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Background Bladder cancer (BC) is a common and deadly disease. Over the past decade, a number of genetic alterations have been reported in BC. Bladder urothelium expresses abundant urea transporter UT-B encoded by Slc14a1 gene at 18q12.3 locus, which plays an important role in preventing high concentrated urea-caused cell injury. Early genome-wide association studies (GWAS) showed that UT-B gene mutations are genetically linked to the urothelial bladder carcinoma (UBC). In this study, we examined whether Slc14a1 gene has been changed in UBC, which has never been reported. Case presentation A 59-year-old male was admitted to a hospital with the complaint of gross hematuria for 6 days. Ultrasonography revealed a size of 2.8 × 1.7 cm mass lesion located on the rear wall and dome of the bladder. In cystoscopic examination, papillary tumoral lesions 3.0-cm in total diameter were seen on the left wall of the bladder and 2 cm to the left ureteric orifice. Transurethral resection of bladder tumor (TURBT) was performed. Histology showed high-grade non-muscle invasive UBC. Immunostaining was negative for Syn, CK7, CK20, Villin, and positive for HER2, BRCA1, GATA3. Using a fluorescence in situ hybridization (FISH), Slc14a1 gene rearrangement was identified by a pair of break-apart DNA probes. Conclusions We for the first time report a patient diagnosed with urothelial carcinoma accompanied with split Slc14a1 gene abnormality, a crucial gene in bladder.

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In this study, we aim at investigating the expression and regulation role of long non-coding RNA (lncRNA) DLX6-AS1 in bladder cancer (BC). DLX6-AS1 was highly expressed in BC tissues and significant negative correlation with the 5-year survival in the BC patients. The results showed that the proliferation, migration and invasion activities of BC cells were promoted by DLX6-AS1 overexpression, while cell apoptosis was repressed. However, knockdown DLX6-AS1 presented an pposite regulatory effect, and DLX6-AS1 knockdown delayed tumor in vivo. The potential target of DLX6-AS1 in BC was predicted and verified by RIP, RNA pull-down, and dual-luciferase reporter assays as miR-195-5p. The results showed that miR-195-5p was down-regulated in BC tissues, the expression of which was significantly negative correlated with DLX6-AS1 expression. In addition, the results also showed that miR-195-5p targeted and down-regulated the VEGFA. Knockdown of DLX6-AS1 up-regulated miR-195-5p expression and down-regulated VEGFA expression. Moreover, down-regulation of VEGFA expression caused by DLX6-AS1 inhibited phosphorylation of Raf-1, MEK1/2, and ERK1/2, while miR-195-5p inhibitors abolished the effect of silencing DLX6-AS1 expression. Our study demonstrated that DLX6-AS1 played an oncogenic role in BC through miR-195-5p-mediated VEGFA/Ras/Raf/MEK/ERK pathway.

Bladder cancer (BCa) is a common urinary tract malignancy with frequent recurrences after initial resection. Submucosal injection of gemcitabine prior to transurethral resection of bladder tumor (TURBT) may prevent recurrence of urothelial cancer. However, the underlying mechanism remains unknown. In the present study, ultra-performance liquid chromatography Q-Exactive mass spectrometry was used to profile tissue metabolites from 12 BCa patients. The 48 samples included pre- and post-gemcitabine treatment BCa tissues, as well as adjacent normal tissues. Principal component analysis (PCA) revealed that the metabolic profiles of pre-gemcitabine BCa tissues differed significantly from those of pre-gemcitabine normal tissues. A total of 34 significantly altered metabolites were further analyzed. Pathway analysis using MetaboAnalyst identified three metabolic pathways closely associated with BCa, including glutathione, purine and thiamine metabolism, while gluta-thione metabolism was also identified by the enrichment analysis using MetaboAnalyst. In search of the possible targets of gemcitabine, metabolite profiles were compared between the pre-gemcitabine normal and post-gemcitabine BCa tissues. Among the 34 metabolites associated with BCa, the levels of bilirubin and retinal recovered in BCa tissues treated with gemcitabine. When comparing normal bladder tissues with and without gemcitabine treatment, among the 34 metabolites associated with BCa, it was observed that histamine change may be associated with the prevention of relapse, whereas thiamine change may be involved in possible side effects. Therefore, by employing a hypothesis-free tissue-based metabolomics study, the present study investigated the metabolic signatures of BCa and found that bilirubin and retinal may be involved in the mechanism underlying the biomolecular action of submucosal injection of gemcitabine in urothelial BCa.

