Enhancer RNAs (eRNAs) have emerged as important regulators of gene expression and may reshape the therapeutic landscape of prostate cancer. However, their global landscape and clinical relevance in prostate cancer remain unclear.

We systematically integrated prostate cancer multi-omics and functional genomics datasets to characterize genome-wide eRNA transcription, construct eRNA-centered regulatory networks, and link eRNA-regulated genes to clinical outcomes. Representative eRNAs and their predicted target genes were further subjected to siRNA-mediated knockdown and functional validation in prostate cancer cell lines.

We identified 13,595 eRNAs and 1,573 eRNA-regulated genes, including 266 transcription factors and 85 RNA-binding proteins. eRNA-regulated genes showed higher expression levels, lower variability, and enrichment in prostate cancer–related pathways. A five-gene eRNA-associated signature stratified patients into high- and low-risk groups with AUCs of 0.85, 0.77, and 0.89 for 1-, 3-, and 5-year survival and remained an independent prognostic factor. The two risk groups exhibited distinct genetic, transcriptomic, and epigenetic features. Functional validation further demonstrated that knockdown of representative eRNAs suppressed prostate cancer cell proliferation, migration, and invasion, accompanied by reduced expression of the target genes NUP93 and BICD1.

These findings indicate that eRNAs may contribute to prostate cancer biology and could serve as useful markers for prognosis and pathway characterization.