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

Abstract

BACKGROUND The 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. METHODS Primary 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. RESULTS While 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. CONCLUSIONS MiR-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.