Human Bladder Epithelial Cells Research Articles

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

Mitochondrial targeting improves the selectivity of singlet-oxygen cleavable prodrugs in NMIBC treatment.

Mitochondria play an essential role in cancer treatment by providing apoptotic signals. Hexyl aminolevulinate, an FDA-approved diagnosis for non-muscle invasive bladder cancer, induces the production of protoporphyrin IX (PpIX) preferentially by mitochondria in cancer cells. Photosensitizer PpIX upon illumination can release active chemotherapy drugs from singlet oxygen-activatable prodrugs. Prodrugs placed close enough to PpIX formed in mitochondria can improve the antitumor efficiency of PpIX-PDT. The preferred uptake of prodrugs by cancer cells and tumors can further enhance the selective damage of cancer cells over non-cancer cells and surrounding normal tissues. Mitochondriotropic prodrugs of anticancer drugs, such as paclitaxel and SN-38, were synthesized using rhodamine, a mitochondrial-targeting moiety. Invitro, the mitochondrial targeting helped achieve preferential cellular uptake in cancer cells. In RT112 cells (human bladder cancer cells), intracellular prodrug concentrations were 2-3 times higher than the intracellular prodrug concentrations in BdEC cells (human bladder epithelial cells), after 2 h incubation. In an orthotopic rat bladder tumor model, mitochondria-targeted prodrugs achieved as much as 34 times higher prodrug diffusion in the tumor area compared to the nontumor bladder area. Overall, mitochondria targeting made prodrugs more effective in targeting cancer cells and tumors over non-tumor areas, thereby reducing nonspecific toxicity.

Probiotic Limosilactobacillus reuteri KUB-AC5 decreases urothelial cell invasion and enhances macrophage killing of uropathogenic Escherichia coli in vitro study.

Bacterial urinary tract infections (UTI) are among the most common infectious diseases worldwide. The rise of multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) UTI cases is a significant threat to healthcare systems. Several probiotic bacteria have been proposed as an alternative to combat MDR UTI. Lactic acid bacteria in the genus Limosilactobacillus are some of the most studied and used probiotics. However, strain-specific effects play a critical role in probiotic properties. L. reuteri KUB-AC5 (AC5), isolated from the chicken gut, confers antimicrobial and immunobiotic effects against some human pathogens. However, the antibacterial and immune modulatory effects of AC5 on UPEC have never been explored. Here, we investigated both the direct and indirect effects of AC5 against UPEC isolates (UTI89, CFT073, and clinical MDR UPEC AT31) in vitro. Using a spot-on lawn, agar-well diffusion, and competitive growth assays, we found that viable AC5 cells and cell-free components of this probiotic significantly reduced the UPEC growth of all strains tested. The human bladder epithelial cell line UM-UC-3 was used to assess the adhesion and pathogen-attachment inhibition properties of AC5 on UPEC. Our data showed that AC5 can attach to UM-UC-3 and decrease UPEC attachment in a dose-dependent manner. Pretreatment of UPEC-infected murine macrophage RAW264.7 cells with viable AC5 (multiplicity of infection, MOI = 1) for 24 hours enhanced macrophage-killing activity and increased proinflammatory (Nos2, Il6, and Tnfa) and anti-inflammatory (Il10) gene expression. These findings indicate the gut-derived AC5 probiotic could be a potential urogenital probiotic against MDR UTI.

