Yulei Li ¹ Lulu Zhang ¹ Gang Xu ² Gang Xu ¹ Jiajun Chen ¹ Fengyan Tian ¹ Mengyao Li ¹ Jing Jin ¹ Chao Peng ¹ Kaifang Wang ³ Shouhua Pan ¹ Ke Zhu ^2*

• ¹ Shaoxing People's Hospital, Shaoxing, Zhejiang Province, China
• ² Nanchang People's Hospital, Jiangxi, China
• ³ University of Macau, Taipa, Macau Region, China

Background: ROS, a hallmark of cancer, is related to prognosis, tumor progression, and treatment response. The correlation of ROS-based molecular signature with clinical outcome and immune cell infiltration has not been thoroughly studied in BLCA. Accordingly, we aimed to thoroughly examine the role and prognostic value of ROS-related genes in BLCA. Methods: We obtained RNA sequencing and clinical data from TCGA for BLCA patients and identified ROS-associated genes using the MSigDB. We then analyzed differential gene expression between BLCA and normal tissues and explored the functions of these ROS-related genes through GO, KEGG, and PPI analysis. Prognostic ROS-related genes were identified using UCR and LASSO analyses, which were further refined in a MCR analysis to develop a PS. This PS was validated in the GSE13507 cohort, assessing its predictive power with Kaplan-Meier survival and time-dependent ROC curves. To forecast BLCA outcomes, we constructed a nomogram integrating the PS with clinical variables. We also investigated the signature's molecular characteristics through GSEA, ICI, and TMB analyses. The GDSC database was used to predict chemotherapy responses based on the PS. Additionally, we screened for SMDs targeting ROS-related genes using the CMAP database. Finally, we validated our findings by checking protein levels of the signature genes in the HPA and confirmed the role of AKR1B1 through in vitro experiments. Results: The constructed and validated PS that comprised 17 ROS-related genes exhibited good performance in predicting OS.The bioinformatics analysis findings showcased the implication of PS in several oncogenic pathways besides tumor ICI regulation. The PS was negatively associated with the TMB. The high-risk group patients had greater chemotherapy sensitivity in comparison to low-risk group patients. Further, 11 candidate SMDs were identified for treating BLCA. The majority of gene expression exhibited a correlation with the protein expression.The expression of most genes was consistent with protein expression. AKR1B1, one of the seventeen genes identified, was used for in-depth validation. In vitro experiments indicate that siRNA-mediated AKR1B1 silencing impeded BLCA cell viability, migration, and proliferation. Conclusions: We identified a PS based on 17 ROS-related genes that represented independent OS prognostic factors and 11 candidate SMDs for BLCA treatment.