Bladder Cancer Tissue Samples Research Articles

Revealing the potential of solute carrier family 31 (copper transporters), member 1: Insights into its role in bladder cancer progression and therapeutic implications.

Introduction: Bladder cancer represents a significant public health concern with diverse genetic alterations influencing disease onset, progression, and therapy response. In this study, we explore the multifaceted role of Solute Carrier Family 31 Member 1 (SLC31A1) in bladder cancer, a pivotal gene involved in copper homeostasis. Methods: Our research involved analyzing the SLC31A1 gene expression via RT-qPCR, promoter methylation via targeted bisulfite sequencing, and mutational status via Next Generation Sequencing (NGS) using the clinical samples sourced by the local bladder cancer patients. Later on, The Cancer Genome Atlas (TCGA) datasets were utilized for validation purposes. Moreover, prognostic significance, gene enrichment terms, and therapeutic drugs of SLC31A1 were also explored using KM Plotter, DAVID, and DrugBank databases. Results: We observed that SLC31A1 was significantly up-regulated at both the mRNA and protein levels in bladder cancer tissue samples, suggesting its potential involvement in bladder cancer development and progression. Furthermore, our investigation into the methylation status revealed that SLC31A1 was significantly hypomethylated in bladder cancer tissues, which may contribute to its overexpression. The ROC analysis of the SLC31A1 gene indicated promising diagnostic potential, emphasizing its relevance in distinguishing bladder cancer patients from normal individuals. However, it is crucial to consider other factors such as cancer stage, metastasis, and recurrence for a more accurate evaluation in the clinical context. Interestingly, mutational analysis of SLC31A1 demonstrated only benign mutations, indicating their unknown role in the SLC31A1 disruption. In addition to its diagnostic value, high SLC31A1 expression was associated with poorer overall survival (OS) in bladder cancer patients, shedding light on its prognostic relevance. Gene enrichment analysis indicated that SLC31A1 could influence metabolic and copper-related processes, further underscoring its role in bladder cancer. Lastly, we explored the DrugBank database to identify potential therapeutic agents capable of reducing SLC31A1 expression. Our findings unveiled six important drugs with the potential to target SLC31A1 as a treatment strategy. Conclusion: Our comprehensive investigation highlights SLC31A1 as a promising biomarker for bladder cancer development, progression, and therapy.

CSF2 Impairs Nrf2 Signaling through the Akt/Mtor Pathway in the Development of Bladder Cancer.

Bladder Cancer (BCa) is one of the most common cancers of the urinary system. Colony-stimulating factor 2 (CSF2) is involved in many cancers, but not BCa. We investigated the effect of CSF2 on BCa in this study and the underlying molecular mechanisms. CSF2 mRNA levels in BCa were analyzed using the Cancer Genome Atlas (TCGA) database. Western blot was conducted to verify CSF2 expression in BCa tissue samples and cell lines. The effect of CSF2 on the growth of BCa cells was assessed by CCK8 and colony formation. To determine the migration and invasion capabilities of BCa cells, transwell analysis and wound healing assays were conducted. Next, western blot was used to explore the underlying mechanism. In the end, a xenografted BCa mouse model was established to examine the effects of CSF2 on tumorigenesis in vivo. Results showed that CSF2 mRNA was upregulated in BCa samples. Knocking down CSF2 significantly inhibited the proliferation and tumorigenesis of BCa cells in vitro and in vivo. Mechanism analysis revealed that CSF2 knockdown inhibited the proliferation and invasion of BCa cells via AKT/mTOR signaling. Based on these results, CSF2 promotes the proliferation and tumorigenesis of BCa.

Uncovering Microbial Composition of the Tissue Microenvironment in Bladder Cancer using RNA Sequencing Data.