The alternation of halobenzoquinone disinfection byproduct on toxicogenomics of DNA damage and repair in uroepithelial cells

Halobenzoquinones (HBQs) were recently discovered as an emerging class of drinking water disinfection byproducts with carcinogenic concern. However, the molecular mechanism underlying HBQs-induced DNA damage is not clear. In this study, we integrated in vitro genotoxicity, computational toxicology, and the quantitative toxicogenomic analysis of HBQs on DNA damage/repair pathways in human bladder epithelial cells SV-HUC-1. The results showed that HBQs could induce cytotoxicity with the descending order as 2,6-DIBQ > 2,6-DCBQ ≈ 2,6-DBBQ. Also, HBQs can increase DNA damage in SV-HUC-1 cells and thus generate genotoxicity. However, there is no significant difference in genotoxicity among the three HBQs. The results of molecular docking and molecular dynamics simulation further confirmed that HBQs had high binding fractions and stability to DNA. Toxicogenomic analysis indicated that HBQs interfered with DNA repair pathways, mainly affecting base excision repair, nucleotide excision repair and homologous recombination repair. These results have provided new insights into the underlying molecular mechanisms of HBQs-induced DNA damage, and contributed to the understanding of the relationship between exposure to DBPs and risks of developing bladder cancer.

β-defensin 2 protects against Escherichia coli-induced acute urinary tract infection by downregulating β-catenin

β-defensin 2 (BD2) is a small cationic peptide that exerts a critical role in host defense against bacterial infections. Here, we aimed to investigate the role of BD2 in protecting against acute urinary tract infection (AUTI) caused by Escherichia coli (UPEC). Here, LPS-induced human urinary bladder epithelial cell (HCV-29) model and UPEC-induced mice model were used for assessing AUTI. Visceral organ lesions of mice following treatment was assessed by HE staining. Cell viability was determined by CCK-8 assay. Permeability in HCV-29 cells was analyzed in Transwell assay. Expression of inflammatory factors (IL-1β, IL-6, and TNF-α) was measured by ELISA assay. The levels of BD2, β-catenin and tight-junction proteins (ZO-1, Occludin, and Claudin-1) were detected by RT-qPCR, western blotting, immunofluorescence or immunohistochemistry. Our results showed that BD2 was lowly expressed and β-catenin showed the reverse trend in response to bacterial infection in vitro and in vivo. BD2 overexpression alleviated the decreased cell viability, increased cell permeability, upregulation of inflammatory factors, downregulation of tight-junction protein and high β-catenin expression in LPS-induced HCV-29 cells, which may contribute to the negative regulation of β-catenin expression. Furthermore, BD2 overexpression attenuated the bacterial infection of tissues, high levels of inflammatory factors and β-catenin, and low levels of tight-junction proteins in mice stimulated with UPEC. This study showed that BD2 played a crucial role in protecting against AUTI caused by gram-negative bacteria through suppressing β-catenin expression. Targeting BD2 may provide a potential therapeutic approach for the prevention and treatment of AUTI.

Oxybutynin ameliorates LPS-induced inflammatory response in human bladder epithelial cells.

Urinary tract infection (UTI) mainly results from bacterial infections in the urinary tract and markedly impacts the normal lives of millions of patients worldwide. The infection and damage to urethral epithelial cells is the first and key step of UTI development and is a critical target for treating clinical UTI. Oxybutynin, an agent for treating urinary incontinence, is recently claimed with protective effects on bladder ultrastructure. Our study will assess the impact of Oxybutynin on inflammation in lipopolysaccharide (LPS)-stimulated bladder epithelial cells. Bladder epithelial T24 cells were treated with 1 μg/mL LPS with or without 10 and 20 μM Oxybutynin for 24 h. Increased levels of oxidative stress (OS) biomarkers, such as reactive oxygen species, 8-hydroxy-2'-deoxyguanosine, malondialdehyde, as well as upregulated inducible nitric oxide synthase and promoted release of nitric oxide, were observed in LPS-managed T24 cells, all of which were signally suppressed by Oxybutynin. Furthermore, severe inflammatory responses, including enhanced release of cytokines, upregulated matrix metallopeptidase-2 (MMP-2) and MMP-9, and raised monocyte chemoattractant protein-1 level, were found in LPS-challenged T24 cells, which were markedly reversed by Oxybutynin. Moreover, the activated toll-1ike receptor 4/nuclear factor-κB pathway observed in LPS-managed T24 cells was repressed by Oxybutynin. Collectively, Oxybutynin mitigated LPS-induced inflammatory response in human bladder epithelial cells.

