Pan-Cancer Analysis of the TRP Family, Especially TRPV4 and TRPC4, and Its Expression Correlated with Prognosis, Tumor Microenvironment, and Treatment Sensitivity.

BACKGROUND: Transient receptor potential (TRP) channels are involved in various physiological, pathological, and tumorigenesis-related processes. However, only a few studies have comprehensively analyzed TRP family members and their association with prognosis and tumor microenvironment (TME) in various cancers. Thus, in this study, we focused on TRP channels in pan-cancer and screened two typical TRP channels, TRPV4 and TRPC4, as examples. METHODS: Based on the latest public databases, we evaluated the expression level and prognostic value of TRP family genes in pan-cancer tissues via various bioinformatic analytical methods, and investigated the relationship between the expression of TRP family genes with TME, stemness score, immune subtype, drug sensitivity, and immunotherapy outcome in pan-cancer tissues. RESULTS: Pan-cancer analysis revealed that the TRP family genes were differentially expressed in tumor and para-carcinoma tissues. A significant correlation existed between the expression of TRP family genes and prognosis. The expression of TRP family genes was significantly correlated with stromal, immune, RNA stemness, and DNA stemness scores in pan-cancer tissues. Our results indicated that the expression of TRP family genes correlated with the sensitivity to various drugs including PLX-4720, SB-590885, and HYPOTHEMYCIN, immunotherapy outcome, and immune-activation-related genes. Immunohistochemical analysis revealed significant differential expression of TRPV4 in bladder and para-carcinoma tissues. CONCLUSIONS: Our study elucidated the possible role of TRP family genes in cancer progression and provided insights for further studies on TRP family genes as potential pan-cancer targets to develop diagnostic and therapeutic strategies.

Prognosis Risk Model Based on Necroptosis-Related Signature for Bladder Cancer.

BACKGROUND: Bladder cancer(BLCA) is the ninth most common cancer. In recent years, necroptosis was found to be related to the occurrence and development of tumors. In this study, we aimed to construct a model based on a necroptosis-related signature to evaluate the potential prognostic application in BLCA. METHODS: A total of 67 necroptosis-related genes were used to select the ideal cluster numbers, and it was found that there were four necroptosis-related patterns. Then, we compared the gene expression levels among all of the groups and established a necroptosis-related prognostic model. We made the following enrichment analysis of function and built a novel scoring system, the NEC score, to evaluate the state of necroptosis according to the expression level of necroptosis-related genes. RESULTS: A total of 67 necroptosis-related genes were used to define four distinct necroptosis-related patterns: NEC cluster1-4. Each NEC cluster exhibited different patterns of survival and immune infiltration. Based on univariate Cox regression analyses and least absolute shrinkage and selection operator (Lasso) regression, 14 necroptosis-related genes were established to develop the NEC score. Patients were divided into two groups based on the NEC score. Patients in the high NEC score group had a significantly poorer overall survival than those in the low NEC score group. We further confirmed the correlation of clinical characteristics, as well as the immunotherapy outcome, with the NEC score, and confirmed the potential of the NEC score to be an independent prognostic factor. Furthermore, we compared the expression levels of eight potential biomarker genes between our own BLCA tissues and para-carcinoma tissue. CONCLUSION: We developed a novel NEC score that has a potential prognostic value in BLCA patients and may help personalized immunotherapy counselling.

Hengli® Chinese Botulinum Toxin Type A for Treatment of Patients With Overactive Bladder: A Multicenter, Prospective, Randomized, Double-Blind, Placebo-Controlled Trial.