Genome-wide association studies (GWAS) of bladder cancer have identified a number of susceptibility loci in European populations but have yet to uncover the genetic determinants underlying bladder cancer incidence among other ethnicities. Therefore, we performed the first GWAS in a Chinese cohort comprising 3,406 cases of bladder cancer and 4,645 controls. We identified a new susceptibility locus for bladder cancer at 5q12.3, located in the intron of CWC27 (rs2042329), that was significantly associated with disease risk (OR = 1.40; P = 4.61 × 10−11). However, rs2042329 was not associated with bladder cancer risk in patients of European descent. The rs2042329 risk allele was also related to significantly increased expression levels of CWC27 mRNA and protein in bladder cancer tissues from Chinese patients. Additional functional analyses suggested that CWC27 played an oncogenic role in bladder cancer by inducing cell proliferation and suppressing apoptosis. In conclusion, the identification of a risk-associated locus at 5q12.3 provides new insights into the inherited susceptibility to bladder cancer in Chinese populations and may help to identify high-risk individuals. Cancer Res; 76(11); 3277–84. ©2016 AACR.

Much evidence show that over-expression of epidermal growth factor receptor (EGFR) plays an important role in regulating carcinogenesis. Genetic variations in 3' untranslated region (3'UTR) of gene have been reported to affect gene expression by interfering with microRNAs (miRNAs), which are thought to function as either tumour suppressors or oncogenes by binding to their target mRNA. In this study, we investigated the association between the EGFR 3'UTR 774T>C polymorphism and bladder cancer risk. We used the TaqMan technology to genotype this genetic variant in a hospital-based case-control study of 908 bladder cancer patients and 1239 controls in a Chinese population. We found that the 774CC genotype was associated with a statistically significantly increased risk of bladder cancer [adjusted odds ratio = 1.29, 95% confidence interval = 1.05-1.58], compared with the 774TT/TC genotype, and this increased risk was more pronounced among subgroups of age > 65 years, non-smokers and patients' tumour invasive stage. Furthermore, luciferase assays in T24 cell showed that EGFR 3'UTR 774 T to C substitution could increase the expression of EGFR, which was consistent with the association study finding. Additionally, we also provide evidence that 774T>C polymorphism increasing EGFR expression was not regulated by hsa-miR-214 binding. These findings suggested that EGFR 3'UTR 774T>C polymorphism may contribute to susceptibility to bladder cancer.

Death receptor 4 (DR4) is an important mediator of apoptosis, and its dysfunction may be related to carcinogenesis and cancer development. We hypothesized that common variants in the DR4 gene are associated with risk of bladder cancer and test this hypothesis in a case-control study of 368 bladder cancer patients and 368 cancer-free controls. We genotyped six tagging single nucleotide polymorphisms (tagSNPs) in these subjects and found a significantly increased risk of bladder cancer associated with the SNP1-397GT/TT genotype (adjusted OR=1.55; 95% CI=1.15-2.09) compared with the GG genotype. This increased risk was more pronounced for superficial bladder cancer. A luciferase assay, performed in vitro, revealed that the -397T allele had a lower transcriptional activity than the -397G allele. Multifactor dimensionality reduction (MDR) analysis indicated that the two-factor model including -397G>T and pack-years of smoking was best for predicting bladder cancer risk. Moreover, a significant additive (but not multiplicative) interaction, was found between the -397G>T polymorphism and smoking on bladder cancer risk. In conclusion, genetic variants of the DR4 gene may be involved in the etiology of bladder cancer, and our findings need further validation by larger studies.

The first genome-wide association study (GWAS) for bladder cancer has identified a susceptibility locus at 3q28 in the European ancestry. However, the causal variant at 3q28 remains unknown. We conducted a three-stage fine mapping study to identify potential causal variants in the region. A total of 41 single nucleotide polymorphisms (SNPs) across 120 kb at 3q28 were tested for association with bladder cancer risk among 3,094 bladder cancer cases and 3,738 controls. Resequencing and functional assays were further evaluated. The SNP rs35592567 in the 3'-UTR of TP63 was consistently associated with bladder cancer risk in all three stages. In the combined analysis, the T allele of rs35592567 was significantly associated with a decreased risk for bladder cancer (OR = 0.82, 95% CI = 0.75-0.90, P = 9.797 × 10(-6) in the additive model). Biochemical assays revealed that the T allele reduced the post-transcriptional levels of TP63 mediated by interfering binding efficiency of miR-140-5p. In addition, overexpression of miR-140-5p inhibited bladder cancer cell proliferation and attenuated cell migration, invasion and G1 cell-cycle arrest. Together, these results suggest that rs35592567 in TP63 may be a novel causal variant contributing to the susceptibility to bladder cancer, which provides additional insight into the pathogenesis of bladder carcinogenesis.

