Wstępne badania dotyczące zróżnicowania ekspresji receptorów kortykotropiny w gruczolakach nadnerczy

Introduction: The ACTH receptor (ACTHR) is primarily expressed in the adrenal cortex. Previous studies focused on the regulatory function of ACTHR in glucocorticoid secretion, but research on adrenal tumours is rare. The aim of this study was to evaluate ACTHR expression in common adrenal adenomas and investigate its influence on adrenal tumorigenesis using adrenocortical H295R cells. Materials and methods: Real-time polymerase chain reaction and western blot were used to detect the expression of ACTHR in 18 aldosterone-producing adenomas, 16 cortisol-producing adenomas, 9 non-functional adenomas, and 12 normal adrenal samples. Lentiviral vector pLVX-mCMV-ACTHR-ZsGreen was transfected into the H295R cells to increase ACTHR expression. WST-1 and cell count were applied to evaluate cell proliferation at different ACTHR levels. TUNEL staining was used to measure cell apoptosis. Results: Compared with normal adrenal samples, the aldosterone-producing adenoma samples had higher ACTHR mRNA and protein levels. However, the mRNA and protein levels of ACTHR in non-functional adenomas and cortisol-producing adenomas were lower than those in the normal adrenal samples. Proliferative activity in the experimental cells was higher than that in the control cells in the first three days. The proliferative activity peaked in the second day. However, this trend was reversed in the fourth day and became more apparent with time. By contrast, TUNEL staining showed that ACTHR overexpression did not induce a significant difference between the two groups. Conclusions: Differential ACTHR expression may be determined by different types of adrenocortical tumours. ACTHR is more likely to have induced the proliferation rather than the apoptosis of H295R cells.Wstęp: Receptor kortykotropiny (adrenocorticotropic hormone receptor, ACTHR) występuje głównie w korze nadnerczy. Prowadzono wcześniej badania dotyczące regulacyjnej funkcji ACTHR w wydzielaniu glikokortykoidów, lecz rzadko obejmowały one chorych z guzami nadnerczy. Badanie przeprowadzono w celu oceny ekspresji ACTHR w najczęstszych gruczolakach nadnerczy i zbadania jej wpływu na rozwój tych nowotworów w komórkach kory nadnerczy ludzkich linii H295R. Materiał i metody: Do wykrycia ekspresji ACTHR w 18 gruczolakach wydzielających aldosteron, 16 gruczolakach wydzielających kortyzol, 9 gruczolakach nieczynnych hormonalnie i 12 próbkach prawidłowych nadnerczy użyto reakcji łańcuchowej polimerazy w czasie rzeczywistym oraz metody Western blot. W celu zwiększenia ekspresji ACTHR transfekowano wektor lentiwirusowy pLVX-mCMV-ACTHR-ZsGreen do komórek H295R. Proliferację komórek przy różnych poziomach ekspresji ACTHR oceniono na podstawie WST-1 oraz liczby komórek. W celu zmierzenia apoptozy komórek zastosowano barwienie metodą TUNEL. Wyniki: W porównaniu z próbkami prawidłowych nadnerczy w próbkach gruczolaków wydzielających aldosteron stwierdzono wyższą ekspresję mRNA i białka ACTHR. Jednak ekspresja mRNA i białka ACTHR w gruczolakach nieczynnych hormonalnie oraz w gruczolakach wydzielających kortyzol była niższa niż w próbkach prawidłowych nadnerczy. W ciągu 3 pierwszych dni aktywność proliferacyjna w komórkach eksperymentalnych była wyższa niż w komórkach kontrolnych. Aktywność proliferacyjna osiągnęła maksymalną wartość w drugim dniu. Jednak w czwartym dniu ta tendencja uległa odwróceniu, co było wyraźniejsze w miarę upływu czasu. Z kolei barwienie metodą TUNEL wykazało, że nad ekspresja ACTHR nie spowodowała istotnej różnicy między grupami. Wnioski: Zróżnicowanie ekspresji ACTHR może wynikać z różnych typów guzów nadnerczy ocenianych w badaniu. Wydaje się, że ACTHR pobudza proliferację, a nie apoptozę komórek H295R

Identification of key genes and pathways in human clear cell renal cell carcinoma (ccRCC) by co-expression analysis

