Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains encode an annotated intimin-like adhesin ( ila ) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that encodes three predicted intimin-like genes sinH , sinI , and ratA . We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin-encoding proteins in CFT073 are more closely related to invasin proteins found in Salmonella . Deletion of the individual sinH , sinI , and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro . On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo . Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro , coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like, rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

Insulin Downregulated the Infection of Uropathogenic Escherichia coli (UPEC) in Bladder Cells in a High-Glucose Environment through JAK/STAT Signaling Pathway

Diabetic individuals have a higher incidence of urinary tract infection (UTI) than non-diabetic individuals, and also require longer treatment. We evaluated the effects of insulin pretreatment on the regulation of JAK/STAT transduction pathways in UPEC-infected bladder cells in a high-glucose environment. A bladder cell model with GFP-UPEC and fluorescent-labeled TLR4, STAT1, STAT3, and insulin receptor antibodies, was used to evaluate the relationship between insulin receptor signaling, TLR-4-mediated, and JAK/STAT-dependent pathways. Pretreatment with 20 and 40 µg/mL insulin for 24 h significantly and dose-dependently reduced UPEC infection in SV-HUC-1 cells. Additionally, the expression levels of STAT1 and STAT3 were downregulated in a dose-dependent manner. However, insulin receptor (IR) expression was not affected by insulin pretreatment. Our results showed that insulin-mediated reduction of UPEC infection in a high-glucose environment was not only due to the downregulation of JAK1/2 and phosphorylated STAT-1/3, but also because of the decreased expression of TLR-4 proteins and pro-inflammatory IL-6. Here, we demonstrated that insulin reduced not only UPEC infection in bladder epithelial cells, but also inhibited the JAK/STAT transduction pathway during infection in a high-glucose environment. This study provides evidence to support the use of insulin in the treatment of UPEC infection in patients with type 2 diabetes (T2D).

Pseudogene OCT4-pg5 Upregulates OCT4B Expression To Promote Bladder Cancer Progression By Competing With miR-145

Background Octamer-binding transcription factor 4 pseudogene 5 (OCT4-pg5) contributes to tumor progression in many cancer types, but contributions to bladder cancer (BC) have not been investigated. Methods Real-time quantity PCR (RT-qPCR) was performed to measure OCT4-pg5 and OCT4B expressions in different bladder cell lines and different grades of cancer. The effects of OCT4-pg5, OCT4B and miR-145 on proliferation and metastasis were determined by in vitro and in vivo experiments. Luciferase reporter assay was carried out to reveal the interaction among OCT4-pg5, OCT4B and miR-145. Flow cytometry was performed to explore the effects of OCT4-pg5 and OCT4B expression on the cell cycle stage distribution of T24 cells. Results OCT4-pg5 expression was significantly increased in BC cell lines, which was correlated with OCT4B expression and advanced tumor grade. Overexpression of OCT4-pg5 and OCT4B promoted the proliferation and invasion of BC cells, while miR-145 suppressed these activities. Mechanically, OCT4-pg5 3’ untranslated region (3’UTR) competed for miR-145, thereby increasing OCT4B expression. In addition, OCT4-pg5 promoted EMT by activating the Wnt/β-catenin pathway and upregulating the expression levels of matrix metalloproteinases (MMPs) 2 and 9 as well as transcription factors zinc finger E-box binding homeobox (ZEB) 1 and 2. Furthermore, elevated expression of OCT4-pg5 and OCT4B reduced the sensitivity of BC cells to cisplatin by reducing apoptosis and increasing the proportion of cells in G1. Conclusions These findings indicate that OCT4-pg5/miR-145/OCT4B axis promotes the progression of BC by inducing EMT via Wnt/β-catenin pathway and enhances the cisplatin resistance. It could be prospect for the therapeutic approaches for BC.

Early signalling processes in roots play a crucial role in the differential salt tolerance in contrasting Chenopodium quinoa accessions

Significant variation in epidermal bladder cell (EBC) density and salt tolerance (ST) exists amongst quinoa accessions, suggesting that salt sequestration in EBCs is not the only mechanism conferring ST in this halophyte. In order to reveal other traits that may operate in tandem with salt sequestration in EBCs and whether these additional tolerance mechanisms acted mainly at the root or shoot level, two quinoa (Chenopodium quinoa) accessions with contrasting ST and EBC densities (Q30, low ST with high EBC density versus Q68, with high ST and low EBC density) were studied. The results indicate that responses in roots, rather than in shoots, contributed to the greater ST in the accession with low EBC density. In particular, the tolerant accession had improved root plasma membrane integrity and K+ retention in the mature root zone in response to salt. Furthermore, superior ST in the tolerant Q68 was associated with faster and root-specific H2O2 accumulation and reactive oxygen species-induced K+ and Ca2+ fluxes in the root apex within 30 min after NaCl application. This was found to be associated with the constitutive up-regulation of the membrane-localized receptor kinases regulatory protein FERONIA in the tolerant accession. Taken together, this study shows that differential root signalling events upon salt exposure are essential for the halophytic quinoa; the failure to do this limits quinoa adaptation to salinity, independently of salt sequestration in EBCs.

