MicroRNA-375 overexpression influences P19 cell proliferation, apoptosis and differentiation through the Notch signaling pathway

Wang,Lihua; Song,Guixian; Liu,Ming; Chen,Bin; Chen,Yumei; Shen,Yahui; Zhu,Jingai; Zhou,Xiaoyu

doi:10.3892/ijmm.2015.2399

MicroRNA-375 overexpression influences P19 cell proliferation, apoptosis and differentiation through the Notch signaling pathway

Authors:
- Lihua Wang
- Guixian Song
- Ming Liu
- Bin Chen
- Yumei Chen
- Yahui Shen
- Jingai Zhu
- Xiaoyu Zhou
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Affiliations: Department of Neonatology, Nanjing Children's Hospital, Affiliated to Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China, Department of Cardiology, Taizhou People's Hospital, Taizhou, Jiangsu 225300, P.R. China, Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China, Department of Children Health Care, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
Published online on: November 2, 2015 https://doi.org/10.3892/ijmm.2015.2399
Pages: 47-55
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Our previous study reported that microRNA-375 (miR-375) is significantly upregulated in ventricular septal myocardial tissues from 22‑week‑old fetuses with ventricular septal defect as compared with normal controls. In the present study, the specific effects of miR‑375 on P19 cell differentiation into cardiomyocyte‑like cells were investigated. Stable P19 cell lines overexpressing miR‑375 or containing empty vector were established, which could be efficiently induced into cardiomyocyte‑like cells in the presence of dimethyl sulfoxide in vitro. miR‑375 overexpression was verified using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Cell proliferation was determined according to total cell counts; cell cycle distribution and apoptosis levels were examined using flow cytometry. Apoptosis‑related morphological changes were observed using Hoechst staining and fluorescence microscopy. During P19 cell differentiation, the cardiomyogenesis‑related mRNAs (cardiac troponin T, GATA binding protein 4, myocyte‑specific enhancer factor 2C) and mRNAs involved in the Notch signaling pathway (Notch2, Delta‑like 1 and hes family bHLH transcription factor 1) were detected at days 0, 4, 6 and 10. Their differential expression was examined using RT‑qPCR; the apoptosis‑related genes BAX and Bcl‑2 were also detected using this method. The corresponding proteins were evaluated by western blotting. Compared with the control group, miR‑375 overexpression inhibited proliferation but promoted apoptosis in P19 cells, and the associated mRNAs and proteins were decreased during differentiation. miR‑375 has an important role in cardiomyocyte differentiation, and can disrupt this process via the Notch signaling pathway. The present findings contribute to the understanding of the mechanisms of congenital heart disease and facilitate the development of new gene therapies.

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