A migration/invasion-related prognostic signature identifies and functionally validates SLC25A5 as a hub associated with spliceosome programs and invasive phenotypes in esophageal squamous cell carcinoma.

Background esophageal squamous cell carcinoma (ESCC) is highly aggressive and associated with poor survival outcomes. Whether migration/invasion-related transcriptional programs are coupled to RNA splicing regulation and distinct tumor immune microenvironment (TME) features, and how these relationships inform prognosis, remain incompletely understood. Methods using TCGA-ESCC RNA-seq data, we developed a migration/invasion gene-based prognostic signature. Candidate genes from GeneCards and CancerSEA underwent univariate Cox screening, LASSO selection, and multivariable Cox modeling, and GSE53625 was used for external validation. Tumor mutational burden (TMB), TIDE scores, and predicted drug sensitivity were analyzed, and random survival forest (RSF) was used to prioritize hub genes. For the top-ranked hub gene, SLC25A5, we performed differential expression, GO and GSEA analyses. Immune features were evaluated using ESTIMATE and CIBERSORT, and paired tumor-normal expression in TCGA was examined. Functional studies included siRNA-mediated knockdown of SLC25A5 in EC9706, EC-1, and TE-1 cells, Western blot validation of SLC25A5, wound-healing and Transwell assays, analysis of spliceosome-related proteins and EMT markers, qRT-PCR of CD44 isoforms, and rescue experiments by SLC25A5 re-expression. Results a five-gene signature stratified overall survival in TCGA and remained independent of clinicopathological factors, with its predictive value externally validated in GSE53625. High-risk tumors showed lower TMB, higher TIDE scores, and distinct survival patterns in TMB-combined strata. OncoPredict suggested lower predicted IC50 values for multiple agents in the low-risk group. RSF identified SLC25A5 as the leading hub gene. GSEA showed enrichment of Cell cycle and Spliceosome pathways in the SLC25A5-high group. By ESTIMATE, SLC25A5-low tumors had higher stromal, immune, and composite scores, and CIBERSORT revealed shifts in immune-cell subsets; activated dendritic cells correlated positively with continuous SLC25A5 expression. SLC25A5 was upregulated in tumors compared with paired normal tissues. SLC25A5 knockdown inhibited migration and invasion, reduced spliceosome-related proteins, shifted EMT markers toward a more epithelial-like pattern, and altered CD44 isoform expression. These molecular and phenotypic changes were partially reversed by SLC25A5 re-expression. Conclusions this migration/invasion-oriented signature provides biologically informed risk stratification in ESCC. SLC25A5 was identified as a functionally relevant hub gene associated with spliceosome-related changes and invasive phenotypes, while invasion-related risk states were accompanied by immune-evasive features, supporting further mechanistic and translational investigation of SLC25A5 in aggressive ESCC.