The pathogenesis and progression of UC is complicated with various genetic abnormalities . Therefore, it is essential to identify appropriate biomarkers for UC. We conducted a series of bioinformatics analysis based on three gene chip databases.
In the present study, 63 common DEGs were identified including 31 that were up-regulated and 32 that were down-regulated, respectively. We focused on the function of differentially expressed DEGs using GO/KEGG pathway enrichment analysis. Our data demonstrated that DEGs were mainly enriched in collagen catabolic process, ECM organization, ECM structural constituent and ECM-receptor interaction. ECM is a key component exerting an active effect in all the marks of tumor and is associated with tumor metastasis . Collagen is recognized as the most important component of the ECM, which provided multiple biochemical and biophysical cues to tumor cells . ECM-receptor interaction is an important part of cancer cell proliferation and invasion . These results indicated that these DEGs were mainly involved in the occurrence, development and metastasis of UC. Among the DEGs, nine potential hub genes were obtained using the STRING and Cytoscape. Notably, all our hub genes were overexpressed in UC. Afterwards, we analyzed the characteristics of hub genes through various databases, aiming to investigate the potential biomarkers of UC.
There were many studies reporting the family of collagen (e.g. COL1A1, COL1A2, COL3A1 and COL5A2) that was involved in carcinogenesis. Li et al.  and Brooks et al.  both found that high COL1A1 expression was associated with shorter survival time in patients with superficial UC. COL1A1 has been considered as an oncogene and promoted cancer migration and invasion by inducing epithelial-mesenchymal transition (EMT) . In addition, increasing evidence confirmed that COL1A1 involved in proliferation, migration, development, and progression of various cancers [37, 39]. This indicated that COL1A1 might be a putative therapeutic target for cancer. Rong et al.  reported high expression of COL1A2 in gastric cancer than in normal tissues and promoted cell proliferation, migration, and invasion. Additionally, Tamilzhalagan et al.  reported that miR-25 could selectively target the COL1A2 gene. MiR-25 silencing increased the expression of COL1A2 and inhibited the expression of E-cadherin. This is related to EMT, revealing the inhibitory effect of miR-25 on diffuse gastric cancer , which provided insights for mechanistic studies and clinical trials . It has been reported that mRNA transcription level of bladder cancer is increased and CpG hypermethylation of COL1A2 contributes to proliferation and migration activity of human bladder cancer . It has been reported that mRNA transcription level of bladder cancer is increased and CpG hypermethylation of COL1A2 contributes to proliferation and migration activity of human bladder cancer. Qiu et al. reported that miR-29a/b promoted cell migration and invasion in nasopharyngeal carcinoma progression by regulating COL3A1 gene expression . While Su et al. indicated that let-7d can at least partially inhibit growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7 . Yuan et al. discovered that COL3A1 played certain roles in the progression of UC and influenced the prognosis probably by regulating MAPK signaling pathway, which contributed to the poor prognosis of UC . These were consistent with our results. In a previous study, the in situ expression of COL5A2 increased in invasive breast cancer and was associated with ECM remodeling, suggesting that COL5A2 is associated with tumor progression in breast cancer . Zeng et al.  demonstrated that COL5A2 was related to the poor clinical outcome and survival rate of patients with UC, and that COL5A2 was associated with poor clinical outcomes and survivals of patients with UC. This implied that it could be regarded as a biomarker of UC. Therefore, we considered that COL1A1, COL1A2, COL3A1 and COL5A2 may serve as promising candidates for the diagnosis, treatment and prognosis of UC.
MMP9 is an important matrix proteinase involving in embryonic development, wound healing, arthritis, angiogenesis, and cancer invasion and metastasis . The mRNA expression of MMP9 was higher in invasive than in superficial UC . Besides, Lei et al.  revealed that MMP9 could promote cancer invasion, metastasis and angiogenesis, while its expression was correlated with UC grade, invasiveness and poor prognosis. Wu et al.  found that the Wnt signaling pathway would regulate UC metastasis through activating MMP9, and showed that MMP9 may be regulated by miR-3713 in UC cells.
POSTN is an ECM protein expressed in many normal tissues, where it regulated cell adhesion and played an important role in the development and maintenance of mechanical stress-bearing structures (e.g. bones, teeth, and heart valves) . POSTN was overexpressed in many cancers, and high POSTN expression was correlated with tumor proliferation, cell motility and invasion, metastasis, angiogenesis, evasion of apoptosis, clinical stage, and survival of patients with UC , which is consistent with our findings. In addition, at the molecular level, POSTN involved in activation of PI3K/Akt and/or MAP kinase pathways via interacting with integrin receptors (i.e. aVb3 and aVb5), which subsequently promoted cell adhesion, motility, and angiogenesis. Furthermore, it could regulate E-cadherin expression and cell invasion through manner via Akt phosphorylation and regulating E-cadherin transcription, as well as negative regulation on Snail and Twist . We identify POSTN expression as a feature associated with poor outcomes.
SPP1, also known as Osteopontin (OPN), is located on chromosome 4 (4q13) including 6 introns and 7 exons . Recent studies have reported that SPP1 was significantly associated with cell growth, adherence, invasion and metastasis [50, 51]. It was overexpressed in lung, colon, breast, and prostate cancers. Our study showed that SPP1 expression was related to tumor stage, progression and survival rate. In addition, high expression of SPP1 was related to poor prognosis. This implied that SPP1 may be a diagnostic and prognostic marker for UC. Meanwhile, SPP1 can enhance cancer cell survival, angiogenesis, and inflammation, and promote metastasis by enhancing EMT. This indicated that SPP1 was a tumor-promoting gene in tumors [51, 52].
