Urothelial Carcinoma Of The Bladder Tissues Research Articles

Chitinase 3-like 1 expression associated with lymphatic metastasis and prognosis in urothelial carcinoma of thebladder.

Lymphatic metastasis, an early stage of the metastasis process, is associated with adverse clinical outcomes in urothelial carcinoma of the bladder (UCB). However, the role of inflammation in triggering lymphatic metastasis remains unclear. We employed an RNA-sequencing cohort (n = 50) from Sun Yat-Sen Memorial Hospital (SYMH) to identify the most highly upregulated inflammatory gene associated with lymphatic metastasis. Using immunohistochemistry and immunofluorescence analyses, we validated the association of the identified molecule with clinical features and prognosis in an independent UCB cohort (n = 244) from SYMH. We also analysed TCGA-BLCA cohort (n = 408) to identify its potential biological pathways and immune landscape. In our study, chitinase 3-like 1 (CHI3L1) emerged as a significantly overexpressed proinflammatory mediator in UCB tissues with lymphatic metastasis compared to those without lymphatic metastasis (81.1% vs. 47.8%, P < 0.001). Within UCB tissues, CHI3L1 was expressed in both stromal cells (52.8%) and tumor cells (7.3%). Moreover, CHI3L1+ stromal cells, but not tumor cells, exhibited independent prognostic significance for both overall survival (P < 0.001) and recurrence-free survival (P = 0.006). CHI3L1+ stromal cells were positively associated with D2-40+ lymphatic vessel density (P < 0.001) and the immunosuppressive PD-L1/PD-1/CD8 axis in UCB tissues (all P < 0.05). A bioinformatics analysis also identified a positive association between CHI3L1 expression and lymphangiogenesis or immunosuppression pathways. Our study established a clear association between stromal CHI3L1 expression and lymphatic metastasis, suggesting that stromal CHI3L1 expression is a potential prognostic marker for bladder cancer patients.

PXDNL activates the motility of urothelial bladder carcinoma cells through the Wnt/β-catenin pathway and has a prognostic value

AimsAlthough aberrant expression of peroxidasin-like (PXDNL) has been associated with carcinogenesis, its potential role in the Urothelial Carcinoma of the Bladder (UCB) remains unknown. The present study aimed to explore the role of PXDNL in UCB carcinogenesis and its potential clinical value. Main methodsBased on The Cancer Genome Atlas (TCGA) data, bioinformatics was used to explore the potential clinical value of PXDNL. Wound healing and Transwell invasion assays were employed for the purpose of assessing the cell motility, while the Western Blotting experiments were utilized for investigating the protein expression pattern of PXDNL in UCB and investigating the Epithelial-to-Mesenchymal Transition (EMT) and Wnt/β-catenin pathways for understanding the probable mechanisms involved. Key findsPXDNL mRNA was overexpressed in UCB tissues and indicated a poor prognosis. High PXDNL mRNA levels were also associated with advanced clinicopathological features and were regarded as independent prognostic factors for UCB. However, PXDNL showed a weak correlation with immune cell infiltration in UCB. In addition, the findings of the study verified that the existing form of the PXDNL protein was 57-kDa and it was upregulated in the UCB cell lines and tissue samples. Furthermore, silencing PXDNL inhibited, while overexpressing PXDNL promoted EMT and motility of UCB cells in vitro. Mechanistic studies showed that PXDNL activated UCB cell motility via the Wnt/β-catenin pathway. SignificanceThe results reveal a novel molecular target that could be further explored for developing preventive, predictive, and individualized treatment strategies for UCB.

Negative Effects of Stromal Neutrophils on T Cells Reduce Survival in Resectable Urothelial Carcinoma of the Bladder.