Recent genome-wide association studies have identified 14 genetic variants associated with bladder cancer in Caucasians. The effects of these risk variants and their cumulative effects in Asian populations are unknown. We genotyped these newly identified variants in a case-control study of 1,050 patients diagnosed with bladder cancer and 1,404 controls in the Chinese population. Odds rations (ORs) and 95% confidence intervals (CIs) were computed by logistic regression, and cumulative effect of risk alleles were evaluated. Overall, seven of the 14 variants were significantly associated with bladder cancer risk (p = 9.763 × 10(-3) for rs9642880 at 8q24.21, p = 3.004 × 10(-3) for rs2294008 at 8q24.3, p = 0.012 for rs798766 at 4p16.3, p = 0.034 for rs1495741 at 8p22, p = 2.306 × 10(-4) for GSTM1, p = 8.507 × 10(-8) for rs17674580 at 18q12.3, p = 7.179 × 10(-4) for rs10936599 at 3q26.2) and the odds ratios (ORs) ranged from 1.13 to 1.65. Moreover, there were a significant increased risk for bladder cancer positively correlated numbers of risk alleles and smoking status (Ptrend = 7.060 × 10(-16) ). However, no allelic interaction effects on bladder cancer risk were observed between cumulative effects of variants and clinical characteristics. These findings suggest that seven bladder cancer risk-associated variants (rs9642880, rs2294008, rs798766, rs1495741, GSTM1 null, rs17674580 and rs10936599) may be used, collectively, to effectively measure inherited risk for bladder cancer.

PURPOSE: miRNAs can regulate the biological processes, including differentiation, proliferation and apoptosis. DICER and DROSHA are two members of RNase III family, playing pivotal roles in the pathway of miRNAs biogenesis. In this study, we hypothesized that genetic variations of the DICER and DROSHA genes were associated with the bladder cancer risk. EXPERIMENTAL DESIGN: We performed a case-control study of 685 bladder cancer cases and 730 controls to investigate the association between the seven functional SNPs of DICER and DROSHA genes and bladder cancer risk. We then evaluated the functionality of the important SNPs. RESULTS: We found that rs10719T>C polymorphism located in 3' untranslated region (UTR) of DROSHA gene was associated with the increased risk of bladder cancer. Stratified analysis suggested that rs10719TC/CC genotype can increase risk of bladder cancer among male patients (Adjusted OR = 1.34, 95% CI = 1.05-1.70, P = 0.018), and ever smokers (1.56, 1.14-2.14, 0.006), compared with TT genotype. Furthermore, DROSHA rs10719T>C polymorphism was predicted to regulate the binding activity of hsa-miR-27a/b. Luciferase reported gene assay confirmed that rs10719 T to G substitution disrupted the binding site for hsa-miR-27b, resulting the increased levels of DROSHA protein. CONCLUSIONS: Taken together, these findings suggested that DROSHA rs10719T>C polymorphism may be associated with bladder cancer risk in a Chinese population, and hsa-miR-27b can influence the expression of DROSHA protein by binding with 3'UTR.

miRNAs play important roles in numerous cellular processes, including development, proliferation, apoptosis, and carcinogenesis. Because altered expression and function of miRNAs has been observed in bladder cancer, we investigated whether genetic variations in miRNAs are associated with bladder cancer risk and prognosis. Using bioinformatics tools, we selected five single-nucleotide polymorphisms located in miRNAs and used these to evaluate miRNA–disease associations in a two-stage model, consisting of 1,019 bladder cancer cases and 1,182 controls (683 cases and 728 controls in the training set and 336 cases and 454 controls in the test set). We found that miR-146a rs2910164 C allele was associated with significantly decreased risk of bladder cancer in both the training and test sets, as well as the combined set [OR = 0.80, 95% confidence interval (CI) = 0.71–0.90, P = 2.92 × 10−4]. Furthermore, the rs2910164 GC/CC genotypes conferred a significantly reduced risk of recurrence, compared with the GG genotype (P = 0.016). Functional analysis revealed that miR-146a rs2910164 C allele inhibited cell proliferation and significantly downregulated expression of IRAK1 and TRAF6 in bladder cancer cells. Additional examination of 64 bladder cancer tissues showed that individuals carrying the C allele had increased expression levels of miR-146a compared with those carrying the G allele (P = 0.010). Taken together, our findings show that miR-146a rs2910164 plays an important role in the risk and recurrence of bladder cancer, suggesting it may represent a biomarker for risk prevention and therapeutic intervention. Further larger and prospective cohorts are needed to validate our findings. Cancer Res; 72(23); 6173–82. ©2012 AACR.

