Mechanism of RhoA regulating benign prostatic hyperplasia: RhoA-ROCK-ß-catenin signaling axis and static & dynamic dual roles.

BACKGROUND: The pathogenesis of benign prostatic hyperplasia (BPH) has not been fully elucidated. Ras homology family member A (RhoA) plays an important role in regulating cell cytoskeleton, growth and fibrosis. The role of RhoA in BPH remains unclear. METHODS: This study aimed to clarify the expression, functional activity and mechanism of RhoA in BPH. Human prostate tissues, human prostate cell lines, BPH rat model were used. Cell models of RhoA knockdown and overexpression were generated. Immunofluorescence staining, quantitative real time PCR (qRT-PCR), Western blotting, cell counting kit-8 (CCK-8), flow cytometry, phalloidine staining, organ bath study, gel contraction assay, protein stability analysis, isolation and extraction of nuclear protein and cytoplasmic protein were performed. RESULTS: In this study we found that RhoA was localized in prostate stroma and epithelial compartments and was up-regulated in both BPH patients and BPH rats. Functionally, RhoA knockdown induced cell apoptosis and inhibited cell proliferation, fibrosis, epithelial-mesenchymal transformation (EMT) and contraction. Consistently, overexpression of RhoA reversed all aforementioned processes. More importantly, we found that ß-catenin and the downstream of Wnt/ß-catenin signaling, including C-MYC, Survivin and Snail were up-regulated in BPH rats. Downregulation of RhoA significantly reduced the expression of these proteins. Rho kinase inhibitor Y-27632 also down-regulated ß-catenin protein in a concentration-dependent manner. However, overexpression of ß-catenin did not affect RhoA-ROCK levels, suggesting that ß-catenin was the downstream of RhoA-ROCK regulation. Further data suggested that RhoA increased nuclear translocation of ß-catenin and up-regulated ß-catenin expression by inhibiting its proteasomal degradation, thereby activating Wnt/ß-catenin signaling. Overexpression of ß-catenin partially reversed the changes in cell growth, fibrosis and EMT except cell contraction caused by RhoA downregulation. Finally, Y-27632 partially reversed prostatic hyperplasia in vivo, further suggesting the potential of RhoA-ROCK signaling in BPH treatment. CONCLUSION: Our novel data demonstrated that RhoA regulated both static and dynamic factors of BPH, RhoA-ROCK-ß-catenin signaling axis played an important role in the development of BPH and might provide more possibilities for the formulation of subsequent clinical treatment strategies.

Glutathione Peroxidase 3 induced mitochondria-mediated apoptosis via AMPK /ERK1/2 pathway and resisted autophagy-related ferroptosis via AMPK/mTOR pathway in hyperplastic prostate.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a common disease in elderly men, mainly resulted from an imbalance between cell proliferation and death. Glutathione peroxidase 3 (GPX3) was one of the differentially expressed genes in BPH identified by transcriptome sequencing of 5 hyperplastic and 3 normal prostate specimens, which had not been elucidated in the prostate. This study aimed to ascertain the mechanism of GPX3 involved in cell proliferation, apoptosis, autophagy and ferroptosis in BPH. METHODS: Human prostate tissues, GPX3 silencing and overexpression prostate cell (BPH-1 and WPMY-1) models and testosterone-induced rat BPH (T-BPH) model were utilized. The qRT-PCR, CCK8 assay, flow cytometry, Western blotting, immunofluorescence, hematoxylin and eosin, masson's trichrome, immunohistochemical staining and transmission electron microscopy analysis were performed during in vivo and in vitro experiments. RESULTS: Our study indicated that GPX3 was localized both in the stroma and epithelium of prostate, and down-regulated in BPH samples. Overexpression of GPX3 inhibited AMPK and activated ERK1/2 pathway, thereby inducing mitochondria-dependent apoptosis and G0/G1 phase arrest, which could be significantly reversed by MEK1/2 inhibitor U0126 preconditioning. Moreover, overexpression of GPX3 further exerted anti-autophagy by inhibiting AMPK/m-TOR and up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4, mitochondrial GPX4 and cytoplasmic GPX4) to antagonize autophagy-related ferroptosis. Consistently, GPX3 deficiency generated opposite changes in both cell lines. Finally, T-BPH rat model was treated with GPX3 indirect agonist troglitazone (TRO) or GPX4 inhibitor RAS-selective lethal 3 (RSL3) or TRO plus RSL3. These treatments produced significant atrophy of the prostate and related molecular changes were similar to our in vitro observations. CONCLUSIONS: Our novel data manifested that GPX3, which was capable of inducing apoptosis via AMPK/ERK1/2 pathway and antagonizing autophagy-related ferroptosis through AMPK/m-TOR signalling, was a promising therapeutic target for BPH in the future.

