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BackgroundNeural stem cells (NSCs) are able to differentiate into neurons and astroglia. miRNAs have been demonstrated to be involved in NSC self-renewal, proliferation and differentiation. However, the exact role of miR-124 in the development of NSCs and its underlying mechanism remain to be explored.MethodsPrimary NSCs were isolated from embryos of Wistar rats. Immunocytochemistry was used to stain purified NSCs. miR-124, Delta-like 4 (DLL4), ki-67, Nestin, β-tubulin III, glial fibrillary acidic protein (GFAP), HES1, HEY2, and cyclin D1 (CCND1) expressions were detected by qRT-PCR and western blot. The interaction between miR-124 and DLL4 was confirmed by luciferase reporter assay. Cell proliferation was assessed by MTT assay.ResultsNSCs could self-proliferate and differentiate into neurons and astrocyte. miR-124 was up-regulated and DLL4 was down-regulated during NSC differentiation. DLL4 was identified as a target of miR-124 in NSCs. Ectopic expression of miR-124 or knockdown of DLL4 promoted the proliferation and the formation of NSCs to neurospheres. Moreover, miR-124 overexpression or DLL4 down-regulation improved β-tubulin III expression but decreased GFAP expression in NSCs. Furthermore, enforced expression of DLL4 partially reversed the effects of miR-124 on NSCs proliferation and differentiation. Elevated expression of miR-124 suppressed the expressions of HES1, HEY2, and CCND1 in NSCs, while these effects were attenuated following the enhancement of DLL4 expression.ConclusionmiR-124 promoted proliferation and differentiation of NSCs through inactivating Notch pathway.

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Neural stem cells (NSCs) are multipotent and undifferentiated cells with the potential to differentiate into neuronal lineages and gliocytes. NSCs have the ability to generate and regenerate the brain, indicating the possibility of cell-based therapies for neurological disorders. miR-124 has been demonstrated as a modulator in the survival, expansion, and differentiation of NSCs. However, the underlying molecular mechanisms of miR-124 in NSC development are still far from being understood. The expressions of miR-124, dishevelled binding antagonist of beta-catenin 1 (DACT1), ki-67, Nestin, β-tubulin III, glial fibrillary acidic protein (GFAP), β-catenin, cyclinD1, and glycogen synthase kinase-3β (GSK-3β) were examined by qRT-PCR or western blot. Bioinformatics and Dual-Luciferase reporter assay were used to identify the interaction between miR-124 and DACT1. MTS analysis was performed to measure the viability of NSCs. Enhanced expression of miR-124 and lowered expression of DACT1 were observed during a 14-day NSC differentiation period. DACT1 was verified as a direct target of miR-124. Moreover, overexpression of miR-124 promoted NSC proliferation and induced neuron-specific differentiation, presented as increased cell viability, higher neurosphere number, elevated ki-67, Nestin, β-tubulin III expressions, and decreased GFAP expression. Similarly, DACT1 downregulation facilitated proliferation and neuronal differentiation of NSCs. Furthermore, DACT1 overexpression impaired miR-124-induced proliferation and differentiation of NSCs. Additionally, miR-124 stimulated Wnt/β-catenin signaling via suppressing DACT1 expression. miR-124 promoted proliferation and induced NSC differentiation to neurons by activation of Wnt/β-catenin pathway via targeting DACT1, providing a potential target and aiding the development of cell-based therapies for neurological disorders.

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