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:HEK293 Tcells were transfected with wild-type and mu...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:HEK293 Tcells 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-2acells.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.SEMA3C-S329 G,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 SEMA3 D mutations in HSCR patients could lead to the inhibition of Neuro-2a cells′migration and axonal growth.The mechanism of SEMA3 DP615T 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.展开更多
基金supported by the National Natural Science Foundation of China(No.81300266)Beijing Natural Science Foundation(No.7142029)+1 种基金Beijing Excellent Scientist Fund(No.2013D003034000007)Beijing Novo Program(No.Z151100000315091)
文摘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:HEK293 Tcells 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-2acells.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.SEMA3C-S329 G,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 SEMA3 D mutations in HSCR patients could lead to the inhibition of Neuro-2a cells′migration and axonal growth.The mechanism of SEMA3 DP615T 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.
