Zhu Shasha, Hu Xiaoshan, Yu Zhangbin, Peng Yuzhu, Zhu Jingai, Liu Xuehua, Li Mengmeng, Han Shuping, Zhu Chun
Department of Cardiology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China.
State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China.
PLoS One. 2015 Apr 21;10(4):e0123519. doi: 10.1371/journal.pone.0123519. eCollection 2015.
To explore the effect of miR-20b on apoptosis, differentiation, the BMP signaling pathway and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.
A miR-20b over-expression vector, a miR-20b silencing vector and their corresponding empty vectors were constructed and transfected into P19 cells, separately. Stably miR-20b overexpressing and silenced P19 cell lines were successfully selected by blasticidin and puromycin, separately. The cells were induced to undergo apoptosis in FBS-free-α-MEM. The induced cells were examined by flow cytometry and measurement of their caspase-3 activities. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the relative expression of marker genes of cardiomyocytes during differentiation, such as cTnT, GATA4 and ANP. QRT-PCR was also used to detect the mitochondrial DNA (mtDNA) copy number. We investigated the cellular ATP production using a luciferase-based luminescence assay. The reactive oxygen species (ROS) was determined by DCFDA (2', 7'-Dichlorofluorescein diacetate) and the mitochondrial membrane potential (MMP) was elucidated by a JC-1 fluorescent probe, both using fluorescence microscopy and flow cytometer. The expression of BMP signaling pathway-related proteins were analyzed by Western blotting.
Stably miR-20b overexpressing and silenced P19 cell lines were successfully obtained. MiR-20b overexpression increased apoptosis and promoted differentiation in P19 cells by promoting the activation of the BMP signaling pathway. In addition, miR-20b overexpression induced mitochondrial impairment in P19 cells during differentiation, which was characterized by lower MMP, raised ATP synthesis and increased ROS levels. The effects of miR-20b silencing were the exact opposite to those of overexpression.
Collectively, these results suggested that miR-20b was very important in apoptosis, differentiation and mitochondrial function of P19 cells. MiR-20b may represent a new therapeutic target for congenital heart diseases and provide new insights into the mechanisms of cardiac diseases.
探讨miR-20b对体外心肌分化P19细胞模型中细胞凋亡、分化、骨形态发生蛋白（BMP）信号通路及线粒体功能的影响。
构建miR-20b过表达载体、miR-20b沉默载体及其相应空载体，分别转染P19细胞。通过杀稻瘟菌素和嘌呤霉素分别成功筛选出稳定过表达和沉默miR-20b的P19细胞系。将细胞在无血清α-MEM中诱导凋亡。通过流式细胞术和检测其半胱天冬酶-3活性对诱导后的细胞进行检测。采用定量实时逆转录聚合酶链反应（qRT-PCR）评估分化过程中心肌细胞标记基因如心肌肌钙蛋白T（cTnT）、GATA结合蛋白4（GATA4）和心钠素（ANP）的相对表达。qRT-PCR还用于检测线粒体DNA（mtDNA）拷贝数。使用基于荧光素酶的发光测定法研究细胞ATP生成。采用2',7'-二氯二氢荧光素二乙酸酯（DCFDA）测定活性氧（ROS），并使用JC-1荧光探针通过荧光显微镜和流式细胞仪阐明线粒体膜电位（MMP）。通过蛋白质印迹法分析BMP信号通路相关蛋白的表达。
成功获得稳定过表达和沉默miR-20b的P19细胞系。miR-20b过表达通过促进BMP信号通路的激活增加P19细胞凋亡并促进其分化。此外，miR-20b过表达在分化过程中诱导P19细胞线粒体损伤，其特征为较低的MMP、升高的ATP合成和增加的ROS水平。miR-20b沉默的作用与过表达的作用完全相反。
总体而言，这些结果表明miR-20b在P19细胞的凋亡、分化和线粒体功能中非常重要。miR-20b可能代表先天性心脏病的新治疗靶点，并为心脏病机制提供新见解。