Urothelial carcinoma of the bladder (UCB) is a major type of bladder cancer with a distinct tumor microenvironment (TME). Although neutrophils are the main component of myeloid cells in the TME, the clinical significance and function of the neutrophils remain unclear in UCB. Here, we observed CD66b+ neutrophils were predominantly enriched in the stroma of UCB tissues and their levels emerged as an independent prognostic factor for overall survival (P = 0.006, n = 237), and were positively associated with age (P = 0.033), tumor stage (P < 0.0001), nodal metastasis (P = 0.045), and histological grade (P < 0.0001). Furthermore, we found that CD66b+ neutrophils were frequently co-localized with CD4+ T cells (R=0.35, P = 0.0067), CD8+ T cells (R=0.52, P<0.0001) and Cleaved Caspase-3+ apoptosis cells (R=0.44, P = 0.0007) in the stroma of UCB tissue. In addition, better effects of T cells on patients’ survival were markedly reduced by neutrophils and T cells co-infiltration. Moreover, we confirmed bladder tumor cell supernatant treated neutrophils suppressed T cell proliferation and activation, and promoted T cell apoptosis through GM-CSF induced PD-L1 in vitro. The expression of PD-L1 by neutrophils was also detected in fresh UCB tissues by using flow cytometric analysis. These data suggested that stromal CD66b+ neutrophils may potentially represent a reliable marker of poor prognosis for UCB patients, and neutrophils might play an immunosuppressive role on T cell immunity partially via the expression of PD-L1.

Cationic amphiphilic drugs as potential anticancer therapy for bladder cancer.

More effective therapy for patients with either muscle-invasive or high-risk non-muscle-invasive urothelial carcinoma of the bladder (UCB) is an unmet clinical need. For this, drug repositioning of clinically approved drugs represents an interesting approach. By repurposing existing drugs, alternative anticancer therapies can be introduced in the clinic relatively fast, because the safety and dosing of these clinically approved pharmacological agents are generally well known. Cationic amphiphilic drugs (CADs) dose-dependently decreased the viability of a panel of human UCB lines in vitro. CADs induced lysosomal puncta formation, a hallmark of lysosomal leakage. Intravesical instillation of the CAD penfluridol in an orthotopic mouse xenograft model of human UCB resulted in significantly reduced intravesical tumor growth and metastatic progression. Furthermore, treatment of patient-derived exvivo cultured human UCB tissue caused significant partial or complete antitumor responses in 97% of the explanted tumor tissues. In conclusion, penfluridol represents a promising treatment option for bladder cancer patients and warrants further clinical evaluation.

Differential Expression and Clinicopathological Significance of HER2, Indoleamine 2,3-Dioxygenase and PD-L1 in Urothelial Carcinoma of the Bladder.

Purpose: Evasion of the immune system by cancer cells allows for the progression of tumors. Antitumor immunotherapy has shown remarkable effects in a diverse range of cancers. The aim of this study was to determine the clinicopathological significance of human epidermal growth factor receptor 2 (HER2), indoleamine 2,3-dioxygenase (IDO), and programmed death ligand-1 (PD-L1) expression in urothelial carcinoma of the bladder (UCB). Materials and Methods: We retrospectively studied 97 patients with UCB. We performed an immunohistochemical study to measure the expression levels of HER2, IDO, and PD-L1 in UCB tissue from these 97 patients. Results: In all 97 cases, the PD-L1 expression of tumor-infiltrating immune cells (ICs) was significantly correlated with higher pathologic tumor stage (pT). In pT2–pT4 cases (n = 69), higher levels of HER2 and IDO expression in invasive tumor cells (TCs) were associated with shorter periods of disease-free survival (DFS). Conclusion: These results imply that the expression of PD-L1 in ICs of the UCB microenvironment is associated with cancer invasion and the expression of HER2 or IDO in the invasive cancer cell and suggestive of the potential for cancer recurrence. We suggest that the expression levels of IDO, HER2, and PD-L1 could be useful as targets in the development of combined cancer immunotherapeutic strategies.

Nucleobindin 2 expression is an independent prognostic factor for bladder cancer.

