Bladder Urothelial Carcinoma Tissues Research Articles

The Oncogenic Potential of IQ Motif-Containing GTPase-Activating Protein3 in Human Bladder Cancer: An In-Silico Analysis

Introduction: IQ motif-containing GTPase-activating protein3 (IQGAP3) contributes to the progression of bladder urothelial carcinoma (BLCA), but its mechanisms are not systematically specified. Due to the oncogenic potential of IQGAP3, the current in-silico study intended to elucidate IQGAP3's role in BLCA progression. Materials and Methods: Many bioinformatics tools, including UALCAN, Kaplan–Meier plotter, TNMplot, cBioPortal, GeneMania, Enrichr, TIMER2, muTarget, and UCSC Xena, were applied in the current study. Results: The IQGAP3 level was more pronouncedly raised in BLCA tissues than in normal bladder tissues, and its increased expression was related to the advanced stage and higher grade. Enhanced IQGAP3 expression could result from its genetic alteration. Moreover, the mutation in P53 and RB1 genes was robustly associated with increased IQGAP3 expression. Besides, IQGAP3 correlative genes were dominantly involved in the cell cycle. On the other hand, IQGAP3 upregulation influenced immune checkpoint levels in the tumor microenvironment. Conclusion: The in-silico findings suggested that IQGAP3 overexpression could be a crucial biomarker in BLCA.

Investigating cellular similarities and differences between upper tract urothelial carcinoma and bladder urothelial carcinoma using single-cell sequencing.

Upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BLCA) both originate from uroepithelial tissue, sharing remarkably similar clinical manifestations and therapeutic modalities. However, emerging evidence suggests that identical treatment regimens may lead to less favorable outcomes in UTUC compared to BLCA. Therefore, it is imperative to explore molecular processes of UTUC and identify biological differences between UTUC and BLCA. In this study, we performed a comprehensive analysis using single-cell RNA sequencing (scRNA-seq) on three UTUC cases and four normal ureteral tissues. These data were combined with publicly available datasets from previous BLCA studies and RNA sequencing (RNA-seq) data for both cancer types. This pooled analysis allowed us to delineate the transcriptional differences among distinct cell subsets within the microenvironment, thus identifying critical factors contributing to UTUC progression and phenotypic differences between UTUC and BLCA. scRNA-seq analysis revealed seemingly similar but transcriptionally distinct cellular identities within the UTUC and BLCA ecosystems. Notably, we observed striking differences in acquired immunological landscapes and varied cellular functional phenotypes between these two cancers. In addition, we uncovered the immunomodulatory functions of vein endothelial cells (ECs) in UTUC, and intercellular network analysis demonstrated that fibroblasts play important roles in the microenvironment. Further intersection analysis showed that MARCKS promote UTUC progression, and immunohistochemistry (IHC) staining revealed that the diverse expression patterns of MARCKS in UTUC, BLCA and normal ureter tissues. This study expands our multidimensional understanding of the similarities and distinctions between UTUC and BLCA. Our findings lay the foundation for further investigations to develop diagnostic and therapeutic targets for UTUC.

SYNPO2 promotes the development of BLCA by upregulating the infiltration of resting mast cells and increasing the resistance to immunotherapy.

Synaptopodin 2 (SYNPO2) plays a pivotal role in regulating tumor growth, development and progression in bladder urothelial Carcinoma (BLCA). However, the precise biological functions and mechanisms of SYNPO2 in BLCA remain unclear. Based on TCGA database‑derived BLCA RNA sequencing data, survival analysis and prognosis analysis indicate that elevated SYNPO2 expression was associated with poor survival outcomes. Notably, exogenous SYNPO2 expression significantly promoted tumor invasion and migration by upregulating vimentin expression in BLCA cell lines. Enrichment analysis revealed the involvement of SYNPO2 in humoral immune responses and the PI3K/AKT signaling pathway. Moreover, increased SYNPO2 levels increased the sensitivity of BLCA to PI3K/AKT pathway‑targeted drugs while being resistant to conventional chemotherapy. In invivo BLCA mouse models, SYNPO2 overexpression increased pulmonary metastasis of 5637 cells. High SYNPO2 expression led to increased infiltration of innate immune cells, particularly mast cells, in both nude mouse model and clinical BLCA samples. Furthermore, tumor immune dysfunction and exclusion score showed that patients with BLCA patients and high SYNPO2 expression exhibited worse clinical outcomes when treated with immune checkpoint inhibitors. Notably, in the IMvigor 210 cohort, SYNPO2 expression was significantly associated with the population of resting mast cells in BLCA tissue following PD1/PDL1 targeted therapy. In conclusion, SYNPO2 may be a promising prognostic factor in BLCA by modulating mast cell infiltration and exacerbating resistance to immune therapy and conventional chemotherapy.