DNA damaged by exposure to exogenous and endogenous carcinogens could be removed effectively by the base excision repair pathway, in which the XRCC1, APE1, and ADPRT genes play a key role. Genetic variations in these important genes may alter repair function and contribute to cancer risk. We hypothesized that XRCC1, APE1, and ADPRT polymorphisms are associated with risk of bladder cancer. In a hospital-based case-control study of 234 patients with bladder cancer and 253 cancer-free controls, we genotyped the XRCC1-77TC, Arg194Trp, Arg280His, Arg399Gln, APE1-656TG, Asp148Glu, ADPRT-442GA, and Val762Ala polymorphisms using polymerase chain reaction-restriction fragment length polymorphism method. We found an increased risk of bladder cancer associated with the XRCC1 194Trp/Trp and 280Arg/His genotypes (adjusted odds ratio = 3.90, 95% confidence interval = 1.69-8.98 for 194Trp/Trp and 2.53, 1.67-3.83 for 280Arg/His) compared with the 194Arg/Arg and 280Arg/Arg genotypes, respectively. In contrast, the APE1-656GG genotype was associated with a decreased risk of bladder cancer (0.57, 0.33-0.98) compared with the TT genotype. When we evaluated these eight polymorphisms together, we found that the combined genotypes with 9-13 variant (risk) alleles were associated with an increased risk of bladder cancer (2.25, 1.48-3.40) compared with those with 3-8 variants. These findings suggest that the XRCC1 and APE1 polymorphisms may contribute to susceptibility to bladder cancer. Larger studies are warranted to verify these findings.

A recent genome-wide association study identified two common variants that confer susceptibility to bladder cancer. We hypothesized that these variants are associated with risk of bladder cancer in Chinese populations. We genotyped rs9642880 G>T on 8q24 and rs710521 A>G on 3q28 in a two-stage case-control study of bladder cancer to evaluate the association and further examined the expression of MYC. We found that the rs9642880 G>T, but not the rs710521 A>G polymorphism, was associated with an increased risk of bladder cancer. Compared with the rs9642880 GG genotype, the GT/TT genotypes were associated with an odds ratio of 1.65 (95% confidence interval = 1.25-2.17), and this risk was more pronounced in young men and for low-risk tumors. Additional experiments revealed that the rs9642880 GT/TT genotypes were associated with enhanced levels of both MYC mRNA and protein in bladder tissues. Our findings suggested that the rs9642880 G>T polymorphism on 8q24 was independently associated with the risk of bladder cancer in Chinese populations.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays important roles in many biological processes, whereas RUNX3 is a target of TGF-beta-mediated tumor suppressor pathway. In humans, RUNX3 inactivation may lead to the cancer development, including bladder cancer. To determine whether the RUNX3 polymorphisms are associated with risk of bladder cancer, we conducted a case-control study of 368 bladder cancer patients and 368 cancer-free controls to assess the associations between the RUNX3 tagging single-nucleotide polymorphisms (tSNPs) and bladder cancer risk. In the single-locus analysis, we found a significantly increased risk of bladder cancer associated with the SNP7 rs760805 AA genotype (adjusted odds ratio = 1.97, 95% confidence interval = 1.44-2.69), compared with the AT/TT genotype. Haplotype-based association analysis revealed that the increased risk of bladder cancer was significantly associated with two haplotypes TATCCCAAAA (2.37, 1.16-4.83) and AGCTTGAGAG (2.70, 1.08-6.72) that included the rs760805 A allele. Multifactor dimensionality reduction (MDR) analysis identified a significant more than multiplicative interaction between the SNP7 rs760805 AA and smoking and an additive interaction between the SNP3 rs11249206 TT and smoking on bladder cancer risk. The SNP3 rs11249206, SNP5 rs1395621, SNP7 rs760805, SNP8 rs2236852 and the trichotomized cumulative smoking were the five factors best predicted by the MDR models. When the variables were combined and dichotomized and fitted into the MDR model, the subjects carrying the combined risk stratum had a significantly increased risk for bladder cancer (6.37, 4.57-8.87, P = 7.03 x 10(-28)). These results suggested that the genetic variants in RUNX3 may modulate the risk of bladder cancer.

