Comprehensive Gene Expression Analysis in NMIBC Using RNA-seq Reveals New Therapy Strategies.

Non-muscle invasive bladder cancer (NMIBC) patients often have fewer treatment options, and suffer the progression of disease due to mechanisms that are not clear, as well as due to its diversity. This study was designed to explore the molecular mechanism of bladder cancer through an RNA-seq. In addition to conventional analyses, we also simplified the network through modularization using the WGCNA algorithm, with the help of the topological overlapping matrix and hierarchical cluster tree, which are based on the PPI network of STRING. Furthermore, the hub genes were confirmed through survival analyses in the independent cohorts (n = 431). Among them, 15 genes were significantly associated with poor prognosis. Finally, we validated the results at mRNA and protein level using qRT-PCR, IHC and western blotting. Taken together, our research is important for the prediction, as well as the prospective clinical development of drug targets and biomarkers.

PKM2 Inhibitor Shikonin Overcomes the Cisplatin Resistance in Bladder Cancer by Inducing Necroptosis.

Cisplatin-based chemotherapy often results in the development of chemo-resistance when used to treat bladder cancer (BC), which is difficult to overcome. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC, and contributes to the cisplatin resistance in BC. However, the underlying mechanisms remain unclear. In this study, we also found that the expression level of PKM2 is also higher in cisplatin resistant BC cells and tumors. Down-regulation of PKM2 by siRNA or inhibition of PKM2 by shikonin re-sensitized the cisplatin resistant T24 cells. Shikonin and cisplatin together exhibit significantly greater killing effects than when used alone. Interestingly, we found shikonin kills the T24 cisplatin resistant cells by inducing necroptosis, as the cell death could not inhibited by apoptosis inhibitor, z-VAD, but compromised by RIP3 inhibitor, GSK872, or RIP3 siRNA. In contrast, shikonin induced apoptosis in T24 parental cells. We further investigate the underlying mechanism, and found that the dysregulation of Bcl-2 family proteins, including Bcl-2, PUMA, Bax, play an important role in deciding that shikonin kills the BC cells by necroptosis or apoptosis. Collectively, our results suggested that inducing necroptosis is an alternative way to overcome the apoptosis resistant in BC therapy, and orchestrating the regulation of Bcl-2, PUMA, and Bax in BC cisplatin resistant cells may improve the therapy effect of cisplatin in BC tumor.

Screening biomarkers of bladder cancer using combined miRNA and mRNA microarray analysis.

Biomarkers, such as microRNAs (miRNAs) may be useful for the diagnosis of bladder cancer. In order to understand the molecular mechanisms underlying bladder cancer, differentially expressed miRNAs (DE-miRNAs) and their target genes in bladder cancer were analyzed. In the present study, miRNA and mRNA expression profiles (GSE40355) were obtained from the Gene Expression Omnibus. These consisted of healthy bladder samples (n=8) and urothelial carcinoma samples (low-grade, n=8 and high-grade, n=8). DE-miRNAs and differentially expressed genes (DEGs) were identified using the limma package and the Benjamin and Hochberg method from the multtest package in R. Target genes of DE-miRNAs were screened. Associations between DEGs were investigated using STRING, and an interaction network was constructed using Cytoscape. Functional and pathway enrichment analyses were performed for DEGs from the interaction network. 87 DE-miRNAs and 2058 DEGs were screened from low-grade bladder cancer samples, and 40 DE-miRNAs and 2477 DEGs were screened from high-grade bladder cancer samples. DE-target genes were significantly associated with the regulation of cell apoptosis. Bladder cancer, non-small cell lung cancer and pancreatic cancer biological pathways were found to be enriched. The results of the present study demonstrated that E2F transcription factor 1, which is targeted by miR-106b, and cyclin-dependent kinase inhibitor 2A (CDKN2A) and V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog-2, which are targeted by miR-125b, participate in the bladder cancer pathway. In conclusion, DE-miRNAs in bladder cancer tissue samples and DE-targeted genes, such as miR-106b and CDKN2A, which were identified in the present study, may provide the basis for targeted therapy for breast cancer and enhance understanding of its pathogenesis.

Microsurgical spermatic-inferior epigastric vein anastomosis for treating nutcracker syndrome-associated varicocele in infertile men: a preliminary experience.