Purpose: Bladder cancer (BC) is one of the top 10 common tumors in the world. It has been reported that microbiota can colonize tissues and play important roles in tumorigenesis and progression. However, the current understanding of microorganisms in the BC tissue microenvironment remains unclear. Methods: In this study, we integrated the RNA-seq data of 479 BC tissue samples from seven datasets combined with a range of bioinformatics tools to explore the landscape of microbiome in the BC tissue microenvironment. Results: The pan-microbiome was estimated to surpass 1,400 genera. A total of seven core microbiota (Bacillus, Corynebacterium, Cutibacterium, Escherichia, Halomonas, Pasteurella, and Streptomyces) were identified. Among them, Bacillus was widely distributed in all datasets with a high relative abundance (10.11% of all samples on average). Moreover, some biological factors, including tissue source and tumor grade, were found significant effects on the microbial composition of the bladder tissue. Pseudomonas, Porphyrobacter, and Acinetobacter were enriched in tumor tissues, while Mycolicibacterium and Streptomyces were enriched in patients who showed durable response to BCG therapy. In addition, we established microbial co-occurrence networks and found that the BCG therapy may attenuate the microbiological interactions. Conclusions: This study clearly provided a microbial landscape of the BC tissue microenvironment, which was important for exploring the interactions between microorganisms and BC tissues. The identified specific taxa might be potential biomarkers for BC.

Promoting effect and immunologic role of secretogranin II on bladder cancer progression via regulating MAPK and NF-κB pathways.

Bladder cancer (BLCA) is ranked among the top ten most prevalent cancers worldwide and is the second most common malignant tumor within the field of urology. The limited effectiveness of immune targeted therapy in treating BLCA, due to its high metastasis and recurrence rates, necessitates the identification of new therapeutic targets. Secretogranin II (SCG2), a member of the chromaffin granin/secreted granin family, plays a crucial role in the regulated release of peptides and hormones. The role of SCG2 in the tumor microenvironment (TME) of lung adenocarcinoma and colon cancer has been established, but its functional significance in BLCA remains uncertain. This study aimed to investigate SCG2 expression in 15 bladder cancer tissue samples and their corresponding adjacent control tissues. The potential involvement of SCG2 in BLCA progression was assessed using various techniques, including analysis of public databases, immunohistochemistry, Western Blotting, immunofluorescence, wound-healing assay, Transwell assay, and xenograft tumor formation experiments in nude mice. This study provided novel evidence indicating that SCG2 plays a pivotal role in facilitating the proliferation, migration, and invasion of BLCA by activating the MEK/Erk and MEK/IKK/NF-κB signaling pathways, as well as by promoting M2 macrophage polarization. These findings propose the potential of SCG2 as a molecular target for immunotherapy in human BLCA.

TGF-β1 dominates stromal fibroblast-mediated EMT via the FAP/VCAN axis in bladder cancer cells

BackgroundBladder cancer is one of the most common malignant tumors of the urinary system and is associated with a poor prognosis once invasion and distant metastases occur. Epithelial-mesenchymal transition (EMT) drives metastasis and invasion in bladder cancer. Transforming growth factor β1 (TGF-β1) and stromal fibroblasts, especially cancer-associated fibroblasts (CAFs), are positive regulators of EMT in bladder cancer. However, it remains unclear how TGF-β1 mediates crosstalk between bladder cancer cells and CAFs and how it induces stromal fibroblast-mediated EMT in bladder cancer. We aimed to investigate the mechanism of TGF-β1 regulation of stromal fibroblast-mediated EMT in bladder cancer cells.MethodsPrimary CAFs with high expression of fibroblast activation protein (FAP) were isolated from bladder cancer tissue samples. Subsequently, different conditioned media were used to stimulate the bladder cancer cell line T24 in a co-culture system. Gene set enrichment analysis, a human cytokine antibody array, and cytological assays were performed to investigate the mechanism of TGF-β1 regulation of stromal fibroblast-mediated EMT in bladder cancer cells.ResultsAmong the TGF-β family, TGF-β1 was the most highly expressed factor in bladder cancer tissue and primary stromal fibroblast supernatant. In the tumor microenvironment, TGF-β1 was mainly derived from stromal fibroblasts, especially CAFs. In stimulated bladder cells, stromal fibroblast-derived TGF-β1 promoted bladder cancer cell migration, invasion, and EMT. Furthermore, TGF-β1 promoted the activation of stromal fibroblasts, inducing CAF-like features, by upregulating FAP in primary normal fibroblasts and a normal fibroblast cell line. Stromal fibroblast-mediated EMT was induced in bladder cancer cells by TGF-β1/FAP. Versican (VCAN), a downstream molecule of FAP, plays an essential role in TGF-β1/FAP axis-induced EMT in bladder cancer cells. VCAN may also function through the PI3K/AKT1 signaling pathway.ConclusionsTGF-β1 is a critical mediator of crosstalk between stromal fibroblasts and bladder cancer cells. We revealed a new mechanism whereby TGF-β1 dominated stromal fibroblast-mediated EMT of bladder cancer cells via the FAP/VCAN axis and identified potential biomarkers (FAP, VCAN, N-cadherin, and Vimentin) of bladder cancer. These results enhance our understanding of bladder cancer invasion and metastasis and provide potential strategies for diagnosis, treatment, and prognosis.

