒体功能,发现miR-19b沉默组线粒体DNA拷贝数的相对含量高于对照组,其活性氧水平显著低于对照组,ATP水平也高于对照组(均P<0.05)。结论:miR-19b沉默可抑制细胞凋亡,抑制心肌细胞分化。
AIM: To investigate the effect of miR-19b on the differentiation and mitochondrial function of P19 cells by silencing microRNA-19b (miR-19b). METHODS: miR-19b silencing plasmid and empty plasmid were transfected into P19 cells to establish a stable cell line. Mitochondrial function assay was used to detect apoptosis. P19 cells were induced to differentiate. Real-time quantitative PCR was used to detect miR-19b silenced cells Key gene expression levels. Results: Target Scan 5.1 software and dual luciferase reporter gene system confirmed that Wnt1 was the target of miR-19b in P19 cells (P <0.05), and that 0,4,6,8,10,12 The mRNA expression levels of cTn T, NKX2.5, GATA4 and other genes showed that the markers of myocardial differentiation increased gradually and the miR-19b silencing group was significantly lower than that of the control group (P <0.05). On the 10th day of differentiation, mitochondrial function The relative content of copy number of mitochondrial DNA in miR-19b silencing group was found to be higher than that in control group. The level of reactive oxygen species in miR-19b silencing group was significantly lower than that in control group, and the ATP level was also higher than that in control group (all P <0.05). Conclusion: miR-19b silencing can inhibit apoptosis and inhibit cardiomyocyte differentiation.