A microRNA-pair signature for prognosis and immune modulation in gastric cancer

MicroRNAs (miRNAs) play critical roles in gastric cancer (GC) progression and patient survival. However, the combined prognostic value of miRNA pairs and their regulatory mechanisms remain largely unexplored. Genome-wide survival analysis of miRNA pairwise ratios identified hsa-miR-1249/hsa-miR-323b as the most significant pair. A risk scoring system based on prognostic pairs stratified patients into high- and low-risk groups. Transcriptomic analysis, functional enrichment, and single-cell RNA sequencing were used to explore biological differences, while Ecotyper assessed tumor microenvironment cell states. miRNA–transcription factor–target gene networks and drug–gene interactions were further analyzed. Patients with a high hsa-miR-1249/hsa-miR-323b ratio or low risk scores showed significantly longer survival. Transcriptomic analysis revealed 156 up- and 6 down-regulated genes in the high-risk group, mainly linked to extracellular matrix organization and muscle contraction. Fibroblast alterations were evident, and single-cell data confirmed fibroblast-specific expression of MYH11, HAND2, and SIX2, which correlated with poor survival and were predicted miR-1249 targets. Preliminary qRT-PCR analysis demonstrated increased expression trends of selected miRNA target genes in GC samples, consistent with the computational analyses. Dual-luciferase reporter assays confirmed the direct binding between miR-1249 and its predicted target gene HAND2. Hormone-related genes (ESR1, PGR, ESR2) were negatively associated with survival, and Mendelian randomization supported a potential association between ESR1 and GC. This study highlights the prognostic potential of miRNA pairs in GC and reveals transcriptional and microenvironmental alterations linked to patient risk, suggesting hormone signaling pathways as potential therapeutic targets.