Pan-cancer analysis of PLAU indicates its potential prognostic value and correlation with neutrophil infiltration in BLCA

BackgroundPLAU is known as a selected serine protease converting plasminogen to plasmin. The role of PLAU in the development of pan-cancer, especially bladder urothelial carcinoma (BLCA) remains unclear. MethodA variety of online tools and cancer databases, including TCGA, GETx, HPA database, GSCALite, UALCAN, ESTIMATE, CIBERSORT, ssGSEA algorithms and SangerBox website, were applied to investigate the associations between PLAU expression and prognosis, genetic alterations, pathway activation, and tumor immunity in pan-cancer. Through cBioPortal and STITCH platforms, the oncogenic role of PLAU and related targeting medicines in BLCA were also explored. We verified the expression of PLAU in pan-cancer cells and its function in bladder cancer cell lines using wet-lab experiments. ResultsPLAU expression levels were significantly higher in most cancer tissues. PLAU had a certain accuracy in the diagnosis of various types of cancers (90 % AUC > 0.700). In BLCA, PLAU has abundant methylated sites and showed statistical differences in clinical features. PLAU was involved in tumor immune infiltration, and especially positively correlated with neutrophil infiltration. High-expressed PLAU indicated poorer prognosis in the BLCA patients receiving Atezolizumab. A high mRNA and protein expression levels of PLAU were observed in pan-cancer cell lines, especially BLCA cells. Knockdown of PLAU inhibited the invasive, proliferative, and aggressive phenotypes of bladder cancer cells. Immunohistochemical staining validated PLAU's higher expression in BLCA tissues than in adjacent non-cancerous tissues. And overexpression of PLAU was associated with more advanced TNM stage, and high infiltrating depth. ConclusionOur study revealed that PLAU can serve as a potential therapeutic target and prognostic marker for various malignancies, especially BLCA.

Identification of tryptophan metabolism- and immune-related genes signature and prediction of immune infiltration landscape in bladder urothelial carcinoma.

Tryptophan metabolism is indirectly involved in immune tolerance and promotes response to anticancer drugs. However, the mechanisms underlying tryptophan metabolism and immune landscape in bladder urothelial carcinoma (BLCA) are not fully understood. A BLCA dataset containing 406 tumor samples with clinical survival information and 19 normal samples were obtained from the Cancer Genome Atlas database. The validation set, GSE32894, contained 223 BLCA tumor samples with survival information, and the single-cell dataset, GSE135337, included seven BLCA tumor samples; both were obtained from the gene expression omnibus database. Univariate and multivariate Cox regression analyses were conducted to evaluate clinical parameters and risk scores. Immune infiltration and checkpoint analyses were performed to explore the immune landscape of BLCA. Single-cell analysis was conducted to further identify the roles of model genes in BLCA. Finally, NAMPT expression in BLCA and adjacent tissues was detected using RT-qPCR, CCK-8 and Transwell assays were conducted to determine the role of NAMPT in BLCA cells. Six crossover genes (TDO2, ACAT1, IDO1, KMO, KYNU, and NAMPT) were identified by overlap analysis of tryptophan metabolism-related genes, immune-related genes, and differentially expressed genes (DEGs). Three biomarkers, NAMPT, IDO1, and ACAT1, were identified using Cox regression analysis. Accordingly, a tryptophan metabolism- and immune-related gene risk model was constructed, and the patients were divided into high- and low-risk groups. There were significant differences in the clinical parameters, prognosis, immune infiltration, and immunotherapy response between the risk groups. RT-qPCR revealed that NAMPT was upregulated in BLCA samples. Knocking down NAMPT significantly inhibited BLCA cell proliferation, migration, and invasion. In our study, we constructed a tryptophan metabolism- and immune-related gene risk model based on three biomarkers, namely NAMPT, IDO1, and ACAT1, that were significantly associated with the progression and immune landscape of BLCA. The risk model could effectively predict patient prognosis and immunotherapy response and can guide individualized immunotherapy.

