Many factors, such as carcinogen exposure, gene mutation, chronic inflammation, and genetic predisposition, are involved in the pathogenesis of bladder cancer as a common malignant tumor [1]. Mutations and abnormal expression of several genes have been found to be closely related to the development and progression of bladder cancer. For example, HER2 overexpression plays an important role in the pathogenesis of bladder cancer by promoting the proliferation and survival of cancer cells, regulating cell cycle, and enhancing cell adhesion [14, 15]. Currently, targeted therapy against HER2 has been widely studied. Drugs targeting HER2, such as vedicizumab, have been used to treat patients with HER2-positive bladder cancer [16, 17]. In addition, TP53 gene mutations are common in bladder cancer, attenuating its inhibitory effect on tumor growth [18]. Similarly, dysfunction of RB1, a classical tumor suppressor gene, may be related to the occurrence and metastasis of bladder cancer. Simultaneous mutation of RB1 and TP53 were found to strongly correlate with genomic biomarkers of response to immune checkpoint inhibitors in human bladder cancer. Therefore, studying gene-level changes is of great significance for understanding the pathogenesis of bladder cancer and finding new therapeutic targets.
As for the causes of gender differences in bladder cancer incidence, the current study indicated that the higher incidence among men may be due to their more frequent exposure to toxic substances, smoking, long-term drinking, and other reasons, but there are few studies on genetic differences in males and females. Currently, there are several reports on the role of immune cell infiltration-related genes [19], pyroptosis-related genes [20], and autophagy-related genes [21], in the development of bladder cancer. However, these studies did not separately analyze data from men and women and could not uncover genetic differences behind male and female bladder cancer. We separately analyzed data from male and female bladder cancer. By analyzing gene expression profiles, we found that the most significant different genes in male and female bladder cancer are not exactly the same. The results of WGCNA analysis indicated that the key modules of males and females were not the same, indicating that the genetic background of male bladder cancer is different from that of female bladder cancer. Considering the intersection of differential genes and module-related genes obtained by WGCNA analysis, we obtained 376 overlapping genes in the male bladder cancer group and 328 overlapping genes in the female bladder cancer group. Using the functional enrichment analysis of these genes, we found that there were gender differences in tumor metabolism-related signaling pathways. For example, RAS signaling pathway was enriched in the male patient group, but not in the female patient group. On the contrary, JAK/STAT signaling pathway was enriched in the female patient group, but not in the male patient group. These metabolic pathways play an important role in the development of bladder cancer. Studies have shown that RAS can activate PI3K/Akt signaling pathway and promote epithelial-mesenchymal transition (EMT) and cell migration [22], thereby promoting bladder cancer cells to cross the basement membrane and spread to the surrounding tissues [23]. In bladder cancer, abnormal activation of JAK/STAT signaling pathway may lead to uncontrolled proliferation and survival of tumor cells, thus promoting tumor development and progression [24]. Studies have also shown that an aberrantly activated JAK/STAT signaling pathway may lead to immune cell dysfunction, thus affecting immune surveillance and helping tumor immune evasion [25]. These differences in metabolic pathways suggest that there may be differences in the underlying molecular mechanisms between male bladder cancer and female bladder cancer.
Using PPI analysis, we identified 10 most important hub genes in male and female bladder cancer. Among them, COL3A1, COL1A2, ACTA2, MYL9, TPM1, TPM2, and MYH11 were common in male and female bladder cancer, while other genes (PDGFRB, VEGFA, CAV1, TAGLN, ACTG2, and CALD1) were different between male and female bladder cancer. According to these genes, we selected the top five genes with the highest scores and constructed a nomogram model to predict the risk of male bladder cancer (model genes: CAV1, VEGFA, COL3A1, MYL9, and COL1A2) and female bladder cancer (model genes: COL3A1, ACTA2 TPM2 TPM1, and COL1A2). ROC and AUC curves showed that our two nomogram models showed excellent performance in predicting the risk of bladder cancer. In view of the large difference between the incidence of male bladder cancer and female bladder cancer, incidence risk prediction models should consider gender-specific differences in gene expression. Although some studies suggested that gender-specific factors should be incorporated into pre-clinical models of bladder cancer [26], to our knowledge, our study is the first that separately built risk prediction models for male bladder cancer and female bladder cancer. These findings are conducive to more accurate risk prediction for patients with bladder cancer.
Immune cell infiltration is affected by age, sex, metabolism, and other factors in bladder cancer [26, 27]. However, there is no report on differences in immune cell infiltration between male and female bladder cancer. We found that immune cell infiltration patterns were not significantly different in male and female bladder cancer. The tissue abundance of Naïve B cells significantly decreased in female bladder cancer, while there was no statistical difference in male bladder cancer. As a powerful angiogenesis inducer, VEGFA can stimulate blood vessel formation in bladder cancer and provide more blood supply for tumor growth [28, 29]. Recently, it has also been shown that VEGFA expression affects the immune microenvironment in bladder cancer. For example, VEGFA overexpression in bladder cancer is related to high infiltration of cd163 + tams [30]. We found that in male bladder cancer, the tissue abundance of Tregs, M0 macrophages, follicular T cells, and activated dendritic cells was positively correlated with VEGFA expression, while the tissue abundance of CD8 + T cells, resting mast cells, M2 macrophages, and CD4 + T cells was negatively correlated with VEGFA expression. VEGFA expression did not affect immune cell infiltration in female bladder cancer. These findings suggest that in male bladder cancer but not in female bladder cancer patients, VEGFA signaling may affect immune cell infiltration.
Based on the importance of VEGFA in bladder cancer, we used data from GWAS to conduct a two-sample MR analysis and explore the causal relationship between VEGFA expression and bladder cancer risk. The results of MR analysis showed that VEGFA expression may be causally related to the increased risk of bladder cancer. MR is conceptually similar to prospective randomized controlled trials (RCTs), but reduces systematic bias of traditional observational studies, such as confounding factors and reverse causality. The high accuracy of genotyping can help effectively avoid detection errors caused by regression dilution. To ensure that SNPs between VEGFA and bladder cancer were not associated with any confounding factors, we selected only participants from European populations. Finally, we also performed the MR Egger regression test to ensure the stability of results, which showed no directional level pleiotropy.
Despite these significant findings, our study has some limitations. Firstly, we only used one dataset for analysis. If more datasets were included for analysis, the results could be more convincing. Secondly, this study only used bioinformatics to analyze the hub genes related to bladder cancer and their potential functions, without molecular biology and cell function experiments. Finally, due to the lack of gender-specific data in MR analysis, this study did not separately conduct MR analysis for male bladder cancer and female bladder cancer.