膀胱癌（BLCA）在全球范围内影响着数百万人，发病率和死亡率均较高。铁死亡证明是一种由氧化应激引发的新型细胞死亡过程。我们从GEO数据库的GSE169379中获取了25个单核RNA测序（snRNA-seq）样本。我们从TCGA和GEO数据库中获取了不同的BLCA患者队列用于模型训练和验证。我们从FerrDb数据库中选择了369个与铁死亡相关的基因（FRGs）。通过AUCell分析为所有细胞类型分配了铁死亡评分。我们进行了加权基因共表达网络分析（WGCNA）、COX和LASSO回归分析，以保留和确定具有预后价值的基因。使用多种生物信息学方法描述了免疫浸润的特征。我们进行了一系列的克隆形成分析、流式细胞术和免疫印迹（WB）分析，以确定SKAP1在BLCA中的作用。我们根据铁死亡活性评分将细胞分为高铁死亡组和低铁死亡组，然后通过差异表达分析筛选了与铁死亡最相关的2150个基因，这些基因与紫外线诱导的DNA损伤、雄性激素反应、脂肪酸代谢和缺氧相关。随后，WGCNA算法从这2150个基因中进一步筛选了741个铁死亡相关基因用于构建预测模型。我们使用Lasso-Cox回归分析构建了预测模型，并获得由JUN、SYT1、MAP3K8、GALNT14、TCIRG1和SKAP1组成的预测模型。接下来，我们构建了一个整合了临床因素的直观图模型，以提高准确性。此外，我们对不同亚组进行了药物敏感性分析，发现Staurosporine、Rapamycin、Gemcitabine和BI-2536可能成为高风险人群药物治疗的候选药物。ESTIMATE结果显示低风险组中间质得分、免疫得分和ESTIMATE得分较高，表明该组的整体免疫水平和肿瘤微环境（TME）的免疫原性更高，而肿瘤免疫功能障碍和排除（TIDE）分析证实了低风险组对免疫疗法的更好应答。最后，我们选择了预测基因中的SKAP1进行体外验证，并发现SKAP1直接调节了BLCA细胞的增殖和凋亡。我们确定了一组六个基因（JUN、SYT1、MAP3K8、GALNT14、TCIRG1和SKAP1），它们在区分具有不同预后的BLCA患者方面表现出显著的潜力。此外，我们揭示了SKAP1在BLCA细胞增殖和凋亡中的直接调控作用，为FRGs在BLCA发病机制中的作用提供了一些线索。© 2023 Wiley Periodicals LLC.

Bladder cancer (BLCA) affects millions of people worldwide, with high rates of incidence and mortality. Ferroptosis proves to be a novel form of cell death process that is triggered by oxidative stress.We procured a total of 25 single nuclear RNA-seq (snRNA-seq) samples from GSE169379 in GEO database. We obtained different cohorts of BLCA patients from the TCGA and GEO databases for model training and validation. A total of 369 ferroptosis-related genes (FRGs) were selected from the FerrDb database. AUCell analysis was performed to assign ferroptosis scores to all the cell types. Weighted Gene Co-Expression Network Analysis (WGCNA), COX, and LASSO regression analysis were conducted to retain and finalize the genes of prognostic values. Various bioinformatic approaches were utilized to depict immune infiltration profile. We conducted a series of colony formation analysis, flow cytometry and western blot (WB) analysis to determine the role of SKAP1 in BLCA.We divided the cells into high ferroptosis group and low ferroptosis group according to ferroptosis activity score, and then screened 2150 genes most associated with ferroptosis by differential expression analysis, which are related to UV-induced DNA damage, male hormone response, fatty acid metabolism and hypoxia. Subsequently, WGCNA algorithm further screened 741 ferroptosis related genes from the 2150 genes for the construction of prognostic model. Lasso-Cox regression analysis was used to construct the prognostic model, and the prognostic model consisting of 6 genes was obtained, namely JUN, SYT1, MAP3K8, GALNT14, TCIRG1, and SKAP1. Next, we constructed a nomogram model that integrated clinical factors to improving the accuracy. In addition, we performed drug sensitivity analyses in different subgroups and found that Staurosporine, Rapamycin, Gemcitabine, and BI-2536 may be candidates for the drugs treatment in high-risk populations. The ESTIMATE results showed higher stromal scores, immune scores, and ESTIMATE scores in the low-risk group, indicating a higher overall immunity level and immunogenicity of tumor microenvironment (TME) in this group, and tumor immune dysfunction and exclusion (TIDE) analysis confirmed a better response to immunotherapy in the low-risk group. Finally, we selected the oncogene SKAP1 in the prognostic gene for in vitro validation, and found that SKAP1 directly regulated BLCA cell proliferation and apoptosis.We identified a set of six genes, JUN, SYT1, MAP3K8, GALNT14, TCIRG1, and SKAP1, that exhibited significant potential in stratification of BLCA patients with varying prognosis. In addition, we uncovered the direct regulatory effect of SKAP1 on BLCA cell proliferation and apoptosis, shedding some light on the role of FRGs in pathogenesis of BLCA.© 2023 Wiley Periodicals LLC.