SEC23A Is an Independent Prognostic Biomarker in Bladder Cancer Correlated With MAPK Signaling

Clinical data mining and bioinformatics analysis can be employed effectively to elucidate the function and underlying mechanisms of the gene of interest. Here, we have proposed a framework for the identification and validation of independent biomarkers in human cancer and for mechanistic profiling using gene sets enrichment analysis and pathway analysis. This is followed by validation with in vitro experiments. Using this framework to analyze the clinical relevance of SEC23A, we have discovered the prognostic potential of SEC23A in different cancers and identified SEC23A as an independent prognostic factor for poor prognosis in bladder cancer, which implicates SEC23A, for the first time, as an oncogene. Bioinformatic analyses have elucidated an association between SEC23A expression and the upregulation of the MAPK signaling pathway. Using the T24 human bladder cell line, we confirmed that knockdown of SEC23A expression could effectively impact the MAPK signaling pathway. Further, through PCR verification, we showed that MEF2A, one of the key genes of the MAPK signaling pathway, might be a downstream factor of the SEC23A gene.

Integrative analysis the characterization of peroxiredoxins in pan-cancer

Background Peroxiredoxins (PRDXs) are an antioxidant enzymes protein family involved in several biological functions such as differentiation, cell growth. In addition, previous studies report that PRDXs play critical roles in the occurrence and development of carcinomas. However, few studies have conducted systematic analysis of PRDXs in cancers. Therefore, the present study sought to explore the molecular characteristics and potential clinical significance of PRDX family members in pan cancer and further validate the function of PRDX6 in bladder urothelial carcinoma (BLCA). Methods A comprehensive analysis of PRDXs in 33 types of cancer was performed based on the TCGA database. This involved an analysis of mRNA expression profiles, genetic alterations, methylation, prognostic values, potential biological pathways and target drugs. Moreover, both the gain and loss of function strategies were used to assess the importance and mechanism of PRDX6 in the cell cycle of BLCA. Result Analysis showed abnormal expression of PRDX1-6 in several types of cancer compared to normal tissues. Univariate Cox proportional hazard regression analysis showed that expression levels of PRDX1, PRDX4 and PRDX6 were mostly associated with poor survival of OS, DSS and PFI, and PRDX2 and PRDX3 with favorable survival. In addition, the expression of PRDX genes were positively correlated with CNV and negatively with methylation. Moreover, analysis based on PharmacoDB dataset showed that the augmented levels of PRDX1, PRDX3 and PRDX6 were significantly correlated with EGFR/VEGFR inhibitor drugs. Furthermore, knocking down of PRDX6 inhibited growth of cancer cells through the JAK2-STAT3 in bladder cell lines. Conclusions PRDXs are potential biomarkers and therapeutic targets for several carcinomas, especially for BLCA. In addition, PRDX6 could regulate proliferation of cancer cell via JAK2-STAT3 pathway and involve into the process of cell cycle in BLCA.

THE EFFECT OF CONDITIONED MEDIUM ADIPOSE DERIVED MESENCHYMAL STEM CELLS IN UROTHELIAL CARCINOMA CULTURE CELLS VIABILITY

Objective: This study aimed to gives a perspective in CM-ADMSCs effect in urothelial bladder cancer viability. Material & Methods: Human bladder cell carcinoma type 5637 was used as the subject of this in vitro study. This study contains four different groups: untreated control group, Culture medium: hADMSCs with 1:1, 1:2, and 1:4 concentration group. Each group consists of 6 replications to prevent bias of the study. Viability was determined with MTT assay methods and evaluation performed after 48 h exposure of conditioned medium. Results: A post hoc test was conducted to analyze the data. The 5637 bladder cancer cell line demonstrated significantly decreased viability after exposure to culture medium: CM-hADMSCs 1:1 (p: 0.002) compared to the negative control group, but there are no significant differences in viability between the control groups with groups that were exposed to culture medium: CM-hADMSCs 1:2 and culture medium: CM-hADMSCs 1:4 with p: 0.480 and p: 0.060 respectively. Conclusion: Decreased viability of urothelial bladder cancer cells after exposure to CM-hADMSCs occurs at a concentration of 1:1 and Dosage addition more than 1:1 concentration doesn’t give any advantages.