The Role of NLRP3 in Regulation of Antimicrobial Peptides and Estrogen Signaling in UPEC-Infected Bladder Epithelial Cells.

The NLRP3 inflammasome, estrogen and antimicrobial peptides have all been found to have a vital role in the protection of the bladder urothelium. However, the interdependence between these protective factors during a bladder infection is currently unknown. Our aim was to investigate the role of NLRP3 in the regulation of antimicrobial peptides and estrogen signaling in bladder epithelial cells during a UPEC infection. Human bladder epithelial cells and CRISPR/Cas9-generated NLRP3-deficient cells were stimulated with the UPEC strain CFT073 and estradiol. The gene and protein expression were evaluated with microarray, qRT-PCR, western blot and ELISA. Microarray results showed that the expression of most antimicrobial peptides was reduced in CFT073-infected NLRP3-deficient cells compared to Cas9 control cells. Conditioned medium from NLRP3-deficient cells also lost the ability to suppress CFT073 growth. Moreover, NLRP3-deficient cells had lower basal release of Beta-defensin-1, Beta-defensin-2 and RNase7. The ability of estradiol to induce an increased expression of antimicrobial peptides was also abrogated in NLRP3-deficient cells. The decreased antimicrobial peptide expression might be linked to the observed reduced expression and activity of estradiol receptor beta in NLRP3-deficient cells. This study suggests that NLRP3 may regulate the release and expression of antimicrobial peptides and affect estrogen signaling in bladder epithelial cells.

Tolterodine ameliorates inflammatory response and ferroptosis against LPS in human bladder epithelial cells.

Bacterial endotoxin lipopolysaccharide (LPS)-induced inflammatory response and ferroptosis play an important role in urinary tract infections. Tolterodine has been used as a urinary tract antispasmodic and anticholinergic agent. However, the effects of Tolterodine against LPS-induced insults in human bladder epithelial cells (hBECs) have not been reported before. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays to determine the cell viability, reactive oxygen species (ROS) and malondialdehyde level detection were used to determine the level of oxidative stress, enzyme-linked immunosorbent assay and Western blot analysis were used to detect the protein level. In the current study, we found that Tolterodine ameliorated LPS-induced production of ROS and lipid oxidation in hBECs. Interestingly, Tolterodine inhibited the production of interleukin 6, interleukin-1β, and tumor necrosis factor α. Also, Tolterodine reduced the levels of Fe2+ and suppressed ferroptosis by reducing the levels of glutathione peroxidase 4, prostaglandin-endoperoxide synthase 2, and acyl-CoA synthetase long-chain family member 4 in LPS-challenged bladder epithelial cells. Mechanistically, it was shown that Tolterodine restored the nuclear factor E2-related factor 2 (Nrf2)/nuclear factor-κB signaling. Importantly, inhibition of Nrf2 with its specific inhibitor ML385 abolished the protective effects of Tolterodine in the inflammatory response and ferroptosis, suggesting that the effects of Tolterodine are mediated by Nrf2. Based on these findings, we conclude that Tolterodine might serve as a promising agent for the treatment of LPS-induced bladder inflammation.

Circ-SFMBT2 sponges miR-224-5p to induce ketamine-induced cystitis by up-regulating metadherin (MTDH).

