Yuyan Zhu

ncRAN, a Newly Identified Long Noncoding RNA, Enhances Human Bladder Tumor Growth, Invasion, and Survival

OBJECTIVES To study the functional significance of ncRAN, a newly identified long noncoding RNA, expression in bladder cancer growth, invasion, and survival. METHODS Expression of ncRAN isoforms was analyzed by reverse transcription-polymerase chain reaction in 40 clinical specimens and four bladder cancer cell lines, respectively. The expression of ncRAN in the clinical specimens was correlated to clinical stage or grade to assess for potential relationship. ncRAN-long form or ncRAN-short form was stably overexpressed in human bladder cancer cell line, RT4(superficial) exhibiting low endogenous expression of ncRAN, respectively. The influence of ncRAN on cancer cell survival was evaluated by silencing with short hairpin RNA in 5637 cells (invasive). Functional assays were performed to study the resulting changes in cell proliferation, motility, invasion, and survival. RESULTS Expression of ncRAN was significantly higher in bladder cancers compared with normal tissues (P < .01) and in invasive tumor compared with superficial ones (P < .01). Consistently, ncRAN expressed significantly higher in invasive bladder tumor cell lines (5637, T24, J82) than that in superficial tumor cell line (RT4). Overexpression of ncRAN in RT4 cells significantly enhanced cell proliferation, migration, and invasion. Silencing of ncRAN improved chemotherapy sensitivity in 5637 cells. CONCLUSIONS Our clinical and experimental data suggest that ncRAN may play an important role in the pathogenesis of human bladder cancer, and may help in designing effective therapy targeting the ncRAN pathway to control bladder cancer growth and invasion.

Dependence receptor UNC5D mediates nerve growth factor depletion–induced neuroblastoma regression

Spontaneous regression of neuroblastoma (NB) resembles the developmentally regulated programmed cell death (PCD) of sympathetic neurons. Regressing tumor cells express high levels of the nerve growth factor (NGF) receptors TRKA and p75NTR and are dependent on NGF for survival; however, the underlying molecular mechanism remains elusive. Here, we show that UNC5D, a dependence receptor that is directly targeted by p53 family members, is highly expressed in favorable NBs. NGF withdrawal strongly upregulated UNC5D, E2F1, and p53 in human primary favorable NBs. The induced UNC5D was cleaved by caspases 2/3, and the released intracellular fragment translocated into the nucleus and interacted with E2F1 to selectively transactivate the proapoptotic target gene. The cleavage of UNC5D and its induction of apoptosis were strongly inhibited by addition of netrin-1. Unc5d(-/-) mice consistently exhibited a significant increase in dorsal root ganglia neurons and resistance to NGF depletion-induced apoptosis in sympathetic neurons compared with wild-type cells. Our data suggest that UNC5D forms a positive feedback loop with p53 and E2F1 to promote NGF dependence-mediated PCD during NB regression.

Corticotropin-independent Cushing's Syndrome in Patients with Bilateral Adrenal Masses

OBJECTIVES To present our institutional experience in the patients with ACTH-independent Cushings syndrome treated for bilateral adrenal masses during the past 8 years. Bilateral adrenal masses are rare in patients with adrenocorticotropic hormone (ACTH)-independent Cushings syndrome. METHODS A retrospective review of 11 patients with ACTH-independent Cushings syndrome of bilateral adrenal masses was performed. Bilateral adrenalectomy or bilateral/unilateral partial adrenalectomy was made for these patients. The steroid replacement was discontinued after 6-12 months postoperatively, and the follow-up information was obtained to evaluate the disease outcome. RESULTS Combining ultrasonography and computed tomography scan with biochemical tests, 6 cases of bilateral adrenal adenomas and 5 cases of ACTH-independent macronodular adrenal hyperplasia (AIMAH) were diagnosed. The median follow-up time was 13 months (range, 9-22). For all patients, both systolic (181 ± 17 vs 145 ± 11 mm Hg; P <.001) and diastolic blood pressure levels (118 ± 13 vs 88 ± 11 mm Hg; P <.001) were significantly reduced postoperatively. The body mass index significantly decreased (28.0 ± 2.4 vs 24.6 ± 1.3 kg/m(2); P <.001). After bilateral adrenalectomy, glucocorticoid therapy was enough to maintain the balance of water and electrolytes metabolism, and the follow-up outcome showed no disorder of serum electrolytes metabolism. CONCLUSIONS Although a feasible method is still needed for determining the lateralization of cortisol secretion of bilateral adrenal masses, ultrasonography and computed tomography scan are useful for the diagnosis and differentiated diagnosis of AIMAH. Rational surgical treatment is important for bilateral adrenal masses, while glucocorticoid therapies could be effective alternation for steroid replacement.

Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylation

Plk3, one of Polo‐like kinase family members, is involved in the regulation of cell cycle progression and DNA damage response. In this study, we found that Plk3 inhibits pro‐apoptotic activity of p73 through physical interaction and phosphorylation. During cisplatin (CDDP)‐mediated apoptosis, Plk3 was transcriptionally induced, whereas its protein level was kept at basal level, suggesting that Plk3 might rapidly degrade in response to CDDP. Immunoprecipitation and in vitro pull‐down experiments demonstrated that Plk3 interacts with p73. Luciferase reporter assays and RT‐PCR experiments revealed that Plk3 inhibits p73‐mediated transcriptional activity. Consistent with these results, pro‐apoptotic activity of p73 was blocked by Plk3. Additionally, Plk3 decreased the stability of p73. Intriguingly, kinase‐deficient Plk3 failed to inhibit p73 function, indicating that kinase activity of Plk3 is required for Plk3‐mediated inhibition of p73. Indeed, in vitro kinase reaction showed that NH2‐terminal portion of p73 is phosphorylated by Plk3. In accordance with these observations, knocking down of Plk3 increased the stability of p73 and promoted CDDP‐mediated apoptosis in association with up‐regulation of p73. Collectively, our present findings suggest that Plk3 plays an important role in the regulation of cell fate determination in response to DNA damage through the inhibition of p73.

miR-130b, an onco-miRNA in bladder cancer, is directly regulated by NF-κB and sustains NF-κB activation by decreasing Cylindromatosis expression

Persistent activation of NF-κB signaling is closely related to chronic inflammation and tumorigenesis. Commonly, NF-κB signaling is tightly controlled by multiple feedback loops and regulators, such as the deubiquitinases (DUBs). However, in cancer cells, NF-κB may override these feedbacks through special pathways and lead to the sustained activation. In the present study, we demonstrate that in transitional cell carcinoma (TCC) of bladder, miR-130b plays an oncogenesis role, it enhanced proliferation, invasion and migration of TCC cell, and was highly correlated with tumor progression. On the other hand, NF-κB directly regulated the transcription of miR-130b by binding with its promoter region. Importantly, we verify that, through deceasing the expression of Cylindromatosis (CYLD), a K63-specific DUB and endogenous blocker of NF-κB signaling, miR-130b can in return sustain the persistent activation of NF-κB, which may promote the malignant progression of TCC. Thus, the present study uncovers a potential signaling transduction in which NF-κB is continuously activated, and may provide a novel therapeutic approach for the clinical management of TCC.

PKC α regulates netrin-1/UNC5B-mediated survival pathway in bladder cancer.

BackgroundNetrin-1 and its receptor UNC5B play important roles in angiogenesis, embryonic development, cancer and inflammation. However, their expression patttern and biological roles in bladder cancer have not been well characterized. The present study aims to investigating the clinical significance of PKC α, netrin-1 and UNC5B in bladder cancer as well as their association with malignant biological behavior of cancer cells.MethodsNetrin-1 and UNC5B expression was examined in 120 bladder cancer specimens using immunohistochemistry and in 40 fresh cancer tissues by western blot. Immunofluorescence was performed in cancer cell lines. PKC α agonist PMA and PKC siRNA was employed in bladder cancer cells. CCK-8, wound healing assays and flow cytometry analysis were used to examine cell proliferation, migration and cell cycle, respectively.ResultsNetrin-1 expression was positively correlated with histological grade, T stage, metastasis and poor prognosis in bladder cancer tissues. Immunofluorescence showed elevated netrin-1 and decreased UNC5B expression in bladder cancer cells compared with normal bladder cell line. Furthermore, cell proliferation, migration and cell cycle progression were promoted with PMA treatment while inhibited by calphostin C. In addition, PMA treatment could induce while calphostin C reduce netrin-1 expression in bladder cancer cells.ConclusionsThe present study identified netrin-1/UNC5B, which could be regulated by PKC signaling, was important mediators of bladder cancer progression.