Synthetic lethality in human bladder cancer cells by curcumin via concurrent Aurora A inhibition and autophagy induction

Combination therapies to induce mixed-type cell death and synthetic lethality have the potential to overcome drug resistance in cancer. In this study, we demonstrated that the curcumin-enhanced cytotoxicity of cisplatin/carboplatin in combination with gemcitabine was associated with Aurora A suppression-mediated G2/M arrest, and thus apoptosis, as well as MEK/ERK-mediated autophagy in human bladder cancer cells. Animal study data confirmed that curcumin combined with cisplatin/gemcitabine reduced tumorigenesis of xenograft in mice and this phenomenon was associated with elevated expressions of p-ERK and reduced p-Aurora A in tumors. Gene analyses using data repositories further revealed that reduced Aurora A expression alone did not significantly elevate the sensitivity of human bladder carcinoma cells to these anticancer drugs. Unlike other major cancer types, human bladder urothelial carcinoma tissue coexpressed higher AURKA and lower MAP1LC3B than normal tissue, and reduced Aurora A and induction of autophagy have been clinically associated with a better prognosis in patients with early but not advanced stage bladder cancer. Therefore, our results suggest that treatment strategies can utilize the synthetic lethal pair to concurrently suppress oncogenic Aurora A and induce autophagy by coadministrating curcumin with anticancer drugs for early-stage bladder cancer with high expression of Aurora A.

Prognostic and immunological role of Asporin across cancers and exploration in bladder cancer

BackgroundAsporin (ASPN) has been identified as a player in tumorigenesis, but its precise roles and modulatory function are largely unknown. Methods: In the present study, ASPN expression was first explored, followed by a prognostic evaluation of ASPN and a comprehensive investigation of the connections between ASPN and immunomodulation, immune cell infiltration and potential compounds on a pancancer level. Finally, ASPN expression was validated in bladder urothelial carcinoma (BLCA) tissues, and the potential function of ASPN, including its effects on migration and invasion capabilities, was investigated in tumor cells. Results: The expression of ASPN exhibited significant variation across cancers and was found to be associated with patient prognosis. In addition, the expression level of APSN was markedly correlated with the abundances of infiltrating immune cells and cancer-associated fibroblasts and the expression levels of immunomodulatory genes based on the results of pancancer analysis. Metastasis and immune-associated signaling pathways were identified in enrichment analysis based on ASPN expression. Finally, we confirmed that ASPN expression increased with the degree of malignancy in BLCA tissues and cell lines and that low expression of ASPN hindered the migration and invasion of cells. Conclusions: ASPN has the potential to be a biomarker of cancer prognosis and a therapeutic target, and it also has predictive capability for the progression of BLCA.

SIRT4 is an independent prognostic factor in bladder cancer and inhibits bladder cancer growth by suppressing autophagy

