Effects And Mechanism Of Propofol On Proliferation,Differentiation,and Migration Of Rat Neural Stem Cells

Background Propofol as an intravenous anesthesia drug can induce neuro-apoptosis and can result in long-term neurocognitive deficits,especially in developing brains.However,advances in obstetric and pediatric surgery have made the use of these anaesthetic agents common in neonates and young children despite their potentially deleterious effects,underscoring the need to understand their mechanisms of action and to prevent neurotoxic effects.Neural stem cells(NSCs)are a subset of undifferentiated precursor cells;they have the capacity to self-renew and can generate the three main cell types in the central nervous system,namely neurons,astrocytes,and oligodendrocytes.Brain development is a very complicated process,which involves the proliferation,differentiation and migration of neural stem cells are key steps.The understanding of the specific role of anesthetics on neural stem cells will help clinical safe drug use.MicroRNAs(miRNAs)are noncoding RNAs of approximately 20-22 nucleotides that inhibit gene expression at the post-transcriptional level by binding to complementary sequences in the 3?-untranslated region(3?-UTR)of target genes.miRNAs play pivotal roles in diverse developmental and physiological processes,including cell growth,differentiation,apoptosis,organ development and the immune response.In recent studies,the variable expression of miRNAs has been shown to play an important role in many different disease processes,including neurological diseases.It has been reported that miR-141-3p is involved in the proliferation,differentiation,and aging of mesenchymal stem cells.Our previous research has found that propofol upregulates miR-141-3p.However,the role and molecular mechanism of miR-141-3p in NSCs is unclear.Objective The purpose of this study is to demonstrate the role and underlying mechanisms of propofol in the proliferation,migration,and neuronal differentiation of NSCs.These results will provide theoretical basis for the clinical medication safety.Methods (1)NSCs were harvested from the hippocampus of embryonic day 15(E15)Sprague-Dawley rat embryos.To verify the identity of NSCs,single neural cells were immunostained with anti-nestin antibody.To induce differentiation,the medium was changed to neuron differentiation medium or astrocyte differentiation medium.Cells were allowed to differentiate for 7 days,followed by immunostaining for neuronal and glial markers: anti-?-tubulin III,or anti-GFAP.NSCs were exposed to the clinically relevant concentrations of propofol(0,5,10 or 20 ?g/mL)or to equal volumes of DMSO as the vehicle control for 6 h or 24 h.Cell proliferation,neuronal differentiation,and migration were assessed using the MTT,western blotting or wound-healing assay,respectively.(2)Our previous research has found that propofol upregulates miR-141-3p.To explore the role of miR-141-3p in propofol-mediated inhibition of NSC proliferation,differentiation and migration,NSCs were transduced with a lentivirus expressing anti-miR-141-3p.Cell proliferation,neuronal differentiation,and migration were assessed inanti-miR-141-3p-transfected NSCs.A search of the TargetScan,miRanda,and miRBase databases identified IGF2BP2 as a potential target of miR-141-3p.To confirm the binding of miR-141-3p to the 3?-UTR of IGF2BP2,luciferase reporter assay,qRT-PCR and western blotting were performed.(3)To determine whether propofol influences NSC neurogenesis by regulating IGF2BP2 expression,we performed a rescue experiment by IGF2BP2 and miR-141-3p overexpression simultaneously.Meanwhile,we performed another rescue experimentby adding the miRNA inhibitor and IGF2BP2 siRNA simultaneously.Results (1)NSCs isolated from rat embryonic hippocampus were cultured,and more than90% of the cells were positive for nestin(a marker for progenitor cells).Culture in specific induction media resulted in the differentiation of NSCs into neurons and astrocytes,as determined by positive immunostaining against the neuronal marker?-tubulin III and the astrocyte marker GFAP.Propofol exposure(20 ?g/mL for 6 h)inhibits NSC proliferation,differentiation and migration.(2)We treated NSCs with 20 ?g/mL propofol for 6 hand then detected that miR-141-3p was upregulated by approximately 5-fold.Knockdown of miR-141-3p abolished the effect of propofol on cell proliferation,differentiation and migration.IGF2BP2 is the direct target of miR-141-3p.Following propofol exposure,IGF2BP2 mRNA and protein levels were significantly downregulated.(3)Overexpression of IGF2BP2 rescued the function of NSCs with regard to the propofol-induced defects in cell proliferation,neuronal differentiation and migration.This effect could be abrogated bymiR-141-3p overexpression.While anti-miR-141-3p promoted NSC neurogenesis in the presence of propofol,this effect was significantly suppressed in NSCs transfected with IGF2BP2 siRNA.Conclusions These results suggest that the effect of propofol on NSC proliferation,neuronal differentiation and migration is mediated by the miR-141-3p/IGF2BP2 axis,revealing the new mechanism of propofol on neural stem cells.