There is increasing evidence that circular RNAs (circRNAs) play significant roles in various biological processes, yet few reports have examined their roles and molecular mechanisms in ketamine-induced cystitis (KIC). This study examines the possible molecular mechanisms underlying the circRNA-microRNA-mRNA regulatory network in the development of KIC. Transcriptome data were collected, and bioinformatics analysis was conducted to create a circRNA-miRNA-mRNA regulatory network (ceRNA network) associated with the occurrence of KIC. Human bladder epithelial cells (SV-HUC-1) were used in in vitro cell assays. The binding affinity among circ-SFMBT2, miR-224-5p, and Metadherin (MTDH) was identified. To investigate the effects of circ-SFMBT2/miR-224-5p/MTDH on bladder function, KIC mouse models were induced by intraperitoneal injection of ketamine, and gain- or loss-of-function experiments were conducted. Our results demonstrate that MTDH may be a key gene involved in the occurrence of KIC. Both bioinformatics analysis and in vitro cell assays verified that circ-SFMBT2 can competitively bind to miR-224-5p, and miR-224-5p can target and inhibit MTDH. In the bladder tissues of KIC mice, circ-SFMBT2 and MTDH were up-regulated, while miR-224-5p was down-regulated. Animal experiments further confirmed that circ-SFMBT2 can up-regulate MTDH expression by sponging miR-224-5p, thereby exacerbating bladder dysfunction in KIC mice. This study proved that circ-SFMBT2 up-regulates MTDH by competitively binding to miR-224-5p, thereby exacerbating the bladder dysfunction of KIC.

Serpina3n/serpina3 alleviates cyclophosphamide-induced interstitial cystitis by activating the Wnt/β-catenin signal

Background/objectiveSerpina3n/Serpina3 has been identified to be implicated in inflammatory diseases, but its role in interstitial cystitis/bladder pain syndrome (IC/BPS) remains unknown. Here, we aimed to reveal serpina3n/serpina3 role in IC/BPS in vivo and in vitro.MethodsThe IC/BPS model in mice was induced by intraperitoneal injection of 150 mg/kg of cyclophosphamide (CYP). HE and toluidine blue staining were used for histology assessment. Serpina3n/serpina3 expression in the bladder tissues from IC/BPS patients and mouse models were determined by qPCR, immunohistochemistry and western blotting. XAV-939 treatment was applied to inhibit β-catenin activation. Serpina3 role in modulating the growth and apoptosis of HBlEpCs, a human primary bladder epithelial cell line, was assessed by CCK-8 and flow cytometry assays.ResultsSerpina3n/serpina3 expression was decreased in both human and mice bladder tissues with IC/BPS. Upregulation of serpina3n significantly alleviated CYP-induced bladder injury, with decreased mast cells and pro-inflammatory factor levels, including IL-1β, IL-6, and TNF-α, while increased IL-10 level. In addition, serpina3 overexpression inhibited the apoptosis of HBlEpCs, and increased cell growth. In mechanism, we found that serpina3 overexpression promoted the activation of wnt/β-catenin signaling. And, the inhibition of wnt/β-catenin signaling with XAV-939 abolished serpina3n/serpina3 role in protecting bladder tissues from CYP-induced cystitis, as well as inhibiting HBlEpC apoptosis.ConclusionSerpina3n/serpina3 expression was decreased in IC/BPS. Overexpression of serpina3n could alleviate CYP-induced IC/BPS by activating the Wnt/β-catenin signal. This study may provide a new therapeutic strategy for IC/BPS.

Rosmarinic acid ameliorates HCl-induced cystitis in rats.

Shiso (Perilla frutescens var crispa f. purprea) is a traditional medicinal herb that exerts anti-inflammatory effects and alleviates lower urinary tract symptoms. In this study, we examined the effects of rosmarinic acid, a major polyphenol in shiso, on urinary function and the bladder in a rat hydrochloric acid-induced cystitis model. Sprague-Dawley rats were administered intravesically with hydrochloric acid or saline solution (control) to induce cystitis. Afterwards, the rats were administered orally with distilled water or rosmarinic acid for three days and then the intravesical pressure was measured, a stretch stimulation test was performed using the harvested bladder, and histological and biochemical analyses were performed. In addition, we investigated the effects of rosmarinic acid on the expression of inflammation-related molecules in normal human bladder epithelial cells. Rosmarinic acid ameliorated hydrochloric acid-induced shortening of micturition interval by 49%. In hydrochloric acid-treated bladders, significantly more prostaglandin E2 was released after stretching; however, rosmarinic acid suppressed its release to control levels. Rosmarinic acid also reduced hydrochloric acid-induced epithelial thickening and the levels of inflammatory molecules in the bladder. Furthermore, rosmarinic acid suppressed interleukin 1β-induced increases in Cox2 and Il6 expression in bladder epithelial cells. These findings indicate that rosmarinic acid can ameliorate hydrochloric acid-induced cystitis in rats and that these effects are due, at least in part, to its anti-inflammatory effects on the bladder and inhibition of stretch-induced prostaglandin E2 release.

