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Purpose: We observed abnormal expression of the microRNA-23b/27b (miR-23b/27b) cluster in our previous study of miRNA expression signatures. However, the relationship between aberrant miRNA expression and clear cell renal cell carcinoma is not well established. We investigated the functional significance of the miR-23b/27b cluster in clear cell renal cell carcinoma cells and evaluated these miRNAs as biomarkers to predict the risk of clear cell renal cell carcinoma., Materials and Methods: Expression levels of miR-23b and miR-27b were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. The association between miRNA expression and overall survival was estimated by the Kaplan-Meier method. Gain of function assays were performed using mature miR-23b and miR-27b in the 786-O and A498 renal cell carcinoma cell lines. Targets regulated by these miRNAs were predicted by in silico analysis., Results: Expression of the miR-23b/27b cluster was significantly decreased in clear cell renal cell carcinoma tissue specimens and associated with pathological grade and stage. Significantly shorter overall survival was observed in patients with lower expression of the miR-23b/27b cluster. Restoration of miR-23b and miR-27b significantly inhibited cancer cell proliferation, migration and invasion., Conclusions: Expression of the miR-23b/27b cluster was frequently decreased in clear cell renal cell carcinoma tissue. Reduced expression of these miRNAs increased the risk of disease progression and predicted poor survival. Thus, miR-23b and miR-27b function as tumor suppressors, targeting several oncogenic genes in clear cell renal cell carcinoma cells., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Our microRNA expression signature of renal cell carcinoma revealed that miR-218 expression was significantly decreased in cancer tissues, suggesting that miR-218 is a candidate tumor suppressor. We investigated the functional significance of miR-218 in cancer cells and identified what are to our knowledge novel miR-218 mediated cancer pathways in renal cell carcinoma., Materials and Methods: Gain of function studies using mature miR-218 were performed to investigate cell proliferation, migration and invasion in the A498 and 786-O renal cell carcinoma cell lines. To identify miR-218 mediated molecular pathways and responsible genes in renal cell carcinoma, we used gene expression and in silico database analyses. Loss of function assays were performed to investigate the functional significance of miR-218 target genes., Results: Restoration of mature miR-218 significantly inhibited RCC cell proliferation, migration and invasion. Gene expression studies and luciferase reporter assays showed that CAV2 involved in the focal adhesion pathway was directly regulated by miR-218. A silencing study of CAV2 revealed significant inhibition of cell proliferation, migration and invasion. CAV2 mRNA and protein expression was significantly up-regulated in renal cell carcinoma clinical specimens., Conclusions: Loss of tumor suppressive miR-218 enhances cancer cell migration and invasion through dysregulation of the focal adhesion pathway, especially CAV2 as an oncogenic function in renal cell carcinoma. Tumor suppressive microRNA mediated cancer pathways and responsible genes provide new insights into the potential mechanisms of renal cell carcinoma oncogenesis and metastasis., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Aberrant DNA hypermethylation has been reported in renal cell carcinoma. We performed microarray analysis in the renal cancer cell line ACHN treated with the demethylating agent 5-aza-2'-deoxycytidine and investigated the UCHL1 gene involved in the regulation of cellular ubiquitin levels., Materials and Methods: We subjected 131 renal cell carcinoma and 61 corresponding normal kidney tissue samples to real-time reverse transcriptase-polymerase chain reaction, quantitative methylation specific polymerase chain reaction and immunohistochemistry. We also established a stable UCHL1 transfectant to evaluate cell growth., Results: We identified 10 genes that were up-regulated more than 2.5-fold in 5-aza-2'-deoxycytidine treated vs untreated ACHN cells. UCHL1 expression was increased 3.41-fold by 5-aza-2'-deoxycytidine treatment. In clinical samples the UCHL1 methylation index was significantly higher in renal cell carcinoma than in normal kidney tissue (p = 0.011). Conversely UCHL1 mRNA expression was significantly lower in renal cell carcinoma than in normal kidney tissue (p <0.0001). There was a negative correlation between mRNA expression and the UCHL1 methylation index (p = 0.017). The immunostaining score for UCHL1 was significantly higher in normal kidney tissue than in renal cell carcinoma (p <0.0001). Kaplan-Meier analysis showed that a positive UCHL1 methylation index had a significant adverse effect on prognosis (p = 0.048). Significant growth inhibition in UCHL1 transfectant compared to that in WT ACHN (p <0.0001) suggests that UCHL1 functions as a potential tumor suppressor gene in human renal cell carcinoma., Conclusions: To our knowledge we report the first study demonstrating that the mechanism of UCHL1 down-regulation in renal cell carcinoma is through CpG hypermethylation of the promoter region and methylation of the UCHL1 gene is associated with a poor prognosis in patients with renal cell carcinoma.

