miR-9 regulates the differentiation of neural stem cells in mouse cerebral cortex

Liu Yingzhao; Ma Yanxia; Lin Yaofa; Zhang Guoqiao; Miao Weiliang; Jia Yanli; Chi Chenshen; Song Wangsheng; Li Di; Liu Chenglong; Zhang Haonan

doi:10.12307/2026.246

Abstract：BACKGROUND:Neural stem cells located in the ventricular zone and subventricular zone are crucial for cortical neurodevelopment and the treatment of neurodegenerative diseases.However,their precise regulatory mechanisms remain incompletely understood.miRNA-9 is one of the most abundantly expressed miRNAs in the vertebrate embryonic and adult brain,playing diverse roles during development.Yet,the function of miRNA-9 in neural stem cell differentiation remains unclear. OBJECTIVE:To investigate the role of miRNA-9 in regulating the differentiation of neural stem cells in the ventricular zone and subventricular zone. METHODS:Neural stem cells were isolated from the ventricular zone and subventricular zone of embryonic day 14.5 ICR mice and cultured in proliferation medium for 3-4 days to form neurospheres.The stemness of these cells was confirmed by Pax6/Nestin immunofluorescence staining.The expression profile of miRNA-9 was assessed using qRT-PCR in forebrain tissues at different developmental stages(embryonic days 12.5,14.5,16.5,18.5,postnatal days 0,and 7)as well as in in vitro cultured neural stem cells at embryonic day 14.5.Neural stem cells were transfected with either a miRNA-9 inhibitor or mimic using a transfection reagent.After 24 hours of transfection,cells were induced to differentiate for 3-4 days(for neurons)or 6-8 days(for glial cells).The differentiation ratios of neuronal and glial lineages were quantified via immunofluorescence staining for Tuj1(a neuronal marker),myelin basic protein(an oligodendrocyte marker),and glial fibrillary acidic protein(an astrocyte marker). RESULTS AND CONCLUSION:qRT-PCR results revealed that miRNA-9 was highly expressed during early embryonic stages(embryonic days 12.5-14.5),with its expression gradually decreasing as development progressed(embryonic day 16.5 to postnatal day 7).The expression level of miRNA-9 in neural stem cells at embryonic day 14.5 was approximately 90%of the expression level of the internal reference RNU6B.Functional experiments demonstrated that compared with the control group,the proportion of Tuj1-positive neurons and myelin basic protein-positive oligodendrocytes decreased in the miRNA-9 inhibition group,while the proportion of glial fibrillary acidic protein-positive astrocytes increased.Conversely,the proportion of Tuj1-positive neurons and myelin basic protein-positive oligodendrocytes increased in the miRNA-9 overexpression group,while the proportion of glial fibrillary acidic protein-positive astrocytes decreased.All differences were significant(P<0.001).Results indicate that miRNA-9 plays a bidirectional regulatory role in neural stem cell differentiation:(1)It participates in the temporal regulation of neurogenesis through a developmental-stage-specific expression pattern(high expression early,downregulation later);(2)It maintains a balance in tri-lineage differentiation by promoting neural stem cell differentiation into neurons and oligodendrocytes while inhibiting astrocyte formation.