Human clear cell renal cell carcinoma (ccRCC) is the most common solid lesion within kidney, and its prognostic is influenced by the progression covering a complex network of gene interactions. In our study, we screened differential expressed genes, and constructed protein-protein interaction (PPI) network and a weighted gene co-expression network to identify key genes and pathways associated with the progression of ccRCC (n = 56). Functional and pathway enrichment analysis demonstrated that upregulated differentially expressed genes (DEGs) were significantly enriched in response to wounding, positive regulation of immune system process, leukocyte activation, immune response and cell activation. Downregulated DEGs were significantly enriched in oxidation reduction, monovalent inorganic cation transport, ion transport, excretion and anion transport. In the PPI network, top 10 hub genes were identified (TOP2A, MYC, ALB, CDK1, VEGFA, MMP9, PTPRC, CASR, EGFR and PTGS2). In co-expression network, 6 ccRCC-related modules were identified. They were associated with immune response, metabolic process, cell cycle regulation, angiogenesis and ion transport. In conclusion, our study illustrated the hub genes and pathways involved in the progress of ccRCC, and further molecular biological experiments are needed to confirm the function of the candidate biomarkers in human ccRCC

Upregulation of Phosphodiesterase type 5 in the Hyperplastic Prostate

Both erectile dysfunction (ED) and lower urinary tract symptoms (LUTS)/benign prostatic hyperplasia (BPH) are common in the aging male. Numerous clinical trials have demonstrated the efficacy and safety of phosphodiesterase type 5 inhibitors (PDE5-Is) for treating LUTS/BPH with/without ED. However, the influence of BPH on prostatic PDE5 expression has never been studied. A testosterone-induced rat model of BPH was developed and human hyperplastic prostate specimens were harvested during cystoprostatectomy. PDE5, nNOS, eNOS and α1-adrenoreceptor subtypes (α1aARs, α1bARs and α1dARs) were determined with real-time RT-PCR for rat tissues whilst PDE5 and α1-adrenoreceptor subtypes were determined in human samples. PDE5 was further analyzed with Western-blot and histological examination. Serum testosterone was measured with ELISA. The rat BPH model was validated as having a significantly enlarged prostate. PDE5 localized mainly in fibromuscular stroma in prostate. Our data showed a significant and previously undocumented upregulation of PDE5 in both rat and human BPH, along with increased expression of nNOS and α1d ARs for rat tissues and α1a ARs for human BPH. The upregulation of PDE5 in the hyperplastic prostate could explain the mechanism and contribute to the high effectiveness of PDE5-Is for treating LUTS/BPH. Fibromuscular stroma could be the main target for PDE5-Is within prostate

Upregulated Interleukin 21 Receptor Enhances Proliferation and Epithelial-Mesenchymal Transition Process in Benign Prostatic Hyperplasia

Background: Interleukins (ILs) and related chronic inflammation have been found to contribute to the development of benign prostatic hyperplasia (BPH) in recent decades. As a late member of the ILs family, IL-21 receptor (IL-21R) can modulate cell proliferation, however, IL-21R activity in the prostate has not been examined. The current study aimed to elucidate a potential role of IL-21R in the development of BPH.Material and Methods: Human prostate tissues, cell lines and rats were used. QRT-PCR, Western blot, and immunohistochemistry, along with hematoxylin and eosin, Masson's trichrome, and immunofluorescent staining were performed. BPH-1 cells with IL-21R silenced were cultured or co-cultured with macrophages (active THP-1, AcTHP-1). Apoptosis and cell cycle phases were determined via flow cytometry. Epithelial-mesenchymal transition (EMT) processes were also examined. In vivo, rat prostatitis was induced with intraprostatic injected lipopolysaccharide (LPS).Results: IL-21R was highly expressed in human as well as rat prostate, mainly in the epithelial compartment. BPH concomitant with prostatitis significantly upregulated the expression of IL-21R. Knockdown of IL-21R induced cell apoptosis and cycle arrest at G0/G1 phase, and blocked the EMT process in BPH-1 cells. When IL-21R silenced BPH-1 cells were co-cultured with AcTHP-1 cells, these aforementioned processes and IL-21R change were completely reversed. Prostatic hyperplasia was observed with IL-21R upregulated in LPS induced prostatitis rats. More specifically, the expression of apoptosis, cyclin, and EMT proteins in this rat model are altered in a manner consistent with that seen in the cell line model.Conclusions: Our novel data demonstrates the expression and functional activities of IL-21R in the mechanism for development of BPH. IL-21R mainly localized in prostate epithelium and it was upregulated in hyperplastic prostate tissues. IL-21R enhanced proliferation of BPH-1 cells, via inhibiting cell apoptosis, and modulating cell cycles, as well as the EMT process, in response to inflammatory stimuli

Co-expression network analysis identified six hub genes in association with progression and prognosis in human clear cell renal cell carcinoma (ccRCC)

