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Effects of miR-19b knockdown on the cardiac differentiation of P19 mouse embryonic carcinoma cells

Xuehua Liu,Liming Yang,Huiyan Wang,Guofeng Xu,Shasha Zhu,Meng-meng Li,Xiao-shan Hu,Jingai Zhu,C. Zhu,Jing Xu,Shuping Han,Zhang-bin Yu

Published 2014 in Molecular Medicine Reports

ABSTRACT

MicroRNA-19b (miR-19b) is part of the miR-17–92 cluster which is associated with cardiac development. It has previously been reported that the overexpression of miR-19b increases proliferation, inhibits apoptosis and promotes differentiation of embryonic carcinoma cells (P19 cells). The aim of the current study was to investigate the effects of miR-19b knockdown on the proliferation, apoptosis, differentiation and regulation of the Wnt/β-catenin signaling pathway in P19 cells. P19 cells were transfected with an miR-19b knockdown plasmid or an empty vector. MiR-19b knockdown or vector control stable cell lines were selected using puromycin. Cell Counting kit-8 and flow cytometry were used to analyze the levels of cellular proliferation, cell cycle progression and the levels of apoptosis, respectively. Caspase-3 activity and mitochondrial function assays were also used to analyze apoptosis. An inverted microscope was used to observe the morphological changes of P19 cells during differentiation. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to detect P19 cell differentiation markers and Wnt/β-catenin signaling pathway-related genes and their corresponding proteins. The results demonstrated that miR-19b knockdown inhibited the proliferation and apoptosis of P19 cells. However, the levels of expression of Wnt and β-catenin increased. MiR-19b knockdown activated the Wnt/β-catenin signaling pathway, which may regulate cardiomyocyte differentiation. The results of this study indicate that miR-19b is a novel therapeutic target for cardiovascular diseases and provide insight into the mechanisms underlying congenital heart diseases.

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