Flavokawain A is a natural inhibitor of PRMT5 in bladder cancer

BackgroundProtein arginine methyltransferases (PRMTs) regulate protein biological activity by modulating arginine methylation in cancer and are increasingly recognized as potential drug targets. Inhibitors targeting PRMTs are currently in the early phases of clinical trials and more candidate drugs are needed. Flavokawain A (FKA), extracted from kava plant, has been recognized as a potential chemotherapy drug in bladder cancer (BC), but its action mechanism remains unclear.MethodsWe first determined the role of a type II PRMT, PRMT5, in BC tissue samples and performed cytological experiments. We then utilized bioinformatics tools, including computational simulation, virtual screening, molecular docking, and energy analysis, to identify the potential use of PRMT5 inhibitors for BC treatment. In vitro and in vivo co-IP and mutation assays were performed to elucidate the molecular mechanism of PRMT5 inhibitor. Pharmacology experiments like bio-layer interferometry, CETSA, and pull-down assays were further used to provide direct evidence of the complex binding process.ResultsAmong PRMTs, PRMT5 was identified as a therapeutic target for BC. PRMT5 expression in BC was correlated with poor prognosis and manipulating its expression could affect cancer cell growth. Through screening and extensive experimental validation, we recognized that a natural product, FKA, was a small new inhibitor molecule for PRMT5. We noticed that the product could inhibit the action of BC, in vitro and in vivo, by inhibiting PRMT5. We further demonstrated that FKA blocks the symmetric arginine dimethylation of histone H2A and H4 by binding to Y304 and F580 of PRMT5.ConclusionsIn summary, our research strongly suggests that PRMT5 is a potential epigenetic therapeutic target in bladder cancer, and that FKA can be used as a targeted inhibitor of PRMT5 for the treatment of bladder cancer.

Identification of ENO1 as a prognostic biomarker and molecular target among ENOs in bladder cancer

BackgroundEnolase is an essential enzyme in the process of glycolysis and has been implicated in cancer progression. Though dysregulation of ENOs has been reported in multiple cancers, their prognostic value and specific role in bladder cancer (BLCA) remain unclear.MethodsMultiple databases were employed to examine the expression of ENOs in BLCA. The expression of ENO1 was also validated in BLCA cell lines and tissue samples by western blotting and immunohistochemistry. Kaplan–Meier analysis, ROC curve, univariate and multivariate Cox regression were performed to evaluate the predictive capability of the ENO1. Gene ontology (GO) and Gene Set Enrichment Analyses (GSEA) analysis were employed to perform the biological processes enrichment. Function experiments were performed to explore the biological role of ENO1 in BLCA. The correlation of ENO1 with immune cell infiltration was explored by CIBERSORT.ResultsBy analyzing three ENO isoforms in multiple databases, we identified that ENO1 was the only significantly upregulated gene in BLCA. High expression level of ENO1 was further confirmed in BLCA tissue samples. Aberrant ENO1 overexpression was associated with clinicopathological characteristics and unfavorable prognosis. Functional studies demonstrated that ENO1 depletion inhibited cancer cell aggressiveness. Furthermore, the expression level of ENO1 was correlated with the infiltration levels of immune cells and immune-related functions.ConclusionsTaken together, our results indicated that ENO1 might serve as a promising prognostic biomarker for prognosticating prognosis associated with the tumor immune microenvironment, suggesting that ENO1 could be a potential immune-related target against BLCA.

MiR-133b regulates the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4.