Nucleobindin 2 (NUCB2) has been reported to play an important role in both tumorigenesis and cancer progression. This study aimed to examine the clinical significance of NUCB2 expression urothelial carcinoma of the bladder (UCB).The expression level of NUCB2 and its correlation with clinicopathological parameters was analyzed in 225 UCB tissues by immunohistochemistry. Kaplan–Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between NUCB2 expression and the prognosis of UCB patients. High NUCB2 expression of UCB patients significantly correlated with aggressive clinicopathological features. Patients with high NUCB2 had shorter overall survival and recurrence-free survival in Kaplan–Meier survival curve (P = .018 and P = .001, respectively).Our results show that high expression of NUCB2 associated with aggressive clinicopathological feature and predicted unfavorable prognosis in patients with UCB might serve as feasible biomarker for clinical outcome of UCB patients after surgery and potential therapeutic target in the future.

Disabled Homolog 2 (DAB2) Protein in Tumor Microenvironment Correlates with Aggressive Phenotype in Human Urothelial Carcinoma of the Bladder.

Disabled homolog-2 (DAB2) has been reported to be a tumor suppressor gene. However, a number of contrary studies suggested that DAB2 promotes tumor invasion in urothelial carcinoma of the bladder (UCB). Here, we investigated the clinical role and biological function of DAB2 in human UCB. Immunohistochemical staining analysis for DAB2 was carried out on UCB tissue specimens. DAB2 expression levels were compared with clinicopathological factors. DAB2 was knocked-down by small interfering RNA (siRNA) transfection, and then its effects on cell proliferation, invasion, and migration, and changes to epithelial-mesenchymal transition (EMT)-related proteins were evaluated. In our in vivo assays, tumor-bearing athymic nude mice subcutaneously inoculated with human UCB cells (MGH-U-3 or UM-UC-3) were treated by DAB2-targeting siRNA. Higher expression of DAB2 was associated with higher clinical T category, high tumor grade, and poor oncological outcome. The knock-down of DAB2 decreased both invasion and migration ability and expression of EMT-related proteins. Significant inhibitory effects on tumor growth and invasion were observed in xenograft tumors of UM-UC-3 treated by DAB2-targeting siRNA. Our findings suggested that DAB2 expression was associated with poor prognosis through increased oncogenic properties including tumor proliferation, migration, invasion, and enhancement of EMT in human UCB.

Microlocalization and clinical significance of stabilin-1+ macrophages in treatment-na\xefve patients with urothelial carcinoma of the bladder

PurposeEmerging evidence has shown that macrophages (Mφs) at different tumor sites have diverse clinical attributes. Stabilin-1 is a multi-functional scavenger marker for specialized tumor-associated Mφs. This study investigates the relationship between the density and microlocalization of stabilin-1+ Mφs within tumors and the clinical outcomes of patients with urothelial carcinoma of the bladder (UCB).MethodsIn this retrospective study, 283 UCB patients who received radical cystectomy or transurethral resection were examined. Immunohistochemistry and immunofluorescence analyses were used to colocalize the expression of stabilin-1 with other markers for Mφs (CD14, CD68, CD163, and CD206). Kaplan–Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS) and recurrence-free survival (RFS).ResultsIn UCB tissues, stabilin-1 was primarily expressed on Mφs, as evident from triple immunofluorescence staining for stabilin-1 and Mφ markers. Stabilin-1+ Mφs were often more prominent in stromal regions rather than intratumoral regions in UCB tissues (P < 0.0001). After dichotomization at the median cell density for stabilin-1+ Mφs, only intratumoral stabilin-1+ Mφ density was a predictor of poor OS (P < 0.001) and RFS (P = 0.026). Moreover, intratumoral stabilin-1+ Mφ density was positively associated with tumor stage (P < 0.01) and histological grade (P < 0.01), and emerged as an independent prognostic factor for OS (HR 2.371; P < 0.0001), but not for RFS (HR 1.491; P = 0.061).ConclusionsOur findings indicate that intratumoral stabilin-1+ Mφs could potentially be used as a pro-tumoral prognostic marker for UCB patients.

Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder.