Objective: To evaluate the efficacy and safety of Hengli® Chinese botulinum toxin type A (BTX-A; 100 U) in Chinese patients with overactive bladder. Methods: This study was a multicenter, randomized, double-blind, placebo-controlled trial in Chinese patients who were inadequately managed with anticholinergic medications. Eligible patients were randomized 2:1 to receive intradetrusor injections of Hengli® BTX-A (n = 144) or placebo (n = 72). The primary endpoint was the change in the number of daily micturition episodes at week 6 from baseline. The secondary efficacy endpoints included the average frequency of urgency and urinary incontinence (UI) episodes per day, urgency score, average micturition volume per day, OABSS, and QoL score. Results: In the Hengli® BTX-A group, there was a significantly greater reduction in the average number of micturition episodes per 24 h compared with the placebo group (3.28 vs. 1.43; p = 0.003). Moreover, there was a significantly greater improvement in the daily number of urgency episodes, micturition volume and OABSS score. An increased post-void residual urine volume, dysuria, and urinary tract infection represented adverse events (AEs) in the Hengli® BTX-A group. Most AEs were mild or moderate in severity. One patient in the BTX-A group initiated clean intermittent catheterization (CIC) during treatment. Conclusion: Hengli® BTX-A treatment was well-tolerated and resulted in significant improvements in OAB symptoms among Chinese patients inadequately managed by anticholinergics. Clinical Trial Registration: http://www.chinadrugtrials.org.cn/clinicaltrials.prosearch.dhtml, Identifier: CTR20131190.

Synergistic inhibitory effects of 5-aza-2'-deoxycytidine and cisplatin on urothelial carcinoma growth via suppression of TGFBI-MAPK signaling pathways.

This study aimed to reveal the gene transcriptional alterations, possible molecular mechanisms, and pathways involved in the synergy of 5-aza-2'-deoxycytidine (DAC) and Cisplatin (CDDP) in urothelial carcinoma (UC). Two UC cell lines, 5637 and T24, were used in the present study. A cDNA microarray was performed to identify critical genes involved in the synergistic mechanism of both agents against UC cells. The results showed that several key regulatory genes, such as interleukin 24 (IL24), fibroblast growth factor 1 (FGF1), and transforming growth factor beta induced (TGFBI), may play critical roles in the synergy of DAC and CDDP in UC. Pathway enrichment suggested that many carcinogenesis-related pathways, such as the ECM-receptor interaction and MAPK signaling pathways, may participate in the synergy of both agents. Our results suggest that TGF-ß1 stimulates the phosphorylation of ERK1/2 and p38 by increasing TGFBI expression, and that the TGFBI-MAPK signaling pathway plays an important role in the synergy of DAC and CDDP against UC. Therefore, we revealed the synergistic mechanism of DAC and CDDP in UC. Several key regulatory genes play critical roles in the synergy of combined treatment, and the TGFBI-MAPK signaling pathway may be an important potential target of these two agents.

A tumor microenvironment preoperative nomogram for prediction of lymph node metastasis in bladder cancer.

Background: Growing evidence suggests that tumor metastasis necessitates multi-step microenvironmental regulation. Lymph node metastasis (LNM) influences both pre- and post-operative bladder cancer (BLCA) treatment strategies. Given that current LNM diagnosis methods are still insufficient, we intend to investigate the microenvironmental changes in BLCA with and without LNM and develop a prediction model to confirm LNM status. Method: "Estimation of Stromal and Immune cells in Malignant Tumors using Expression data" (ESTIMATE) algorithm was used to characterize the tumor microenvironment pattern of TCGA-BLCA cohort, and dimension reduction, feature selection, and StrLNM signature construction were accomplished using least absolute shrinkage and selection operator (LASSO) regression. StrLNM signature was combined with the genomic mutation to establish an LNM nomogram by using multivariable logistic regression. The performance of the nomogram was evaluated in terms of calibration, discrimination, and clinical utility. The testing set from the TCGA-BLCA cohort was used for internal validation. Moreover, three independent cohorts were used for external validation, and BLCA patients from our cohort were also used for further validation. Results: The StrLNM signature, consisting of 22 selected features, could accurately predict LNM status in the TCGA-BLCA cohort and several independent cohorts. The nomogram performed well in discriminating LNM status, with the area under curve (AUC) of 75.1% and 65.4% in training and testing datasets from the TCGA-BLCA cohort. Furthermore, the StrLNM nomogram demonstrated good calibration with p >0.05 in the Hosmer-Lemeshow goodness of fit test. Decision curve analysis (DCA) revealed that the StrLNM nomogram had a high potential for clinical utility. Additionally, 14 of 22 stably expressed genes were identified by survival analysis and confirmed by qPCR in BLCA patient samples in our cohort. Conclusion: In summary, we developed a nomogram that included an StrLNM signature and facilitated the preoperative prediction of LNM status in BLCA patients.

