Comprehensive and Combined Omics Analysis Reveals Immune–Infiltration‐Associated Alternative Splicing and RBP Regulators Contributing to Ischemia–Reperfusion Injury in Liver Transplantation

Abstract

Background Hepatic ischemia–reperfusion injury (HIRI) is a major cause of early allograft dysfunction after liver transplantation, but its post‐transcriptional regulation remains poorly understood. This study aimed to characterize alternative splicing (AS) events in HIRI and explore a potential association network involving RNA‐binding proteins (RBPs), AS, and immune responses. Methods Using the GSE151648 dataset (80 paired samples from 40 patients), we identified regulated alternative splicing (RAS) events and analyzed dynamic changes in immune cell populations. An RBP‐RAS co‐expression network was constructed, and single‐cell data (GSE171539, one donor, three time points) were used to explore cell‐type‐specific expression of key RBPs in a qualitative, hypothesis‐generating capacity. RBP expression changes were validated in an independent microarray cohort (GSE12720, 20 paired biopsies) and cross‐validated with Human Protein Atlas liver proteomic data. Results Mild IRI (IRI−) samples mainly exhibited alternative 5 ′/3 ′ splice site events, whereas severe IRI (IRI+) samples were characterized by intron retention and overlapping exons, suggesting that severe injury may involve spliceosomal dysfunction. Post‐transplantation, activated mast cells and NK cells increased, with a shift toward M0 macrophages and a decrease in M2 macrophages. Thirty immune‐related RAS events significantly correlated with immune cell proportions (|r| ≥ 0.5, p < 0.01). An RBP‐RAS network comprising 43 differentially expressed RBPs was constructed, showing coordinated co‐variation between splicing factors and immune‐associated splicing events. Single‐cell analysis further revealed cell‐type‐specific expression patterns of multiple RBPs across hepatocytes, endothelial cells, and immune populations. External validation in GSE12720 confirmed that 39 of 42 detected RBPs (93%) were consistently upregulated post‐reperfusion, and the Human Protein Atlas confirmed protein‐level expression for 25 of 43 RBPs (58%) in normal liver tissue. Conclusions This study suggests that mild and severe HIRI are associated with distinct splicing programs and that these changes are closely associated with immune microenvironment remodeling, supporting the hypothesis of an RBP‐AS‐immune regulatory network. These findings, in which the RBP expression changes were independently validated in an external cohort, provide a basis for generating hypotheses regarding post‐transcriptional regulation in HIRI.