The Regulatory Effect And Mechanism Of Notch Signaling Pathway On Proliferation And Self-renewal Of Neural Stem Cells Through MiR-582-5p

Genetic information is expressed as traits through the process of development,and stem cells play a pivotal role in the development.Neural stem cells(NSCs)are a class of pluripotent stem cells in the central nervous system(CNS).They can maintain stemness through self-renewal and can also differentiate into various types of nerve cells,such as neurons,astrocytes,and oligodendrocytes.NSCs exist not only in developing embryos,but also in the brain of adult mammals,playing an important role in the development of the nervous system and the repair of nerve cell damage.In addition,the expansion and transplantation of NSCs in vitro can treat neurodegenerative diseases and repair nervous system injury.Therefore,it is particularly important and urgent to clarify and improve the regulatory mechanism of stemness maintenance and directional differentiation of NSCs.Current research shows that NSCs are regulated by transcription factors,epigenetic mechanisms,signaling pathways,and other factors in the process of stemness maintenance,proliferation,and differentiation.However,the complex and subtle regulation of the key molecules of various mechanisms is still unclear,and further research on the development regulation network of NSCs is needed.The canonical Notch signaling pathway mediated by Notch-RBP-J(recombination signal binding protein for immunoglobulin kappa J region)plays an important regulatory role in NSCs development,but its downstream effector molecules and epigenetic mechanisms have not been completely elucidated.In this paper,we analyzed the results of Nestin-specific RBP-J knockout mouse NSCs,and obtained miR-582-5p with reduced expression,which may be the downstream molecule of Notch signaling pathway regulating the development of NSCs.Subsequently,we isolated and cultured the primary NSCs of mice in vitro,transfected miR-582-5p mimic and inhibitor and their corresponding negative controls(NC),and detect the changes in the proliferation and self-renewal ability of NSCs.The results showed that the number of spheres formed by NSCs transfected with miR-582-5p mimic increased,the number of Ed U-positive cells increased,and the expression of m RNA and protein levels of stem cell markers such as Sox2,Nestin,Pax6 also increased.After transfected with miR-582-5p inhibitor,the number of spheres and Ed U-positive cells decreased,and the expression of stem cell markers decreased.In vitro experiments have proved that miR-582-5p can regulate the proliferation and self-renewal of NSCs and enhance the stemness of NSCs.Next,we constructed miR-582 knockout(KO)mice,and tested the regulatory effect of miR-582-5p on NSCs in vivo.We prepared the tissue sections of the subventricular zone(SVZ)of adult male mice by freezing section,and performed immunofluorescence staining.The staining results showed that the number of GFAP+Sox2 double positive cells and EGFR positive cells in SVZ decreased in miR-582 KO mice compared with wild mice.The number of DCX positive cells and Ki67 positive cells increased,and the number of CD24 positive cells did not change significantly.These results indicated that in SVZ of miR-582 KO mice,adult neural stem cells(a NSCs)and transient amplifying progenitors(TAPs)decreased,neuroblasts(NB)and proliferating cells increased,and ependymal cells did not change.In vivo experiments have proved that miR-582-5p maintained the stemness of adult neural stem cells.After knocking out miR-582,the stemness of a NSCs decreased and converted to NB.Then we explored the specific molecular mechanism of miR-582-5p regulating NSCs.After transfecting miR-582-5p mimic and its negative control(NC-mimic)in NSCs,we extracted RNA for transcriptome sequencing(RNA-seq).The analysis of differentially expressed genes(DEGs)in the sequencing results revealed a decrease in the expression of8 genes.Through RT-q PCR and Western blotting detection,the expression of family with sequence similarity 19 member A1(FAM19A1)decreased in NSCs transfected with miR-582-5p mimic and increased in NSCs transfected with miR-582-5p inhibitor,indicating that the expression of FAM19A1 was regulated by miR-582-5p.There is a specific binding site of miR-582-5p in the 3’UTR of FAM19A1,so we constructed the FAM19A1-3’UTR reporter gene plasmid,and verified whether FAM19A1 is a downstream target gene of miR-582-5p through a dual luciferase reporter gene experiment.The experimental results showed that the fluorescence intensity in HEK293 T cells decreased with the increase of miR-582-5p level after transfection of the reporter gene plasmid,indicating that miR-582-5p could inhibit the expression of FAM19A1.In order to further verify that miR-582-5p promotes the proliferation and self-renewal,maintains the stemness of NSCs by inhibiting the expression of FAM19A1,we designed the si RNA of FAM19A1.After transfection of FAM19A1-si RNA in NSCs,the changes of proliferation and self-renewal ability were detected.The results showed that after NSCs transfected with FAM19A1-si RNA,the number of spheres increased,the number of Ed U-positive cells increased,and the expression of m RNA and protein levels of stem cell markers increased.In addition,we designed a rescue experiment of co-transfecting miR-582-5p-inhibitor and FAM19A1-si RNA.The results of rescue experiment showed that compared with the control group,the number of spheres and the number of Ed U-positive cells decreased after NSCs transfected with inhibitor,and the expression of m RNA and protein levels of stem cell markers decreased.After co-transfection of miR-582-5p-inhibitor and FAM19A1-si RNA,the corresponding phenotypes were rescued to some extent.These results showed that miR-582-5p promoted the proliferation and self-renewal of NSCs and maintained the stemness of NSCs by inhibiting the expression of FAM19A1.Furthermore,we verified the regulatory effect of Notch signal on miR-582-5p.After blocking Notch signal by using gamma-secretase inhibitor(GSI)in NSCs,the expression of miR-582-5p decreased and the expression of Pde4 d,the host gene of miR-582-5p,increased,indicating that blocking Notch signal can inhibit the expression of miR-582-5p.The dual luciferase reporter gene experiment showed that Notch signal activation could not increase the expression of Pde4 d,which indicated that Notch signal did not directly regulate the expression of Pde4 d.In summary,this paper confirmed that the role of miR-582-5p in the development of nervous system is to maintain the stemness of NSCs.miR-582-5p can promote the proliferation and self-renewal of NSCs and maintain the stemness of NSCs by inhibiting the expression of FAM19A1.The expression of miR-582-5p is regulated by Notch signaling pathway.Blocking Notch signal can inhibit the expression of miR-582-5p and reduce the stemness of NSCs.miR-582-5p can promote the proliferation of NSCs in vivo and in vitro,providing a new approach and new target for the treatment of neural stem cell transplantation and the repair of nervous system injury in vivo.