Glutamate Transporters GltS, GltP and GltI Are Involved in Escherichia coli Tolerance In Vitro and Pathogenicity in Mouse Urinary Tract Infections.

To verify the roles of GltS, GltP, and GltI in E. coli tolerance and pathogenicity, we quantified and compared the relative abundance of gltS, gltP, and gltI in log-phase and stationary-phase E. coli and constructed their knockout mutant strains in E. coli BW25113 and uropathogenic E. coli (UPEC) separately, followed by analysis of their abilities to tolerate antibiotics and stressors, their capacity for adhesion to and invasion of human bladder epithelial cells, and their survival ability in mouse urinary tracts. Our results showed that gltS, gltP, and gltI transcripts were higher in stationary phase E. coli than in log-phase incubation. Furthermore, deletion of gltS, gltP, and gltI genes in E. coli BW25113 results in decreased tolerance to antibiotics (levofloxacin and ofloxacin) and stressors (acid pH, hyperosmosis, and heat), and loss of gltS, gltP, and gltI in uropathogenic E. coli UTI89 caused attenuated adhesion and invasion in human bladder epithelial cells and markedly reduced survival in mice. The results showed the important roles of the glutamate transporter genes gltI, gltP, and gltS in E. coli tolerance to antibiotics (levofloxacin and ofloxacin) and stressors (acid pH, hyperosmosis, and heat) in vitro and in pathogenicity in mouse urinary tracts and human bladder epithelial cells, as shown by reduced survival and colonization, which improves our understanding of the molecular mechanisms of bacterial tolerance and pathogenicity.

Insightful Improvement in the Design of Potent Uropathogenic E. coli FimH Antagonists

Selective antiadhesion antagonists of Uropathogenic Escherichia coli (UPEC) type-1 Fimbrial adhesin (FimH) are attractive alternatives for antibiotic therapies and prophylaxes against acute or recurrent urinary tract infections (UTIs) caused by UPECs. A rational small library of FimH antagonists based on previously described C-linked allyl α-D-mannopyranoside was synthesized using Heck cross-coupling reaction using a series of iodoaryl derivatives. This work reports two new members of FimH antagonist amongst the above family with sub nanomolar affinity. The resulting hydrophobic aglycones, including constrained alkene and aryl groups, were designed to provide additional favorable binding interactions with the so-called FimH "tyrosine gate". The newly synthesized C-linked glycomimetic antagonists, having a hydrolytically stable anomeric linkage, exhibited improved binding when compared to previously published analogs, as demonstrated by affinity measurement through interactions by FimH lectin. The crystal structure of FimH co-crystallized with one of the nanomolar antagonists revealed the binding mode of this inhibitor into the active site of the tyrosine gate. In addition, selected mannopyranoside constructs neither affected bacterial growth or cell viability nor interfered with antibiotic activity. C-linked mannoside antagonists were effective in decreasing bacterial adhesion to human bladder epithelial cells (HTB-9). Therefore, these molecules constituted additional therapeutic candidates' worth further development in the search for potent anti-adhesive drugs against infections caused by UPEC.

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.

Selective inhibition of integrin αvβ6 leads to rapid induction of urinary bladder tumors in cynomolgus macaques.