BackgroundNucleosome-localized sirtuin 4 (SIRT4) was found to function as an oncogene and tumor suppressor gene in different tumors. However, the clinical significance of SIRT4 in bladder urothelial carcinoma (BLCA) has not been assessed, nor has the function of SIRT4 in BLCA been analyzed.MethodsIn this study, we assessed the levels of SIRT4 protein in BLCA tissues and its association with clinicopathological parameters and overall survival time of BLCA patients by immunohistochemical staining of tissue microarrays containing 59 BLCA patients. Then, we constructed BLCA cell lines (T24) with overexpression or interference of SIRT4 by lentiviral infection. The effects of SIRT4 on the proliferation, migration and invasive ability of T24 cells were investigated using cell counting kit-8 (CCK-8) assays, wound healing assays, and migration and invasion assays. Moreover, we also investigated the effect of SIRT4 on the cell cycle and apoptosis of T24 cells. Mechanistically, we explored the relationship between SIRT4 and autophagy and its role in the inhibition of BLCA.ResultsWe found by immunohistochemistry that SIRT4 protein levels were reduced in BLCA and that lower SIRT4 levels were associated with larger tumor volumes, later T-staging and later AJCC staging in BLCA patients and were an independent prognostic factor in BLCA patients. Overexpression of SIRT4 significantly inhibited the proliferative viability, scratch healing capacity, migratory capacity, and invasive capacity of T24 cells, while interference with SIRT4 had the opposite effect. Moreover, overexpression of SIRT4 significantly inhibited the cell cycle and increased the apoptosis rate of T24 cells. Mechanistically, SIRT4 inhibits BLCA growth by suppressing autophagic flow.ConclusionsOur study suggests that SIRT4 is an independent prognostic factor for BLCA and that SIRT4 plays a tumor suppressor role in BLCA. This suggests a potential target for SIRT4 in the diagnosis and treatment of BLCA.

Comprehensive analysis reveals the value of the expression of chromobox family members for bladder urothelial carcinoma prognosis.

Bladder urothelial carcinoma (BLCA) accounts for 95% of all cases of bladder cancer worldwide, with a high incidence and poor prognosis. Chromobox (CBX) proteins play a key role in numerous malignant tumors; however, the role of CBX in BLCA remains unknown. Herein, the present study found that, compared with in normal bladder tissues, the expression levels of CBX1, CBX2, CBX3, CBX4 and CBX8 were markedly increased in BLCA tissues, as determined by Tumor Immune Estimation Resource, UALCAN and ONCOMINE analyses, whereas CBX6 and CBX7 were decreased in BLCA tissues. Furthermore, evident hypomethylation in the promoters of CBX1, and CBX2, as well as significant hypermethylation in the promoters of CBX5, CBX6 and CBX7, was detected in BLCA tissues compared with in normal bladder tissues. The expression of CBX1, CBX2 and CBX7 was involved in the prognosis of patients with BLCA. Low CBX7 expression was strongly associated with poorer overall survival in patients with BLCA, whereas high CBX1 and CBX2 expression was associated with poorer progression-free survival. Besides, significant associations were determined between the expression of CBXs and immune cell infiltration, including dendritic cells, neutrophils, macrophages, CD4+ T cells, CD8+ T cells and B cells. Overall, the current results may provide a rationale for developing new targets and prognostic markers for BLCA therapy.

KDELR2 as a diagnostic and prognostic biomarker of bladder urothelial carcinoma and its correlation with immune infiltration.

KDELR2 has been reported as a promotive factor for the genesis and progression of several malignancies. However, it is uncertain how it affects bladder urothelial carcinoma (BLCA). Using data extracted from online databases, an enhanced expression of KDELR2 in BLCA tissues was verified. Overexpression of KDELR2 was correlated with advanced clinicopathologic characteristics and unfavourable prognosis of BLCA. Receiver operating characteristic analysis highlighted the potential diagnostic value of KDELR2. Univariate and multivariate logistic regression analyses further revealed the predictive effect of KDELR2 for the prognosis of BLCA. KDELR2 was primarily enriched in biological functions related to organization of the extracellular matrix. TIMER, ssGSEA and GEPIA analyses suggested that KDELR2 expression is positively related to the infiltration of macrophages, Th2 cells and neutrophils. Finally, knocking-down of KDELR2 in T24 cells resulted in reduced proliferation, migration and macrophages recruitment. These results suggest that KDELR2 overexpression is an indicator for poor prognosis of BLCA and it has the potential to be employed as an immunotherapy target for BLCA.

Effects of Holliday Junction-Recognition Protein-Mediated C-Jun N-Terminal Kinase/ Signal Transducer and Activator of Transcription 3 Signaling Pathway on Cell Proliferation, Cell Cycle and Cell Apoptosis in Bladder Urothelial Carcinoma.