Simvastatin Improves Benign Prostatic Hyperplasia: Role of Peroxisome-Proliferator-Activated Receptor-γ and Classic WNT/ß-Catenin Pathway.

Benign prostatic hyperplasia (BPH) is a common disease in elderly men with an uncertain etiology and mechanistic basis. Metabolic syndrome (MetS) is also a very common illness and is closely related to BPH. Simvastatin (SV) is one of the widely used statins for MetS. Peroxisome-proliferator-activated receptor gamma (PPARγ), crosstalking with the WNT/ß-catenin pathway, plays important roles in MetS. Our current study aimed to examine SV-PPARγ-WNT/ß-catenin signaling in the development of BPH. Human prostate tissues and cell lines plus a BPH rat model were utilized. Immunohistochemical, immunofluorescence, hematoxylin and eosin (H&E) and Masson's trichrome staining, construction of a tissue microarray (TMA), ELISA, CCK-8 assay, qRT-PCR, flow cytometry, and Western blotting were also performed. PPARγ was expressed in both prostate stroma and epithelial compartments and downregulated in BPH tissues. Furthermore, SV dose-dependently triggered cell apoptosis and cell cycle arrest at the G0/G1 phase and attenuated tissue fibrosis and the epithelial-mesenchymal transition (EMT) process both in vitro and in vivo. SV also upregulated the PPARγ pathway, whose antagonist could reverse SV produced in the aforementioned biological process. Additionally, crosstalk between PPARγ and WNT/ß-catenin signaling was demonstrated. Finally, correlation analysis with our TMA containing 104 BPH specimens showed that PPARγ was negatively related with prostate volume (PV) and free prostate-specific antigen (fPSA) and positively correlated with maximum urinary flow rate (Qmax). WNT-1 and ß-catenin were positively related with International Prostate Symptom Score (IPSS) and nocturia, respectively. Our novel data demonstrate that SV could modulate cell proliferation, apoptosis, tissue fibrosis, and the EMT process in the prostate through crosstalk between PPARγ and WNT/ß-catenin pathways.

Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 (CXCL13) in Hyperplastic Prostate.

BACKGROUND: C-X-C motif chemokine ligand 13 (CXCL13), a member of the CXC subtype in chemokine superfamily, affects numerous biological processes of various types of cells and the progress of a great number of clinical diseases. The purpose of the current study was to reveal the internal mechanism between CXCL13 and benign prostatic hyperplasia (BPH). METHODS: Human serum, prostate tissues and human prostate cell lines (BPH-1, WPMY-1) were utilized. The effect of recombinant human CXCL13 (rHuCXCL13) protein and the influences of the knockdown/overexpression of CXCL13 on two cell lines were studied. Rescue experiments by anti-CXCR5 were also conducted. In vivo, rHuCXCL13 was injected into the ventral prostate of rats. Additionally, a tissue microarray of hyperplastic prostate tissues was constructed to analyze the correlations between CXCL13 and clinical parameters. RESULTS: CXCL13 was highly expressed in the prostate tissues and upregulated in the BPH group. It was observed that CXCL13 modulated cell proliferation, apoptosis, and the epithelial-mesenchymal transition (EMT) through CXCR5 via AKT and the ERK1/2 pathway in BPH-1, while it contributed to inflammation and fibrosis through CXCR5 via the STAT3 pathway in WPMY-1. In vivo, rHuCXCL13 induced the development of rat BPH. Additionally, CXCL13 was positively correlated with the prostate volume and total prostate specific antigen. CONCLUSIONS: Our novel data demonstrated that CXCL13 modulated cell proliferation, cell cycle, the EMT of epithelial cells, and induced the fibrosis of prostatic stromal cells via a variety of inflammatory factors, suggesting that CXCL13 might be rediscovered as a potential therapeutic target for the treatment of BPH.