BackgroundTaurine-upregulated gene 1 (TUG1) has been documented to be implicated in carcinogenesis and chemoresistance in solid tumors. Here, we explored the biological role and regulatory mechanism of TUG1 in progression and chemoresistance of urothelial carcinoma of the bladder (UCB).MethodsNuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) mRNA and TUG1 expression was determined by quantitative reverse transcription polymerase chain reaction. Western blot was performed to determine the protein levels of Nrf2, p-glycoprotein (p-gp), Ki-67 (Ki67), matrix metalloproteinase (MMP)-2 and MMP-9 and cleaved caspase-3. The effects of either Nrf2 or TUG1 knockdown on the proliferation, invasion, apoptosis and adriamycin (ADM) resistance of UCB cells were evaluated by CCK-8 assay, transwell invasion assay and flow cytometry analysis. Xenograft tumor assay was carried out to confirm the role of Nrf2 and TUG1 in ADM resistance of UCB cells in vivo.ResultsNrf2 and TUG1 were upregulated in UCB tissues and cell lines. A positive correlation between Nrf2 and TUG1 expression was discovered in UCB tissues. Moreover, Nrf2 and TUG1 expression levels were higher in ADM-resistant cells compared with those in parental cells. Furthermore, Nrf2 positively regulated the expression of TUG1 in UCB cells. Knockdown of either Nrf2 or TUG1 led to the inhibition of cell proliferation and invasion and promotion of cell apoptosis, accompanying with down-regulation of Ki67, MMP-2 and MMP-9 and up-regulation of cleaved caspase-3. Knockdown of either Nrf2 or TUG1 enhanced the sensitivity of BIU-87/ADM and T24/ADM cells to ADM, as indicated by decreased expression of p-gp. Besides, knockdown of either Nrf2 or TUG1 inhibited tumor growth in the absence or presence of ADM in vivo.ConclusionsNrf2 induces the up-regulation of TUG1 to promote progression and ADM resistance in UCB.

LGALS4 as a Prognostic Factor in Urothelial Carcinoma of Bladder Affects Cell Functions.

Background:To identify the hub genes related to urothelial carcinoma of the bladder prognosis and to understand their underlying mechanism.Methods:The expression profiles of 18 pairs of urothelial carcinoma of the bladder patient tissue and paired adjacent tissue obtained from the Cancer Genome Atlas were performed. Weighted gene coexpression network analysis was employed to screen gene modules and hub genes with significant differential expressions in urothelial carcinoma of the bladder. The hub genes expression in urothelial carcinoma of the bladder tissues was validated by reverse transcription-quantitative polymerase chain reaction. The overall survival curve and disease-free survival curve of prognostic factor (LGALS4) were plotted using the Kaplan-Meier method. Furthermore, LGALS4 messenger RNA and protein expression were also assessed in 2 urothelial carcinoma of the bladder cell lines (T24 and 5637) by quantitative reverse transcription–polymerase chain reaction and Western blot. The functions of urothelial carcinoma of the bladder cells with transfected pcDNA3.1-LGALS4 were identified through MTT assay, plate clone formation assay, flow cytometry, and cell migration experiments.Results:LGALS4 was the hub gene of pink module and it was related to prognosis. Higher LGALS4 expression predicted higher probabilities of overall survival and disease-free survival. Overexpression of LGALS4 in urothelial carcinoma of the bladder cells suppressed cell viability and migration but induced apoptosis.Conclusion:LGALS4 played a critical role in the progression of urothelial carcinoma of the bladder and held a promise to be the biomarker for diagnosis and treatment of urothelial carcinoma of the bladder. It predicted good prognosis of urothelial carcinoma of the bladder and restrained the growth and migration of urothelial carcinoma of the bladder cells.

PRMT5 Circular RNA Promotes Metastasis of Urothelial Carcinoma of the Bladder through Sponging miR-30c to Induce Epithelial-Mesenchymal Transition.

