Schwann Cell-like Cells Promote Peripheral Nerve Regeneration Through A MicroRNA-214/c-Jun Pathway

Objective: To investigate the effect of human amniotic mesenchymal stem cell-derived schwann cell-like cells on peripheral nerve regeneration via microrna-214 /c-jun pathwayMethods :1)When the isolated and cultured hAMSCs were passaged to P3,the induction solution was added to induce the differentiation into schwan-like cells(SCLCs).After significant morphological changes,Western blot(WB)and RT-PCR were used to test the expression of s-100 \P75\ GFAP in cells of the two groups.ELISA detected the concentration of BDNF and NGF in the supernatant on0 d,7 d,13 d and19 d during the induction of hAMSCs into SCLCs.hAMSCs and SCLCs were transplanted to the injured site.The experiment was falled into four tranches,including the control group,the sciatic nerve injury model group,the sciatic nerve injury model group,the hAMSCs graft group,and the SCLCs graft group.Footprint method was used to evaluate the functional recovery of sciatic nerve injury.2)Overexpression or inhibition of c-jun was constructed in SCLCs by liposome transduction,SCLCs mobility was measured by cell scratch assay,and the expression levels of myelin marker MBP zinc finger transcription factor krox-20(Egr2)and myelin sheath protein0(MPZ0)were tested by WB.Mi RWalk was used to predict the potential miRNAs regulating c-jun,and lentivirus was used to construct the vector of overexpression or inhibition of mir-214 in SCLCs.Rt-pcr was used to analyze the changes of mir-214 in SCLCs,and WB was used to detect the protein expression of c-jun in SCLCs.Meanwhile,mir-214 and c-jun were overexpressed in SCLCs.Cell scratch assay was used to measure the migration rate of transfected SCLCs,and WB was used to detect the expression levels of myelin sheath generating factors Egr2 MBP and MPZ0.The whole 3'-UTR of c-jun was cloned into luciferase 3'-UTR,and the luciferase signal of mir-214 binding to c-jun's 3'-UTR was measured.The lentiviral vector of mir-214 was constructed,the SCLCs overexpressing mir-214 was constructed,and the sciatic nerve injury model of rats was established.The SCLCs and the overexpressed SCLCs of mir-214 were transplanted to the site of sciatic nerve injury,which was the same as the above method,and the SFI value was quantified.Results :1)on day 0,7,13 and 19 of hAMSCs-induced differentiation,WB showed a gradual increase in the content of s-100,GFAP and p75.Immunofluorescence staining showed the expression changes of s-100,GFAP and p75.ELISA showed that the levels of BDNF and GDNF in the supernatant of culture continued to increase,which confirmed that hAMSCs could induce differentiation into SCLCs and damaged sciatic nerve repair.The SFI value of the model group increased to-80 immediately after injury and to-57 slowly after 6 weeks Transplantation of hAMSCs did not significantly improve functional recovery.SFI value in the transplanted SCLCs group was significantly increased from-80 to-36,demonstrating that SCLCs can promote functional recovery of sciatic nerve injury.2)Cell scratch test showed that overexpression of c-jun significantly inhibited the migration of SCLCs,while the inhibition of c-jun enhanced the migration of SCLCs;WB showed that overexpression of c-Jun significantly down-regulated the expression levels of Egr2,MBP and MPZ,while inhibition of c-Jun up-regulated the expression level.WB results showed that miR-214 significantly up-regulated the expression of myelin-producing factor,while overexpression of c-Jun prevented the increase of myelination markers in SCLCs,indicating that miR-214 promotes the migration and medullary of SCLCs by targeting c-Jun.WB show that miR-214 had significantly lower c-Jun protein expression level,cut in miR-214 c-Jun protein levels increased significantly;Luciferase report showed that miR-214 set of induction of c-Jun's 3'-UTR luciferase signal is significantly reduced,and anti-miR-214 set of induction of c-Jun's 3'-UTR luciferase signal results show that the marked increase in c-Jun negative regulation SCLCs migration and myelin repair sciatic nerve injury model shows that compared with SCLCs group,miR-214 through express SCLCs set foot claws coordination,anatomical repair after immunohistochemical study further confirmed.In conclusion,overexpression of miR-214 in SCLCs can further promote functional recovery of PNR and sciatic nerve injury.Conclusion: hAMSCs-derived SCLCs can promote peripheral nerve regeneration through microrna-214 / c-jun pathway...