M2a macrophage can rescue proliferation and gene expression of benign prostate hyperplasia epithelial and stroma cells from insulin-like growth factor 1 knockdown.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a common disease in elderly men and is often accompanied by chronic inflammation. Macrophages (several subtypes) are the main inflammatory cells that infiltrate the hyperplastic prostate and are found to secrete cytokines and growth factors. The current study aims to explore the effect of M2a macrophages on the development of BPH via insulin-like growth factor 1 (IGF-1). METHODS: Human prostate tissues, prostate, and monocyte cell lines (WPMY-1, BPH-1, and THP-1) were used. THP-1 was polarized into several subtypes with cytokines. The expression and localization of IGF-1 and M2 macrophages were determined via immunofluorescent staining, quantitative real-time polymerase chain reaction, and Western blot analysis. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to investigate the effects of different subtypes of macrophages on prostate cells. IGF-1 in WPMY-1 and BPH-1 cells was silenced and cocultured with or without M2a macrophages. Cell proliferation, apoptosis, cell cycle, epithelial-mesenchymal transition (EMT), and fibrosis processes were examined. RESULTS: The polarized subtypes of macrophages were verified by amplifying their specific markers. M2a macrophages enhanced prostate cell proliferation more significantly and CD206 was more expressed in hyperplastic prostate. IGF-1 was localized in both epithelial and stromal components of prostate and upregulated in BPH tissues. M2a macrophages expressed more IGF-1 than other subtypes. Knockdown of IGF-1 in WPMY-1 and BPH-1 cells attenuated cell proliferation, promoted cell apoptosis, retarded cell cycle at the G0/G1 phase, and suppressed the EMT process in BPH-1 cells as well as the fibrotic process in WPMY-1 cells, which was reversible when cocultured with M2a macrophages. CONCLUSION: These data demonstrated that knockdown of IGF-1 expression in cultured BPH epithelial and stromal cells reduces proliferation and increases apoptosis. These effects are reversed by coculture with M2a macrophages.

Upregulated bone morphogenetic protein 5 enhances proliferation and epithelial-mesenchymal transition process in benign prostatic hyperplasia via BMP/Smad signaling pathway.

BACKGROUND: Benign prostatic hyperplasia (BPH) is one of the most common illnesses in aging men. Recent studies found that bone morphogenetic protein 5 (BMP5) is upregulated in BPH tissues, however, the role of BMP5 in the development of BPH has not been examined. The current study aims to elucidate the potential roles of BMP5 and related signaling pathways in BPH. METHODS: Human prostate cell lines (BPH-1, WPMY-1) and human/rat hyperplastic prostate tissues were utilized. Western blot, quantitative real-time polymerase chain reaction, immunofluorescent staining, and immunohistochemical staining were performed. BMP5-silenced and -overexpressed cell models were generated and then cell cycle progression, apoptosis, and proliferation were determined. The epithelial-mesenchymal transition (EMT) was also quantitated. And rescue experiments by BMP/Smad signaling pathway agonist or antagonist were accomplished. Moreover, BPH-related tissue microarray analysis was performed and associations between clinical parameters and expression of BMP5 were analyzed. RESULTS: Our study demonstrated that BMP5 was upregulated in human and rat hyperplastic tissues and localized both in the epithelial and stromal compartments of the prostate tissues. E-cadherin was downregulated in hyperplastic tissues, while N-cadherin and vimentin were upregulated. Overexpression of BMP5 enhanced cell proliferation and the EMT process via phosphorylation of Smad1/5/8, while knockdown of BMP5 induced cell cycle arrest at G0/G1 phase and blocked the EMT process. Moreover, a BMP/Smad signaling pathway agonist and antagonist reversed the effects of BMP5 silencing and overexpression, respectively. In addition, BMP5 expression positively correlated with prostate volume and total prostate-specific antigen. CONCLUSION: Our novel data suggest that BMP5 modulated cell proliferation and the EMT process through the BMP/Smad signaling pathway which could contribute to the development of BPH. However, further studies are required to determine the exact mechanism. Our study also indicated that BMP/Smad signaling may be rediscovered as a promising new therapeutic target for the treatment of BPH.

Changes in the expression and functional activities of Myosin II isoforms in human hyperplastic prostate.

Benign prostatic hyperplasia (BPH) is a common disease among aging males with the etiology remaining unclear. We recently found myosin II was abundantly expressed in rat and cultured human prostate cells with permissive roles in the dynamic and static components. The present study aimed to explore the expression and functional activities of myosin II isoforms including smooth muscle (SM) myosin II (SMM II) and non-muscle myosin II (NMM II) in the hyperplastic prostate. Human prostate cell lines and tissues from normal human and BPH patients were used. Hematoxylin and Eosin (H&E), Masson's trichrome, immunohistochemical staining, in vitro organ bath, RT-polymerase chain reaction (PCR) and Western-blotting were performed. We further created cell models with NMM II isoforms silenced and proliferation, cycle, and apoptosis of prostate cells were determined by cell counting kit-8 (CCK-8) assay and flow cytometry. Hyperplastic prostate SM expressed more SM1 and LC17b isoforms compared with their alternatively spliced counterparts, favoring a slower more tonic-type contraction and greater force generation. For BPH group, blebbistatin (BLEB, a selective myosin II inhibitor), exhibited a stronger effect on relaxing phenylephrine (PE) pre-contracted prostate strips and inhibiting PE-induced contraction. Additionally, NMMHC-A and NMMHC-B were up-regulated in hyperplastic prostate with no change in NMMHC-C. Knockdown of NMMHC-A or NMMHC-B inhibited prostate cell proliferation and induced apoptosis, with no changes in cell cycle. Our novel data demonstrate that expression and functional activities of myosin II isoforms are altered in human hyperplastic prostate, suggesting a new pathological mechanism for BPH. Thus, the myosin II system may provide potential new therapeutic targets for BPH/lower urinary tract symptoms (LUTS).