Bladder cancer is one of the most common urothelial tumors with high incidence and mortality rates. Although it has been reported that microRNA (miR)-133b can regulate tumorigenesis of bladder cancer, the mechanism remains unclear. Sex-determining region Y-box transcription factor 4 (SOX4) exhibits an important role in tumorigenesis, but it is unclear whether SOX4 and miR-133b are associated with regulation of pathogenesis of bladder cancer. This study aims to determine the expressions of SOX4 and miR-133b in bladder cancer tissues and cells, investigate their effects on the proliferation, colony formation, and invasion of bladder cancer cells, and to explore the association between miR-133b and SOX4 in regulating biological featurss of bladder cancer cells. The bladder cancer and adjacent tissue samples of 10 patients who underwent surgical resection in the Second Xiangya Hospital of Central South Universty from Januray to June 2015 were obtained. The levels of miR-133b were tested by real-time PCR, and the protein levels of SOX4 were evaluated using Western blotting in bladder cancer tissues, matched adjacent tissues, and cell lines. The correlation between miR-133b expression and SOX4 expression in bladder cancer tissues was analyzed. Using the online database TargetScan, the relationship between SOX4 and miR-133b was predicted. MiR-133b mimics, miR-133b inhibitor, and short hairpin RNA (shRNA)-SOX4 were transfected into T24 cells by Lipofectamine 2000. The relationship between miR-133b and SOX4 was also verified by a dual-luciferase reporter assay. The proliferation of T24 cells cultured for 0, 12, 48, 72, and 96 h was evaluated by cell counting kit-8 (CCK-8) assay. The colony formation capacity of bladder cancer cells was tested after 14-day culture, and cell invasion capacity was evaluated with Transwell invasion assay. Bladder cancer tissue and bladder cancer cells had low level of miR-133b but high level of SOX4, compared with matched adjacent tissues and normal bladder epithelial cells. A negative correlation between miR-133b mRNA and SOX4 protein levels in bladder cancer tissues was also found (r=-0.84). The results of online database TargetScan showed that miR-133b targets at SOX4, and overexpression of miR-133b significantly attenuated the expression of SOX4 in T24 cells. Both overexpression of miR-133b and knockdown of SOX4 significantly inhibited the proliferation, colony formation, and invasion capacity of bladder cancer cells in vitro. SOX4 down-regulation restored the effects of miR-133b inhibitor on the proliferation, colony formation, and invasion capacity of T24 cells. The up-regulation of SOX4 contributes to the progression of bladder cancer, and miR-133b can regulate the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4.

MiR-148a-3p inhibits the proliferation and migration of bladder cancer via regulating the expression of ROCK-1.

PurposeTo investigate the mechanism of miR-148a-3p regulating the proliferation and migration of bladder tumor cells.Materials and MethodsWe conducted a preliminary study to detect the relative expression of miR-148a-3p in bladder cancer and para-cancerous tissue samples. Three bladder tumor cell lines, T24, 5,637 and UM-UC-3, were selected. The expression levels of miR-148a-3p were artificially regulated with miR-148a-3p mimics and the miR-148a-3p inhibitor. The relative expression levels of miR-148a-3p in the samples of each cell line were determined. Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation, while the effect of the miR-148a-3p mimics and inhibitor on tumor cell migration was detected by wound healing assay. Flow cytometry assay was carried out to explore the effect of miR-148a-3p on cell apoptosis. Dual-luciferase reporter assay was performed in order to verify miR-148a-3p’s target gene. The expressions of ROCK-1 and Bcl-2 were analyzed by western blot.ResultsThe relative expression of miR-148a-3p in tumor and adjacent tissues was assessed with qRT-PCR (P < 0.05) and found to be significantly lower in the tumor tissues than the adjacent tissues. The data obtained from the CCK-8 and wound healing assay showed that intracellular transfection of miR-148a-3p mimics could inhibit cell proliferation and migration, while the miR-148a-3p inhibitor promoted them. Overexpression of miR-148a-3p promoted cell apoptosis in the T24 and 5,637 cell lines. The dual-luciferase reporter assay verified that ROCK-1 is a direct target of miR-148a-3p. Western blot showed that miR-148a-3p overexpression downregulated the expression of ROCK-1 and Bcl-2, while miR-148a-3p knockdown upregulated the expression of ROCK-1 and Bcl-2.ConclusionsWe confirmed that miR-148a-3p was significantly decreased in bladder cancer cells. miR-148a-3p overexpression inhibited bladder cancer cell proliferation and migration, whereas miR-148a-3p knockdown promoted bladder cancer cell proliferation and migration. Moreover, we found that ROCK-1 was a downstream target of miR-148a-3p. We also found that miR-148a-3p induced cell apoptosis by regulating the expression of Bcl-2. However, the deeper mechanism of this regulatory relationship needs further study.