Diagnostic value comparison of CellDetect, fluorescent in situ hybridization (FISH), and cytology in urothelial carcinoma.

BACKGROUND: To evaluate the clinical effectiveness of a novel CellDetect staining technique, compared with fluorescent in situ hybridization (FISH), and urine cytology, in the diagnosis of urothelial carcinoma (UC). METHODS: A total of 264 patients with suspicious UC were enrolled in this study. All tissue specimens were collected by biopsy or surgery. Urine specimen was obtained for examinations prior to the surgical procedure. CellDetect staining was carried out with CellDetect kit, and FISH was performed with UroVysion detection kit, according to the manufacturer's instructions. For urine cytology, all specimens were centrifuged using the cytospin method, and the slides were stained by standard Papanicolaou stain. RESULTS: In this study, there were 128 cases of UC and 136 cases of non-UC, with no significant difference in gender and age between the two groups. Results for sensitivity of CellDetect, FISH, and urine cytology were 82.8%, 83.6%, and 39.8%, respectively. The specificity of the three techniques were 88.2%, 90.4%, and 86.0%, respectively. The sensitivity of CellDetect and FISH are significantly superior compared to the conventional urine cytology; however, there was no significant difference in specificity among three staining techniques. In addition, the sensitivity of CellDetect in lower urinary tract UC, upper urinary tract UC, non-muscle-invasive bladder cancer (NMIBC), and muscle-invasive bladder cancer (MIBC) were 83.3%, 81.8%, 83.5%, and 72.0%, respectively. The screening ability of CellDetect has no correlation with tumor location and the tumor stage. The sensitivity of CellDetect in low-grade UC and high-grade UC were 51.6 and 92.8%. Thus, screening ability of CellDetect in high-grade UC is significantly superior compared to that in low-grade UC. CONCLUSIONS: CellDetect and FISH show equal value in diagnosing UC, both are superior to conventional urine cytology. Compared to FISH, CellDetect is cost effective, easy to operate, with extensive clinical application value to monitor recurrence of UC, and to screen indetectable UC.

Exosome-Transmitted miR-128 Targets CCL18 to Inhibit the Proliferation and Metastasis of Urothelial Carcinoma.

Objective: To investigate the regulatory function of exosome-transmitted miR-128 and chemokine (C-C motif) ligand 18 (CCL18) on urothelial carcinomas (UCs). Methods: Tumor tissues, paracancerous tissues, and serum were collected from 20 patients with UCs (diagnosed at Beijing Friendship Hospital, Capital Medical University). CCL18 was detected by immunohistochemistry and ELISA. PCR was used to measure the expression levels of CCL18 and mir-183, miR-128, mir-33a in UCs. We acquired exosomes from mesenchymal stem cells and synthesized exosomes overexpressing miR-128 (HMSC-128-EV). The effects of miR-128 on the migration and invasion abilities, apoptosis and epithelial-mesenchymal transition of BUC T24 cells were investigated by co-culturing HMSC-128-EV. The therapeutic potential of miR-128 on disease models was explored by injecting HMSC-128-EV into nude mice. Results: The expression of CCL18 in UCs was significantly higher than that in normal tissues (p < 0.05), and the serum level of CCL18 in patients with UC was significantly increased compared with those in healthy controls (p < 0.05). CCL18 overexpression or downregulation enhanced or suppressed the proliferation, migration and invasion of BUC T24 cells, resectively (p < 0.05). The exosome-transmitted miR-128 can inhibit cell proliferation (p < 0.05), invasion (p < 0.05), and migration (p < 0.05) in UCs, and these effects can be reversed by CCL18. In terms of apoptosis, miR-128 was able to promote the occurrence of BUC T24 apoptosis (p < 0.05), which can also be reversed by CCL18. In addition, miR-128 can inhibit the proliferation (p < 0.05) and metastasis (p < 0.05) of UCs in nude mice. Conclusion: The miR-128 inhibits the proliferation, invasion, migration of UCs, and promotes its apoptosis by regulating CCL18 secretion.

Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/ß-catenin signaling.