NELL2 modulates cell proliferation and apoptosis via ERK pathway in the development of benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is a quite common illness but its etiology and mechanism remain unclear. Neural epidermal growth factor-like like 2 (NELL2) plays multifunctional roles in neural cell growth and is strongly linked to the urinary tract disease. Current study aims to determine the expression, functional activities and underlying mechanism of NELL2 in BPH. Human prostate cell lines and tissues from normal human and BPH patients were utilized. Immunohistochemical staining, immunofluorescent staining, RT-polymerase chain reaction (PCR) and Western blotting were performed. We further generated cell models with NELL2 silenced or overexpressed. Subsequently, proliferation, cycle, and apoptosis of prostate cells were determined by cell counting kit-8 (CCK-8) assay and flow cytometry analysis. The epithelial-mesenchymal transition (EMT) and fibrosis process were also analyzed. Our study revealed that NELL2 was up-regulated in BPH samples and localized in the stroma and the epithelium compartments of human prostate tissues. NELL2 deficiency induced a mitochondria-dependent cell apoptosis, and inhibited cell proliferation via phosphorylating extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Additionally, suppression of ERK1/2 with U0126 incubation could significantly reverse NELL2 deficiency triggered cell apoptosis. Consistently, overexpression of NELL2 promoted cell proliferation and inhibited cell apoptosis. However, NELL2 interference was observed no effect on EMT and fibrosis process. Our novel data demonstrated that up-regulation of NELL2 in the enlarged prostate could contribute to the development of BPH through enhancing cell proliferation and inhibited a mitochondria-dependent cell apoptosis via the ERK pathway. The NELL2-ERK system might represent an important target to facilitate the development of future therapeutic approaches in BPH.

Alterations in the phosphodiesterase type 5 pathway and oxidative stress correlate with erectile function in spontaneously hypertensive rats.

To explore how alterations in the phosphodiesterase type 5 (PDE5) signalling pathway and oxidative stress correlate with changes in the expression of relaxation and contraction molecules and erectile dysfunction (ED) in the corpus cavernosum smooth muscle (CCSM) of spontaneously hypertensive rats (SHR). In this study, SHR and Wistar-Kyoto (WKY) rats were used. Erectile function was determined by apomorphine test and electrical stimulation (ES) of cavernous nerve. Masson's trichrome staining and confocal microscopy were performed. Nitric oxide synthase (NOS), PDE5, phosphorylated-PDE5 and α1-adrenergic receptor (α1AR) were determined by RT-PCR and Western blotting while oxidative stress in CC was determined by colorimetric analysis. SHR exhibited obvious ED. CC of SHR showed less SM but more collagen fibres. The expression of NOS isoforms in SHR was significantly decreased while all α1AR isoforms were increased. In addition, PDE5 and phosphorylated-PDE5 were down-regulated and its activity attenuated in the hypertensive rats. Meanwhile, the SHR group suffered oxidative stress, which may be modulated by endoplasmic reticulum stress and NADPH oxidase up-regulation. Dysregulation of NOS and α1AR, histological changes and oxidative stress in CC may be associated with the pathophysiology of hypertension-induced ED. In addition, PDE5 down-regulation may lead to the decreased efficacy of PDE5 inhibitors in some hypertensive ED patients and treatment of oxidative stress could be used as a new therapeutic target for this type of ED.

Changes in the expression and function of the PDE5 pathway in the obstructed urinary bladder.

Our study aims to explore changes in bladder contractility and the phosphodiesterase type 5 (PDE5) signalling pathway in response to partial bladder outlet obstruction (PBOO). A surgically induced male rat PBOO model and human obstructed bladder tissues were used. Histological changes were examined by H&E and Masson's trichrome staining. Bladder strip contractility was measured via organ bath. The expressions of nitric oxide synthase (NOS) isoforms, PDE5, muscarinic cholinergic receptor (CHRM) isoforms and PDE4 isoforms in bladder were detected by RT-PCR and Western blotting. The immunolocalization of the PDE5 protein and its functional activity were also determined. PBOO bladder tissue exhibited significant SM hypertrophy and elevated responsiveness to KCl depolarization and the muscarinic receptor agonist carbachol. NOS isoforms, PDE5, CHRM2, CHRM3 and PDE4A were up-regulated in obstructed bladder tissue, whereas no change in PDE4B and PDE4D isoform expression was observed. With regard to PDE5, it was expressed in the SM bundles of bladder. Interestingly, obstructed bladder exhibited less relaxation responsiveness to sodium nitroprusside (SNP), but an exaggerated PDE5 inhibition effect. The up-regulation of PDE5 could contribute to the lack of effect on Qmax for benign prostatic hyperplasia/lower urinary tract symptom (BPH/LUTS) patients treated with PDE5 inhibitors. Moreover, PDE5 (with presence of NO) and PDE4 may serve as new therapeutic targets for bladder diseases such as BPH-induced LUTS and overactive bladder (OAB).