Circ_0000658 knockdown inhibits epithelial-mesenchymal transition in bladder cancer via miR-498-induced HMGA2 downregulation

BackgroundEpithelial-mesenchymal transition (EMT) has been associated with the angiogenesis and oncogenic phenotypes of multiple malignant tumors including bladder cancer (BCa). Circular RNAs (circRNAs) are recognized as crucial regulators in the EMT. This study aims to illustrate the possible role of circular RNA_0000658 (circ_0000658) in BCa and the underlying molecular mechanism.MethodsThe expression of circ_0000658, microRNA (miR)-498, and high mobility group AT-hook 2 (HMGA2) was assessed in cancer and adjacent normal tissue collected from BCa patients and human BCa cell lines (MGH-U3, T24, 5637 and SW780). BCa cells were transduced with a series of overexpression or shRNA plasmids to clarify the function of circ_0000658 and miR-498 on the oncogenic phenotypes and EMT of BCa cells. Further, we established nude mice xenografted with BCa cells to validate the roles of circ_0000658 on tumor growth in vivo.ResultsCirc_0000658 was highly expressed in BCa tissue samples and cell lines, which indicated a poor prognosis of BCa patients. Circ_0000658 competitively bound to miR-498 and thus restricted miR-498 expression. Meanwhile, circ_0000658 weakened the binding of miR-498 to the target gene HMGA2 and upregulated the HMGA2 expression. Circ_0000658 elevation or miR-498 knockdown augmented oncogenic phenotypes and EMT of BCa cells, corresponding to a reduction in the expression of β-catenin and E-cadherin as well as an increase in the expression of N-cadherin, Slug, Snail, ZEB1 and Twist. Inhibition of HMGA2 reversed the effects of circ_0000658 overexpression on tumor growth in vivo.ConclusionAltogether, our study uncovered the tumor-promoting role of circ_0000658 in BCa via the miR-498/HMGA2 axis.

Implications of targeted next-generation sequencing for bladder cancer: report of four cases

BackgroundBladder cancer is considered heterogeneous diseases with two major subgroups: non-muscle- invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). It is a major healthcare problem, and it is one of the leading causes of mortality worldwide. Genetic mutations are not only a cause for carcinogenesis but are also a way for treatment strategy. The present study aimed to investigate breast cancer (BRCA genes) tumor suppressor gene mutations in bladder cancer tissue and combined blood samples for patients who developed secondary tumor after or during trimodal therapy. Fresh tissue samples and their matched blood samples were collected from four patients with bladder cancer. The objective regions for the examined genes (BRCA1 and BRCA2) were sequenced using next-generation sequencing (NGS); generated BAM files were uploaded to the cloud-based Ionreporter server, and the Oncomine BRCA-specific plugin was used to analyze the paired normal and tumor sample for each patient using the default plugin parameters. ResultsIntronic BRCA1 mutation c.5050-104 C >T was reported among the four investigated bladder cancer patients, and three somatic mutations were reported as follows: two of them were found to be benign rs1064793056 and rs28897679 on the Clinivar database and one nonsense pathogenic variant rs80357006. BRCA 2 gene mutation reported an exonic synonymous mutation rs397507876 in the tissue and germline DNA. Patients were treated with trimodal; however, three bladder cancer patients who reported BRCA mutations developed secondary tumors. ConclusionIdentification of mutational BRCA changes in bladder cancer is a promising marker for better treatment strategy. Further studies are encouraged on a large cohort of bladder cancer patients to confirm our findings.

Identification and validation of a hypoxia-related prognostic and immune microenvironment signature in bladder cancer

BackgroundBladder cancer is the leading causes of cancer-associated mortality and seriously affects population health. Hypoxia plays a key role in tumor development and immune escape, which contributes to malignant behaviors.MethodsIn this study, we analyzed the RNA-seq and clinical information of bladder cancer patients from The Cancer Genome Atlas (TCGA) database. To investigate the hypoxia-related prognostic and immune microenvironment in bladder cancer, we constructed a hypoxia-related risk model for overall survival (OS). The RNA-seq and clinical data of bladder cancer patients from the Gene Expression Omnibus (GEO) database were used as validation sets.ResultsThe hypoxia-related risk signature was significantly correlated with clinical outcomes and could independently predict OS outcomes. Furthermore, the hypoxia-related risk signature could effectively reflected the levels of immune cell type fractions and the expression of critical immune checkpoint genes were higher in the high-risk group compared to the low-risk group. We also validated the expression levels of the prognostic genes in bladder cancer and paracancerous tissue samples through qRT-PCR analysis.ConclusionWe established a 7 hypoxia-related gene (HRG) signature that can be used as an independent clinical predictor and provided a potential mechanism in bladder cancer immunotherapy.