Modulating Intra-Nuclear LC3 with Small Molecules Rescues Cells from a Docetaxel-Induced Phenotype

Nucleus-associated autophagy has been described as a cellular metabolic response by which nuclear material is actively degraded. This degradation occurs after stress, such as nuclear damage or the onset of tumorigenesis. Here we describe a nucleus-associated autophagic process distinct from other forms of selective autophagy in human cell lines. We found that although nuclear localization of MAP1LC3B (LC3) is not dependent on particular nuclear importins, knockdown of nuclear importins, which causes nuclear stress, can induce a nuclear autophagic response. Our characterization of this autophagic phenomenon was facilitated by chemical modulation of the process via two compounds discovered previously in a high content analysis. These small molecules bidirectionally regulate nuclear LC3 in human renal, pancreatic, and bladder cell lines. One molecule (NSC31762 or DTEP) enhances nuclear LC3 puncta and increases lysosomal targeting of LC3. This compound also decreases the nuclear envelope protein LaminB1. Another molecule (NSC279895 or DIHI) reduces the nuclear localization of LC3. Finally, we applied these chemical tools in the setting of mitotic-disruptor induced nuclear stress. The compound DIHI, shown to reduce nuclear autophagic puncta, diminished the mitotic disruptor effect. These new tools will allow for deeper exploration of nucleus-associated autophagies, and could serve as proof-of-principle in guiding new therapies for diseases involving nuclear stress.

C1QTNF6 Overexpression Acts as a Predictor of Poor Prognosis in Bladder Cancer Patients

Background. Bladder cancer is one of the most common urinary malignancies. This study is aimed at providing some promising molecular biomarkers for bladder cancer (BC) by investigating the correlation between C1QTNF6 expression and clinical characteristics as well as prognosis in patients with bladder cancer. Methods. Sequencing profiles of C1QTNF6 mRNA in BC patients were collected to evaluate the distinctive gene expression, between normal bladder mucosa and BC, according to the TCGA and GEO databases. The association between C1QTNF6 expression and the clinical features as well as the disease prognosis was evaluated using two independent cohorts. The expression of C1QTNF6 in normal bladder and BC cells was examined by western blotting and PCR, so the underlying molecular mechanism could be further investigated. Results. C1QTNF6 mRNA levels were found to be differentially expressed in two independent public cohorts, including the TCGA database and GSE13507 dataset from GEO. The protein and RNA levels of C1QTNF6 in BC cells were both elevated when compared to normal bladder cell lines. High C1QTNF6 expression was detected in advanced T/M stages, pathological grade, and AJCC stage when compared to the low C1QTNF6 expression group. The underlying mechanism related to this differential expression could be explained by cell migration and invasion assays, where bladder cancer cells 5637 and T24 had a significant reduction on migration and invasion ability upon knockdown of C1QTNF6 expression. The low C1QTNF6 expression group presented a more prominent OS advantage over the high-expression group in both TCGA and GSE13507 cohorts. Moreover, the protein content in tissues was further validated using the HPA database and TMA. Survival analyses also indicated that the high C1QTNF6 expression group had an unfavorable OS when compared to the low-expression group. Conclusions. High C1QTNF6 expression may serve as a predictor of poor prognosis in bladder cancer patients, and the underlying mechanism is possibly associated with changes on cancer cell migration and invasion ability.

MicroRNA-340 Inhibits Bladder Cancer Cell Growth by Downregulating Glucose Transporter-1

Background: MicroRNA (miRNA)-340 is emerging as a critical regulator for the development and progression of various cancers, such as oral squamous cell carcinoma and prostate cancer. However, little was known about the role of miR-340 in bladder cancer. Methods: Bladder cancer and adjacent non-tumor tissue samples were collected from thirty patients undergoing surgical resection at Shaoxing People's Hospital. The expression of miR-340 and Glut-1 were studied via real-time quantitative PCR (RT-qPCR) or western blots. Bladder cancer T24 cells were transfected with miR-340 mimics to explore cell proliferation and apoptosis via MTT assay, flow cytometer, RT-qPCR and western blots. Results: Compared with that of normal tissues, miR-340 expression was significantly lower, while both mRNA and protein expression of Glut-1 were higher in bladder cancer tissues. The miR-340 could negatively regulate Glut-1 expression in bladder cells. Moreover, bladder cell proliferation could be inhibited by miR-340 and the corresponding antitumor effect could partially reverse by the overexpression of Glut-1. In view of the complexity of gene networks, some other multiple pathways might also confer to miR-340 inducing bladder cell apoptosis, including downregulating PCNA, upregulating Bax and decreasing the phosphorylation levels of PI3K and AKT.Conclusion: This work suggested an inverse correlation between miR-340 and glucose transporter-1 (Glut-1) expression in bladder cancer. miR-340/Glut-1 axis might be a potential and novel therapeutic target for the treatment of bladder cancer. More investigations need to further explore the applications of miR-340 in bladder cancer.