Circular RNAs (circRNAs), a novel class of noncoding RNAs, have recently drawn lots of attention in the pathogenesis of human cancers. However, the role of circRNAs in cancer cells epithelial-mesenchymal transition (EMT) remains unclear. In this study, we aimed to identify novel circRNAs that regulate urothelial carcinoma of the bladder (UCB) cells' EMT and explored their regulatory mechanisms and clinical significance in UCBs. We first screened circRNA expression profiles using a circRNA microarray in paired UCB and normal tissues, and then studied the clinical significance of an upregulated circRNA, circPRMT5, in a large cohort of patients with UCB. We further investigated the functions and underlying mechanisms of circPRMT5 in UCB cells' EMT. Moreover, we evaluated the regulation effect of circPRMT5 on miR-30c, and its target genes, SNAIL1 and E-cadherin, in two independent cohorts from our institute and The Cancer Genome Atlas (TCGA). We demonstrated that upregulated expression of circPRMT5 was positively associated with advanced clinical stage and worse survival in patients with UCB. We further revealed that circPRMT5 promoted UCB cell's EMT via sponging miR-30c. Clinical analysis from two independent UCB cohorts showed that the circPRMT5/miR-30c/SNAIL1/E-cadherin pathway was essential in supporting UCB progression. Importantly, we identified that circPRMT5 was upregulated in serum and urine exosomes from patients with UCB, and significantly correlated with tumor metastasis. CircPRMT5 exerts critical roles in promoting UCB cells' EMT and/or aggressiveness and is a prognostic biomarker of the disease, suggesting that circPRMT5 may serve as an exploitable therapeutic target for patients with UCB.

TRIM65 supports bladder urothelial carcinoma cell aggressiveness by promoting ANXA2 ubiquitination and degradation

Clinically, most of human urothelial carcinoma of the bladder (UCB)-related deaths result from tumor metastasis, but the underlying molecular mechanisms are largely unknown. Recently, a growing number of tripartite motif (TRIM) family members have been suggested to be important regulators for tumorigenesis. However, the impact of most TRIM members on UCB pathogenesis is unclear. In this study, TRIM65 was first screened as an important oncogenic factor of UCB from the Cancer Genome Atlas (TCGA) database and was validated by a large cohort of clinical UCB tissues. By in vitro and in vivo experiments, we demonstrated that TRIM65 promotes UCB cell invasive and metastatic capacities. Notably, we showed that TRIM65 modulates cytoskeleton rearrangement and induces UCB cells epithelial-mesenchymal transition by the ubiquitination of ANXA2, ultimately leading to an enhanced invasiveness of UCB cells. Importantly, UCBs with high expression of TRIM65 and low expression of ANXA2 showed the poorest outcome. Collectively, our results suggest that the overexpression of TRIM65 has an essential oncogenic role via ubiquitination of ANXA2 in UCB pathogenesis, and that such could be used as a novel prognostic marker and/or therapeutic target for UCB.

Upregulation of long non-coding RNA HNF1A-AS1 is associated with poor prognosis in urothelial carcinoma of the bladder.

Long noncoding RNAs (lncRNAs) play important roles in the pathogenesis of bladder cancer. A recent study reported that lncRNA HNF1A-AS1 (HNF1A-AS1) was upregulated in urothelial carcinoma of the bladder (UCB) and served as a tumor promoter. However, the clinical significance of HNF1A-AS1 in the prognosis of patients with UCB was poorly understood. This work was designed to investigate the relationship between HNF1A-AS1 expression level and the prognosis of UCB. Relative expression levels of HNF1A-AS1 in UCB tissues were determined by qRT-PCR. Then, the associations between HNF1A-AS1 expression and clinical pathological parameters were further evaluated. Survival and Cox proportional-hazards regression analyses were performed to determine the correlation between HNF1A-AS1 expression levels and prognosis in the patients. Data showed that the majority of UCB tissues showed higher HNF1A-AS1 levels than the corresponding normal tissues controls (p < 0.01). Statistical assay revealed that high HNF1A-AS1 expression was significantly correlated with histological grade (p = 0.008), tumor stage T (p = 0.003) and lymph nodes metastasis (p = 0.007). In addition, the overall survival time of patients with high HNF1A-AS1 expression was significantly shorter compared to those with low HNF1A-AS1 expression. Furthermore, multivariate analysis confirmed that relative HNF1A-AS1 expression was an independent predictor of overall survival in patients with UCB. HNF1A-AS1 expression was upregulated in UCB, and it may be a useful prognostic biomarker for patients with UCB.