The Holliday Junction-Recognition Protein (HJURP) was upregulated in several tumors, which was associated with poor outcome. This study investigated the effects of the HJURP-mediated c-Jun N-terminal kinase (JNK)/ signal transducer and activator of transcription 3 (STAT3) pathway on bladder urothelial carcinoma (BLUC). Online databases were used to analyze HJURP expression in BLUC and the correlation of HJURP to JNK1 [mitogen-activated protein kinase 8 (MAPK8)], JNK2 (MAPK9), STAT3, marker of proliferation Ki-67 (MKI67), proliferating cell nuclear antigen (PCNA), cyclin dependent kinase 2 (CDK2), CDK4 and CDK6. HJURP expression was detected in BLUC cells and human normal primary bladder epithelial cells (BdECs). BLUC cells were treated with HJURP lentivirus activation /shRNA lentivirus particles or JNK inhibitor SP600125. HJURP was upregulated in BLUC tissues and correlated with poor prognosis of patients (all P < 0.05). HJURP in tumor positively correlated with MAPK8 (R = 0.30), MAPK9 (R = 0.30), STAT3 (R = 0.15), MKI67 (R = 0.60), PCNA (R = 0.46), CDK2 (R = 0.39), CDK4 (R = 0.24) and CDK6 (R = 0.21). The JNK inhibitor SP600125 decreased p-JNK/JNK and p-STAT3/STAT3 in BLUC cells, which was reversed by HJURP overexpression (P < 0.05). The HJURP-mediated JNK/STAT3 pathway promoted BLUC cell proliferation and inhibited cell apoptosis (P < 0.05). HJURP reversed the arrested G0/G1 phase of BLUC cells by SP600125. HJURP acted as an oncogene to regulate BLUC cell proliferation, apoptosis and the cell cycle by mediating the JNK/STAT3 pathway. Therefore, HJURP targeting might be an attractive novel therapeutic target for early diagnosis and treatment in BLUC.

ANLN Serves as an Oncogene in Bladder Urothelial Carcinoma via Activating JNK Signaling Pathway

Introduction: To understand the significance of ANLN (anillin, actin-binding protein)-mediated c-Jun N-terminal kinase (JNK) signal pathway on the progression of bladder urothelial carcinoma (BLCA). Methods: The Cancer Genome Atlas (TCGA) database was utilized to perform the clinical significance of ANLN in BLCA. Then, ANLN expression was determined in human normal primary bladder epithelial cells (BdEC) and BLCA cells. Later, ANLN knockdown was performed in BLCA cells, where the expression of MAPK8, MAPK9, and p-JNK/JNK was detected. BLCA cells were divided into the Mock, siNC, siANLN, SP600125 (a selective JNK inhibitor), and ANLN + SP600125 group, followed by measurements of real-time quantitative polymerase chain reaction, 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Annexin V-FITC/PI, Wound-healing, Transwell, and immunofluorescence assays. Results: ANLN was upregulated in the BLCA tissues, which showed a relation with the stage of patients. Besides, BLCA patients with high expression of ANLN had a worse prognosis than those with low expression of ANLN. Besides, the expression of ANLN in the BLCA tissues was positively correlated with MAPK8 and MAPK9. SP600125 suppressed the JNK signal pathway, reduced the proliferation, and increased BLCA cell apoptosis, with the reductions in the invasion and migration and the upregulation of phospho-histone H3 Ser-10 (pHH3), which was abolished by the overexpression of ANLN. Conclusion: ANLN, as an oncogene of BLCA, may associate with the activation of JNK signal pathway. Inhibiting ANLN could deactivate the JNK signal pathway, thereby suppressing the proliferation, invasion, and migration while promoting the apoptosis of BLCA cells.

