Zhu Shasha, Hu Xiaoshan, Han Shuping, Yu Zhangbin, Peng Yuzhu, Zhu Jingai, Liu Xuehua, Qian Lingmei, Zhu Chun, Li Mengmeng, Song Guixian, Guo Xirong
1. State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China.
2. Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China.
Int J Med Sci. 2014 Mar 28;11(5):500-7. doi: 10.7150/ijms.7849. eCollection 2014.
Many long non-coding RNAs (lncRNAs) are species specific and seem to be less conserved than protein-coding genes. Some of them are involved in the development of the lateral mesoderm in the heart and in the differentiation of cardiomyocytes. The purpose of the study was to investigate the expression profiles of lncRNAs during the differentiation of P19 cells into cardiomyocytes, with a view to studying the biological function of lncRNAs and their involvement in the mechanism of heart development. First, we observed the morphology of P19 cells during differentiation using an inverted microscope. Then, cardiac troponin T (cTnT) expression was detected to validate that the cells had successfully differentiated into cardiac myocytes by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) and western blotting. Lastly, the expression profile of lncRNA genes was obtained using an lncRNA microarray and real-time RT-PCR analyses. The microarray results showed that 40 lncRNAs were differentially expressed, of which 28 were upregulated and 12 were downregulated in differentiated cardiomyocytes. The differentially expressed lncRNAs were further validated. Our results illustrated a critical role of lncRNAs during the differentiation of P19 cells into cardiac myocytes, which will provide the foundation for further study of the biological functions of lncRNAs and the mechanism of heart development.
许多长链非编码RNA(lncRNA)具有物种特异性,并且似乎比蛋白质编码基因的保守性更低。其中一些参与心脏中胚层的发育以及心肌细胞的分化。本研究的目的是调查lncRNA在P19细胞向心肌细胞分化过程中的表达谱,以研究lncRNA的生物学功能及其在心脏发育机制中的作用。首先,我们使用倒置显微镜观察P19细胞在分化过程中的形态。然后,通过实时逆转录聚合酶链反应(实时RT-PCR)和蛋白质印迹法检测心肌肌钙蛋白T(cTnT)的表达,以验证细胞是否成功分化为心肌细胞。最后,使用lncRNA微阵列和实时RT-PCR分析获得lncRNA基因的表达谱。微阵列结果显示,40种lncRNA在分化的心肌细胞中差异表达,其中28种上调,12种下调。对差异表达的lncRNA进行了进一步验证。我们的结果说明了lncRNA在P19细胞向心肌细胞分化过程中的关键作用,这将为进一步研究lncRNA的生物学功能和心脏发育机制提供基础。