Upregulation of NFKBIZ affects bladder cancer progression via the PTEN/PI3K/Akt signaling pathway.

NF-κB inhibitor ζ (NFKBIZ), a member of the IκB family that interacts with NF-κB, has been reported to be an important regulator of inflammation, cell proliferation and survival. However, the role of NFKBIZ in bladder cancer (BC) remains unknown. The present study aimed to investigate the functions of NFKBIZ in BC. First, the expression levels of NFKBIZ and the associations between NFKBIZ expression and the clinical survival of patients were determined using BC tissue samples, BC cell lines and datasets from different databases. Two BC cell lines (T24 and 5637) were selected to overexpress NFKBIZ, and the proliferative, migratory and invasive abilities of cells were determined; additionally, tumor growth following transplantation in in vivo mouse models was analyzed using T24 cells overexpressing NFKBIZ. Subsequently, the association between NFKBIZ and PTEN was determined using data from databases and immunohistochemistry analysis of clinical and nude mice tumor tissues. Finally, the interactions between NFKBIZ, PTEN and the downstream PI3K/AKT/mTOR signaling pathway were evaluated using western blotting. In conclusion, the present results indicated that NFKBIZ expression was low in BC, and NFKBIZ inhibited the proliferation of BC cells through the PTEN/PI3K/Akt signaling pathway, suggesting that NFKBIZ may represent a novel prognostic biomarker in BC and may provide a potential therapeutic tumor-associated antigen for BC.

Characteristics and Clinical Significance of CD163+/CD206+M2 Mono-macrophage in the Bladder Cancer Microenvironment

The tumor microenvironment may recruit monocytes, with a protumoral macrophage phenotype (M2) that plays an important role in solid tumor progression and metastasis. Therefore, it is necessary to understand the characteristics of these cells for cancer prevention and treatment. Bladder cancer tissue samples and paracarcinoma tissues samples were collected, and the expression of CD163+ cells in tumor tissues was observed. Then, we observed the expression of infiltrating CD45+CD14+CD163+ cell subset and analyzed the molecular expressions related to immunity and angiogenesis. C57/BL6 mice were inoculated subcutaneously, and dynamic changes of CD11b+F4/80+CD206+ mononuclear macrophages expression for tumor-bearing mice were detected. The results showed that the proportion of CD45+CD14+CD163+ mono-macrophage subset infiltrated by tumor tissue was significantly higher than that in paracarcinoma tissues. In bladder cancer tissue, the expression rate of CD40 in CD45+CD14+CD163- mono-macrophage subset was significantly lower than that in CD45+CD14+CD163+ mono-macrophage subset. Similar results were found in the paracarcinoma tissues. We found that, as the proportion of CD11b+F4/80+CD206+ mono-macrophages increased gradually, the difference was statistically significant. CD163+/CD206+ mono-macrophages in bladder cancer microenvironment are abnormally elevated, and these cells are closely related to tumor progression. CD40 may be an important molecule that exerts biological function in this subset.

O-GlcNAcylation Enhances NUSAP1 Stability and Promotes Bladder Cancer Aggressiveness.

ObjectiveNUSAPl and O-GlcNAcylation were reported to be hyper-activated in many kinds of cancers and involved in the advanced progression of cancers. In bladder cancer, O-GlcNAc transferase (OGT) expresses in patients’ urine samples, with no expression in healthy individuals, indicating O-GlcNAcylation might involve in the occurrence and development of bladder cancer. Therefore, the present study aims to investigate the effects of O-GlcNAcylation in bladder cancer and if it can regulate NUSAP1 protein.Materials and MethodsWestern blot, immunohistochemistry, and PCR were used to evaluate the protein expression and mRNA level of NUSAP1; CCK-8 and flow cytometry used to evaluate the proliferation and inhibited the apoptosis of bladder cancer.ResultsThe results showed that NUSAP1 was highly expressed in bladder cancer cells and tissue samples. NUSAP1 up-regulation significantly promoted the proliferation and inhibited the apoptosis of bladder cancer HT-1376 and T24 cells. Besides, the expression of O-GlcNAc was elevated in bladder cancer tissues and cells, and up-regulation of O-GlcNAc with GlcNAc and PuGNAc obviously increased NUSAP1 protein expression and stability. Moreover, knockdown OGT significantly inhibited the proliferation and tumorigenesis and promoted the apoptosis of bladder cancer cells, confirmed by CCK-8, in vivo xenotransplantation, and flow cytometry, whereas these roles were impaired when NUSAP1 was up-regulated.ConclusionOverall, our study makes clear that hyper-O-GlcNAcylation accelerates bladder cancer progression through promotion of NUSAP1 expression and its stability.

