BC is one of the most common cancers and patients with early or limited BC can be treated by surgical resection, while patients with advanced BC are usually treated with radiotherapy or chemotherapy. Despite effective treatment, the outcome is still unsatisfactory . Bladder cancer is considered to be one of the most frequently mutated cancers in humans, with a mutation rate second only to lung and skin cancers [20, 21]. The major mutation among these mutations is the promoter mutation in the gene encoding telomerase reverse transcriptase (TERT), which occurs at a frequency of 70–80% in bladder cancer patients [22–25]. Therefore, it is essential to find potential therapeutic targets for bladder cancer as soon as possible. And a large number of studies have shown that regular aspirin consumption can reduce the risk of cancer [26–28].
In the present study, we used comprehensive bioinformatics analysis to elucidate the molecular roles of aspirin and its target proteins in BC. First, we analyzed aspirin by  identifying the primary DPT of aspirin using Drug Bank . Protein-protein interaction (PPI) networks and signaling pathways of aspirin DPT were analyzed using STRING . Detection and testing of genetic alterations using the cBio portal.  Identification of genes associated with 4 aspirin target genes in BC using STRING. We identified 16 action targets of aspirin: PTGS1, PTGS2, AKR1C1, PRKAA1, EDNRA, TP53, HSPA5, RPS6KA3, NFKBIA, TNFAIP6, CASP1, CASP3, CCND1, MYC, PCNA, CCNA2. Among them, CCND1, MYC, and TP53 were associated with BC. Subsequently, we determined that the alteration of CCND1 showed gene amplification. the alteration of MYC mainly showed gene amplification. the alteration of TP53 mainly included missense mutation, splice mutation and truncation mutation. finally, we constructed a PPI pathway consisting of three target genes of BC to predict the potential target genes of aspirin in BC. Meanwhile, we identified the top 50 overexpressed genes of BC using Oncomine. Finally, we identified the co-expressed genes (GSK3B, CDC20, TPX2, AURKA and CCNE1) among the genes interlinked with the 3 aspirin target genes in BC samples as potential targets for aspirin treatment of BC.
GSK-3β, GSK3B, is a positive regulator of NF-κB transcriptional activity [29, 30]. It has been shown that NF-κB plays a role in human cancer progression and chemoresistance [31, 32] through positive regulation of its target genes XIAP  and Bcl-2 .Levidou et al showed that nuclear expression of NF-κB correlates with histological grading and staging of bladder cancer.Sei Naito et al. found that urothelial epithelial carcinoma cells and abnormal nuclear accumulation of GSK-3β in most human bladder cancers. nuclear expression of GSK-3β was associated with high malignancy, metastasis and poorer survival in bladder cancer patients. They suggested that GSK-3 is a positive regulator of bladder cancer cell proliferation and survival . cdc20 is usually considered as an oncogenic factor that promotes tumor development [36, 37]. Moreover, it has been demonstrated that increased CDC20 expression in bladder cancer patients is associated with poor prognosis .AURKA and AURKB, of the AURKA kinase family, are closely associated with the development of malignancy.AURKA is a cell cycle-associated serine-threonine kinase that is overexpressed in various types of cancer and is strongly associated with poor prognosis .Mobley et al.  found that knockdown AURKA had little effect on bladder cancer cell proliferation but prevented tumor cell invasion, and that overexpression of AURKA was associated with poor prognosis.AURKB is a key regulator of malignant mitosis and is involved in chromosome segregation and cytoplasmic division.Bufo et al  found that high expression of AURKB may be involved in bladder carcinogenesis and hypothesized that bladder cancer could be treated in the future by targeting AURKB expression and specific antimitotic agents. dysregulation of CCNE1/2 activity is present in various cancers [42–45], leading to disruption of the G1-S transition and uncontrolled cell proliferation. involvement of the CCNE-CDK2 complex in cell cycle regulation has been demonstrated to play an important role in tumor development [46, 47]. the E2F transcription factor strongly activates CCNE1 and CCNE2, the CCNE-CDK2 complex phosphorylates and inactivates Rb, and phosphorylated Rb releases the E2F transcription factor, thus promoting cell cycle progression from G1 to S phase . In addition, it has been demonstrated that MNX1 induces bladder cancer proliferation and tumorigenicity by targeting promoters to upregulate CCNE1 and CCNE2 expression .
As for the TPX2 gene, by String prediction, we learned that no complete biological process, signaling pathway related to TPX2 has been found so far. However, there are many literatures that have demonstrated that TPX2 is significantly associated with bladder cancer. liang Yan et al. demonstrated that overexpression of TPX2 promotes bladder cancer growth, while overexpression of GLIPR1 or p53 inhibits bladder cancer growth. Increasing evidence supports the role of TPX2 as a tumor promoter in human tumor development, with bladder cancer tissues expressing high TPX2 levels having upregulated p53 expression and downregulated GLIPR1 expression. In addition, TPX2 and p53 expression was lower in non-muscle-infiltrating bladder cancer cells than in muscle-infiltrating bladder cancer cells, while the opposite pattern of GLIPR1 expression was observed . overexpression of GLIPR1 suppressed TPX2. meanwhile, SP1 and c-Myb expression were negatively correlated with GLIPR1 expression. [51–53]. Yan et al. demonstrated that TPX2 is highly expressed in human bladder cancer tissues and that upregulation of TPX2 predicts poor prognosis in patients with bladder cancer. In addition, TPX2 promotes T24 cell proliferation and tumorigenesis and blocks apoptosis .
In conclusion, we believe that these five target genes (GSK3B, CDC20, TPX2, AURKA and CCNE1) may promote the occurrence of bladder cancer and lead to poor prognosis. We explored the potential therapeutic targets of aspirin for bladder cancer by comprehensive bioinformatics analysis. We suggest that aspirin acts through cell cycle and signaling pathways in bladder cancer cells, and our results aim to provide new clues to elucidate the mechanisms of aspirin's action in bladder cancer.
However, there are still challenges in applying Web-based data to the study of drugs such as aspirin. Identifying drug-target interactions is important in the drug discovery process. Although microarrays, proteomics and other high-throughput screening analyses have been applied, experimental methods for drug-target interaction identification remain challenging.