TAG1/APP Inhibits Embryonic Neural Stem Cells Differentiation Into Neruons Through Regulating Mir-218-5p And The Underlying Mechanism

Objective:At present,Alzheimer's disease(AD)is one of the research hotspots in the health care field of world,but until now the definite cause is still unclear.It is reported that Amyloid precursor protein(APP)intracellular C-terminal domain(AICD)has a role as transcription factors that can regulate a variety of genes expression,and AICD-transgenic mice has typical AD symptoms,so AICD has become a new focus in AD research.Previous study found that TAG 1 was a functional ligand of APP,which could promote APP to release AICD and finally inhibited the differentiation of embryonic neural stem cells(eNSC)into neurons.One pathological feature of AD is the decrease of neurons,so this TAG 1-APP signaling pathway may have a relationship with AD,so this pathway related molecular could be potential drug for treatment of AD in the future.However,the exactly mechanism underlying the regulation of eNSC differentiation into neurons by AICD remains unclear until now.It is known miRNA can participate effectively in neural stem cells proliferation,differentiation,apoptosis and so on.So we wondered that whether TAG 1-APP signaling pathway through AICD to regulate the expression of miRNA finally inhibits eNSC differentiation into neurons.In addition,miRNAs are known to have functions by regulating mRNA degradation or transcription of target genes.So we further screened and confirmed the functional target gene of miRNA in the TAG1-APP signaling pathway in eNSC.Methods:First,we optimized the exactly embryonic day and brain tissue to isolate and culture eNSC through cellular immunofluorescence staining and real-time PCR assay.Then,by analyzing the AICD ChIP-Seq experiments data and miRNA microarray experiments data of TAG1 KO and APP KO adult mouse SVZ tissue respectively,we screened the candidate miRNA associated with AICD,TAG1 and APP.furthermore we mainly used real time PCR to test which candidate miRNA can be regulated by AICD,TAG1 and APP in eNSC.Then,we tested the expression profile of candidate miRNA in nervous system,their functions in eNSC proliferation,differentiation and apoptosis and whether they could effectively rescue the function of AICD,TAG1 and APP by using real time PCR,cellular immunofluorescence staining and cell counting analysis.Finally,we screened and confirmed the target genes of candidate miRNA through mRNA microarray,bioinformatic prediction,real time PCR and dual-luciferase report assay.What's more,we also tested target genes mRNA and protein expression in nervous system and their function in eNSC differentiation.Results:We found that the forebrain tissue of embryonic 14-day(E14)mouse could be used to isolate and culture high quality NSC which had proliferation and differentiation ability for the later experiments.There were 10 candidate miRNAs related to AICD,TAG1 and APP by analyzing AICD ChIP-Seq and miRNA microarray data.In addition,we found only miR-218-5p of the 10 candidate miRNAs was simultaneously regulated by AICD,TAG1 and APP in eNSC.Then we used real time PCR experiment and found that miR-218-5p was expressed in the mouse nervous system which expression had a spatial,temporal and cellular-specific feature.And miR-218-5p could promote eNSC proliferation and inhibit eNSC differentiation into neurons,but had no effect on the apoptosis of eNSC.Meanwhile,miR-218-5p could effectively reverse the function of AICD,TAG1,and APP on eNSC differentiation into neurons.Finally,through mRNA microarray,bioinformatic prediction,real time PCR and dual-luciferase report assay we found that Phc3 was a direct target gene of miR-218-5p,the expression of Phc3 mRNA was temporal-specific,while the expression of Phc3 protein was subcellular-specific.Besides,we also found Phc3 siRNA could inhibit eNSC differentiation into neurons,what's more,Phc3 siRNA could effectively rescue the effect of miR-218-5p inhibitor on eNSC differentiation into neurons.Conclusion:In this project,we mainly studied that TAG1 interacted with APP to produce AICD which inhibited eNSC differentiation into neurons by regulating the expression of miR-218-5p,and in this process miR-218-5p degraded Phc3 mRNA to achieve this function.The purpose of us to study TAGl-APP signaling pathway in detail is to further research the effect of this pathway on the process of the occurrence and development in AD,and the potential therapeutic value of the pathway related molecular in treatment with AD.