Administration of a novel and selective small molecule integrin αvβ6 inhibitor, MORF-627, to young cynomolgus monkeys for 28 days resulted in the rapid induction of epithelial proliferative changes in the urinary bladder of 2 animals, in the absence of test agent genotoxicity. Microscopic findings included suburothelial infiltration by irregular nests and/or trabeculae of epithelial cells, variable cytologic atypia, and high mitotic rate, without invasion into the tunica muscularis. Morphologic features and patterns of tumor growth were consistent with a diagnosis of early-stage invasive urothelial carcinoma. Ki67 immunohistochemistry demonstrated diffusely increased epithelial proliferation in the urinary bladder of several monkeys, including those with tumors, and αvβ6 was expressed in some epithelial tissues, including urinary bladder, in monkeys and humans. Spontaneous urothelial carcinomas are extremely unusual in young healthy monkeys, suggesting a direct link of the finding to the test agent. Inhibition of integrin αvβ6 is intended to locally and selectively block transforming growth factor beta (TGF-β) signaling, which is implicated in epithelial proliferative disorders. Subsequent in vitro studies using a panel of integrin αvβ6 inhibitors in human bladder epithelial cells replicated the increased urothelial proliferation observed in monkeys and was reversed through exogenous application of TGF-β. Moreover, analysis of in vivo models of liver and lung fibrosis revealed evidence of epithelial hyperplasia and cell cycle dysregulation in mice treated with integrin αvβ6 or TGF-β receptor I inhibitors. The cumulative evidence suggests a direct link between integrin αvβ6 inhibition and decreased TGF-β signaling in the local bladder environment, with implications for epithelial proliferation and carcinogenesis.

Construction of an lncRNA model for prognostic prediction of bladder cancer

ObjectiveWe aimed to investigate the role and potential mechanisms of long non-coding RNAs (lncRNAs) in bladder cancer (BC), as well as determine their prognostic value.MethodsLncRNA expression data and clinical data from BC patients were downloaded from The Cancer Genome Atlas (TCGA) database. R software was used to carry out principal component analysis (PCA), differential analysis, and prognostic analysis. Lasso regression and multivariate Cox regression analyses were performed to identify potential prognostic genes. The expression of five identified genes and their correlation with prognosis were verified using TCGA and GSE13507 datasets. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to confirm the expression of these five genes in cell lines (two human BC cell lines and one human bladder epithelial cell line) and tissues (84 pairs of BC tissues and the corresponding paracancerous tissues). Risk scores that had been generated from the five genes and their prognostic ability were assessed by receiver operating characteristic (ROC) and Kaplan–Meier (KM) curves. Co-expressed genes were screened by WGCNA and analyzed by GO and KEGG, while functional enrichment and immune infiltration analyses were performed using STRING (https://cn.string-db.org/) and TIMER2.0 (http://timer.cistrome.org/) online tools, respectively.ResultsCYP4F8, FAR2P1, LINC01518, LINC01764, and DTNA were identified as potential prognostic genes. We found that these five genes were differentially expressed in BC tissue, as well as in BC cell lines, and were significantly correlated with the prognosis of BC patients. KM analysis considering risk scores as independent parameters revealed differences in overall survival (OS) by subgroups. The ROC curve revealed that a combined model consisting of all five genes had good predictive ability at 1, 3, and 5 years. GO and KEGG analyses of 567 co-expressed genes revealed that these genes were significantly associated with muscle function.ConclusionLncRNAs can be good predictors of BC development and prognosis, and may act as potential tumor markers and therapeutic targets that may be beneficial in helping clinicians decide the most effective treatment strategies.

Low-dose arsenite causes overexpression of EGF, TGFα, and HSP90 through Trx1-TXNIP-NLRP3 axis mediated signaling pathways in the human bladder epithelial cells

