MiR-140-5p targets Prox1 to regulate the proliferation and differentiation of neural stem cells through the ERK/MAPK signaling pathway.

Abstract

BackgroundThe expression of miR-140-5p increased in the brain tissue of a bilateral common carotid artery ligation model, while the overexpression of miR-140-5p significantly decreased the number of neurons. The luciferase report experiment in the previous study proved that miR-140-5p negatively regulated one of the potential targets of Prospero-related homeobox 1 (Prox1). Therefore, we want to investigate the effect of miR-140-5p on the proliferation and differentiation of neural stem cells (NSCs) and the underlying mechanism.MethodsPrimary NSCs were extracted from pregnant ICR mice aged 16–18 days and induced to differentiate. After transient transfection with miR-140-5p mimic and inhibitor into NSCs, the cells were divided into five groups: blank, mimic normal control, mimic, inhibitor normal control, and inhibitor. Cell Counting Kit-8 (CCK-8) and 5-Bromo-2-deoxyUridine (BrDU), Ki-67 were used, and the diameter of neural spheres was measured to observe proliferation ability 48 h later. Doublecortin (DCX), glial fibrillary acidic protein (GFAP), microtubule-associated proteins 2 (MAP-2), synapsin I (SYN1), and postsynaptic density protein-95 (PSD-95) were stained to identify the effect of miR-140-5p on the differentiation ability of NSCs into neural precursor cells, astrocytes, and neurons and the expression of synapse-associated proteins. The expression of miR-140-5p, Prox1, p-ERK1/2, and ERK1/2 was analyzed by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis.ResultsWhile the expression of miR-140-5p decreased after NSC differentiation (P<0.05), the results of CCK-8, BrDU, and Ki-67 staining showed no significant difference in cell viability and the percentage of NSCs with proliferation ability (P>0.05). However, the neural spheres were shorter in the miR-140-5p overexpression group (P<0.05) and the expression of DCX, MAP2, synapsin I, and PSD-95 decreased, while the expression of GFAP increased after differentiation in the mimic group (P<0.05). In addition, the expression of Prox1 decreased and the expression of p-ERK1/2 protein increased (P<0.05), but the expression of ERK1/2 showed no significant difference (P>0.05) in the miR-140-5p overexpression group.ConclusionsMiR-140-5p reduced the proliferation rate of NSCs, inhibited their differentiation into neurons, produced synapse-associated proteins, and promoted their differentiation into astrocytes. MiR-140-5p negatively regulated downstream target Prox1 and activated the ERK/MAPK signaling pathway.