Promoting Integration With Astrocytes And Inhibiting Characteristic Of Astrocytes By Overexpression Of MiR-124 In Schwann Cells

Spinal cord injury?SCI?as a traumatic disease with high morbidity and disability has not been completely cured by current treatments and the patients will suffer from neurological disorders.The difficulties of self-repair after SCI due to that astrocytes are activated into reactive astrocytes and form glial scar ultimately that become barriers to hinder axons regeneration.On the other hand,the lack of supporting for regenerative axons makes it hard to guide axonal nerve fibers through the cavity which creates an abrupt obstacle for regenerating and repair for SCI.With the development of cellular engineering and medicine,cell therapy has become an effective strategy for SCI treatments.Schwann cells?SCs?become one of the appropriate seed cells of autologous transplantation for SCI which are capable to fill the cavities to provide scaffolds for regenerative axons,secrete trophic factors and cytokines to protect neurons and myelinate the demyelinated axons to enhance the repair of damage nerve.However,transplanted Schwann cells can hardly intermingle and mix with host astrocytes freely and therefore restrict migration from transplant regions which leads to the formation of boundary between the Schwann cells of grafts and the host astrocytes.This results in axons remaining at the graft-host interface and failing to exit the Schwann cells graft back to the CNS.The reduced ability of regeneration after SCI is the result of the formation of characteristic glial scar which inhibits axonal regeneration and restricts Schwann cells graft to grow into astrocytic CNS regions.According to researches,the expression of microRNA-124?miR-124?in CNS neurons was specific higher than other non-neuronal cells.MiR-124 can regulate hundreds of target genes to alter cell properties and cell phenotypes which differentiate various cells into neurons.Previous studies found that overexpression of miR-124could differentiate astrocytes into neurons and the expression of GFAP was downregulated in astrocytes which meant the tendency of inhibiting the activation of astrocytes and the formation of glial scar.We found that the expression levels of miR-124 in Schwann cells and astrocytes were significantly lower than normal spinal cord.To modify the phenotypes of Schwann cells to promote integration with astrocytes,improve the extracellular environment of the Schwann cells grafts and host astrocytes,and inhibit activation and scar formation of astrocytes,we applied miR-124 to modify the phenotypes of Schwann cells to enhance the intermingling of Schwann cells with astrocytes,inhibit astrogliosis and eventullaly promote the repair for SCI after Schwann cell transplantation.Schwann cells derived from rat sciatic nerve and astrocytes derived from neonatal rat cerebral cortex,we applied cell immunofluorescence to identify properties of primary cells.Lentiviral vectors of miR-124 were infected into Schwann cells and RT-PCR,qRT-PCR and Western blot were applied to detect the expression of miR-124 in Schwann cells.Cell proliferation after overexpression of miR-124 were detected by EdU and cck-8.Integration of modified Schwann cells and astrocytes were detected by boundary assay and we investigated the mechanisms by detecting the expression of integrated relative markers EphA4,N-Cadherin and Krox20.Dual-luciferase assay was applied to verify whether Krox20 is the targetgene of miR-124.To simulate the microenvironment of the Schwann cell graft and the host spinal cord,Schwann cells and astrocytes were seeded into upper and below chambers in the Transwell permeable support inserts.The influence of the modified Schwann cells on phenotypes of astrocytes were detected by qRT-PCR and Western blot.Here comes to the conclusions:?1?Schwann cells and astrocytes were isolated and cultured in vitro successfully and the purity of cells were about 95%.The expression of Schwann cell specific markers GFAP,Sox10 and p75^NTR and the expression of astrocyte specific markers GFAP and S100?were positive by cell immunofluorescence.?2?Construction of overexpression of miR-124 in Schwann cells?miR-SC?were employed by transfection of lentivirus,the expression of miR-124 in Schwann cells after transfection for 48h,96h and 7d increased nearly fourfold at each time points.?3?The gene expression of GFAP and Sox10 were down-regulated in miR-SC while the gene expression of neurotrophic factors NT-3 and BDNF increased significantly.The protein expression of GFAP were down-regulated obviously and the protein expression of Sox10 had no change.Cell proliferation was not affected.?4?Integration of miR-SC and astrocytes was detected by boundary assay.Normal Schwann cells and astrocytes were separated and formed a sharp boundary between them while miR-SC and astrocytes integrated and mixed freely.?5?The expression of integrative related molecule EphA4 and N-Cadherin had no significant change while the expression of Krox20 was down-regulated obviously according to Western blot.Dual-luciferase assay was applied and the results indicated that Krox20 is not the targetgene of miR-124 and the indirect mechanisms of miR-124 on Krox20 was unclear.?6?Transwell assay was applied to detect the effect of miR-SC on the phenotypes of astrocytes.The expression of astrocytes specific genes GFAP,Sox9 and S100?had no change.The expression of proteins GFAP and p-STAT3 in the formation of scar- related signaling pathway were down-regulated significantly.This leaded to the assumption that miR-SC inhibits the formation of glial scar.In conclusions,overexpression of miR-124 in Schwann cells restrained the properties of Schwann cells and the expression of GFAP and Krox20 without affecting proliferation.This study confirmed that miR-124 can not target but regulate Krox20indirectly to enhance the integration of Schwann cells and astrocytes.miR-SC can suppress the activation of astrocytes and formation of glial scar which may improve the damaged microenvironment and promote the repair of Schwann cells treatments for SCI.