Human clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignant adult kidney tumors. We constructed a weighted gene co-expression network to identify gene modules associated with clinical features of ccRCC (n = 97). Six hub genes (CCNB2, CDC20, CEP55, KIF20A, TOP2A and UBE2C) were identified in both co-expression and protein-protein interaction (PPI) networks, which were highly correlated with pathologic stage. The significance of expression of the hub genes in ccRCC was ranked top 4 among all cancers and correlated with poor prognosis. Functional analysis revealed that the hub genes were significantly enriched in cell cycle regulation and cell division. Gene set enrichment analysis suggested that the samples with highly expressed hub gene were correlated with cell cycle and p53 signaling pathway. Taken together, six hub genes were identified to be associated with progression and prognosis of ccRCC, and they might lead to poor prognosis by regulating p53 signaling pathway

CIRBP is a novel oncogene in human bladder cancer inducing expression of HIF-1 alpha

Cold-inducible RNA binding protein (CIRBP) has been reported to be associated with distinct tumorigenesis. In this study, we investigated the role of CIRBP in human bladder cancer (BCa), indicating that CIRBP is overexpressed in BCa tissues and cell lines to promote proliferation and migration. Moreover, CIRBP could induce expression of HIF-1 alpha via binding to the 3'-UTR of its mRNA to increase the mRNA stability in BCa cells. Furthermore, we demonstrated that PTGIS is a HIF-1 alpha targeted gene, a major regulator in hypoxic cancer progression by activating transcription of various oncogenes. Our results also suggested that overexpression of HIF-1 alpha may suppress the expression of PTGIS in BCa cells, by binding to HRE sequence at the promoter region of PTGIS. In addition, we found a strongly downregulation of PTGIS in BCa tissue and transcriptionally inhibited by HIF-1 alpha in BCa cells, which could be triggered by its DNA methylation. Further result suggested that knockdown of CIRBP could promote the expression of PTGIS, meanwhile knockdown of PTGIS could partially rescue CIRBP-deficiency induced inhibition of migration and proliferation in BCa cells. Taken together, our study indicated that CIRBP could be a novel oncogene in human bladder cancer inducing transcription of HIF-1 alpha, which could inhibit expression of methylated PTGIS

Upregulation of Oxytocin Receptor in the Hyperplastic Prostate

Background: The etiology of benign prostatic hyperplasia (BPH) is complex, both age and androgen are thought to be important. However, the failure of androgen blockade treatments suggests other paracrine/autocrine factors involved in BPH. Oxytocin was found to have a paracrine/autocrine role in prostate in recent years. The influence of BPH on prostatic oxytocin receptor (OTR) expression has never been studied.Material and methods: A testosterone-estradiol induced rat model of BPH was employed and human hyperplastic prostate specimens were harvested. Expressions of OTR, α1-adrenoreceptor subtypes and nitric oxide synthase isoforms were determined via real-time RT-PCR. OTR was further analyzed with Western-Blotting and histological examination. Subsequently, rat epithelial cells, human stromal cells and epithelial cells were cultured in vitro and treated with gradient concentrations of OT from 1 to 5 days. Cell proliferation was tested by Cell Counting Kit-8 and Flow Cytometry.Results: The rat BPH model was validated with significant increased prostate weight. H-E stain revealed a different histopathology between human and rat BPH. Masson's trichrome staining demonstrated that smooth muscle (SM) cells, epithelium cells and collagen fibers were simultaneously augmented in this rat BPH model and human BPH samples. OTR mainly localized in epithelium in rat prostate whereas it mainly localized in stroma in human prostate. OTR gene was upregulated 3.3-fold in rat BPH and 3.0-fold in human BPH, along with increased expression of 2.0-fold α1aARs and 3.0-fold eNOS for rat BPH and 5.0-fold α1aARs for human BPH. The expression of OTR protein was upregulated 1.4-fold in rat BPH and 3.9-fold in human BPH, respectively. Increased concentrations of exogenous OT can accelerate proliferation of rat epithelial cells and human stromal cells but has no impact on human epithelial cells in vitro. Flow Cytometry showed oxytocin could significantly increase G2/M period cell number.Conclusions: Our novel data demonstrates a significant and previously undocumented upregulation of OTR in both rat and human BPH. Moreover, exogenous OT accelerates proliferation of rat prostate epithelial cells and human prostate stromal cells. It is suggested OTR is involved in the development of BPH and OT regulatory system could be a potential new target for the BPH treatment

Identification of Biomarkers Associated With Pathological Stage and Prognosis of Clear Cell Renal Cell Carcinoma by Co-expression Network Analysis