A prognostic immune predictor, HLA-DRA, plays diverse roles in non-muscle invasive and muscle invasive bladder cancer

BackgroundThere is an increasing demand for prognostic immune biomarkers of cancer. The prognostic significance of immune markers has been shown for various cancers, but biomarkers of bladder cancer (BCa) have not been fully evaluated. To clarify the role of human leukocyte antigen DR alpha chain (HLA-DRA) in BCa development, we examined expression of HLA-DRA mRNA in tissue samples of non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Materials and MethodsTissues of 96 NMIBC, 43 MIBC and 59 controls comprising noncancerous BCa surrounding tissues were used to examine the expression of HLA-DRA gene by real-time polymerase chain reaction. The expression of up-stream genes regulating HLA-DRA were also measured to explain the role of HLA-DRA in BCa. ResultsPatients with high grade NMIBC showed higher expression of HLA-DRA than those with low grade NMIBC (P < 0.05). In addition, NMIBC patients who progressed to MIBC showed high expression of HLA-DRA mRNA. Kaplan-Meier analysis showed that NMIBC patients with low expression of HLA-DRA had better progression-free survival than those with high expression (P = 0.004). Moreover, the expression of genes regulating HLA-DRA varied in NMIBC and MIBC, indicating a different immunoregulation effect of HLA-DRA in both cancers. ConclusionsHigh expression of HLA-DRA in NMIBC patients has implications for patient stratification strategies, as well as for BCa tumor immunology.

Human Papillomavirus Prevalence and Integration Status in Tissue Samples of Bladder Cancer in the Chinese Population.

Human papillomavirus (HPV) infection is associated with multiple types of cancer, but the evidence has not yet been fully elucidated in bladder cancer. Frozen tissue samples collected from 146 patients aged 32 to 89 years with bladder cancer pathological diagnosis between 2015 and 2019 were analyzed. HPV genotyping and integration status determination were performed by capture-based next generation sequencing. Statistical analysis of HPV type distributions was performed according to stage, grade, sex, and age group of patients. Mean (SD) age of the 146 patients was 66.64 ± 10.06 years and 83.56% were men. Overall HPV infection rate was 28.77% (37.50% in women and 27.05% in men), with 11.90% HPV integration events. Among them, 17.12% single and 11.65% coinfections were observed. HPV18 (24.66%) was the most prevalent genotype, followed by HPV33, 16, and 39. All HPV were European lineage (A). HPV16 was more prevalent in women (P = .04). HPV infection may contribute to the etiology both in men and women with bladder cancer. HPV18, followed by HPV33, 16, and 39 genotypes, potentially represent the predominant oncogenic risk types for bladder carcinogenesis.

Prognostic model of 10 immune-related genes and identification of small molecule drugs in bladder urothelial carcinoma (BLCA).

BackgroundWe aimed to establish an immune-related gene (IRG) based signature that could provide guidance for clinical bladder cancer (BC) prognostic surveillance.MethodsDifferentially expressed IRGs and transcription factors (TFs) between BCs and normal tissues were extracted from transcriptome data downloaded from the TCGA database. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to identify related pathways based on differently expressed IRGs. Then, univariate Cox regression analysis was performed to investigate IRGs with prognostic values and LASSO penalized Cox regression analysis was utilized to develop the prognostic index (PI) model.ResultsA total of 411 BC tissue samples and 19 normal bladder tissues in the TCGA database were enrolled in this study and 259 differentially expressed IRGs were identified. Networks between TFs and IRGs were also provided to seek the upstream regulators of differentially expressed IRGs. By means of univariate Cox regression analysis, 57 IRGs were analyzed with prognostic values and 10 IRGs were finally identified by LASSO penalized Cox regression analysis to construct the PI model. This model could significantly classified BC patients into high-risk group and low-risk group in terms of OS (P=9.923e-07) and its AUC reached 0.711. By means of univariate and multivariate COX regression analysis, this PI was proven to be a valuable independent prognostic factor (HR =1.119, 95% CI =1.066–1.175, P<0.001). CMap database analysis was also utilized to screen out 10 small molecules drugs with the potential for the treatment of BC.ConclusionsOur study successfully provided a novel PI based on IRGs with the potential to predict the prognosis of BC and screened out 10 small molecules drugs with the potential to treat BC. Besides, networks between TFs and IRGs were also displayed to seek its upstream regulators for future researches.

