Integrative microRNA and transcriptome analysis reveals sex-specific molecular divergence in human bladder cancer

Bladder cancer affects men and women differently: men are diagnosed more frequently, but women often present with advanced disease and have worse survival. The biological mechanisms underlying these disparities remain unclear. This study aimed to identify sex-specific molecular features and regulatory interactions that shape tumor biology and outcomes. We performed an integrative multi-omics analysis combining bulk messenger RNA and microRNA expression, survival modeling, and single-cell transcriptomic profiling. Data were obtained from The Cancer Genome Atlas, Gene Expression Omnibus, and the Genome Sequence Archive. Differential expression analyses were conducted separately in tumors and in normal samples to compare males and females. Experimentally validated microRNA–mRNA target pairs were tested for correlation, and survival associations were evaluated using Kaplan–Meier and Cox models. Single-cell RNA-seq data were analyzed to assess sex-biased expression across tumor and immune cell populations. We identified 48 tumor-specific sex-biased microRNAs and 456 tumor-specific sex-biased genes, the majority located on autosomes rather than sex chromosomes. Correlation analysis revealed 82 experimentally supported, negatively correlated microRNA–mRNA pairs, including 63 discordant pairs with opposite sex-biased expression, suggesting sex-specific regulatory interactions. Several of these features were significantly associated with overall survival in a sex-dependent manner. For example, the male-upregulated microRNA miR-1270 showed repression of the female-biased targets CYP26B1 and FAM180A, both of which were associated with poor survival, highlighting potential prognostic and therapeutic relevance. Single-cell analysis revealed widespread sex-biased expression across epithelial, stromal, and immune cells, with female tumors showing stronger signals in stromal and immune compartments, which may contribute to the more aggressive clinical course observed in females. Our findings indicate that sex disparities in bladder cancer are largely driven by post-transcriptional regulation of autosomal genes, rather than sex chromosome dosage. By linking sex-biased microRNAs, target genes, and patient survival with cell type–specific expression, this study provides new insight into the biological basis of sex differences in bladder cancer. These results underscore the importance of incorporating sex as a critical variable in biomarker development, therapeutic targeting, and clinical trial design. Bladder cancer affects men and women differently. Men are more likely to develop the disease, but women who are diagnosed often have more advanced cancer and worse survival. The reasons for these differences are not fully understood. In this study, we looked for biological explanations for these sex differences. Using data from large cancer research programs, we examined the activity of thousands of genes and small molecules called microRNAs (miRNAs), which help control gene activity. We also analyzed single cells from bladder tumors to see which genes were active in different cell types. We found 48 miRNAs and 456 genes that differ between male and female bladder tumors. Most of these differences were not on sex chromosomes, but on other chromosomes, showing that regulation of gene activity plays a major role. Some of these miRNAs and genes were also linked to survival, but only in men or only in women. At the single-cell level, we found that sex-biased genes were especially active in immune cells and stromal cells that support tumor growth. This pattern may help explain why female bladder tumors can behave more aggressively. Overall, our results show that sex is an important factor in bladder cancer biology. Taking these differences into account could improve diagnosis, guide treatment choices, and ensure that future clinical trials benefit both men and women. We performed an integrative analysis of bulk mRNA, miRNA, survival data, and single-cell RNA-seq to investigate sex differences in human bladder cancer. A total of 48 miRNAs and 456 genes showed sex-biased activity in tumors, the vast majority located on autosomes rather than sex chromosomes. We identified 82 validated miRNA–mRNA pairs, including discordant pairs in which male-upregulated miRNAs target female-upregulated genes (or vice versa), consistent with post-transcriptional repression that differs by sex. Several sex-biased miRNAs and genes showed sex-stratified associations with overall survival, suggesting potential prognostic biomarkers. Single-cell analysis revealed that sex-biased genes were especially active in immune and stromal cells, providing insight into why female bladder tumors often present as more aggressive.