FAM83H and Nectin1 expression are related with survival and relapse of bladder urothelial carcinoma patients

BackgroundFAM83H was originally reported to be essential for dental enamel formation. However, FAM83H has recently been implicated in tumorigenesis and tumor progression. Analysis of a publicly available gene expression database revealed a significant correlation between FAM83H and Nectin1 mRNA expression and bladder urothelial carcinoma (BUC). Therefore, we investigated the association between FAM83H and Nectin1 expression levels and the survival and recurrence of BUC in BUC patients using a tissue microarray.MethodsWe performed immunohistochemical staining of FAM83H and Nectin1 in 165 human BUC tissue sections, and analyzed the prognostic significance of FAM83H and Nectin1 expression.ResultsBoth FAM83H and Nectin1 were mainly expressed in the cytoplasm, and their expression was significantly associated. FAM83H expression was significantly correlated with higher histologic grade, higher T stage, higher TNM stage, and recurrence. Nectin1 expression was significantly associated with higher histologic grade and recurrence. Univariate analysis showed FAM83H expression and Nectin1 expression were significantly associated with worse overall survival (OS) and shorter relapse-free survival (RFS) of BUC patients. In multivariate analysis, levels of FAM83H and Nectin1 were independent indicators of shorter survival of BUC patients.ConclusionsOur results suggest that FAM83H and Nectin1 are important in the progression of BUC, and that expression patterns of these two proteins can be used as prognostic indicators of survival in BUC patients.

Knockdown of microRNA-130b improves doxorubicin sensitivity in bladder urothelial carcinoma by negatively regulating cylindromatosis expression.

IntroductionChemotherapeutic resistance reduces the sensitivity of bladder urothelial carcinoma (BUC) to chemotherapeutic drugs and contributes a barrier leading to treatment failure. The purpose of this research project is to investigate the regulatory effects of miR-130b on chemotherapeutic drug resistance of BUC and its mechanism.Material and methodsThe relative expression of miRNA-130b and cylindromatosis (CYLD) was examined using real-time quantitative PCR. The cell proliferation and doxorubicin sensitivity were detected with the enhanced CCK-8 assay. The specific combination of miR-130b and CYLD was verified with the luciferase reporter gene assay. Protein expression was detected by Western blot.ResultsOur study found that miR-130b was up-regulated in doxorubicin-insensitive BUC tissues and cell lines, and its high expression was negatively related to doxorubicin sensitivity in BUC. The miR-130b knockdown reduced the IC50 of doxorubicin and improved doxorubicin sensitivity of J82/Dox and T24/Dox cells. For the regulation mechanism analysis of miR-130b, bioinformatics analysis software was used to predict the potential targets of miR-130b, including the CYLD gene. The following luciferase activities assay, quantitative real time-PCR and western blot identified the CYLD gene as a target of miR-130b. Knockdown of CYLD reversed miR-130b’s regulatory roles in doxorubicin sensitivity in J82/Dox and T24/Dox cells.ConclusionsHigh expression of miR-130b is negatively related to doxorubicin sensitivity in BUC, and knockdown of miR-130b improves doxorubicin sensitivity in BUC by negatively regulating CYLD expression. Our findings will provide guidance for the clinical chemotherapy of BUC.

Role of NRP1 in Bladder Cancer Pathogenesis and Progression.

Bladder urothelial carcinoma (BC) is a fatal invasive malignancy and the most common malignancy of the urinary system. In the current study, we investigated the function and mechanisms of Neuropilin-1 (NRP1), the co-receptor for vascular endothelial growth factor, in BC pathogenesis and progression. The expression of NRP1 was evaluated using data extracted from GEO and HPA databases and examined in BC cell lines. The effect on proliferation, apoptosis, angiogenesis, migration, and invasion of BC cells were validated after NRP1 knockdown. After identifying differentially expressed genes (DEGs) induced by NRP1 silencing, GO/KEGG and IPA® bioinformatics analyses were performed and specific predicted pathways and targets were confirmed in vitro. Additionally, the co-expressed genes and ceRNA network were predicted using data downloaded from CCLE and TCGA databases, respectively. High expression of NRP1 was observed in BC tissues and cells. NRP1 knockdown promoted apoptosis and suppressed proliferation, angiogenesis, migration, and invasion of BC cells. Additionally, after NRP1 silencing the activity of MAPK signaling and molecular mechanisms of cancer pathways were predicted by KEGG and IPA® pathway analysis and validated using western blot in BC cells. NRP1 knockdown also affected various biological functions, including antiviral response, immune response, cell cycle, proliferation and migration of cells, and neovascularisation. Furthermore, the main upstream molecule of the DEGs induced by NRP1 knockdown may be NUPR1, and NRP1 was also the downstream target of NUPR1 and essential for regulation of FOXP3 expression to activate neovascularisation. DCBLD2 was positively regulated by NRP1, and PPAR signaling was significantly associated with low NRP1 expression. We also found that NRP1 was a predicted target of miR-204, miR-143, miR-145, and miR-195 in BC development. Our data provide evidence for the biological function and molecular aetiology of NRP1 in BC and for the first time demonstrated an association between NRP1 and NUPR1, FOXP3, and DCBLD2. Specifically, downregulation of NRP1 contributes to BC progression, which is associated with activation of MAPK signaling and molecular mechanisms involved in cancer pathways. Therefore, NRP1 may serve as a target for new therapeutic strategies to treat BC and other cancers.