Epidemiological studies have demonstrated an increased incidence of bladder cancer in arseniasis- endemic areas; however, the precise molecular mechanisms remain unknown. Our previous results have shown that the protein levels of EGF, TGFα, and HSP90 in arsenite-treated bladder uroepithelial cells increased markedly and contributed to hyperactivation of EGF receptors. The aim of this study was to further explore the regulatory ways underlying overexpression of EGF, TGFα, and HSP90 in these cells. The present results showed that both Trx and GSH systems were stimulated in arsenite-treated cells, and ROS levels in 2 μM arsenite-treated cells did not changed obviously; however, ROS levels in 4 μM arsenite-treated cells increased significantly. By using the antioxidant and specific inhibitors, we found that in 2 μM arsenite-treated cells, JNK/NF-κB signaling pathway was involved in overexpression of EGF and TGFα, and ERK/NF-κB signaling pathway contributed to HSP90 overexpression, however in 4 μM arsenite-treated cells, both ERK/ and JNK/NF-κB signaling pathways were involved in overexpression of EGF, TGFα, and HSP90, and PI3K/AKT/NF-κB signaling pathway contributed to overexpression of EGF and TGFα. Furthermore, our results also showed that the Trx1-TXNIP-NLRP3 axis was activated in arsenite-treated cells, and played a pivotal role in activation of the signaling pathways involved in overexpression of EGF, TGFα, and HSP90. In conclusion, the Trx1-TXNIP-NLRP3 axis might be activated by arsenite-induced redox imbalance in bladder uroepithelial cells, and mediate the activation of signaling pathways involved in overexpression of EGF, TGFα, and HSP90.

Targeting Host Tyrosine Kinase Receptor EPHA2 Signaling Affects Uropathogen Infection in Human Bladder Epithelial Cells.

Urinary tract infections (UTIs) affect a major proportion of the world population but have limited non-antibiotic-based therapeutic and preventative strategies against UTIs. Facultative intracellular uropathogens such as strains of uropathogenic E. coli, K. pneumoniae, E. faecalis, E. cloacae are well-known uropathogens causing UTIs. These pathogens manipulate several host-signaling pathways during infection, which contributes to recurrent UTIs and inappropriate antibiotic application. Since host cell receptor tyrosine kinases (RTKs) are critical for the entry, survival and replication of intracellular pathogens, we investigated whether different uropathogens require host EPHA2 receptors for their intracellular survival using a cell culture model of intracellular infection in human bladder epithelial cells (BECs). Infection of BECs with seven different uropathogens enhanced the expression levels and activation of EPHA2. The significance of EPHA2 signaling for uropathogen infection was investigated by silencing EPHA2 expression using RNA interference or by inhibiting the kinase activity of EPHA2 using small-molecule compounds such as dasatinib or ALW-II-41-27. Both preventive and therapeutic tyrosine kinase inhibition significantly reduced the intracellular bacterial load. Thus, our results demonstrate the involvement of host cell EPHA2 receptor during intracellular uropathogen infection of BECs, and targeting RTK activity is a viable non-antibiotic therapeutic strategy for managing recurrent UTIs.

Chronic exposure to microcystin-leucine-arginine induces epithelial hyperplasia and inflammation in the mouse bladder.

Microcystin-leucine-arginine (MC-LR) is a cyclic heptapeptide compound produced by cyanobacteria with strong cytotoxicity. Previous studies have confirmed that MC-LR could exert toxic effects on the genitourinary system, but there are few reports about its toxicity to the bladder. In this study, we investigated the effects of MC-LR on mouse bladder and human bladder epithelial cells (SV-HUC-1 cells). We observed that the bladder weight and the number of bladder epithelial cells were markedly increased in mice following chronic low-dose exposure to MC-LR. Further investigation showed that MC-LR activates AKT/NF-kB signaling pathway to induce the production of proinflammatory cytokines TNF-α and IL-6. In addition, the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in bladder tissue was increased and the relative migration and invasion capacities of SV-HUC-1 cells were enhanced upon exposure to MC-LR. In conclusion, these results suggest that chronic exposure to MC-LR induced epithelial hyperplasia and inflammation, upregulated the expression of matrix metalloproteinases (MMPs) and promoted the migration and invasion of bladder epithelial cells, which provides a basis for further exploring the potential mechanism by which environmental factors increasing the risk of bladder cancer.

Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus invitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.