Advantages of robot-assisted resection of large mediastinal tumors: a single-center preliminary study.

Current study aims to assess the safety and efficacy of robot-assisted thoracoscopic surgery (RATS) for sizable mediastinal masses with a minimum diameter ≥6 cm, compared with video-assisted thoracoscopic surgery (VATS) and open surgery. This study enrolled 130 patients with mediastinal tumors with no less than 6 cm diameter in Zhongnan Hospital, Wuhan University, including 33 patients who underwent RATS, 52 patients who underwent VATS and 45 patients who underwent open surgery. After classifying based on mass size and whether it has invaded or not, we compared their clinical characteristics and perioperative outcomes. There was no significant difference in age, gender, mass size, myasthenia gravis, mass location, pathological types (p > 0.05) in three groups. Patients undergoing open surgery typically presenting at a more advanced stage (p < 0.05). No obvious difference was discovered in the average postoperative length of stay, operation duration, chest tube duration and average postoperative day 1 drainage output between RATS group and VATS group (p > 0.05), while intraoperative blood loss in RATS group was significantly lower than VATS group (p = 0.046). Moreover, the postoperative length of stay, operation duration, chest tube duration and intraoperative blood loss in RATS group were significantly lower than open surgery group (p < 0.001). RATS is a secure and efficient approach for removing large mediastinal masses at early postoperative period. In comparison with VATS, RATS is associated with lower intraoperative blood loss. Compared with open surgery, RATS is also associated with shorter postoperative length of stay, operation duration, chest tube duration and intraoperative blood loss.

S100A4 modulates cell proliferation, apoptosis and fibrosis in the hyperplastic prostate.

Benign prostatic hyperplasia (BPH) is one of the most common diseases in elderly men worldwide that may result in lower urinary tract symptoms (LUTS). At present, the specific pathophysiological mechanism for BPH/LUTS LUTS remains unclear. S100 calcium binding protein A4 (S100A4), a member of the calcium binding protein family, regulates a variety of biological processes including cell proliferation, apoptosis and fibrosis. The aim of the current study was to explore and clarify the possible role of S100A4 in BPH/LUTS. The human prostate stromal cell line (WPMY-1), rat prostate epithelial cells, human prostate tissues and two BPH rat models were employed in this study. The expression and localization of S100A4 were detected by quantitative real time PCR (qRT-PCR), immunofluorescence microscopy, Western blotting and immunohistochemistry analysis. Also, S100A4 knockdown or overexpression cell models were constructed and a BPH rat model was induced with testosterone propionate (T) or phenylephrine (PE). The BPH animals were treated with Niclosamide, a S100A4 transcription inhibitor. Results demonstrated that S100A4 was mainly localized in human prostatic stroma and rat prostatic epithelium, and showed a higher expression in BPH. Knockdown of S100A4 induced cell apoptosis, cell proliferation arrest and a reduction of tissue fibrosis markers. Overexpression of S100A4 reversed the aforementioned changes. We also demonstrated that S100A4 regulated proliferation and apoptosis mainly through the ERK pathway and modulated fibrosis via Wnt/ß-catenin signaling. In conclusion, our novel data demonstrate that S100A4 could play a crucial role in BPH development and may be explored as a new therapeutic target of BPH.

Upregulated X-C motif chemokine ligand 2 (XCL2) is associated with poor prognosis and increased immune infiltration in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the most popular malignant carcinomas in the genitourinary system. As a novel tumor-related gene, X-C Motif Chemokine Ligand 2 (XCL2) was up-regulated in ccRCC. The current study aims to reveal the functional activity of XCL2 in ccRCC. METHODS: The transcriptome profiling, clinical parameters, and simple nucleotide variation profiles of ccRCC samples were obtained from the Cancer Genome Atlas (TCGA) database. The survival analysis, multivariate/univariate Cox analysis, correlation analysis, gene set enrichment analysis (GSEA), and tumor mutation burden (TMB) analysis were performed. Next, immune cell infiltration and immune functions were analyzed. Finally, the functions of XCL2 were investigated in Caki-1 and 786-O cells. RESULTS: Upregulated XCL2 was associated with worse overall survival of ccRCC and correlated to age, grade, stage, and T stage. Age, grade, and XCL2 were independent prognostic factors. Significant enrichment in apoptosis, DNA replication, and immune response was demonstrated by GSEA. XCL2 was not only tightly associated with immune cell infiltration, but also significantly linked with several immune functions. Moreover, patients, who had higher XCL2 expression, owned higher levels of TMB. Interestingly, XCL2 was positively correlated with common immune checkpoints. In vitro, XCL2 could inhibit apoptosis, and promote proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of Caki-1 and 786-O cells. CONCLUSIONS: In general, the current study suggested that XCL2 may participate in the progression of ccRCC. Importantly, XCL2 may be a potential new target of immunotherapy.

