The Study On Methylation Differential Genes Related With Axon Regeneration Between Normol Schwann Cells And Activated Schwann Cells

ObjectiveSchwann cells(SCs)is one of the most important cells in the peripheral nervous system.Mature SCs coating neurons form myelin,these SCs were called normal Schwann cells(NSCs).After peripheral nerve injury,injuryed axon of the distal nerve fiber and myelin sheath were full-length damaged,and then completely disintegrated,this phenomenon is known as Wallerian degeneration.After nerve injury SCs showed high biological activity,there SCs called activated Schwann cells(ASCs).ASCs promotes axonal regeneration.However,the current researches have focused on the protein level and miRNA level,and studies have shown that there are significant differences between the ASCs and the NSCs protein and the level of miRNA,however,the two root causes SCs difference is not clear.In recent years,with the development of genetic engineering technology,the research of gene DNA methylation has been paid more and more attention by researchers.At the same time,it was found that some of the genes(such as Shh and Olig1)were methylated in the peripheral nervous system,and the methylation of these genes played a key role in the regeneration of axons.However,there is no genome-wide comparison of the differences between ASCs and NSCs axon regeneration related genes.To explore the difference of DNA methylation levels between normal and activated Schwann cells in rats,and screen the genes related with axon regeneration and analysis their functions.MethodsThe adult Wistar rats experienced sciatic nerve ligation and were feeded for 7 days.The activated Schwann cells(ASCs)and normal Schwann cells(NSCs)were separated from ligated sciatic nerves and brachial plexus respectively.Immunocytochemical staining of S-100 antibody was used to identify the cells.The growth condition was detected by CCK-8 method.DNA immunoprecipitation sequencing(MeDIP-Seq)was applied to filter the differentially methylated region between ASCs and NSCs.Annotation and statistical analysis of differentially methylated regions were conducted with KOBAS.The distribution of differentially methylated genes related with axonal regeneration in chromosome was analyzed by interProScan.Gene ontology(GO)and PATHWAY analysis were also conducted.ResultsHigh purity of ASCs and NSCs were obtained successfully,which were both positive for S-100 antibody.In the same culture condition,ASCs showed a faster proliferation than NSCs.A total of 176 610 differentially methylated regions were found by MeDIP-Seq.Among them,1 097 were located in the promoter(?1 kb),1 136 in the promoter(1-2 kb)and 567 on the CpG.After functional annotation of differentially methylated genes,214 differentially methylated genes related wih axonal regeneration were found in ASCs and NSCs.These genes were located on different chromosomes,most of which on chromosome 12(22 genes)and the least on chromosomes M(2 genes).Gene ontology analysis indiacted that the differential methylated genes were involved in axon growth,axon formation,axon elongation,axon guidance.The MAPK,cell adhesion molecules,Ras signaling pathway maybe related with the differential methylated genes.ConclusionThere are obvious differences in the methylation level of ASCs and NSCs,214 differentially methylated genes related with axon regeneration,which relates to the growth of axon,axon formation,extension of axons,axon guidance processes,MAPK signaling pathway,cell adhesion molecules and Ras signaling pathway.