Comprehensive analysis of m6A methylation modification in chronic spinal cord injury in mice

Abstract

Chronic spinal cord injury (CSCI) is a catastrophic disease of the central nervous system (CNS), resulting in partial or complete loss of neurological function. N6-methyladenosine (m6A) is the most common form of reversible posttranslational modi cation at the RNA level. However, the role of m6A modi cation in CSCI remains unknown. In this study, we established a CSCI model using a water-absorbable polyurethane polymer, with behavioral assessment, electrophysiological analysis, and histochemical staining for validation. Methylated RNA immunoprecipitation sequencing (meRIP-seq) and mRNA sequencing (mRNA-seq) were jointly explored to compare the differences in CSCI spinal tissue and normal spinal tissue. Furthermore, qRT-PCR, western blotting, and immuno uorescence staining were used to analyze m6A modi cation-related proteins. We found that water-absorbable polyurethane polymer well simulated chronic spinal cord compression. BMS scores and electrophysiological analysis showed continuous neurological function decline after chronic compression of the spinal cord. meRIP-seq identi ed 642 differentially modi ed m6A genes, among which 263 genes were downregulated and 379 genes were upregulated. mRNA-seq showed that 1544 genes were upregulated and 290 genes were downregulated after CSCI. Gene Ontology (GO) terms and enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were also identi ed. qRT-PCR, western blotting, and immuno uorescence staining showed that Mettl14 was signi cantly upregulated after CSCI. Our study revealed a comprehensive pro le of m6A modi cations in CSCI and may provide a valuable tool for further research on CSCI. study showed that mettl14 was signi cantly overexpressed after CSCI and mainly expressed in neurons, which was further validated by qRT-PCR, western blotting, and immuno uorescence.Our study is not without limitations. First, we obtained whole spinal tissue for meRIP-seq and mRNA-seq. Although the total m6A modi cations were assessed, it was di cult to distinguish m6A methylation changes in speci c cell types. In further in-depth research, extracting speci c cells for veri cation of m6A modi cation and exploring its function would be meaningful. Second, we performed preliminary analyses of differentially expressed genes and enriched GO and KEGG pathways, but their roles in CSCI require further validation through extensive experiments.Collectively, our study analyzed m6A methylation modi cations in spinal cord tissue after CSCI. The results indicated that m6A plays an important role in the process of CSCI. Our research may provide new insights for further study of the CSCI pathological process and potential treatment options. DeclarationsAuthor contributions JH, YC and HL designed the study. CL and JZ carried out most of the experiments and data analysis. YJ, TQ, CD and TW assisted in experiments and data analysis. CL drafted the manuscript, and JH and YC revised the manuscript.