Expression and functional activity of myosin II in hyperplastic prostates of varying volumes.

Prostate volume (PV) differs dramatically among benign prostatic hyperplasia (BPH) patients. Estimation of PV is important to guide the most appropriate pharmacologic or interventional treatment approach. However, the underlying pathophysiological mechanisms for the differences in PV remain unknown. We recently found that the myosin II system might participate in the etiology and development of BPH via static and dynamic factors. Our present study aims to explore the expression and functional activities of myosin II isoforms including smooth muscle (SM) myosin II (SMM II) and non-muscle myosin II (NMM II) in hyperplastic prostates with varied PV. Human hyperplastic prostates and the testosterone-induced rat BPH model were employed for this study. Hematoxylin and Eosin (H&E), Masson's trichrome, immunohistochemical staining, in vitro organ bath, RT-polymerase chain reaction (PCR) and Western-blotting were performed. Also, a BPH tissue microarray (TMA) was constructed to determine the correlations between myosin II isoforms with clinical parameters of BPH patients. With the increase of PV, the expression of NMMHC-A, NMMHC-C, SM-A and LC17b isoforms were increased, and the contractility of prostate smooth muscle was enhanced but force developed more slowly. Consistently, NMMHC-A, NMMHC-C, SM-A and LC17b were correlated positively with PV. Similar outcomes were also observed in the BPH rat model with different PVs. Alterations in the expression and function of myosin the II system may be involved in the pathophysiological mechanism of PV differences between BPH patients.

Fibronectin-1: A Predictive Immunotherapy Response Biomarker for Muscle­Invasive Bladder Cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is characterized as bladder tumors that infiltrate into the muscle layer, along with multiple metastasis and poor prognosis. Numerous research studies have been performed to identify the underlying clinical and pathological alterations that occur. However, few studies have revealed the molecular mechanism of its progression based upon the immunotherapy response. Our present study was designed to identify biomarkers which may predict the immunotherapy response by investigating the tumor microenvironment (TME) in MIBC. METHODS: The transcriptome and clinical data of MIBC patients were obtained and analyzed with R version 4.0.3 (POSIT Software, Boston, MA, USA) ESTIMATE package. Differentially expressed immune-related genes (DEIRGs) were identified and further analyzed via the protein-protein interaction network (PPI). Meanwhile, univariate Cox analysis was utilized to screen out the prognostic DEIRGs (PDEIRGs). Then, the PPI core gene was matched with PDEIRGs to obtain the target gene-fibronectin-1 (FN1). Human MIBC and control tissues were collected and FN1 was measured with Quantitative Reverse Transcription PCR (qRT-PCR) and Western-Blot. Finally, the relationship between FN1 expression level and MIBC was validated through survival, univariate Cox, multivariate Cox, Gene Set Enrichment Analysis (GSEA) and correlation analysis of tumor infiltrating immune cells. RESULTS: TME DEIRGs were identified and the target gene FN1 was obtained. The higher expression of FN1 was confirmed in MIBC tissues via bioinformatics analysis, qRT-PCR and Western-Blot. Moreover, higher FN1 expression correlated with reduced survival time and FN1 expression was further favorably correlated with clinic-pathological features (grade, TNM stage, invasion, lymphatic and distant metastasis). Additionally, the genes in the high FN1 expression group were mainly enriched in immune-related activities and macrophage M2, T cell CD4, T cell CD8 and T cell follicular helper cells were correlated with FN1. Finally, it was observed that FN1 was closely related to key immune checkpoints. CONCLUSIONS: FN1 was identified as a novel and independent prognostic factor for MIBC. Our data also suggests FN1 can predict MIBC patients' response to immune checkpoints inhibitors.

DHCR7 promotes tumorigenesis via activating PI3K/AKT/mTOR signalling pathway in bladder cancer.

