miR-19b沉默对P19细胞分化及线粒体功能的影响-东南大学学报医学版

miR-19b沉默对P19细胞分化及线粒体功能的影响

作者：刘雪华¹ 2 朱莎莎² 余章斌² 朱春² 李萌萌² 韩树萍² 胡晓山² 朱金改² 彭宇竹¹ 2

单位：1. 南京医科大学, 江苏南京 210029;
2. 南京医科大学附属南京妇幼保健院儿科, 江苏南京 210004

分类号：R329.2

出版年·卷·期（页码）：2015·34·第五期（704-709）

摘要：

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:TargetScan 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cTnT、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。

Objective:To explore the function of microRNA-19b(miR-19b) knockdown on differentiation in P19 cells. Methods: We transfect miRNA-19b knockdown plasmid and vector into P19 cells and stable cell lines were selected by puromycin and conformed by dual luciferase reporter gene system. Mitochondria function assays were adopted to analyze apoptosis respectively. Real time PCR detected P19 cell differentiation markers. Results: TargetScan 5.1 software and dual luciferase reporter gene system confirmed that Wnt1 was the target gene of miR-19b(P<0.05);Expressions of all the marker genes gradually grew during the process of differentiation; the genes showed significantly lower degrees of expression in miR-19b-knockdow cells. Compared with the vector group, mtDNA copy number was significantly higher in the miR-19b knockdown group, so as to ATP levels. ROS levels in miR-19b-knockdown cells were much lower than those in control cells (all P<0.05).Conclusion: miR-19b knockdown could inhibit apoptosis and differentiation in P19 cells.

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