Propofol Affects Cell Fate Of Neural Stem Cell Via MiR-124-3p/Sp1/cdkn1b Axis

BackgroundPropofol is the most appreciated and widely used intravenous anesthetics worldwide,which possess rapid onset,clear-headed recovery and lower postoperative nausea and vomiting incidence.However,propofol is still an off-label choice in most clinical pediatric practice.The dilemma is not only due to lacking sufficient evidence to support propofol has no harmful effect on developmental brain,but accumulating preclinical studies indicated neurotoxic effect caused by propofol,e.g.neurogenesis impairment.However,the effect of propofol on the neurodevelopment is not fully understood.NSCs are critical within neurogenesis whereby they can self-renew and differentiate into neurons or glial cells.Some previous studies have shown that propofol affecting nervous system is related to the regulation of microRNAs(miRNAs).We previously demonstrated that propofol could modulate the expression of microRNAs in neural stem cells(NSCs)but the effects of this interaction on cell fate were unknown.miRNAs are enriched in nervous system and play a vital role in neurodevelopment.MiR-124-3p has been discovered to owns a compelling potential to regulate a diverse range of genes expression that determine neural fate and affect development.Blocking the function of miR-124-3p during embryotic development causes neuron dysfunction and brain size loss due to more neuron apoptosis which finally leads to behavioral abnormalities.However,the effect of down-regulated miR-124-3p after propofol exposure in neural stem cells is still unclear.ObjectivesThe main purpose of this study is to verify the effect propofol has on neural stem cells and the specific mechanism of propofol affecting neural stem cells by down-regulating miR-124-3p.Material and MethodsPerforming immunofluorescence staining,cell scratch test,and EDU cell proliferation detection on primary cultured neural stem cells to determine the effect of propofol on neural stem cells.Using qRT-PCR to determine the regulation of propofol on miR-124-3p in neural stem cells.Then analyze and identify the target of miR-124-3p by MiRWalk2.0 and Gene Ontology analysis.Using qRT-PCR,western blot,and RNA interference to confirm that propofol affects neural stem cells by regulating miR-124-3p targeting Sp1.Using chromatin co-precipitation to clarify that propofol affects neural stem cells through Spl targets the cdknlb promoter.Result1.Propofol tends to induce neural stem cells differentiating into GFAP+cells.2.Propofol down-regulates miR-124-3p in neural stem cells.Sp1 is the direct target of miR-124-3p.3.Propofol induces neural stem cells differentiating into GFAP+cells through miR-124-3p targeting Sp1.4.Propofol alters cell fate of neural stem cells via enhancing Sp1 binding to the promoter of cdknlb.ConclusionPropofol alters the cell fate of neural stem cells through down-regulating the expression of miR-124-3p,which leads to the up-regulation of Spl,consequently enhanced binding of Spl to the cdknlb promoter.