Clear cell renal cell carcinoma (ccRCC) is the most common subtype among renal cancer whose prognostic is affected by the tumor progression associated with complex gene interactions. However, there is currently no available molecular markers associated with ccRCC progression and used or clinical application. In our study, microarray data of 101 ccRCC samples and 95 normal kidney samples were analyzed and 2,425 differentially expressed genes (DEGs) were screened. Weighted gene co-expression network analysis (WGCNA) was then conducted and 11 co-expressed gene modules were identified. Module preservation analysis revealed that two modules (red and black) were found to be most stable. In addition, Pearson's correlation analysis identified the module most relevant to pathological stage(patho-module) (r = 0.44, p = 3e-07). Functional enrichment analysis showed that biological processes of the patho-module focused on cell cycle and cell division related biological process and pathway. In addition, 29 network hub genes highly related to ccRCC progression were identified from the stage module. These 29 hub genes were subsequently validated using 2 other independent datasets including GSE53757 (n = 72) and TCGA (n = 530), and the results indicated that all hub genes were significantly positive correlated with the 4 stages of ccRCC progression. Kaplan-Meier survival curve showed that patients with higher expression of each hub gene had significantly lower overall survival rate and disease-free survival rate, indicating that all hub genes could act as prognosis and recurrence/progression biomarkers of ccRCC. In summary, we identified 29 molecular markers correlated with different pathological stages of ccRCC. They may have important clinical implications for improving risk stratification, therapeutic decision and prognosis prediction in ccRCC patients

Application of the Pipeline Embolization Device for Giant Vertebrobasilar Dissecting Aneurysms in Pediatric Patients

Objective: To evaluate the feasibility and effectiveness of the pipeline embolization device (PED) for the treatment of pediatric giant vertebrobasilar dissecting aneurysms (VBDAs).Methods: We retrospectively reviewed our institutional clinical database and identified 2,706 patients who presented with a diagnosis of intracranial aneurysms from January 2016 to June 2018. Among this group, 153 patients were diagnosed with VBDAs, and 54 of these patients underwent PED therapy. The PED technique was used in four patients who were 18 years old or younger at the time of presentation (two males, two females; mean age 9.25 years; age range 8–11 years).Results: All four included pediatric patients were managed with the PED. One patient (25%) was treated with the PED alone, while three (75%) were treated with the PED and coils. One patient died from brainstem infarction or compression of the brainstem, while follow-up of the other three patients revealed favorable outcomes. The mass effect was reduced in cases 1, 2, and 3 on follow-up MRI performed 6 months after the PED procedure.Conclusions: PEDs could be feasible in the treatment of pediatric giant VBDAs. However, the safety and efficacy of this method have not been clarified in this special pediatric population, and long-term follow-up is still necessary

Efficacy and safety of a novel 450 nm blue diode laser versus plasmakinetic electrocautery for the transurethral resection of non-muscle invasive bladder cancer: The protocol and result of a multicenter randomized controlled trial

ObjectivesTo be the first to apply a novel 450 nm blue diode laser in transurethral resection of bladder tumor (TURBt) to treat patients with non-muscle invasive bladder cancer (NMIBC) and evaluate its efficacy and safety during the preoperative period compared to the conventional plasmakinetic electrocautery.Materials and MethodsRandomized controlled trial (RCT) in five medical centers was designed as a non-inferiority study and conducted from October 2018 to December 2019. Patients with NMIBC were randomized to the blue laser or plasmakinetic electrocautery group for TURBt. As the first study to evaluate this novel blue laser device, the primary outcome was the effective resection rate of bladder tumors, including effective dissection and hemostasis. The secondary outcomes were the perioperative records, including surgical time, postoperative indwelling catheter time, hospital stay length, blood loss, reoperation rate, wound healing and adverse events.ResultsA total of 174 patients were randomized to either the blue laser group (85 patients) or plasmakinetic electrocautery group (89 patients). There was no statistical significance in the clinical features of bladder tumors, including tumor site, number and maximum lesion size. Both the blue laser and plasmakinetic electrocautery could effectively dissect all visible bladder tumors. The surgical time for patients in the blue laser group was longer (p=0.001), but their blood loss was less than that of patients in the control group (p=0.003). There were no differences in the postoperative indwelling catheter time, hospital stay length, reoperation rate or other adverse events. However, the patients undergoing TURBt with the blue laser showed a faster wound healing at 3 months after operation.ConclusionThe novel blue laser could be effectively and safely used for TURBt in patients with NMIBC, and this method was not inferior to plasmakinetic electrocautery during the perioperative period. However, TURBt with the blue laser may provide the benefit to reduce preoperative blood loss and accelerate postoperative wound healing. Moreover, longer follow-up to confirm recurrence-free survival benefit was required