THBS2, also known as TSP2, is a disulfide-linked glycoprotein that mediates ECM assembly, cell-to-matrix interactions, degradation of MMP2 and MMP9, and inhibition of angiogenesis . THBS2 mainly participates in tumors by inhibiting angiogenesis and negatively regulating MMP-2 and MMP-9 . THBS2 is generally considered a tumor suppressive gene. However, there are disputes on expression of THBS2 in various tumors. High THBS2 immunoreactivity was associated with the tumor response to neoadjuvant chemoradiotherapy. Besides, it was also an independent and good prognostic factor for DFS in patients with rectal cancer . THBS2 gene is down-regulated in prostate cancer tissues and cell lines . In addition, Chijiwa found that the transcription level of THBS2 in lung adenocarcinoma was significantly higher than that of normal lung tissue . In our study, THBS2 was up-regulated in UC, which played important roles in UC progression.
VCAN, also known as chondroitin sulfate proteoglycan 2 (CSPG2), is a highly conserved structural component in ECM expressed in invasive and metastatic cancer . To date, four subtypes (i.e.V0, V1, V2, and V3) have been identified, and all of which contribute to the proliferation, adhesion, and migration of tumor cells, as well as interaction with tumor microenvironment (TME) . In a recent study, VCAN has been reported to be correlated with low RhoGDI2 expression, high VCAN expression and poor clinical outcomes . Our data showed VCAN expression was related to grade and prognosis of UC patients. The highly inflammatory microenvironment induced high expression of VCAN, and increased macrophage infiltration. In turn, infiltration of macrophage exacerbated VCAN overexpression along with the secretion of other cytokines and inflammatory mediators. In this context, VCAN appeared to mediate a dialogue between inflammatory cells, cytokines and cancer cells in TME . Therefore, we proposed that VCAN may serve as the first step in the amplification of inflammation.
Increasing evidence indicated that immunocyte infiltration may affect tumor progression and recurrence . In recent years, immunotherapy has been preferred in treating aggressive or advanced cancers with promising efficiency [7, 10]. As immune microenvironment affects tumor progression, we intended to explore an immune-related gene signature associated with prognosis of patients with UC. TME is composed of cancer cells and non-cancer cell components such as immunocyte (B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils and dendritic cells), mesenchymal cells, endothelial cells, ECM molecules, and inflammatory mediators . Tumor gene expression profile could quantify the immune activity in TME, such as CD4 + T recognizing cancer antigens, and then activated M1 macrophages may inhibit tumor growth . In addition, fibroblasts were associated with exocrine phenotype of T cells in bladder cancer, and the secreting transforming growth factor-β (TGF-β) can lead to the efflux of immunocyte or the resistance of chemotherapy drugs . Therefore, it is necessary to screen biomarkers for hub genes in the immune responses.
Although the effects of CD4 + T cells on the clinical outcome were controversial, high infiltration of CD8 + cytotoxic T cells and memory T cells were clearly shown with a longer survival. There was increase in expression of T-helper 1 and cytotoxicity-related genes in UC patients . Mariathasan et al. also shown that invasive UC patient with a high number of CD8 + T cells within the tumor showed a longer survival than those with fewer CD8 + T cells . Immunocyte has complex biological relationships with tumor cells, which then triggers the progression or repression . Therefore, we speculated that the diversity of immunocyte in TME might be one of the causes leading to different immunotherapeutic responses. In this study, we found that hub genes expressions were negatively correlation with B cell infiltration (P < 0.05), and was positive correlation wit the infiltration of CD4 + T cells, CD8 + T cell, macrophages, neutrophils and dendritic cells. In addition, our research results also showed that B cells, T cells, macrophages, neutrophils, and dendritic cells were factors related to the cumulative survival rate of UC over time and the immunocyte infiltration levels seemed to associate with hub genes CNV in UC. These findings together suggest that these hub genes were prognostic indicators and may involve in activation and recruitment of immunocytes in UC.
Our study focused on the exploration of therapeutic targets and prognostic biomarkers in hub genes of UC microenvironment by the screening of multiple databases. There are some limitations in our study. First, multiple different databases are utilized, and it is difficult to guarantee the unity among different databases. Second, we did not verify the data from these databases. In dependent cohort and in vitro or in vivo studies are required to validate our results. Meanwhile, functional and molecular mechanisms investigations on the hub genes in UC should be further performed.
In conclusion, 63 differentially expressed genes were identified in the GEO datasets of UC. Nine of them (i.e. COL1A1, COL1A2, COL3A1, COL5A2, MMP9, POSTN, SPP1, THBS2 and VCAN) were identified to be important for the pathogenesis and progression of UC. In the database, the expression levels of hub genes in invasive UC tissues were significantly higher than in superficial UC tissues. The expression of hub genes cannot only be used as a prognostic indicator of UC, but it is also related to the immunocyte infiltration. The present study provided novel insights into the occurrence and progression of UC. However, the diagnostic and prognostic value of these genes required further validation. Therefore, we hope our results can provide novel insights for the design of new immunotherapeutic drugs, help clinicians choose appropriate drugs for their UC patients and prognostic biomarkers, and to more accurately predict the survival of patients with UC.