Tumor suppressive functions of hsa­miR­34a on cell cycle, migration and protective autophagy in bladder cancer.

Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa­microRNA­34a (miR­34a) presents anti­tumor function in several types of cancer. However, the functional mechanism of miR­34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. First, transfected BC cells with miR­34a mimic exhibited LC3­II and p62 accumulation through immunofluorescence staining. It was demonstrated that syntaxin 17 (STX17), which is required for autophagosome­lysosome fusion, was downregulated upon miR­34a mimic treatment. Mechanistically, miR­34a reduced the expression of STX17 proteins that directly bind on STX17 3'­untranslated regions and thus suppressed STX17 mRNA translation to eventually inhibit protective autophagy in BC. Cell viability and colony formation assays revealed that overexpression of miR­34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin and mitomycin C. Furthermore, miR­34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR­34a suppressed cell motility through the downregulation of epithelial­mesenchymal transition. In summary, miR­34a inhibits cell proliferation, motility and autophagy activity in BC, which can benefit BC treatment.

The Application of Wireless Underground Sensor Networks to Monitor Seepage inside an Earth Dam.

Earth dams or embankments are susceptible to instability due to internal seepage, piping, and erosion, which can lead to catastrophic failure. Therefore, monitoring the seepage water level before the dam collapses is an important task for early warning of dam failure. Currently, there are hardly any monitoring methods that use wireless underground transmission to monitor the water content inside earth dams. Real-time monitoring of changes in the soil moisture content can more directly determine the water level of seepage. Wireless transmission of sensors buried underground requires signal transmission through the soil medium, which is more complex than traditional air transmission. Henceforth, this study establishes a wireless underground transmission sensor that overcomes the distance limitation of underground transmission through a hop network. A series of feasibility tests were conducted on the wireless underground transmission sensor, including peer-to-peer transmission tests, multi-hop underground transmission tests, power management tests, and soil moisture measurement tests. Finally, field seepage tests were conducted to apply wireless underground transmission sensors to monitor the internal seepage water level before an earth dam failure. The findings show that wireless underground transmission sensors can achieve the monitoring of seepage water levels inside earth dams. In addition, the results supersede those of a conventional water level gauge. This could be crucial in early warning systems during the era of climate change, which has caused unprecedented flooding events.

Autophagy blockade potentiates cancer-associated immunosuppression through programmed death ligand-1 upregulation in bladder cancer.

A high basal level of autophagic flux in bladder cancer (BC) cells prevents cell death and weakens chemotherapy efficacy. However, how autophagy influences cancer-associated immunosuppression in BC remains undetermined. In this study, we observed a negative correlation between the autophagy-related markers LC3-II and programmed death ligand-1 (PD-L1) in BC cells. The autophagy inhibitors chloroquine (CQ) and bafilomycin A1 (Baf-A1) increased PD-L1 expression in BC cells through the ERK-JNK-c-Jun signal-transduction pathway. Moreover, the treatment of BC cells with CQ and Baf-A1 inhibited hsa-microRNA-34a (miR-34a) expression and miR-34a overexpression in BC cells prevented the autophagy blockade-induced PD-L1 expression; a negative correlation between miR-34a and PD-L1 expression was observed during treatment with autophagy inhibitors. Furthermore, miR-34a overexpression induced the cytotoxic activity of natural killer cells against BC cells. Our results provide evidence that autophagy blockade and its regulatory pathway affect cancer-associated immunosuppression through PD-L1 elevation. Thus, the coadministration of autophagy inhibitors and a PD-L1 immune checkpoint blockade provides a potential therapeutic approach for treating BC.

Hsa-miR-30a-3p overcomes the acquired protective autophagy of bladder cancer in chemotherapy and suppresses tumor growth and muscle invasion.

Bladder cancer (BC) is the second most common urologic cancer in western countries. New strategies for managing high-grade muscle-invasive bladder cancer (MIBC) are urgently required because MIBC has a high risk of recurrence and poor survival. A growing body of evidence indicates that microRNA has potent antitumorigenic properties in various cancers, and thus, therapeutic strategies based on microRNA may show promising results in cancer therapy. Analysis of The Cancer Genome Atlas (TCGA) database indicated that hsa-miR-30a-3p is downregulated in human BC. Our in vitro investigation demonstrated that hsa-miR-30a-3p suppresses the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 and reduces the cell invasive potential of BC cells. Furthermore, hsa-miR-30a-3p directly targets ATG5, ATG12, and Beclin 1; this in turn improves the chemosensitivity of BC cells to cisplatin through the repression of protective autophagy. In a tumor-xenograft mice model, hsa-miR-30a-3p suppressed muscle invasion. Cotreatment with hsa-miR-30a-3p enhanced the antitumor effect of cisplatin in reducing tumor growth in BC. The current study provides a novel strategy of using hsa-miR-30a-3p as an adjuvant or replacement therapy in future BC treatment.

Cardiovascular Disease Risk in Children With Chronic Kidney Disease: Impact of Apolipoprotein C-II and Apolipoprotein C-III.

Cardiovascular disease (CVD) is an evolving process that begins in the early stages of chronic kidney disease (CKD) in children. Several surrogate markers, such as ambulatory blood pressure monitoring (ABPM), left ventricular (LV) mass, and arterial stiffness assessment, allow for the early detection of subclinical CVD in pediatric CKD. Four groups of plasma samples (n = 3/group) from congenital anomalies of the kidney and urinary tract (CAKUT), as well as non-CAKUT patients with or without BP abnormalities, were studied to screen differentially expressed proteins using isobaric tags for relative and absolute protein quantification (iTRAQ)-based proteomics. As a result, 20 differentially expressed proteins associated with hypertension in children with CKD were discovered. Among them, apolipoprotein C-II (apoC-II) was found to have the highest abundance among the CKD patients with hypertension. As such, we hypothesized that apoC-II and apolipoprotein C-III (apoC-III) levels were related to BP abnormalities and CVD in children suffering from mild-to-moderate CKD. We examined their associations with surrogate markers of CV risk in 88 pediatric patients with CKD stages G1-G4. Children with CKD stages G2-G4 had a higher plasma apoC-II level than G1 patients (6.35 vs. 5.05 mg/dl, p < 0.05). We observed that ABPM abnormalities, LV mass, and arterial stiffness parameters were greater in CKD children who had stages G2-G4 than in those who had stage G1 (all p < 0.05). Plasma levels of apoC-II and apoC-III were positively correlated with total cholesterol, triglyceride, and low-density lipoprotein (LDL) (all p < 0.001). In multivariate linear regression analyses, apoC-II was correlated with a high LV mass index and an abnormal ABPM profile, and apoC-III was correlated with 24-h hypertension (r = 0.303, p = 0.003) and asleep hypertension (r = 0.379, p < 0.001). Early evaluations of apoC-II and apoC-III, ABPM, and surrogate markers of CV risk will aid in early preventative interventions to reduce the risk of CV in youths suffering from CKD.

Attenuation of chloroquine and hydroxychloroquine on the invasive potential of bladder cancer through targeting matrix metalloproteinase 2 expression.

Bladder cancer (BC), one of the most common urological neoplastic disorders in men, has an extremely low survival rate because of its tendency to metastasize. The anticancer drugs chloroquine (CQ) and hydroxy CQ (HCQ) might inhibit tumor progression and invasiveness. However, the mechanism by which CQ and HCQ influence BC is undetermined. In this study, CQ and HCQ treatments inhibited the migration and invasion of two BC cell types (5637 and T24) through expression modulation of matrix metalloproteinase-2 (MMP-2), which belongs to the matrix MMP family and is a key mediator of cancer progression. Moreover, additional data revealed that the migrative and invasive effects of BC cells treated with CQ or HCQ were abolished after treatment with rapamycin, which induces autophagy, demonstrating that CQ and HCQ functions in BC are based on autophagy inhibition. In conclusion, our research demonstrated that CQ and HCQ regulated cell motility in BC through MMP-2 downregulation by targeting autophagy functions, providing a novel therapeutic strategy for BC treatment.

Thrombospondin-4 promotes bladder cancer cell migration and invasion via MMP2 production.

Bladder cancer (BC) is the second most common urological tumour in Western countries. Approximately, 80% of patients with BC will present with non-muscle invasive bladder cancer (NMIBC), whereas a quarter will have muscle invasive disease (MIBC) at the time of BC diagnosis. However, patients with NMIBC are at risk of BC recurrence or progression into MIBC, and an MIBC prognosis is determined by the presence of progression and metastasis. Matrix metalloproteinase 2 (MMP2), a type of matrix metalloproteinase (MMP), plays a major role in tumour invasion and is well-characterized in BC prognosis. In BC, the mechanisms regulating MMP2 expression, and, in turn, promote cancer invasion, have hardly been explored. Thrombospondin-4 (THBS4/TSP4) is a matricellular glycoprotein that regulates multiple biological functions, including proliferation, angiogenesis, cell adhesion and extracellular matrix modelling. Based on the results of a meta-analysis in the Gene Expression Profiling Interactive Analysis 2 database, we observed that TSP4 expression levels were consistent with overall survival (OS) rate and BC progression, with the highest expression levels observed in the advanced stages of BC and associated with poor OS rate. In our pilot experiments, incubation with recombinant TSP4 promoted the migration and invasion in BC cells. Furthermore, MMP2 expression levels increased after recombinant TSP4 incubation. TSP4-induced-MMP2 expression and cell motility were regulated via the AKT signalling pathway. Our findings facilitate further investigation into TSP4 silencing-based therapeutic strategies for BC.

MicroRNA­34a­5p serves as a tumor suppressor by regulating the cell motility of bladder cancer cells through matrix metalloproteinase­2 silencing.

Bladder cancer (BC), a common urologic cancer, is the fifth most frequently diagnosed tumor worldwide. hsa­miR­34a displays antitumor activity in several types of cancer. However, the functional mechanisms underlying hsa­miR­34a in BC remains largely unknown. We observed that hsa­mir­34a levels were significantly and negatively associated with clinical disease stage as well as regional lymph node metastasis in human BC. In a series of in vitro investigations, overexpression of hsa­miR­34a inhibited cell migration and invasion in BC cell lines 5637 and UMUC3 as detected by Transwell assays. We further found that hsa­miR­34a inhibited cell migration and invasion by silencing matrix metalloproteinase­2 (MMP­2) expression and thus interrupting MMP­2­mediated cell motility. Our analysis of BC datasets from The Cancer Genome Atlas database revealed a negative correlation between hsa­miR­34a and MMP­2. Moreover, higher MMP­2 protein expression was observed in the BC tissues when compared with that noted in the normal tissue. MMP­2 levels were also significantly associated with clinical disease stage and poor survival rate in human BC. These findings indicate that MMP­2 plays a critical role in regulating BC progression. Therefore, hsa­miR­34a is a promising treatment to target MMP­2 for the prevention and inhibition of cell migration and invasion in BC.

Miconazole induces protective autophagy in bladder cancer cells.

Autophagy plays a dual function in cancer progression; autophagy activation can support cancer cell survival or contribute to cell death. Miconazole, a Food and Drug Administration-approved antifungal drug, has been implicated in oncology research recently. Miconazole was found to exert antitumor effects in various tumors, including bladder cancer (BC). However, whether it provokes protective autophagy has been never discussed. We provide evidence that miconazole induces protective autophagy in BC for the first time. The results indicated that 1A/1B-light chain 3 (LC3)-II processing and p62 expression were elevated after miconazole exposure. Also, adenosine monophosphate-activated protein kinase phosphorylation was increased after miconazole treatment. We also confirmed the autophagy-promoting effect of miconazole in the presence of bafilomycin A1 (Baf A1). The result indicates that a combination treatment of miconazole and Baf A1 improved LC3-II processing, confirming that miconazole promoted autophagic flux. The acridine orange, Lysotracker, and cathepsin D staining results indicate that miconazole increased lysosome formation, revealing its autophagy-promoting function. Finally, miconazole and autophagy inhibitor 3-methyladenine cotreatment further reduced the cell viability and induced apoptosis in BC cells, proving that miconazole provokes protective autophagy in BC cells. Our findings approve that miconazole has an antitumor effect in promoting cell apoptosis; however, its function of protective autophagy is needed to be concerned in cancer treatment.

Trends in Antimicrobial Susceptibility of Escherichia coli Isolates in a Taiwanese Child Cohort with Urinary Tract Infections between 2004 and 2018.

The aim of this study was to investigate the annual incidence of Escherichia coli isolates in urinary tract infections (UTIs) and the antimicrobial resistance of the third-generation cephalosporin (3GCs) to E. coli, including the factors associated with the resistance in hospitalized children in Taiwan. A large electronic database of medical records combining hospital admission and microbiological data during 2004-2018 was used to study childhood UTIs in Taiwan. Annual incidence rate ratios (IRR) of E. coli in children with UTIs and its resistant rate to the 3GCs and other antibiotics were estimated by linear Poisson regression. Factors associated with E. coli resistance to 3GCs were assessed through multivariable logistic regression analysis. E. coli UTIs occurred in 10,756 unique individuals among 41,879 hospitalized children, with 92.58% being community associated based on urine culture results reported within four days after the hospitalization. The overall IRR E. coli UTI was 1.01 (95% confidence interval (CI) 0.99-1.02) in community-associated (CA) and 0.96 (0.90-1.02) in healthcare-associated infections. The trend in 3GCs against E. coli increased (IRR 1.18, 95% CI 1.13-1.24) over time in CA-UTIs. Complex chronic disease (adjusted odds ratio (aOR), 2.04; 95% CI, 1.47-2.83) and antibiotics therapy ≤ 3 months prior (aOR, 1.49; 95% CI, 1.15-1.94) were associated with increased risk of 3GCs resistance to E. coli. The study results suggested little or no change in the trend of E. coli UTIs in Taiwanese youths over the past 15 years. Nevertheless, the increase in 3GCs-resistant E. coli was substantial. Interventions for children with complex chronic comorbidities and prior antibiotic treatment could be effective in reducing the incidence of 3GCs-resistant E. coli in CA-UTIs in this region and more generally.

Miconazole Contributes to NRF2 Activation by Noncanonical P62-KEAP1 Pathway in Bladder Cancer Cells.

PURPOSE: Nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, a transcription factor capable of upregulating antioxidant response element (ARE)-mediated expression and cytoprotective proteins, plays critical roles in chemoprevention, inflammation and aging. NRF2 has recently been proposed as a novel target for cancer chemoprevention. The fungicide miconazole has shown promising antiproliferative effects in cancer cells. MATERIALS AND METHODS: After miconazole treatment, the p62-KEAP1-NRF2 activation was analyzed by qPCR and Western blot. The nuclear translocation indicating NRF2 activation was further confirmed by immunofluorescence. Finally, the ROS production was detected by CM-H2DCFDA staining. RESULTS: We demonstrate in this study that miconazole dramatically increases NRF2 activation in bladder cancer cells, in a dose- and time-dependent manner. Interestingly, levels of expression of p62, a noncanonical pathway that mediates NRF2 activation, appeared to increase in accordance with NRF2. We also investigated levels of the negative regulator kelch-like ECH-associated protein 1 (KEAP1), which is involved in NRF2 activation. As expected, a decrease in KEAP1 expression was found after miconazole exposure. Confirmation of NRF2 nuclear translocation was monitored by immunofluorescence. Miconazole-induced generation of reactive oxygen species (ROS) promoted NRF2 activation. Pretreatment of bladder cancer cells with ROS scavengers abolished NRF2 expression and nuclear translocation, indicating that miconazole activates the noncanonical p62-KEAP1-NRF2 pathway, which is regulated by ROS production. CONCLUSION: Our study elucidates the mechanisms through which miconazole stimulates NRF2 which may contribute to cancer chemopreventive effects.

Benzyl isothiocyanate promotes miR-99a expression through ERK/AP-1-dependent pathway in bladder cancer cells.

Benzyl isothiocyanate (BITC), a bioactive natural product present in cruciferous vegetables, has been proved to prevent cancer progression through various mechanisms. In our previous report, we proved that BITC exhibits antitumor effects in bladder cancer by suppressing IGF1R, FGFR3, and mTOR, which is mediated by miR-99a expression. In this study, we identified the signal pathway involved in regulating miR-99a expression after BITC exposure in bladder cancer. Treatment with different BITC concentrations resulted in induction of miR-99a expression in bladder cancer cell lines. Activation of extracellular signal-regulated protein kinase (ERK) and c-jun N-terminal kinase was observed in bladder cancer after BITC treatment for 24 hours. Interestingly, by using a chemical inhibitor of candidate pathways, we found that only the ERK signal pathway is required for miR-99a expression. Furthermore, we evaluated the transcription factor that may contribute to miR-99a expression in response to BITC treatment. The results indicated that c-Jun/AP-1 was activated after BITC treatment. Moreover, we confirmed c-Jun/AP-1 activation through immunofluorescence and the luciferase reporter assay. The results showed that BITC treatment markedly improved nuclear translocation of c-Jun/AP-1 and luciferase activity dose dependently. Finally, pretreatment with the ERK inhibitor U0126 diminished c-Jun phosphorylation and transcriptional activation, suggesting that BITC elicits ERK/c-Jun signal transduction, which is responsible for miR-99a expression in bladder cancer. The present work identifies the mechanism involved in upregulation miR-99a after BITC treatment, which provides an explanation for BITC biological function in our previous work.

Cisplatin contributes to programmed death-ligand 1 expression in bladder cancer through ERK1/2-AP-1 signaling pathway.

Bladder cancer (BC) is the second most common urologic malignancy and the ninth most common malignancy worldwide. Surgical resection is the mainstay of treatment for patients with early-stage disease, whereas therapeutic options are limited for patients with advanced-stage or residual BC. Programmed cell death ligand-1 (PD-L1) is an important target for immunotherapy. It is known that PD-L1 is overexpressed in BC; a clinical trial involving PD-L1 immune checkpoint inhibitors in advanced BC is ongoing. In the present study, we used Western blot and quantitative real-time PCR (qPCR) to define the expression level of PD-L1 after cisplatin treatment in BC-derived cell lines. The signal activation was also evaluated by Western blot in BC-derived cell lines. We found that chemotherapeutic drug cisplatin can induce PD-L1 but not PD-L2 expression in BC-derived cell lines. Furthermore, the expression level of PD-L1 was increased in a dose- and time-dependent manner after cisplatin treatment. The cisplatin-induced PD-L1 expression is mainly mediated by ERK1/2 but not Akt/mTOR signal pathway. Moreover, we found that cisplatin activates transcription factor activator protein-1 (AP-1) to regulate PD-L1 expression. The chemotherapy drug such as cisplatin may trigger resistance of BC through PD-L1 up-regulation. The present study suggests that PD-L1 antibody should be used concomitantly with chemotherapy in the setting of advanced and metastatic BC.

Resveratrol prevents combined prenatal NG-nitro-L-arginine-methyl ester (L-NAME) treatment plus postnatal high-fat diet induced programmed hypertension in adult rat offspring: interplay between nutrient-sensing signals, oxidative stress and gut microbiota.

Oxidative stress, nutrient-sensing signals, high-fat (HF) intake and dysbiosis of gut microbiota are involved in the development of hypertension, a disorder that can originate in early life. We examined whether postnatal HF diet can aggravate maternal NG-nitro-L-arginine-methyl ester (L-NAME) treatment-induced programmed hypertension and whether resveratrol therapy can prevent it. Pregnant Sprague-Dawley rats received L-NAME administration at 60 mg/kg/day subcutaneously during pregnancy alone, or with additional resveratrol (R) 50 mg/L in drinking water during the pregnancy and lactation. The offspring were onto either regular chow or HF diet (D12331) from weaning to 16 weeks of age. Male offspring rats were assigned to five groups (N=8/group): control, L-NAME, HF, L-NAME+HF and L-NAME+HF + R at weaning at 3 weeks of age. Rats were sacrificed at 16 weeks of age. We observed that postnatal HF diet exacerbates maternal L-NAME treatment-induced programmed hypertension in male adult offspring, which resveratrol attenuated. Combined L-LAME and HF diet-induced hypertension is related to increased oxidative stress, inhibiting AMP-activated protein kinase (AMPK)/ peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) pathway and altered gut microbiota compositions. L-NAME+HF caused an increase of the Firmicutes to Bacteroidetes ratio, which resveratrol therapy prevented. Additionally, the abundances of phylum Verrucomicrobia and genus Akkermansia were amplified by resveratrol therapy. Conclusively, our data highlighted the interactions between maternal NO deficiency, HF diet, AMPK/PGC-1α pathway and gut microbiota in which the blood pressure of adult offspring can be modified by resveratrol. Resveratrol might be a useful reprogramming strategy to prevent L-NAME and HF diet-induced hypertension of developmental origin.

Benzyl isothiocyanate suppresses IGF1R, FGFR3 and mTOR expression by upregulation of miR-99a-5p in human bladder cancer cells.

Benzyl isothiocyanate (BITC) is known for its pharmacological properties against malignant neoplasm, including bladder cancer (BC). The current study investigated microRNAs (miRNA or miR) expression profiles with an emphasis on the role of miR­99a­5p in BITC­treated BC cells. A quantitative polymerase chain reaction (qPCR) microarray containing 79 aberrantly expressed miRNAs in BC was used to detect miRNA expression in BITC­treated cells. Several dysregulated miRNAs were identified and further confirmed using miRNA stem­loop reverse transcription (RT)­qPCR in 5637 cells. Insulin­like growth factor 1 receptor (IGF1R), fibroblast growth factor receptor 3 (FGFR3) and mammalian target of rapamycin (mTOR) expression were determined by RT­qPCR and western blotting. Cell viability was evaluated using WST­1 reagent and apoptosis was monitored by determining the levels of cleaved­poly ADP­ribose polymerase and cleaved­caspase­3. BITC treatment significantly upregulated miR­99a­5p levels in a dose­dependent manner. miR­99a­5p overexpression decreased IGF1R, mTOR and FGFR3 expression, predicted targets of miR­99a­5p. In addition, antisense miR­99a­5p sequences inhibited BITC­induced miR­99a­5p overexpression, resulting in the restoration of protein expression and decreased cell viability. The current study identified multiple miRNAs responsive to BITC treatment, including miR­99a­5p. In addition, the induction of miR­99a­5p decreased IGF1R, mTOR and FGFR3 expression in BITC­treated BC cells. The current study provided novel insight into the antitumor mechanism by which BITC restores miR­99a­5p expression and decreases cancer cell survival.

Allyl Isothiocyanate Induces Autophagy through the Up-Regulation of Beclin-1 in Human Prostate Cancer Cells.

Allyl isothiocyanate (AITC), one of the most widely studied phytochemicals, inhibits the survival of human prostate cancer cells while minimally affecting normal prostate epithelial cells. Our study demonstrates the mechanism of AITC-induced cell death in prostate cancer cells. AITC induces autophagy in RV1 and PC3 cells, judging from the increased level of LC3-II protein in a dose- and time-dependent manner, but not in the normal prostate epithelial cell (PrEC). Inhibition of autophagy in AITC-treated cells decreased cell viability and enhanced apoptosis, suggesting that the autophagy played a protective role. There are several pathways activated in ATIC-treated cells. We detected the phosphorylation forms of mTOR, ERK, AMPK, JNK and p38, and ERK AMPK and JNK activation were also detected. However, inhibition of AITC-activated ERK, AMPK and JNK by pre-treatment of specific inhibitors did not alter autophagy induction. Finally, increased beclin-1 expression was detected in AITC-treated cells, and inhibition of AITC-induced beclin-1 attanuated autophagy induction, indicating that AITC-induced autophagy occurs through upregulating beclin-1. Overall, our data show for the first time that AITC induces protective autophagy in Rv1 and PC3 cells through upregulation of beclin-1. Our results could potentially contribute to a therapeutic application of AITC in prostate cancer patients.

miR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells.

INTRODUCTION: miR-99a-5p, known to play an important role in mammalian target of rapamycin (mTOR) regulation, is downregulated in human bladder cancer. The study aimed to investigate the anticancer activity of miR-99a-5p and the possible mechanism associated with mTOR in bladder cancer cells. MATERIALS AND METHODS: Vectors expressing miR-99a-5p were transfected into human urinary bladder urothelial carcinoma (5637 and T24) cells. The level of miR-99a-5p was monitored by microRNA (miRNA) quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the direct binding of miR-99a-5p to mTOR transcripts. The mTOR transcripts and protein levels were measured by QPCR and Western blot, respectively. Cell viability of miR-99a-5p-transfected cells was detected by tetrazolium salt (WST-1). Inhibition of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling was detected by the phosphorylation of mTOR and AKT using Western blot. The ability of miR-99a-5p to enhance RAD001-induced apoptosis was determined as the expression of cleaved caspase 3 and levels of DNA fragmentation. RESULTS: Transfection of miR-99a-5p-expressing vector elevated the expression level of miR-99a-5p up to sixfold compared to vector-only controls. The results from luciferase assay verified that miR-99a-5p directly binds to the predicted sequence in the 3' untranslated region (3'-UTR) of mTOR. The levels of mTOR RNA and protein were decreased in miR-99a-5p-transfected cells. Dual inhibition of mTORC1 and mTORC2 by miR-99a-5p was confirmed by the decreased phosphorylation of mTOR (at Ser2448 and Ser2481), phospho-rpS6 and phospho-4EBP1. The phosphorylation of AKT was significantly inhibited in miR-99a-5p-transfected cells upon RAD001 treatment. Enforced expression of miR-99a-5p potentiated RAD001-induced apoptosis in these cells. CONCLUSION: This is the first study showing that miR-99a-5p markedly inhibits the growth of bladder cancer cells via dual inhibition of mTORC1 and mTORC2. Our data demonstrated that forced expression of miR-99a-5p inhibits the feedback of AKT survival pathway and enhances the induction of apoptosis in RAD001-treated bladder cancer cells.

Fiber-Optic Thermal Sensor for TiN Film Crack Monitoring.

The study focuses on the thermal and temperature sensitivity behavior of an optical fiber sensor device. In this article, a titanium nitride (TiN)-coated fiber Bragg grating (FBG) sensor fabricated using an ion beam sputtering system was investigated. The reflection spectra of the FBG sensor were tested using R-soft optical software to simulate the refractive index sensitivity. In these experiments, the temperature sensitivity of the TiN FBG was measured at temperatures ranging from 100 to 500 °C using an optical spectrum analyzer (OSA). The results showed that the temperature sensitivity of the proposed TiN FBG sensor reached 12.8 pm/°C for the temperature range of 100 to 300 °C and 20.8 pm/°C for the temperature range of 300 to 500 °C. Additionally, we found that the produced oxidation at temperatures of 400-500 °C caused a crack, with the crack becoming more and more obvious at higher and higher temperatures.

Acridine orange exhibits photodamage in human bladder cancer cells under blue light exposure.

Human bladder cancer (BC) cells exhibit a high basal level of autophagic activity with accumulation of acridine-orange(AO)-stained acidic vesicular organelles. The rapid AO relocalization was observed in treated BC cells under blue-light emission. To investigate the cytotoxic effects of AO on human BC cell lines under blue-light exposure, human immortalized uroepithelial (SV-Huc-1) and BC cell lines (5637 and T24) were treated with indicated concentrations of AO or blue-light exposure alone and in combination. The cell viability was then determined using WST-1, time-lapse imaging with a Cytosmart System and continuous quantification with a multi-mode image-based reader. Treatment of AO or blue-light exposure alone did not cause a significant loss of viability in BC cells. However, AO exhibited a dose-dependent increment of cytotoxicity toward BC cells under blue-light exposure. Furthermore, the tumor formation of BC cells with treatment was significantly reduced when evaluated in a mouse xenograft model. The photodamage caused by AO was nearly neglected in SV-Huc-1 cells, suggesting a differential effect of this treatment between cancer and normal cells. In summary, AO, as a photosensitizer, disrupts acidic organelles and induces cancer cell death in BC cells under blue-light irradiation. Our findings may serve as a novel therapeutic strategy against human BC.