The inhibitory effect of silencing CDCA3 on migration and proliferation in bladder urothelial carcinoma

BackgroundCDCA3 is an important component of the E3 ligase complex with SKP1 and CUL1, which could regulate the progress of cell mitosis. CDCA3 has been widely identified as a proto-oncogene in multiple human cancers, however, its role in promoting human bladder urothelial carcinoma has not been fully elucidated.MethodsBioinformatic methods were used to analyze the expression level of CDCA3 in human bladder urothelial carcinoma tissues and the relationship between its expression level and key clinical characteristics. In vitro studies were performed to validate the specific functions of CDCA3 in regulating cell proliferation, cell migration and cell cycle process. Alterations of related proteins was investigated by western blot assays. In vivo studies were constructed to validate whether silencing CDCA3 could inhibit the proliferation rate in mice model.ResultsBioinformatic analysis revealed that CDCA3 was significantly up-regulated in bladder urothelial carcinoma samples and was related to key clinical characteristics, such as tumor grade and metastasis. Moreover, patients who had higher expression level of CDCA3 tend to show a shorter life span. In vitro studies revealed that silencing CDCA3 could impair the migration ability of tumor cells via down-regulating EMT-related proteins such as MMP9 and Vimentin and inhibit tumor cell growth via arresting cells in the G1 cell cycle phase through regulating cell cycle related proteins like p21. In vivo study confirmed that silencing CDCA3 could inhibit the proliferation of bladder urothelial carcinoma cells.ConclusionsCDCA3 is an important oncogene that could strengthen the migration ability of bladder urothelial carcinoma cells and accelerate tumor cell growth via regulating cell cycle progress and is a potential biomarker of bladder urothelial carcinoma.

Proteomic comparison between different tissue preservation methods for identification of promising biomarkers of urothelial bladder cancer

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1—non-muscle invasive bladder cancer (NMIBC), and T2/T3—muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.

MAFG-AS1/MAFG positive feedback loop contributes to cisplatin resistance in bladder urothelial carcinoma through antagonistic ferroptosis

Though promoting ferroptosis can reduce cisplatin resistance in tumor cells, ferroptosis and cisplatin resistance in bladder urothelial carcinoma (BUC) following long non-coding RNAs (lncRNAs) is largely unknown. Here, we found the highly expressed lncRNA MAF transcription factor G antisense RNA 1 (MAFG-AS1) in BUC, and its inhibition increased the sensitivity of BUC cells to cisplatin by promoting ferroptosis. Mechanically, binding to iron chaperone poly(rC)-binding protein 2 (PCBP2) facilitated the recruitments of MAFG-AS1 to deubiquitinase ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5), thus stabilizing PCBP2 protein itself. Then PCBP2 was confirmed to interact with ferroportin 1 (FPN1), an iron export protein, leading to inhibition of ferroptosis. Moreover, the expression of MAFG-AS1 was regulated by the transcriptional factor MAFG. Interestingly, MAFG-AS1 stimulated MAFG transcription by recruiting histone acetyltransferase p300 (EP300) to promote the histone 3 at lysine 27 (H3K27ac) at genomic locus of MAFG, forming a MAFG-AS1/MAFG positive feedback loop. In patient samples, higher expression of MAFG-AS1 and MAFG in BUC tissues was significantly correlated with T status and N status, such that MAFG-AS1, MAFG, and the combination of the two were independent prognostic indicators and chemotherapy sensitivity predictive biomarkers for BUC patients. These findings suggest that inhibition of MAFG-AS1 and MAFG can increase the sensitivity of BUC cells to cisplatin through promoting ferroptosis, indicating the novel chemotherapy sensitivity biomarkers and therapeutic target for BUC.

