Changes In DNA Methylation Of Axon Regeneration-related Genes After Peripheral Nervous System And Spinal Cord Injury

Objective Spinal Cord Injury(SCI)is one of the world’s medical problems.Its treatment strategy has not been able to make breakthrough progress because of the study of the pathophysiological mechanism of spinal cord injury is not yet clear.Studies have shown that,neurons after peripheral nervous system injury can maintain their regenerative ability and can promote the formation of neuron growth cones in the allowed growth environment,affecting axon growth,unlikely the central nervous system,which is related to the obvious changes in DNA methylation.A large amount of DNA methylation or demethylation changes in the promoter region after nerve damage,mediating axonal regeneration,glial cell activation,vascular regeneration and so on.Therefore,it is very important to reveal the mechanism of DNA methylation modification regulating nerve regeneration after central and peripheral nerve injury,which is expected to provide a new theoretical basis and therapeutic target after SCI.Methods 1.Study design.A total of 36 Wistar rats were used in this study,divided into 4 groups.Group 1: sciatic nerve exposed group(n = 9);Group 2: sciatic nerve injury group(n = 9);Group 3: laminectomy group(n = 9);Group 4: SCI group(n = 9).Rats in group 1 underwent sciatic nerve exposure surgery.Rats in group 2,their bilateral sciatic nerves were exposed and ligated with silk thread at the proximal end of the sciatic nerve.Rats in group 3 underwent laminectomy without spinal cord injury.Rats in group 4 completely removed 2 mm of spinal cord tissue at T10-11.2.Establish a DNA methylation differential gene database with high-throughput sequencing technology.The bilateral sciatic nerves of Group 1 and Group 2 were separated,and activated Schwann cells(ASCs)and normal Schwann cells(NSCs)were extracted and cultured to the third generation for Me DIP-seq.And the spinal cord tissue was collected for WGBS.Establish a database of differential gene methylation of DNA after peripheral nerve and spinal cord injury.3.Bioinformatics analysis the changes in DNA methylation of genes related to axon regeneration after peripheral nervous system and spinal cord injury.GO analysis,KEGG analysis and PPI network were performed on differentially expressed genes,and q RT-PCR further verified the results of bioinformatics analysis.Results 1.Detection of DNA methylation gene changes after peripheral nerve injury using Me DIP-seq.In this study,ASCs and NSCs with high purity were obtained.Both were positive with S-100 and found that ASCs grew faster and adhered faster in the same culture conditions.A number of 177,176 differentially methylated regions found by Me DIP-Seq.A total of 221 differentially methylated genes related to axon regeneration were obtained with gene annotation classification.These genes are mainly involved in axon guidance,axon injury response,axon extension,axon formation and axon growth and so on.2.Reveal DNA methylation changes after spinal cord injury by WGBS.After spinal cord injury,a large number of cavities,necrotic tissue areas and a large number of inflammatory cells were observed.There are 235 differential DNA methylation genes,of which 96 genes were significantly different(P<0.001),50 were high methylation genes,46 were low methylation genes,mainly enriched in the hippo signaling pathway,Endocytosis pathway,calcium signaling pathway and other signal transduction pathways.3.Screening and analysis of key differential genes in DNA methylation.In this study,compared of the above two databases,a total of 8 DNA methylation differential genes related to axon regeneration were screened out,namely Ntrk3,Reln,Tenm2,Numbl,Dpysl2,Smurf1,Lmx1 a,Kalrn.GO analysis showed that it is mainly involved in neuronal regeneration,signal transduction,nervous system development in biological processes;mainly enriched in signal receptor binding,protein binding,Metal ion binding in molecular functions;mainly concentrated in cytoplasm and plasma membrane in cell components.The results of KEGG enrichment analysis showed that differentially methylated genes were mainly enriched in calcium signaling pathway,Endocytosis signaling pathway,Hippo signaling pathway.Conclusion In this study,identifing and verifing different methylation genes after nervous system injury by bioinformatics and q RT-PCR,revealing common changes in DNA methylation genes after spinal cord injury and peripheral nerve injury,and getting 8 axonal regeneration-related DNA methylation differential genes,namely Ntrk3,Reln,Tenm2,Numbl,Dpysl2,Smurf1,Lmx1 a,Kalrn.These common genes with epigenetic changes are considered candidate targets or biomarkers for nerve regeneration and modeling after central and peripheral nerve injury.This study provides new insights into the molecular mechanism of axon regeneration in subacute nerve injury.However,further research is still needed to verify the function and relationship between these epigenetic changes.