The Role of Fascin in Migration and Invasion of Urothelial Carcinoma of the Bladder

Introduction: To investigate the roles of fascin in migration and invasiveness in bladder urothelial carcinoma. Materials and Methods: Immunohistochemical detection of fascin in urothelial carcinoma samples and inhibition the expression of fascin in the urothelial carcinoma cell line were performed, then the differences in cell behaviors before and after silencing of the fascin gene were tested. Results: In our study, we found that overexpression of fascin was more frequent in urothelial carcinoma tissues (p < 0.001). Fascin expression was positively correlated with histological grade (p = 0.024) and pT stage (p < 0.001). After transfection of fascin shRNA, the expressions of fascin in 5637 cells and BIU87 cells were efficiently decreased according to real-time RT-PCR and Western blot analysis. When fascin was inhibited, a significant decrease in migration and invasion, and increase in adhesion were observed in 5637 cells and BIU87 cells. However, there was no significant change in the proliferation of 5637 cells or BIU87 cells with or without inhibition of the fascin gene. Conclusions: Fascin expression can be used as a predictor for transformation and progression of urothelial carcinoma, and reduction of fascin levels may represent a novel therapeutic strategy for urothelial carcinoma of the bladder.

Reduction of protein kinase C α (PKC-α) promote apoptosis via down-regulation of Dicer in bladder cancer.

In clinic, we examined the expression of protein kinase C (PKC)‐α and Dicer in the samples of bladder cancer patients, and found that the two proteins have a line correlation. Our study confirmed this correlation existing by clearing the decreasing expression of Dicer after the PKC‐α knockdown. Treatment of bladder cancer cell lines (T24, 5637) with the PKC‐α or Dicer knockdown and the PKC inhibitors (Calphostin C and Gö 6976) can promote the apoptosis. Inhibition of PKC can increase the activities of caspase‐3 and PARP, however, decrease the expression of Dicer. And knockdown of the PKC‐α or Dicer can also activate the caspase‐3 or the PARP. Considering the reduction of PKC‐α can induce the Dicer down‐regulation, we make the conclusion that the reduction of PKC‐α can promote the apoptosis via the down‐regulation of Dicer in bladder cancer.

Increased expression of lung resistance‐related protein in lower grade urothelial carcinoma of the renal pelvis and ureter

Abstract Background: Lung resistance‐related protein (LRP), like multidrug resistance gene 1 (MDR1) and multidrug resistance‐associated proteins (MRP), has been associated with intrinsic therapeutic resistance in various malignancies. To date, there has been no study on the expression of LRP in urothelial carcinomas of the renal pelvis and ureter. We investigated the protein and mRNA expression levels of LRP, MDR1 and MRP1 in this malignancy and the clinical significance of their expression was evaluated.

Overexpression of UNC5B in bladder cancer cells inhibits proliferation and reduces the volume of transplantation tumors in nude mice

BackgroundThe netrin-1 receptor UNC5B plays vital roles in angiogenesis, inflammation, embryonic development and carcinogenesis. However, the functional significance of UNC5B overexpression in bladder cancer remains unclear. In this study, we investigated the role of UNC5B in bladder cancer in vitro and in vivo.MethodsStable transfection of the human bladder cancer cell line 5637 with UNC5B (5637-U) was confirmed by real-time RT-PCR, western blot and immunofluorescence assays. UNC5B expression in 5637 and 5637-U cells and mice tumor specimens derived from these cell lines was analyzed by immunohistochemistryand western blotting. Changes in the levels of cell cycle proteins were evaluated by western blotting. Flow cytometry, CCK-8 and scratch tests were used to examine cell cycle distribution, proliferation and migration, respectively.ResultsUNC5B overexpression in 5637 cells inhibited cell multiplication and migration and induced cell cycle arrest at the G2/M phase, meanwhile exhibited changes in the expression of cell cycle-associated proteins, showing that UNC5B may inhibit metastatic behaviors in bladder cancer cells. In addition, tumors generated from 5637-U cells were smaller than tumors generated from control 5637 cells.ConclusionsOur findings suggest that UNC5B is a potential anti-neoplastic target in bladder cancer progression.