OBJECTIVE: To assess the therapeutic effectiveness of microsurgical spermatic-inferior epigastric vein anastomosis for the treatment of nutcracker syndrome (NCS)-associated varicocele in infertile men. METHODS: We prospectively analyzed 5 infertile men with NCS-associated varicocele between April 2010 and January 2012. All patients underwent microsurgical spermatic-inferior epigastric vein anastomosis. RESULTS: The mean operation time was 85.0 ± 13.2 minutes, and the mean postoperative hospital stay was 6.0 ± 0.7 days. During a 1-year follow-up, hematuria completely resolved in 66.7% of patients (2/3) and flank pain resolved in the single patient affected. The peak velocity (PV) at the aortomesenteric portion of the left renal vein (LRV) significantly decreased after surgery (167.24 ± 41.68 cm/s vs 46.98 ± 4.22 cm/s). The PV ratio between the aortomesenteric and hilar portion of the LRV also significantly decreased (12.28 ± 2.32 preoperatively vs 3.40 ± 0.67 postoperatively). The mean sperm count and motility at 6 months (24.38 × 10(6)/mL ± 1.58 × 10(6)/mL and 53.96% ± 6.28%, respectively) and 12 months (30.02 × 106/mL ± 3.52 × 106/mL and 59.40% ± 8.59%, respectively) postoperatively were significantly higher than their preoperative values (15.8 × 106/mL ± 4.53 × 106/mL and 26.76% ± 8.68%, respectively). Overall, 80% of the spouses of patients (4/5) went on to conceive naturally. The complications observed were scrotal edema in 1 patient (20%) and wound infection in 1 patient (20%). CONCLUSION: Microsurgical spermatic-inferior epigastric vein anastomosis is a safe and efficient surgical treatment for infertile men with NCS-associated varicocele.

Percutaneous nephrolithotomy versus ureteroscopic lithotomy for large (>15 mm) impacted upper ureteral stones in different locations: is the upper border of the fourth lumbar vertebra a good indication for choice of management method?

Abstract To determine whether treatment of patients with large (>15 mm) impacted upper ureteral stones depended on stone location, we prospectively evaluated the therapeutic outcomes, complications, safety, and effectiveness of percutaneous nephrolithotomy (PCNL) and ureteroscopic lithotomy (URSL) in patients with stones higher and lower than the upper border of the fourth lumbar vertebra. Of the 174 patients analyzed, 83 (47.7%) underwent PCNL and 91 (52.3%) underwent URSL; all patients were followed up 1 month later and every 6 months for 18 months. Mean operation time (108.76±19.36 vs. 63.56±16.38 minutes, p<0.05) and postoperative hospital stay (2.49±1.23 vs. 5.36±1.98 days, p<0.05) were significantly longer in the PCNL than in the URSL group. The overall stone-free rates after 1 month were 96.4% and 75.8%, respectively, differing significantly for stones higher (97.8% vs. 57.5%, p<0.05) but not lower (94.7% vs. 90.2%) than the upper border of the fourth lumbar vertebra. The stone-retropulsion rate of URSL differed significantly for stones higher and lower than the upper border of the 4th lumbar vertebra (47.5% vs. 9.8%, p<0.05). Postprocedural complication rates were comparable in the URSL and PCNL groups, although the rate of auxiliary or salvage procedures was higher in the URSL group. The efficiency quotients (EQ) for PCNL and URSL were 0.93 and 0.59, respectively, with EQs in the URSL group differing significantly for stones higher and lower than the upper border of the fourth lumbar vertebra (0.40 vs. 0.82, p<0.05). Our findings indicate that treatment of impacted upper ureteral stones is dependent on stone location relative to the upper border of the fourth lumbar vertebra. URSL is unsuitable for stones at a higher location, whereas URSL and PCNL were equally effective for stones at a lower location.

[Bilateral ureteral polyps in children: a case report].

Fibroepithelial polyp of the ureter is a benign tumor of mesodermal origin that rarely occurs in children. The most common presenting symptoms are hematuria and flank pain by obstruction of the urinary tract. The etiology of this tumor is still not clear. It occurs more frequently in boys and often arises in the proximal ureter and the ureteropelvic junction. The preoperative diagnosis is difficult. We present here the case of a 11-year-old boy who had fibroepithelial polyps as the cause of the left flank ureteropelvic junction obstruction at pyeloplasty, and had the same condition on the right flank 5 years ago. We used polypectomy and pyeloureterostomy to treat the boy. No major intraoperative or preoperative complications developed.

Identification of natural coumarin compounds that rescue defective DeltaF508-CFTR chloride channel gating.

1. Deletion of phenylalanine at position 508 (DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the DeltaF508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects. 2. We identified a class of natural coumarin compounds that can correct the defective DeltaF508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing DeltaF508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L). 3. Dose-dependent potentiation of defective DeltaF508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTR(inh)-172. Two potent compounds, namely imperatorin and osthole, have activation K(d) values of approximately 10 micromol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of DeltaF508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of DeltaF508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout. 4. In conclusion, the natural coumarin DeltaF508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the DeltaF508 mutation.

Assessment of released acrosin activity as a measurement of the sperm acrosome reaction.

AIM: To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction (AR). METHODS: Human semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization (WHO) criteria. Calcium ionophore A23187 and progesterone (P4) were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA). The percentage of sperm undergoing AR (AR%) was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis. RESULTS: The AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimulation with A23187 (P < 0.001). There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining (r=0.916, P < 0.001). CONCLUSION: Spectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR.