TNF-α-induced exosomal miR-146a mediates mesenchymal stem cell-dependent suppression of urethral stricture.

The effective treatment of urethral stricture remains a medical problem. The use of proinflammatory cytokines as stimuli to improve the reparative efficacy of mesenchymal stem cells (MSCs) towards damaged tissues represents an evolving field of investigation. However, the therapeutic benefits of this strategy in the treatment of urethral stricture remain unknown. Here, we enriched exosomes derived from human umbilical cord-derived MSCs pretreated with or without tumor necrosis factor alpha (TNF-α) to evaluate their therapeutic effects in an in vivo model of TGFß1-induced urethral stricture. Male Sprague-Dawley rats received sham (saline) or TGFß1 injections to urethral tissues followed by incisions in the urethra. Animals in the TGFß1 injection (urethral fibrosis) cohort were subsequently injected with vehicle control, or with exosomes derived from MSCs cultured with or without TNF-α. After 4 weeks, rats underwent ultrasound evaluation and, following euthanasia, urethral tissues were harvested for histological and molecular analysis. In vitro, the effects of MSC-derived exosomes on fibroblast secretion of collagen and cytokines were studied by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. Exosomes derived from MSCs pretreated with TNF-α were more effective in suppressing urethral fibrosis and stricture than exosomes from untreated MSCs. We found that miR-146a, an anti-inflammatory miRNA, was strongly upregulated in TNF-α-stimulated MSCs and was selectively packaged into exosomes. Moreover, miR-146a-containing exosomes were taken up by fibroblasts and inhibited fibroblast activation and associated inflammatory responses, a finding that may underlie the therapeutic mechanism for suppression of urethral stricture. Inhibition of miR-146a in TNF-α-treated MSCs partially reduced antifibrotic effects and increased the release of proinflammatory factors of exosomes derived from these cells. Together these findings demonstrate that exosomes derived from TNF-α-treated MSCs are of therapeutic benefit in urethral fibrosis, suggesting that this strategy may have utility as an adjuvant therapy in the treatment of urethral stricture diseases.

LIMK1 depletion enhances fasudil-dependent inhibition of urethral fibroblast proliferation and migration.

LIM kinase 1 (LIMK1) is an important regulator of the cell cytoskeleton. This study aimed to examine the role of LIMK1 in mediating the effects of the Rho kinase (ROCK) inhibitor fasudil. In vitro cultures of urethral fibroblasts were divided into LIMK1 knockdown (LIMK1 KD) and LIMK1 control (LIMK1 NC) experimental groups. Each group was incubated with fasudil (50 µmol/L) with or without transforming growth factor ß1 (10 ng/mL) for 24 hours. Wound healing and Transwell assays were performed to determine cell migration. Flow cytometry was used to determine apoptosis. LIMK1, collagen I, collagen III, phospho-myosin light chain (p-MLC), alpha smooth muscle actin (α-SMA), and phospho-Cofilin (p-Cofilin) expression was examined by Western blot analysis. The expression of LIMK1 was further validated in human urethral scar tissues. Transwell and wound healing assays revealed that the cells of the LIMK1 KD group exhibited significantly attenuated migration, when compared with those of the LIMK1 NC group ( P < 0.05). Cell migration was also attenuated in the LIMK1 KD group treated with fasudil ( P < 0.05). Flow cytometry analysis revealed that apoptosis was higher in the LIMK1 KD group than that in LIMK1 NC group ( P < 0.05). Apoptosis was also enhanced in the LIMK1 KD group treated with fasudil ( P < 0.05). Western blot analysis demonstrated that LIMK1, collagen I, collagen III, p-MLC, α-SMA, and p-Cofilin expression was significantly attenuated in both the fasudil-treated and untreated LIMK1 KD groups ( P < 0.05). LIMK1 was positively expressed in human urethral scar tissues while it was negatively expressed in normal urethra tissues. In conclusion, loss of LIMK1 expression inhibits the Rho/ROCK pathway-dependent proliferation and migration via downregulation of collagen I, collagen III, p-Cofilin, and α-SMA. LIMK1 loss can also enhance the inhibitory effects of fasudil on the proliferation and migration of urethral fibroblasts.

Identification of key DNA methylation-driven genes in prostate adenocarcinoma: an integrative analysis of TCGA methylation data.

BACKGROUND: Prostate cancer (PCa) remains the second leading cause of deaths due to cancer in the United States in men. The aim of this study was to perform an integrative epigenetic analysis of prostate adenocarcinoma to explore the epigenetic abnormalities involved in the development and progression of prostate adenocarcinoma. The key DNA methylation-driven genes were also identified. METHODS: Methylation and RNA-seq data were downloaded for The Cancer Genome Atlas (TCGA). Methylation and gene expression data from TCGA were incorporated and analyzed using MethylMix package. Methylation data from the Gene Expression Omnibus (GEO) were assessed by R package limma to obtain differentially methylated genes. Pathway analysis was performed on genes identified by MethylMix criteria using ConsensusPathDB. Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were also applied for the identification of pathways in which DNA methylation-driven genes significantly enriched. The protein-protein interaction (PPI) network and module analysis in Cytoscape software were used to find the hub genes. Two methylation profile (GSE112047 and GSE76938) datasets were utilized to validate screened hub genes. Immunohistochemistry of these hub genes were evaluated by the Human Protein Atlas. RESULTS: A total of 553 samples in TCGA database, 32 samples in GSE112047 and 136 samples in GSE76938 were included in this study. There were a total of 266 differentially methylated genes were identified by MethylMix. Plus, a total of 369 differentially methylated genes and 594 differentially methylated genes were identified by the R package limma in GSE112047 and GSE76938, respectively. GO term enrichment analysis suggested that DNA methylation-driven genes significantly enriched in oxidation-reduction process, extracellular exosome, electron carrier activity, response to reactive oxygen species, and aldehyde dehydrogenase [NAD(P)+] activity. KEGG pathway analysis found DNA methylation-driven genes significantly enriched in five pathways including drug metabolism-cytochrome P450, phenylalanine metabolism, histidine metabolism, glutathione metabolism, and tyrosine metabolism. The validated hub genes were MAOB and RTP4. CONCLUSIONS: Methylated hub genes, including MAOB and RTP4, can be regarded as novel biomarkers for accurate PCa diagnosis and treatment. Further studies are needed to draw more attention to the roles of these hub genes in the occurrence and development of PCa.

Building a Competing Endogenous RNA Network to Find Potential Long Non-Coding RNA Biomarkers for Pheochromocytoma.

BACKGROUND/AIMS: Accumulating evidence has shown that long non-coding RNAs (lncRNAs) in competing endogenous RNA (ceRNA) networks play crucial roles in tumor survival and patient prognosis; however, studies investigating ceRNA networks in pheochromocytoma (PCC) are lacking. In this study, we investigated the pathogenesis of PCC and whether lncRNAs acting through ceRNAs networks were associated with prognosis. METHODS: A total of 183 PCC samples and 3 control samples from The Cancer Genome Atlas database were analyzed. The Empirical Analysis of Digital Gene Expression Data package in R (edgeR) was used to analyze differentially expressed RNAs. Biological processes and pathways functional enrichment analysis were performed based on the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. LncRNA/mRNA/miRNA ceRNA network was constructed by Cytoscape v3.0 software based on the differentially expressed RNAs Survival package in R was used to perform survival analysis. RESULTS: In total, 554 differentially expressed lncRNAs, 1775 mRNAs and 40 miRNAs were selected for further analysis. Subsequently, 23 lncRNAs, 22 mRNAs, and 6 miRNAs were included in the constructed ceRNA network. Meanwhile, two of the 23 lncRNAs (C9orf147 and BSN-AS2) were identified as independent predictors of overall survival in PCC patients (P< 0.05). CONCLUSION: This study improves the understanding of lncRNA-related ceRNA networks in PCC and suggests that the lncRNAs C9orf147 and BSN-AS2 could be independent prognostic biomarkers and potential therapeutic targets for PCC.

Prognosis and clinicopathological characteristics of renal cell carcinoma: does bilateral occurrence influence overall and cancer-specific survival?

BACKGROUND: To compare clinicopathological characteristics and prognosis of bilateral and unilateral renal cell carcinoma. METHODS: Data from patients who had been diagnosed with renal cell carcinoma (RCC) and undergone radical nephrectomy (RN) from 2004 to 2014 were extracted from the Surveillance, Epidemiology, and End Results (SEER) registry. The χ2 test was used to compare relevant characteristics between patients with bilateral and unilateral RCC. Survival curves were generated by the Kaplan-Meier method and the log-rank test was used to compare overall survival (OS) and cancer-specific survival (CSS). Cox proportional hazards regression analysis was used to determine the risk factors for OS and CSS. RESULTS: The study cohort comprised 41,573 patients. A nonlinear relationship between age and OS and CSS was identified with and without adjusting for potential factors. Threshold effect analysis revealed that ages 54 and age 74 were the turning points associated with changes in OS and CSS, respectively. Multivariate Cox regression analysis demonstrated that age category, race, grade, and T, N, and M stage were prognostic factors for OS and CSS of patients with RCC. Additionally, sex and pathology were significantly associated with OS. CONCLUSIONS: Bilateral occurrence does not influence OS and CSS in patients with RCC who have undergone RN. The risk of poor OS and CSS was higher with greater age category, tumor grade, and T stage. Patients in different age categories (<54, ≥54 and <74, ≥74 years) may benefit from individualized attention and therapeutic strategies.

The study on analysis of risk factors for severity of white matter lesions and its correlation with cerebral microbleeds in the elderly with lacunar infarction.

This study aimed to explore the risk factors for severity of white matter lesions and its correlation with in the elderly with lacunar infarction.Patients (range, 70-85 years) with lacunar infarction treated in a hospital in China from 2016 to 2017were enrolled. Fazekas rating scale (0-6 points) was used to assess severity of white matter lesions. Risk factors for the severity of white matter lesions and correlation between cerebral microbleeds and white matter lesions in the elderly with lacunar infarction were studied.The elderly (81-85 years' old, odds ratio [OR]: 2.423, 95% confidence interval [CI]: 1.795-3.271, P = .018; 76∼80 years' old, OR: 3.113, 95% CI: 1.723-5.625, P = .043), carotid atherosclerosis (OR: 3.062, 95% CI:1.715-5.468, P < .001), history of hypertension (OR: 3.694, 95% CI: 2.031-6.717, P < .001) were risk factors for the severity of white matter lesions. The white matter lesions score increased corresponding to increase in the cerebral microbleeds grade (P < .001). The white matter lesions score was higher in the cerebral microbleeds combined with the white matter lesions group than in the white matter lesions group (P < .01). After correcting the effects of age, there was a correlation between white matter lesions and cerebral microbleeds (P < .001). Logistic analysis revealed that the patients' age (81-85 years' old, OR: 2.722, 95% CI: 1.985-3.734, P = .019; 76∼80 years' old, OR: 1.857, 95% CI: 1.075-3.207, P = .031), history of hypertension (OR: 2.931, 95% CI: 1.136-7.567, P = 0.0.036), systolic blood pressure (OR: 1.049, 95% CI: 1.015-1.084, P = .007), high-sensitivity C-reactive protein (OR: 1.504, 95% CI: 1.254-1.803, P < .001), homocysteine (OR: 1.076, 95% CI: 1.020-1.136, P = .009), and carotid atherosclerosis (OR: 1.389, 95% CI: 1.103-1.748, P = .010) were significant risk factors for combined cerebral microbleeds with white matter lesions in patients with lacunar infarction.The elderly, carotid atherosclerosis, history of hypertension were risk factors for the severity of white matter lesions. Cerebral microbleeds were positively correlated with the severity of white matter lesions.

Risk factors of developing visceral metastases at diagnosis in prostate cancer patients.

BACKGROUND: Risk factors of visceral metastases in prostate cancer (PCa) patients are unclear. The aim of this study is to investigate the risk factors of developing visceral metastases at diagnosis and the impact of these risk factors on the survival of patients with visceral metastatic PCa. METHODS: Patients with visceral metastases at the time of diagnosis of [2010-2015] PCa were identified using the Surveillance, Epidemiology, and End Results (SEER) database. Visceral metastatic distribution data were provided for liver, lung, and brain. The overall survival (OS) was calculated by the Kaplan-Meier method. Multivariable logistic and Cox regression models were performed to identify risk factors and analyze survival outcomes. RESULTS: A total of 13,092 eligible patients with stage IV PCa were identified from SEER database. A total of 598 patients developed visceral metastases at diagnosis among these patients. In multivariable analyses, patients with PSA >80 ng/mL had 1.545-fold higher risk of developing visceral metastases compared with those with PSA <20 ng/mL (P<0.001). The presence of bone metastasis and lymph node (LN) metastases were represented as risk factors of visceral metastases in stage IV PCa patients. Patients with two or three metastatic sites had 1.604-fold higher risk of shorter OS compared with those with one metastatic site (P<0.05). And patients with bone plus visceral metastases had 1.410-fold higher risk of shorter OS compared with those with visceral metastasis only (P<0.05). Age ≥70 had 1.621-fold higher risk of shorter OS compared with those with age <70 (P<0.05). T4 stage had 1.476-fold higher risk of shorter OS compared with those with T1 stage (P<0.05). CONCLUSIONS: The incidence rate was increased among patients with visceral metastases in stage IV PCa at diagnosis. PSA over 80 ng/mL, the presence of bone metastasis and the presence of LN metastases were risk factors associated with a higher rate of development of visceral metastases in stage IV PCa patients. The presence of visceral plus bone metastases, two or three sites, age over 70, and T4 stage represent prognostic factors on survival outcomes in visceral metastatic PCa patients.

Fasudil inhibits actin polymerization and collagen synthesis and induces apoptosis in human urethral scar fibroblasts via the Rho/ROCK pathway.

PURPOSE: To examine the effects and mechanism of action of fasudil on cytoskeletal polymerization, collagen synthesis, and apoptosis in fibroblasts derived from human urethral scar tissue. MATERIALS AND METHODS: Fibroblasts treated with or without transforming growth factor ß1 (TGF-ß1, 10 ng/mL) were incubated with fasudil (12.5, 25, 50 µmol/L) for 24 hours. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression of Arp2, Arp3, WASP, and WAVE2. Collagen I and III protein levels were also evaluated by Western blotting. The filamentous actin cytoskeleton was examined by immunofluorescence and epifluorescence microscopy. An Annexin V-FITC/PI staining assay was used to investigate apoptosis. RESULTS: TGF-ß1-dependent induction of actin polymerization and collagen synthesis and promotion of apoptosis were dose dependent. When compared with untreated controls, fasudil significantly decreased the expression of Arp2, Arp3, WASP, WAVE2, Collagen I, and Collagen III in cells treated with or without TGF-ß1. Fasudil also promoted apoptosis in cells, irrespective of TGF-ß1 treatment. CONCLUSION: Irrespective of TGF-ß1 activation status, fasudil suppressed actin polymerization and collagen synthesis and induced apoptosis in human urethral scar fibroblasts via the Rho/ROCK signaling pathway.

Potential role of recombinant adeno-associated virus human thioredoxin-PR39 in cell and vascular protection against hypoxia.

The aim of the present study was to successfully construct a recombinant adeno-associated virus (rAAV) vector containing the human thioredoxin (hTRX)-PR39 chimeric gene (rAAV/hTRX-PR39), and verify that the vector was able to maintain a sustained, stable and efficient expression to achieve protein production in the cell. In the present study, a chicken embryo model was utilized to analyze the therapeutical effect of rAAV/hTRX-PR39 in cerebral ischemia diseases. ECV304 cells were transfected with rAAV/hTRX-PR39 and incubated under conditions of 20, 5 and 1% O2. Subsequently, the expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, fibroblast growth factor receptor (FGFR)-1 and syndecan-4 were detected by reverse transcription-quantitative polymerase chain reaction. Under hypoxic conditions, the mRNA expression levels of VEGF, VEGFR-1, VEGFR-2, FGFR-1 and syndecan-4 were found to increase in the PR39-transfected group when compared with the control group, while no statistically significant difference was observed between the PR39-transfected group and the control group under conditions of 20% O2. In addition, hTRX-PR39 was shown to increase the density of the vasculature and the survival rate of the chick embryos. Under hypoxic conditions, it was hypothesized that rAAV/hTRX-PR39 was capable of promoting angiogenesis, which may subsequently protect the cells from impairment by hypoxia. In conclusion, rAAV/hTRX-PR39 was demonstrated to promote vascularization and cell survival in hypoxia; thus, rAAV/hTRX-PR39 may have potential for use in therapy targeting cerebral ischemia.

Adsorption of tripeptide RGD on rutile TiO(2) nanotopography surface in aqueous solution.

Molecular dynamics simulations were carried out to investigate the adsorption mechanisms of tripeptide Arg-Gly-Asp (RGD) on the nanotopography and perfect rutile TiO(2) (110) surfaces in aqueous solution. It is shown that the amino groups (NH(2) and NH3+) and carboxyl group (COO(-)) of RGD are the main groups bonding to hydrophilic TiO(2) surface by electrostatic and van der Waals interactions. It is also demonstrated that RGD adsorbs much more rapidly and stably on the nanotopography surface than the perfect surface. On the hydrophilic TiO(2) surface, the water molecules occupy the adsorption sites to form hydration layers, which have a significant influence on RGD adsorption. On the perfect surface, since the fivefold titanium atom is surrounded by surface bridging oxygen atoms above it and has a water molecule bonding to it, the amino group NH(2) is the adsorption group. However, because the pit surface exposes more adsorption sites and has higher surface energy, RGD can adsorb rapidly on the surfaces by amino groups NH(2) and NH3+, and the carboxyl group COO(-) may edge out the adsorbed water molecules and bond to the surface titanium atom. Moreover, the surface with higher surface energy has more adsorption energy of RGD.