LncRNA AFAP1-AS1 promotes proliferation ability and invasiveness of bladder cancer cells.

It was the aim of this study to explore the role and mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 in the progression of bladder cancer (BCa) by in vitro experiments. AFAP1-AS1 levels in 40 pairs of clinical BCa tissue samples and normal ones collected from BCa patients were determined, and paired sample t-test was applied to compare the differences between groups. The prognosis data of patients with BCa were collected, and survival analysis and t-test were performed to specify the interplay between AFAP1-AS1 and the prognosis of BCa patients. Subsequently, AFAP1-AS1 expression level in BCa and normal cells were further confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), and transwell assays were performed to figure out the influence of this lncRNA on the proliferation ability and invasiveness of BCa cells. Meanwhile, the interaction between AFAP1-AS1 and its sense mRNA was analyzed. We used co-transfection technology to simultaneously transfect si-AFAP1-AS1 and pcDNA3.1-AFAP1 or their corresponding negative controls into BCa cells, and cell proliferation and invasion ability in different subgroups were determined to explore the underlying mechanism through which AFAP1-AS1 plays a role in BCa progression. No matter in BCa tissues or in cell samples, compared to the corresponding normal controls, AFAP1-AS1 was found highly expressed; at the same time, in invasive bladder cancer tissues, the expression level of AFAP1-AS1 was also higher than that in non-invasive tissues. Meanwhile, survival analysis revealed that patients with BCa with high expression of AFAP1-AS1 owned a shorter overall survival rate than those with low expression, indicating a negative interplay between AFAP1-AS1 expression and patients' prognosis. In addition, in BCa cell lines, according to the results of CCK-8, EDU, and transwell assays, the proliferative capacity, as well as the invasive ability of BCa cells, were found weakened after downregulation of AFAP1-AS1. Meanwhile, a negative interplay was discovered between AFAP1-AS1 and its sense mRNA. Finally, the results of cell reversal experiment using co-transfection technique revealed that overexpression of AFAP1 can reverse the inhibitory impact of lncRNAAFAP1-AS1 on the malignant ability of BCa cells. AFAP1-AS1 may enhance the proliferation ability as well as the invasiveness of BCa cells so as to aggravate the degree of BCa malignancy.

A novel circular RNA (hsa_circRNA_102336), a plausible biomarker, promotes the tumorigenesis by sponging miR-515-5p in human bladder cancer

Bladder cancer (BC) ranks as the ninth common human tumor in the world with an increasing incidence. Circular RNAs (circRNAs) have been revealed to exhibit promotive or suppressive impacts on tumor progression of various human cancers, including BC. However, due to the variety of circRNAs, the whole circRNAs network in BC remains unclear. In our study, differentially expressed circRNAs between BC and normal samples in GSE92675 dataset was analyzed by microarray. Circ_102336 was identified to be sharply increased in both BC tissue samples and cell lines, and increased circ_102336 is correlated with a worse overall survival rate of BC patients. Overexpression of circ_102336 dramatically increased the cell proliferation viability of T24 and 5637 cells, while knockdown of circ_102336 exhibited opposite effects. Moreover, circ_102336 knockdown could increase the sensitivity of T24 and 5637 cells to CDDP. In mechanism, circ_102336 was demonstrated to directly bind to and negatively regulate miR-515-5p. Inhibition of miR-515-5p reversed the repressive effects of si-circ_102336 on BC cell proliferation viability. KEGG analysis showed that ATP-binding cassette (ABC) transporters and apoptosis were the major two pathways associated with circ_102336/miR-515-5p axis. therefore, we suggested that circ_102336 indirectly regulate apoptosis and ABC transport pathways through miR-515-5p to finally modulate BC cell proliferation and chemo-resistance.