Long Non-Coding RNA Cancer Susceptibility Candidate 2a (CASC2a) Is a Marker of Early Recurrence After Radical Cystectomy in Patients with Urothelial Carcinoma of the Bladder.

BackgroundThe aim of this study was to investigate the expression of long non-coding RNAs (lncRNA) cancer susceptibility candidate 2a (CASC2a) in patients with urothelial carcinoma of the bladder (UCB) and its predictive value in the recurrence of UCB after radical cystectomy (RC).Material/MethodsTumor and paired adjacent normal tissues were obtained from 112 patients with UCB who underwent RC in our hospital from March 2010 to March 2012. The expression of CASC2a was evaluated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization (FISH).ResultsCASC2a was down-regulated in UCB tissues, and was highly negatively correlated with the pT, pN, tumor size, and lymphovascular invasion (LVI). The sensitivities of CASC2a for diagnosing UCB and its recurrence after RC were 89.30% and 81.55%, respectively, and the specificities were 71.43% and 58.21%, respectively. Patients with a high expression of CASC2a had a higher 5-year recurrence-free survival rate than those with low expression of CASC2a. Kaplan-Meier survival analysis demonstrated that the pT, pN, tumor grade, tumor size, concomitant carcinoma in situ (CIS), LVI, soft tissue surgical margin (STSM), and CASC2a expression were related to the recurrence in patients undergoing RC for UCB. Cox proportional hazard model analysis showed that CASC2 expression, pT4, lymph node metastasis, and CIS were independent risk factors.ConclusionsCASC2a was down-regulated in patients with UCB, and was associated with the risk of recurrence among patients undergoing RC, indicating that lncRNAs could act as predictive biomarkers and potential therapeutic targets in bladder cancer, including CASC2a.

Low expression of TMEM67 is a critical predictor of poor prognosis in human urothelial carcinoma of the bladder

ObjectivesThe aim of the study was to evaluate the expression of TMEM67 in urothelial carcinoma of the bladder (UCB) tissues and to determine the potential relevance between the expression of TMEM67 and prognosis of UCB. Material and methodsIn this study, the expression of TMEM67 mRNA was performed by quantitative real-time PCR in 80 UCB and 54 noncancerous tissues. The expression of TMEM67 protein was identified by immunohistochemistry and western blotting. Chi-square test was conducted to verify the relevance between the expression of TMEM67 and clinical parameters. Kaplan-Meier survival analysis was demonstrated between high or low expression level of TMEM67 mRNA and recurrence-free survival probability. Cox regression analysis was conducted to evaluate the relevance between the expression of TMEM67 and the prognosis in UCB. ResultsLow expression of TMEM67 mRNA and protein was detected in most of UCB tissues using quantitative real-time polymerase chain reaction and western blotting, compared with noncancerous tissues. Low expressions of TMEM67 were associated with TNM stage, grade, and lymph node metastasis (P<0.05). Kaplan-Meier analysis showed that the low expression of TMEM67 mRNA had significantly shorter recurrence-free survival probability (P = 0.018). Cox regression analysis confirmed that low expression of TMEM67 mRNA predicted poor prognosis of patients with UCB (HR = 2.950, P = 0.029, 95% CI: 1.116–7.796). ConclusionsTMEM67 expression is low in UCB tissues, and the TMEM67 low expression predicted poor prognosis of patients with UCB.

Suppression of HBXIP Reduces Cell Proliferation, Migration and Invasion In Vitro, and Tumorigenesis In Vivo in Human Urothelial Carcinoma of the Bladder.