Bladder cancer (BCa) is a common malignancy with uncertain molecular mechanism. 7-dehydrocholesterol reductase (DHCR7), the enzyme of mammalian sterol biosynthesis, plays important roles in several types of cancers but its specific function in BCa is still unknown. The current study aimed to determine the bioinformatic characteristics and biological functions of DHCR7 in BCa. Sequencing results and clinical data from online public databases, human BCa tissues and matched noncancerous tissues, xenograft nude mice, DHCR7 deficiency and overexpression BCa cell (T24 and EJ) models were used. Several bioinformatics analyses were made, qRT-PCR, Western-blotting, flow cytometry, immunohistochemistry (IHC), MTT assay, wound healing and cell invasion assays were performed. It was found that DHCR7 was upregulated in BCa as an independent risk factor, and the expression of DHCR7 was associated with BCa grade and stage, finally resulted in poor prognosis. We further demonstrated that DHCR7 overexpression could accelerate the G0/G1 phase to accelerate the growth of tumor cells, antagonize cell apoptosis, and enhance the invasion and migration capacity, as well as EMT process via PI3K/AKT/mTOR signalling pathway, which could be completely reversed by DHCR7 knockdown. Finally, DHCR7 deficiency significantly decreased tumorigenesis in vivo. Our novel data demonstrated that DHCR7 could modulate BCa tumorigenesis in vitro and in vivo via PI3K/AKT/mTOR signalling pathway. It is suggested that DHCR7 might become a molecular target for the diagnosis and treatment of BCa.

Identification of Hub Genes Associated with Progression and Prognosis of Bladder Cancer by Integrated Bioinformatics Analysis.

BACKGROUND: Bladder cancer (BLCA) is an extremely common carcinoma of the urinary system that has a high incidence of relapse. Although intensive studies have investigated its pathology in the past decades, there are significant knowledge gaps regarding the characterization of the molecular processes underlying the progression of disease and consequently its prognosis. The purpose of current research was to identify significant genes that could serve as prognostic and progression biomarkers. METHODS: Gene expression profiles were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. Differential gene expression analysis (DGEA) and weighted gene co-expression network analysis (WGCNA) were conducted to recognize differential co-expression genes. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to explore gene function. Moreover, protein-protein interactions (PPI) network, overall survival (OS) and disease-free survival (DFS) were used to identify survival-related hub genes. Additionally, associations between these gene's expression and clinical parameters were determined. Finally, the Human Protein Atlas (HPA) database and qRT-PCR were used to validate gene's expression. RESULTS: About 124 differential co-expression genes were identified. These genes were mainly enriched in muscle system process and muscle contraction (biological process, BP), contractile fiber, myofibril, sarcomere, focal adhesion and cell-substrate junction (cellular component, CC) and actin binding (molecular function, MF) in GO enrichment analysis, while enriched in vascular smooth muscle contraction, focal adhesion, cardiac muscle contraction, hypertrophic cardiomyopathy, dilated cardiomyopathy and regulation of actin cytoskeleton in KEGG analysis. Furthermore, five survival-related hub genes (MYH11, ACTA2, CALD1, TPM1, MYLK) were identified via OS and DFS. In addition, these survival-related gene's expression was correlated with grade, stage and TNM stage. Finally, all survival-related hub genes were determined to be down-regulated in BLCA tissues by qRT-PCR and HPA databases. CONCLUSIONS: Our current study verified five new key genes in BLCA, which may participate in the prognosis and progression and serve as novel biomarkers of BLCA.

The Prostate-Associated Gene 4 (PAGE4) Could Play a Role in the Development of Benign Prostatic Hyperplasia under Oxidative Stress.

Benign prostatic hyperplasia (BPH) is a common disease in elderly men with uncertain molecular mechanism, and oxidative stress (OS) has also been found associated with BPH development. Recently, we found that prostate-associated gene 4 (PAGE4) was one of the most significantly changed differentially expressed genes (DEGs) in BPH, which can protect cells against stress stimulation. However, the exact role of PAGE4 in BPH remains unclear. This study is aimed at exploring the effect of PAGE4 in BPH under OS. Human prostate tissues and cultured WPMY-1 and PrPF cells were utilized. The expression and localization of PAGE4 were determined with qRT-PCR, Western blotting, and immunofluorescence staining. OS cell models induced with H2O2 were treated with PAGE4 silencing or PAGE4 overexpression or inhibitor (N-acetyl-L-cysteine (NAC)) of OS. The proliferation activity, apoptosis, OS markers, and MAPK signaling pathways were detected by CCK-8 assay, flow cytometry analysis, and Western blotting. PAGE4 was shown to be upregulated in human hyperplastic prostate and mainly located in the stroma. Acute OS induced with H2O2 increased PAGE4 expression (which was prevented by OS inhibitor), apoptosis, cell cycle arrest, and reactive oxygen species (ROS) accumulation in WPMY-1 and PrPF cells. siPAGE4 plus H2O2 potentiated H2O2 effect via reducing the p-ERK1/2 level and increasing p-JNK1/2 level. Consistently, overexpression of PAGE4 offset the effect of H2O2 and partially reversed the PAGE4 silencing effect. However, knocking down and overexpression of PAGE4 alone determined no significant effects. Our novel data demonstrated that augmented PAGE4 promotes cell survival by activating p-ERK1/2 and decreases cell apoptosis by inhibiting p-JNK1/2 under the OS, which could contribute to the development of BPH.