This study is to investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) in the progression of urothelial carcinoma (UC) after renal transplants (RT). A total of 114 UC patients were gathered, including 60 cases of primary UC and 54 cases UC after RT. RT-PCR was used to detect the mRNA expression of the 54 patients with UC after RT, and immunohistochemistry and western blot were used to examine the protein expression. The proliferative ability of two UC cell lines, and 5637, were measured by WST-1 assay. Transwell system was used to analyze the migration and invasion of UC cells. PPARγ agonist Rosiglitazone and the antagonist GW9662 were used to alter the PPARγ expression. siRNA targeting LEF1 and expression vector containing full-length cDNA of LEF1 regulated the expression of LEF1. Pathway analysis indicated that PPARγ expression was significantly down regulated. Compared with normal urothelium and primary UC, the expression of PPARγ in UC was significantly decreased in RT group. PPARγ expression was correlated with tumor size, clinical stage, pathological and recurrence. PPARγ inactivates LEF1/ß-catenin signaling in UC cells. PPARγ decreased the protein expression of MMP2, and calpain-2. PPARγ suppresses the proliferation, and invasion of UC cells depending on the expression of LEF1. PPARγ inhibited tumor proliferation and metastasis by inhibiting LEF1/ß-catenin signaling, and the expression of PPARγ in UC after RT decreased significantly. Our findings also suggested that PPARγ may be a potential biomarker for the diagnosis of UC after RT.

ZSCAN16 promotes proliferation, migration and invasion of bladder cancer via regulating NF-kB, AKT, mTOR, P38 and other genes.

BACKGROUND: As one of the most common genitourinary malignancies worldwide, bladder cancer affects about 3.4 million people globally, with 430,000 new cases a year since 2015. Despite the advances in bladder cancer diagnosis and therapy, there has been little progress in the patients' overall survival in nearly 30 years. Therefore, investigating novel molecular therapeutic targets is required to gain insight into the tumorigenesis of bladder cancer, which ultimately may be used to develop more effective therapeutic strategies. METHODS: Herein, we used gene knockdown in vitro and in vivo to unveil the unknown roles of ZSCAN16 in bladder cancer. Afterward, to decipher the unknown regulatory role of ZSCAN16 in tumor progression, we verified that a bunch of genes including NF-κB, AKT, mTOR, and P38 were the key downstream regulators of ZSCAN16 by western blot and rescue experiments. RESULTS: We found high expression of ZSCAN16 transcripts in bladder cancer cells and tumor samples from the TCGA database and tissue microarray bank, demonstrated in correlation with poor prognosis for bladder cancer patients. The in vitro experiments indicated that the silencing of ZSCAN16 by shRNA lentivirus promoted apoptosis and inhibited proliferation, colony formation, as well as migration and invasion in T24 cells. By investigating the signaling pathways, we proved ZSCAN16 play a novel role as oncogenic gene in bladder cancer by regulating NF-κB, AKT, mTOR, P38 and other genes. Furthermore, the in vivo experiments identified that ZSCAN16 knockdown retarded the tumor growth in nude mice. CONCLUSIONS: In summary, these findings revealed that ZSCAN16 is a potential novel oncogene in the development and progression of bladder cancer. This study will shed light on developing novel therapeutic targets in the future treatment of bladder cancer.

Transforming growth factor-ß1 enhances proliferative and metastatic potential by up-regulating lymphoid enhancer-binding factor 1/integrin αMß2 in human renal cell carcinoma.

Renal cell carcinoma (RCC) is a kind of malignant tumor with high recurrence, and it is urgent to find molecular markers for diagnosis and prognosis of RCC. Our study investigated the expression and function of integrin αMß2 in RCC cells, aiming to understand the role of integrin αMß2 in RCC and develop new therapeutic target for RCC. Overexpression and knockdown of lymphoid enhancer-binding factor 1 (LEF1) were performed using vector containing full-length cDNA and via siRNA technology, respectively. The expressions of mRNA and protein were detected by RT-PCR and Western blot, respectively. Proliferation of RCC cell was analyzed using WST-1 assay, and metastasis of RCC cell was evaluated using the transwell system. Our results demonstrated that LEF1 and integrin αMß2 were up-regulated in RCC cells via TGF-ß1-dependent mechanism, and LEF1 together with ß-catenin directly increased integrin αMß2 level. On the other hand, TGF-ß1-induced proliferation, migration and invasion were suppressed by function-blocking antibody against integrin αMß2 in RCC cells. In addition, integrin αMß2 is crucial for LEF1 mediated cell invasion by regulating MMP-2, MMP-9 and calpain-2 secretion in RCC cells. LEF1/integrin αMß2 expression was regulated by TGF-ß1, and LEF1/integrin αMß2 was involved in TGF-ß1's improvement effects on the proliferation and metastasis of RCC. Blocking integrin αMß2 activity could be a therapeutic option for patients with advanced RCC.

