RNA processing factors are associated with microglial heterogeneity and intercellular communication in ischemic stroke

Abstract Ischemic stroke (IS) is a leading cause of death and long-term disability globally, with dysregulated RNA processing implicated in its pathogenesis. This study aimed to identify the key genes associated with RNA processing factors (RNAPF) in IS using integrated transcriptomic analyses. Microarray data from human blood (GSE16561 and GSE58294) and single-cell RNA sequencing data from mouse brain tissue (GSE174574) were analyzed. Candidate genes were screened via differential expression analysis and intersected with RNAPF-related genes. Machine learning algorithms identified feature genes, followed by diagnostic validation and functional characterization. Immune infiltration was characterized at the transcriptomic level, whereas cellular heterogeneity was examined at single-cell resolution. To avoid pseudoreplication, all statistical inferences from single-cell data were elevated to the biological replicate level (n = 3 per group). Cell type and microglial subtype proportions were compared using sample-aggregated Wilcoxon rank-sum tests. Using RT-qPCR technology, we conducted a preliminary assessment of expression differences in key genes in a small-scale clinical sample set (n = 5 per group), providing preliminary support for this bioinformatics finding. A total of 12 candidate RNA processing-related genes were identified, with RNASEL and SRPK1 established as potentially key genes via machine learning and validation cohorts. A nomogram integrating both genes demonstrated high diagnostic accuracy. Functional enrichment linked key genes to neuroinflammation pathways, such as Toll-like receptor and chemokine signaling. Immune dysregulation was observed, with RNASEL/SRPK1 positively correlating with M0 macrophages and neutrophils. Single-cell analysis revealed microglia as a cell type of interest. At the sample level (n = 3 per group), microglia showed a decreasing trend in IS (24.40% vs. 21.51%,). Microglial subtype analysis revealed a marked reduction in MG_Homeostatic_Slc2a5 (88.50% → 15.60%) and significant expansion of MG_IEG_Egr1 (3.75% → 46.74%) and MG_Postnatal-PAM_Spp1 (0.05% → 21.22%) in IS. Pseudobulk DESeq2 analysis demonstrated that RNASEL was significantly downregulated (log2FC = − 1.127, padj < 0.001) while SRPK1 was significantly upregulated (log2FC = 0.599, padj < 0.001) in IS microglia. Cell–cell communication analysis highlighted enhanced monocyte–microglia interactions. RT-qPCR analysis in a small but clinically characterized cohort (n = 5 per group) preliminarily supported the key gene-expression patterns observed in the bioinformatics analyses, although these findings require validation in larger cohorts. RNASEL and SRPK1 may serve as candidate RNAPFs whose altered expression is associated with immune dysregulation and microglial reprogramming in IS. Microglial subtype-specific changes and altered intercellular communication networks provide hypotheses for further mechanistic investigation.