Purpose: SKP2 and CKS1 promote aggressive tumor behavior via the regulation of p27 degradation. Our previous DNA microarray analysis of human urothelial carcinoma and normal urothelial epithelium showed that in urothelial carcinoma the 2 most highly up-regulated genes among SKP2-p27 interaction related genes are SKP2 (4.7-fold) and CKS1 (2.2-fold). We hypothesized that SKP2 and CKS1 gene expression is associated with urothelial carcinoma invasiveness and prognosis., Materials and Methods: A total of 84 urothelial carcinoma specimens from patients with bladder (71) and upper urinary tract (13) cancer were examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemical study., Results: Real-time reverse transcriptase-polymerase chain reaction showed that the average mRNA expression level of SKP2 and CKS1 significantly correlated with tumor stage, that is superficial vs invasive urothelial carcinoma (SKP2 and CKS1, p<0.001 and 0.006) and grade (p<0.001 and 0.009, respectively). Of the superficial urothelial carcinomas examined the SKP2 and CKS1 expression level was significantly higher in pT1 than in pTa samples (p=0.005 and 0.017, respectively). Immunohistochemical expression patterns of SKP2 and CKS1 also significantly correlated with tumor stage (p<0.001 and 0.048) and grade (p=0.003 and 0.025, respectively). In contrast, p27 expression inversely correlated with tumor stage and grade (p<0.001 and 0.011, respectively). Logistic regression analysis revealed that while SKP2 mRNA expression was a significant dependent predictor of p27 expression (p=0.021), there was no correlation between CKS1 mRNA expression and p27 (p=0.748). Kaplan-Meier curves and log rank tests revealed that the high mRNA expression levels of SKP2 and CKS1 had a significant adverse effect on prognosis (p=0.043 and 0.003, respectively)., Conclusions: Our results suggest that SKP2 has a major role in the regulation of p27 degradation and CKS1 has a supporting role for SKP2 function in human urothelial carcinoma.

Purpose: Vascular endothelial growth factor (VEGF) is known as a multifunctional protein with roles in angiogenesis stimulation and apoptosis inhibition. We hypothesized that intracavernous administration of VEGF would recover erectile dysfunction due to diabetes by protection from apoptosis in the penile cavernosum., Materials and Methods: A total of 30, 6-month-old male Sprague-Dawley rats were divided into 2 large groups, namely 20 with diabetes and 10 healthy controls. The diabetic group received intraperitoneal injection of streptozotocin (STZ) to induce diabetes. Intracavernous injection of VEGF was administered to randomly selected STZ diabetic rats 6 weeks after STZ injections. Erectile functional studies were performed in 10 STZ and 10 STZ plus VEGF rats at 12 weeks. After completion of the functional study the penile crura were collected for molecular and immunohistochemical studies., Results: Mean intracavernous pressure in the diabetic group was significantly lower than in controls and low pressure was significantly recovered by VEGF treatment. Gene expression of pro-apoptotic and anti-apoptotic factors were present in the control, diabetic and VEGF treated groups. However, anti-apoptotic protein expression was lacking in the diabetic group and it was recovered by VEGF treatment. The apoptotic index in the diabetic group was significantly higher than in controls and this index was significantly decreased in the VEGF treated group., Conclusions: The decrease in and recovery of intracavernous pressure correlated significantly with a variation in anti-apoptotic protein expression in the diabetic and VEGF treated groups. To our knowledge this is the first study to show that intracavernous injection of VEGF restores erectile dysfunction through the inhibition of apoptosis in diabetic rats.