The Role And Mechanism Of Semaphorin 7A In The Development And Progression Of Pterygium

PurposeTo study the expression of Sema 7A in pterygium tissue,and to further clarify the effects of Sema 7A on the occurrence and development of pterygium and its molecular mechanism.MethodTwo sets of data GSE51995 and GSE2513 were collected from NCBI database,and bioinformatics techniques were used to analyze the differential genes in pterygium tissues with normal conjunctiva as control.Immunohistochemistry,real-time fluorescence quantitative PCR and western blot were used to further verify the differential genes identified by bioassay,namely target genes.The effects of target genes on proliferation,migration,cell cycle and apoptosis of primary pterygium fibroblasts were determined by MTT assay,scratch assay,Transwell assay and flow cytometry.The effects of target genes on effector proteins(VEGFA,MMP-2,Integrin-β1)and upstream FAK and ERK proteins were determined by western blot and specific inhibitors of FAK.ResultsThe first part1.Two sets of data,GSE51995 and GSE2513,were collected,and bioinformatics analysis showed that there was a different gene,namely Sema 7A gene,in primary pterygium tissue compared with normal conjunctiva tissue.2.Immunohistochemical results of sections showed that compared with normal conjunctiva tissue,the proportion of Sema 7A positive cells in primary and recurrent pterygium tissue was significantly different(P<0.05).3.qPCR results showed that compared with normal conjunctiva tissue,Sema 7A expression in primary and recurrent pterygium tissue was significantly different(P<0.05).Compared with primary pterygium,Sema 7A expression was increased in recurrent pterygium,and the difference was statistically significant(P<0.05).4.Western blot results also showed that Sema 7A expression was significantly up-regulated in primary and recurrent pterygium compared with normal conjunctiva tissue,with statistical significance(P<0.05).The second part1.After silencing Sema 7A gene in pterygium fibroblasts with siRNA,the results of MTT assay,scratch assay and Transwell migration assay showed that the proliferation and migration ability of pterygium fibroblasts decreased after silencing Sema 7A gene.Further flow cytometry showed that the proportion of pterygium fibroblasts in G2 and M phases increased significantly after Sema 7A gene silencing,and the difference was statistically significant(P<0.05).2.Sema 7A gene was overexpressed in pterygium fibroblasts by plasmid transfection.MTT assay,scratch assay and Transwell migration assay showed that proliferation and migration of pterygium fibroblasts increased after Sema 7A gene was overexpressed.Flow cytometry showed that the proportion of G1 phase in pterygium fibroblasts decreased and that of S phase increased after overexpression of Sema 7A gene,and the difference was statistically significant(P<0.05).3.In addition,flow cytometry was used to detect the apoptosis of pterygium fibroblasts.After Sema 7A silencing/overexpression3 there was no significant difference in the apoptosis of pterygium fibroblasts among all groups(P>0.05).The third part1.After silencing or overexpression of Sema 7A,the expression of MMP-2 and VEGFA were down-regulated or up-regulated in pterygium fibroblasts by western blot.2.Further western blotting revealed increased phosphorylation levels of FAK and ERK and increased Integrin-β1 expression after overexpression of Sema 7A.3.The addition of FAK inhibitor in Sema 7A overexpressed cells inhibited FAK phosphorylation,and the expression of effector proteins VEGFA and MMP-2 were also inhibited.Conclusion1.The expression of Sema 7A gene in pterygium tissue and pterygium fibroblasts showed an upward trend.2.Sema 7A gene can promote proliferation and migration of pterygium fibroblasts and regulate cell cycle,but does not affect cell apoptosis.3.Sema 7A can promote the expression of effector proteins VEGFA and MMP-2 in pterygium fibroblasts.Furthermore,Integrin-β1 expression and FAK/ERK phosphorylation can be promoted upstream,which may affect cell behavior by regulating Integrin-β1/FAK/ERK pathway to activate downstream effector proteins VEGFA and MMP-2.