The inhibitory effect of SEMA3C/3D mutations on Neuro-2a cells migration and axonal growth in patients with Hirschsprung's disease

摘要：Objective:To explore the effect and mechanism of SEMA3C/3D mutations on axonal growth of neurons and cell migration in Hirschsprung's disease (HSCR) patients.Methods:HEK293T cells were transfected with wild-type and mutant SEMA3C/3D plasmids.The supernatants that contained SEMA3C/3D-AP fusion proteins were collected and added into the Neuro-2a cells.The changes in the cell morphology were observed by immunofluorescence staining.The expression and phosphorylation levels of cofilin,ERM and CRMP2 were determined by western blotting.The cell migration rate was measured by transwell assay.Results:Compared with wild type SEMA3D,SEMA3D-P615T mutant suppressed cofilin phosphorylation in Neuro-2a cells (P ＜ 0.05).The neural cells treated by five mutant SEMA3C/3D-AP fusion proteins presented different levels of axon atrophy,growth cone collapse,and sometimes,loss of normal structure.SE MA3C-S329G,SEMA3C-V337M and SEMA3D-P615T mutants cells exhibited a significantly reduced migration rate as compared with wild-type SEMA3C/3D treated cells (P ＜ 0.01).Conclusion:SEMA3C and SEMA3D mutations in HSCR patients could lead to the inhibition of Neuro-2a cells' migration and axonal growth.The mechanism of SEMA3D-P615T mutant might be related to down-regulation of the expression of p-cofilin,which consequently lead to cytoskeleton structure collapse and migrating ability decrease.Our study might provide important clues for the pathogenesis of HSCR.