Purpose: MDM2 is believed to regulate the p53 level in modulating DNA repair, cell cycle control, cell growth, and apoptosis. We hypothesize that genetic variants in the MDM2 gene are associated with risk of bladder cancer. Experimental Design: We first conducted a case-control study of 234 bladder cancer cases and 253 cancer-free controls, using the haplotype-based tagging single nucleotide polymorphism (SNP) approach involving 13 common SNPs initially identified in 100 control subjects. We then examined the functionality of the important SNP. Results: We found that the C1797G polymorphism in the MDM2 promoter region is an important SNP because its homozygous variant genotype, but none of the haplotypes, was associated with risk of bladder cancer. Electrophoretic mobility shift assay indicated that the 1797C to 1797G transition within the CAAT/enhancer binding protein α (C/EBP α) core sequence greatly enhanced the C/EBPα binding affinity to the promoter region. The in vitro luciferase assays in various cell lines further showed an increased transcriptional activity of the 1797G allele compared with the 1797C allele. Additional experiments with tumor tissues revealed that the transcriptional activator C/EBPα containing the 1797G allele increased levels of the MDM2 mRNA and protein in bladder tumor tissues. Conclusions: These data suggested that the novel MDM2 promoter C1797G polymorphism may affect the MDM2 activity by altering the C/EBPα binding affinity to the promoter and, thus, may be a marker for genetic susceptibility to bladder cancer in Chinese populations. Further validation of the functionality of the MDM2 C1797G polymorphism and its association with risk of bladder and other cancers in other ethnic populations is warranted.

Folate deficiency due to the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) variants leads to carcinogenesis by affecting DNA synthesis, repair, and methylation. We hypothesized that the MTHFR C677T, A1298C, and MS A2756G polymorphisms are associated with risk of bladder cancer. In a case–control study of 239 bladder cancer cases and 250 cancer-free controls, we found that the MTHFR 677TT genotype was statistically significantly associated with an increased risk of bladder cancer compared with the 677CC genotype (OR = 2.06, 95% CI = 1.16–3.64). Furthermore, the TA haplotype was associated with a significantly increased bladder cancer risk (OR = 1.38, 95% CI = 1.05–1.81) than was the most common haplotype, CA (e.g., CA denotes MTHFR 677C -1298A). We also found that the combined genotypes with 4–6 variant (risk) alleles (i.e., MTHFR 677T, 1298A, and MS 2756G alleles) were associated with an increased risk of bladder cancer (OR = 1.62, 95% CI = 1.03–2.53) compared with those with 0–3 variants, and this increased risk was more pronounced among subgroup of older people (OR = 1.71, 95% CI = 1.03–2.83). A meta-analysis of seven studies did not show a significant risk of bladder cancer in the MTHFR polymorphisms. The MTHFR polymorphisms and their haplotypes appear to jointly contribute to risk of bladder cancer.

X-ray repair cross-complementing 5 (XRCC5) is a gene involved in repair of DNA double-strand breaks. Abnormal expression of the XRCC5 protein is associated with genomic instability and an increased incidence of cancers. In our study, a polymorphism with a variable number of tandem repeats (21-bp repeat elements at position −201 to −160 relative to the initiation of transcription) in the promoter of XRCC5 was identified. As determined with gel-shift and super-shift assays, the binding affinity of the transcription factor Sp1 to the allele with two 21-bp repeats was greater than that for the allele with one 21-bp repeat. As established with a reporter assay, plasmids containing zero or one repeat element had higher transcriptional activities than plasmids containing two repeat elements. Furthermore, fewer tandem repeats in the promoter of XRCC5 was associated with enhanced levels of the XRCC5 protein in bladder cancer patients. Although, in a case–control study, the different genotypes were not associated with the risk of bladder cancer, individuals not carrying the two tandem repeats allele had an increased risk of bladder cancer compared with those carrying the allele with two repeats. These results indicated that, at least in a population in southeastern China, this polymorphism in the promoter of XRCC5 could regulate the expression of XRCC5 and thereby contribute to susceptibility to bladder cancer.