Urine cell-free RNA captures tumor-reflective long intergenic noncoding and circular RNA programs in bladder cancer

Introduction: Long intergenic noncoding RNAs (lincRNAs) and circular (circ) RNAs have been shown to play key roles in carcinogenesis and tumor progression. Urine represents a promising source of tumor-reflective signals shed directly from the urothelial microenvironment in bladder cancer (BC) patients. Here, we evaluate the urine cell-free (cf) RNA transcriptome in a cohort of 50 patients with localized BC. Methods: A total of 50 localized BC patients and 24 healthy adults were enrolled. Pre-operative urine (collected on the day of radical cystectomy) was processed to isolate cfRNA from urine supernatant. Libraries were prepared using an Illumina tagmentation-based RNA enrichment workflow and sequenced to 100M paired-end reads per sample on a NovaSeq X. FASTQ files were concatenated per sample and QC-checked with RNA-SeQC and Samtools. Reads were aligned to GRCh38/hg38 with STAR, and gene counts were generated using Feature Counts. Linear differential expression analysis (DEA) was performed with DESeq2. For discovery we used random halves of TCGA-BLCA and healthy urine cfRNA samples, alongside a second DEA with random halves of urine BC cfRNA vs. controls; overlapping upregulated genes were validated in the remaining samples. For circular (circ) RNAs, annotations from MiOncoCirc were used; junctions with ≥5 supporting reads were retained, and DEA was conducted with edgeR. Results: In this study, the median age was 68 years, and 80% (40/50) were male. Among the cohort, 66% (33/50) had muscle-invasive bladder cancer (MIBC), of whom 54% received neoadjuvant chemotherapy. Differential expression analysis of TCGA-BLCA tumors and BC urine cfRNA, identified 27 genes commonly upregulated, with 23 of the 27 shared genes strikingly being long intergenic noncoding RNAs (lincRNAs). These BC/urine-specific lincRNAs-including LINC00930, LINC01004, DLG5-AS1, APTR, PSORS1C3, and SERPINB9P1-have known roles in proliferation, EMT, metabolism, and immune signaling. Outside of lincRNAs, tumor-reflective coding transcripts in BC/urine were enriched for autophagy, driven by MAP1LC3B2.TCGA-BLCA survival analysis showed that LINC00930 and PSORS1C3 were associated with worse overall survival. Expanding beyond linear RNA sequences, analysis of urine circRNAs from BC patients revealed a strong cancer-specific dysregulation pattern, with 77 circRNAs significantly upregulated compared with healthy controls. Upregulated urine circRNAs arose from host genes involved in BC-relevant pathways, including invasion ( DOCK1 , ARHGAP5 ), autophagy/ER stress ( VMP1 , ERN1 ), and luminal differentiation ( GRHL2 , ESRP1 ). Conclusion: Cell-free RNA profiling reveals a distinct transcriptomic signature including differential lincRNA- and circRNA-driven autophagy, EMT, proliferation, metabolic, differentiation, and immune signaling programs in the urine of bladder cancer patients.