MiR-25 regulates cell proliferation and metastasis in bladder urothelial carcinoma.

Background: Bladder urothelial carcinoma (BC) is a common malignant tumor with a high incidence. This study aims to explore the role of miR-25 in BC tumorigenesis.Material and Methods: The expression of miR-25 and PTEN were detected in clinical BC tissues. BC cell lines T24 and 5637 were used to transfect miR-25 mimics or inhibitors. Luciferase reporter gene detection confirmed the correlation between miR-25 and PTEN. CCK-8 method and flow cytometry were used to detect cell viability and apoptosis. Cell migration and invasion ability were examined by transwell assays. Western blotting detects the protein levels of PTEN, β-catenin, GSK-3β and p-GSK-3β.Results: MiR-25 and PTEN expression are found to be negatively correlated in BC tissues. Further research confirmed that PTEN is a direct target of miR-25. In addition, the overexpression of miR-25 down-regulates the expression of PTEN, induces cell survival and inhibits apoptosis, while the knockout of miR-25 leads to the opposite result. miR-25 also inhibits the phosphorylation of GSK-3β and β-catenin without changing the total level of GSK-3β. In vivo experiments confirmed that miR-25 plays an oncogene's role by regulating the PTEN and Wnt/β-catenin signaling pathways.Conclusion: Our research shows that miR-25 has a negative regulatory effect on the expression of PTEN in clinical specimens and in vitro. miR-25 can promote the proliferation of BC cells and induce cell invasion. Therefore, miR-25 may be used as a biomarker to predict the progression of BC.

High expression of EMP1 predicts a poor prognosis and correlates with immune infiltrates in bladder urothelial carcinoma.

Epithelial membrane protein 1 (EMP1) is a key gene that regulates cell proliferation and metastatic capability in various types of cancer, and serves an important role in tumor-immune interactions. However, the association between EMP1 and clinical prognosis, as well as the presence of tumor-infiltrating lymphocytes in bladder urothelial carcinoma (BLCA) remains unclear. The present study aimed to explore the relationship between EMP1 expression and tumor immune cell infiltration in BLCA. In the present study, EMP1 expression in BLCA was analyzed using the Oncomine database, The Cancer Genome Atlas (TCGA) and the Tumor Immune Estimation Resource (TIMER). The effects of EMP1 on clinical prognosis were evaluated using the Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis. The correlations between EMP1, cancer immune infiltrates and lymphocyte abundance were determined using the TIMER and Tumor immune system interaction database. In addition, correlations between EMP1 expression and gene markers in immune infiltrates were analyzed using cBioportal. The results demonstrated that, compared with adjacent normal tissues, EMP1 was downregulated in BLCA tissues. High expression of EMP1 was significantly associated with poor overall survival (OS) in BLCA cases obtained from TCGA. Multivariate Cox analysis revealed that EMP1 was an independent predictor of OS in patients with BLCA. Gene set enrichment analysis revealed that EMP1 was associated with cancer-related pathways and was positively correlated with the levels of infiltrating CD8+ T cells, macrophages, neutrophils and dendritic cells in BLCA. Further analysis demonstrated that EMP1 was significantly associated with the enrichment of multiple types of lymphocyte. EMP1 expression exhibited a strong correlation with a range of immune markers in BLCA. In conclusion, the results of the present study demonstrated that EMP1 was associated with a poor prognosis in patients with BLCA, and that the levels of immune infiltration and multiple immunomarker groups were associated with EMP1 expression. These results suggested that EMP1 may be used as a predictive biomarker to determine the prognosis and immune infiltration in BLCA.