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Updated: Jun 6, 2026
Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry
Published on: June 24, 2025
Comprehensive analysis of m6A RNA methylation regulators and the immune microenvironment in spinal cord injury
Xiaoqin Liu1, Jiating Hu1, Guodong Shi1
1Yan'an Medical College of Yan'an University, Yan'an, China.
Background:
Spinal cord injury (SCI) is devastating neurological disorder that leads to severe physical disabilities, reduced quality of life, and a substantial socioeconomic burden. N6-methyladenosine (m6A) RNA modification has emerged as an important regulator of RNA metabolism and immune responses; however, its role in SCI remains poorly understood.
Methods:
Transcriptomic datasets were obtained from the Gene Expression Omnibus (GEO) to identify differentially expressed m6A regulators in SCI. Hub genes were screened using multiple machine learning algorithms and further validated in an independent dataset. Immune cell infiltration was assessed using single-sample gene set enrichment analysis (ssGSEA), and miRNA-gene-TF interaction networks were constructed using NetworkAnalyst. Single-cell RNA sequencing (scRNA-seq) data were analyzed to characterize the cellular distribution of candidate genes. Finally, the expression of candidate genes was validated in a rat SCI model using quantitative real-time PCR (qRT-PCR) and immunofluorescence staining.
Results:
Fourteen differentially expressed m6A regulators were identified, among which eight candidate genes were selected using machine learning approaches. FTO and YTHDC1 were further identified as hub genes through validation in an independent dataset. Immune infiltration analysis revealed significant alterations in immune cell composition in SCI, and both FTO and YTHDC1 were significantly associated with multiple immune cell subsets. Consistent with increased m6A activity in microglia and astrocytes observed in scRNA-seq analysis, Fto and Ythdc1 were highly expressed in these cell types as well as in granulocytes. Furthermore, in vivo experiments validated the downregulation of FTO and YTHDC1 in injured spinal cord tissue.
Conclusion:
These findings suggest that FTO and YTHDC1 may play important roles in the pathogenesis of SCI and represent potential biomarkers and therapeutic targets for further investigation.
