Effects of microRNA-30c knockdown on proliferation and differentiation of P19 cells

Abstract

Objective To explore the effects of microRNA(miRNA)-30c knockdown on proliferation, diffe-rentiation of P19 cells. Methods miRNA-30c knockdown plasmid(miRNA-30c knockdown group) or no-load vector(negative control group) was transfected into P19 cells by lipo2000 and stable cell lines were selected by Blasticidin; Dual luciferase reporter gene system was used to confirm miRNA-30c knockdown.Cell counting kit-8(CCK-8) assay was adopted to detect cell proliferation activity.An inverted microscope was used to observe morphological changes of P19 cell differentiation.Cells were induced to differentiated to myocardiocyte with dimethyl sulfoxide(DMSO). Differentiation marker genes including cTnT, NKX2.5, GATA4 relative mRNA expression levels were detected with real-time quantitative polymerase chain reaction, respectively. Results Observation of green fluorescent protein expression under a fluorescence microscope indicated similar transfection efficiencies, and miRNA-30c knockdown released the activity of target gene Gli2.As a result, miRNA-30c knockdown vector was constructed successfully(P<0.001). During differentiation of mouse P19 cells into myocardial cells, the beating cell clusters in miRNA-30c knockdown cells were much lower than those in the control cells, and cTnT, NKX2.5, GATA4 in miRNA-30c knockdown cells showed significantly lower expression than those in the control cells(all P<0.05). Conclusions miRNA-30c inhibits the P19 cell proliferation and differentiation.This study gives us a new insight of heart development and we need more efforts on exploring the deep function of heart diseases. Key words: microRNA-30c; P19 cell; Proliferation; Differentiation