Hepatitis B X-interacting protein (HBXIP) has been found overexpressed in several types of human cancer, however, the status of HBXIP expression in urothelial carcinoma of the bladder (UCB) has not been explored. In this study, the authors used real-time polymerase chain reaction and Western blot to test the expression of HBXIP in UCB and adjacent tissues. The expression of HBXIP was significantly increased in UCB tissues. In addition, they showed that suppression of HBXIP induced cell cycle arrest and increased cell apoptosis in T24 cells. Also, suppression of HBXIP also decreased T24 and PC3 cell proliferation, migration, and invasion. More importantly, the authors found that inhibition of HBXIP reduced the tumorigenesis in vivo, suggesting that HBXIP plays an important role in UCB progression. These data for the first time showed that HBXIP acts as an oncoprotein in UCB, suggesting that HBXIP may become a potential novel therapeutic target for the treatment of UCB.

Downregulation of miR‐133b predict progression and poor prognosis in patients with urothelial carcinoma of bladder

We found microRNA‐133b (miR‐133b) was downregulated in urothelial carcinoma of the bladder (UCB) tissues, and it could inhibit the proliferation and induce apoptosis in UCB cells. Consequently, we intend to explore the clinical significance of miR‐133b in UCB patients. Expression of miR‐133b in 146 UCB specimens and matched adjacent non‐neoplastic bladder tissues were measured by quantitative real‐time polymerase chain reaction. The overall survival (OS) curve and progression‐free survival (PFS) curve were plotted using the Kaplan–Meier method. Prognostic factors for OS and PFS were identified by univariate and multivariate analyses using the Cox proportional hazards regression model. The expression of miR‐133b was significantly downregulated in UCB tissues compared with those in adjacent non‐neoplastic bladder tissues (P < 0.001). Among UCB patients, low expression of miR‐133b significantly correlated with aggressive clinicopathological features. Multivariate analysis indicated that the expression of miR‐133b was the independent prognostic factors for predicting PFS (RR: 2.97; 95% CI: 1.78–6.44; P = 0.009) and OS (RR: 4.23; 95% CI: 1.51–11.8; P = 0.011) in patients with UCB. Our study demonstrated that downregulation of miR‐133b associated with aggressive clinicopathological features and predicted unfavorable prognosis in patients with UCB, might serve as feasible biomarker for clinical outcome of UCB patients after surgery and potential therapeutic target in the future.

MicroRNA-30a as a prognostic factor in urothelial carcinoma of bladder inhibits cellular malignancy by antagonising Notch1.

To explore the relation between microRNA-30a (miR-30a) and Notch1, and to evaluate the potential prognostic role of miR-30a in invasive urothelial carcinoma of the bladder (UCB). In all, 50 invasive UCB tissue specimens, along with the adjacent bladder tissue specimens were obtained, and the clinical parameters of the 50 patients were analysed. Bioinformatics analysis was performed and miR-30a was selected as a potential miRNA targeting Notch1, with a luciferase assay performed to verify the binding site between miR-30a and Notch1. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to assess the RNA expressions of miR-30a and Notch1, while Western Blotting and immunohistochemical staining were used to assess the protein expression of Notch1. Finally, cell proliferation, cell cycle, cell migration and invasion assays were used to evaluate the cellular effects of miR-30a and Notch1 on the UCB cell lines T24 and 5637. MiR-30a was downregulated in tumour tissues when compared with adjacent bladder tissues (P < 0.001), negatively correlating with Notch1 messenger RNA (R(2) 0.106, P = 0.021) in invasive UCB, and miR-30a expression further decreased in patients with shorter overall survival and disease-free survival (P = 0.039 and P = 0.037, respectively). The luciferase assay showed that miR-30a inhibited the Notch1 3'-untranslated region reporter activities in the T24 and 5637 cell lines (both P < 0.001). Both miR-30a and small interfering RNA Notch1 negatively regulated cell proliferation (P = 0.002 and P = 0.035 in T24; P = 0.029 and P = 0.037 in 5637 cell lines), activated cell cycle arrest (both P < 0.001 in T24; both P < 0.001 in 5637 cell lines), and prevented cellular migration (both P < 0.001 in T24; P = 0.003 and P < 0.001 in 5637 cell lines) and invasion (P = 0.009 and P = 0.006 in T24; P = 0.006 and P = 0.002 in 5637 cell lines) abilities. Ectopic Notch1 without the 3'untranslated region partially rescued the above-mentioned cellular effects of over-expressed miR-30a on T24 and 5637 cells. MiR-30a lessens cellular malignancy by antagonising oncogene Notch1 and plays an effective prognostic role in invasive UCB.