Glucose-regulated protein 78 modulates cell growth, epithelial-mesenchymal transition, and oxidative stress in the hyperplastic prostate.

Benign prostatic hyperplasia (BPH) is a chronic condition which mainly affects elderly males. Existing scientific evidences have not completely revealed the pathogenesis of BPH. Glucose-regulated protein 78 (GRP78) is a member of the heat shock protein 70 superfamily, which serves as an important regulator in many diseases. This study aims at elucidating the role of GRP78 in the BPH process. Human prostate tissues, cultured human prostate cell lines (BPH-1 and WPMY-1) and clinical data from BPH patients were utilized. The expression and localization of GRP78 were determined with quantitative real time PCR (qRT-PCR), Western blotting and immunofluorescence staining. GRP78 knockdown and overexpression cell models were created with GRP78 siRNA and GRP78 plasmid transfection. With these models, cell viability, apoptosis rate, as well as marker levels for epithelial-mesenchymal transition (EMT) and oxidative stress (OS) were detected by CCK8 assay, flow cytometry analysis and Western blotting respectively. AKT/mTOR and MAPK/ERK pathways were also evaluated. Results showed GRP78 was localized in the epithelium and stroma of the prostate, with higher expression in BPH tissues. There was no significant difference in GRP78 expression between BPH-1 and WPMY-1 cell lines. In addition, GRP78 knockdown (KD) slowed cell growth and induced apoptosis, without effects on the cell cycle stage of both cell lines. Lack of GRP78 affected expression levels of markers for EMT and OS. Consistently, overexpression of GRP78 completely reversed all effects of knocking down GRP78. We further found that GRP78 modulated cell growth and OS via AKT/mTOR signaling, rather than the MAPK/ERK pathway. Overall, our novel data demonstrates that GRP78 plays a significant role in the development of BPH and suggests that GRP78 might be rediscovered as a new target for treatment of BPH.

The Immune-Related Gene HCST as a Novel Biomarker for the Diagnosis and Prognosis of Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor worldwide. Analysis of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases showed that the immune-related gene (IRG) hematopoietic cell signal transducer (HCST) could provide guidance for the diagnosis, prognosis, and treatment of ccRCC. The RNA-seq data of ccRCC tissues were extracted from two databases: TCGA (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) and GEO (https://www.ncbi.nlm.nih.gov/geo/). Corresponding clinical information was downloaded from TCGA. Immune-related gene data were extracted from the IMMPORT website (https://www.immport.org/). Differential analysis with R software (https://www.r-project.org/) was used to obtain a prognosis model of ccRCC IRGs. The differences were combined with the clinical data to assess the usefulness of the HCST as a prognostic biomarker. Based on data obtained from the Oncomine (https://www.oncomine.org/), Human Protein Atlas (https://www.proteinatlas.org/), and PubMed (https://pubmed.ncbi.nlm.nih.gov/) databases, the expression levels of the HCST in ccRCC, clinical-pathological indicators of relevance, and influence on prognosis were analyzed. Regulation of the HCST gene in ccRCC was assessed by gene set enrichment analysis (GSEA). In TCGA/GEO databases, the high HCST expression in tumor tissues was significantly correlated to the TMN stage, tumor grade, invasion depth, and lymphatic metastasis (p < 0.05). The overall survival (OS) of patients with high HCST gene expression was significantly lower than that of patients with low HCST gene expression (p < 0.001). Multivariate Cox regression analysis suggested that the HCST expression level [hazard ratio (HR) = 1.630, 95% confidence interval (CI) = 1.042-2.552], tumor cell grade (HR = 1.829, 95% CI = 1.115-3.001), and distant metastasis (HR = 2.634, 95%, CI = 1.562-4.442) were independent risk factors affecting the OS of ccRCC patients (all, p < 0.05). The GSEA study showed that there was significant enrichment in cell adhesion, tumorigenesis, and immune and inflammatory responses in HCST high expression samples. Hematopoietic cell signal transducer expression was closely associated with the levels of infiltrating immune cells around ccRCC tissues, especially dendritic cells (DCs). In conclusion, the present study suggested that the HCST was interrelated to the clinicopathology and poor prognosis of ccRCC. High HCST expression was also closely correlated with the levels of tumor-infiltrating immune cells, especially DCs.