Transcriptome Profiling of a Multiple Recurrent Muscle-Invasive Urothelial Carcinoma of the Bladder by Deep Sequencing

Urothelial carcinoma of the bladder (UCB) is one of the commonly diagnosed cancers in the world. The UCB has the highest rate of recurrence of any malignancy. A genome-wide screening of transcriptome dysregulation between cancer and normal tissue would provide insight into the molecular basis of UCB recurrence and is a key step to discovering biomarkers for diagnosis and therapeutic targets. Compared with microarray technology, which is commonly used to identify expression level changes, the recently developed RNA-seq technique has the ability to detect other abnormal regulations in the cancer transcriptome, such as alternative splicing. In this study, we performed high-throughput transcriptome sequencing at ∼50× coverage on a recurrent muscle-invasive cisplatin-resistance UCB tissue and the adjacent non-tumor tissue. The results revealed cancer-specific differentially expressed genes between the tumor and non-tumor tissue enriched in the cell adhesion molecules, focal adhesion and ECM-receptor interaction pathway. Five dysregulated genes, including CDH1, VEGFA, PTPRF, CLDN7, and MMP2 were confirmed by Real time qPCR in the sequencing samples and the additional eleven samples. Our data revealed that more than three hundred genes showed differential splicing patterns between tumor tissue and non-tumor tissue. Among these genes, we filtered 24 cancer-associated alternative splicing genes with differential exon usage. The findings from RNA-Seq were validated by Real time qPCR for CD44, PDGFA, NUMB, and LPHN2. This study provides a comprehensive survey of the UCB transcriptome, which provides better insight into the complexity of regulatory changes during recurrence and metastasis.

PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

BackgroundPIN2/TRF1-interacting telomerase inhibitor1 (PinX1) was recently suggested as a putative tumor suppressor in several types of human cancer, based on its binding to and inhibition of telomerase. Moreover, loss of PinX1 has been detected in many human malignancies. However, the possible involvement of PinX1 and its clinical/prognostic significance in urothelial carcinoma of the bladder (UCB) are unclear.MethodsThe PinX1 expression profile was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry (IHC) in UCB tissues and adjacent normal urothelial bladder epithelial tissues. PinX1 was overexpressed and silenced in UCB cell lines to determine its role in tumorigenesis, development of UCB, and the possible mechanism.ResultsPinX1 expression in UCB was significantly down-regulated at both mRNA and protein level as compared with that in normal urothelial bladder epithelial tissues. PinX1 levels were inversely correlated with tumor multiplicity, advanced N classification, high proliferation index (Ki-67), and poor survival (P < 0.05). Moreover, overexpression of PinX1 in UCB cells significantly inhibited cell proliferation in vitro and in vivo, whereas silencing PinX1 dramatically enhanced cell proliferation. Overexpression of PinX1 resulted in G1/S phase arrest and cell growth/proliferation inhibition, while silencing PinX1 led to acceleration of G1/S transition, and cell growth/proliferation promotion by inhibiting/enhancing telomerase activity and via the p16/cyclin D1 pathway.ConclusionsThese findings suggest that down-regulation of PinX1 play an important role in the tumorigenesis and development of UCB and that the expression of PinX1 as detected by IHC is an independent molecular marker in patients with UCB.