New mechanistic insights of clear cell renal cell carcinoma from integrated miRNA and mRNA expression profiling studies.

Differentially expressed (DE) microRNAs (miRNAs) in clear cell renal cell carcinoma (ccRCC) tissues from pooled samples were reported to affect the tumorigenesis and progression of ccRCC. However, systematic studies on the miRNA-mRNA regulatory networks involved in various pathways in all four stages of the disease are lacking. In this study, we applied microarray technology to perform an integrated analysis of the miRNome and transcriptome in ccRCC tissues from patients at different stages of ccRCC. A total of 604 DEmiRNAs and 6892 DEgenes (DEGs) were identified by comparison with corresponding adjacent normal tissues. The pairing of miRNAs with DEGs were searched using validated miRWalk module, and the pairs were confirmed by comparing with DEmiRNAs in our study. Our results demonstrated that different stages of ccRCC had distinct miRNA/mRNA profiles. However, four common pathways (the complement and coagulation cascades, the pathway for the metabolism of xenobiotics by cytochrome P450, the PPAR signaling pathway, and the pathway for aldosterone-regulated sodium reabsorption) were enriched by targets of DEmiRNAs at all stages of ccRCC. We carried out an extensive analysis of data on miR-16, which had the most target genes, and found that its differential expression was validated in The Cancer Genome Atlas dataset. We also verified the correlation between miR-16 expression and target pathways by gene set enrichment analysis and in vitro experiments. High miR-16 level was also associated with shorter survival time in ccRCC. Our work presents a systematic profiling of miRNA, mRNA and pathways regulated by miRNAs in different stages of ccRCC. Our cross-omics results also identify four common pathways that function aberrantly in all stages of the disease. These pathways are likely to be critical in occurrence and progression of ccRCC. These common dysfunctional pathways have the potential to serve as therapeutic targets and diagnostic biomarkers, whereas miRNAs (miR-20, 484, 497) differentially expressed in only stage I tissues and in blood could be used to diagnose early-stage ccRCC patients.

The link between FOXJ1 expression level in bladder carcinoma and tumor recurrence.

[This retracts the article DOI: 10.3892/ol.2017.7504.].

The link between FOXJ1 expression level in bladder carcinoma and tumor recurrence.

We studied the expression level of FOXJ1 in bladder epithelial carcinoma and its relation to clinical features and tumor recurrence. From January 2014 to June 2015, 66 patients with bladder epithelial carcinoma were enrolled in this study and their tumor and para-carcinoma tissue samples were collected. FOXJ1 positive expression rate was evaluated using immunohistochemical staining, and FOXJ1 mRNA expression level was measured by RT-PCR. RT-PCR and immunohistochemistry results showed that FOXJ1 expression level in tumor samples was significantly lower than that in para-carcinoma tissue samples. The median survival time in patients with positive expression of FOXJ1 was significantly longer than that of patients with negative expression of FOXJ1. We also showed that FOXJ1 expression level was negatively correlated with neoplasm staging and tumor recurrence rate. We concluded that FOXJ1 was expressed in low quantities in bladder epithelial carcinoma, which was closely correlated with the biological characteristics of the tumor. FOXJ1 expression presents a promising application prospect for further exploration of the specific biological mechanism of FOXJ1 in regulating the occurrence and development of bladder epithelial carcinoma. FOXJ1 may be used as a new marker for early diagnosis and prediction of recurrence.

Epirubicin suppresses proliferative and metastatic potential by downregulating transforming growth factor-ß-induced expression in urothelial carcinoma.

Transforming growth factor-ß-induced (TGFΒI) is considered to be a vital gene in several carcinomas. In this study we determined the effect of TGFBI on the proliferative and metastatic potential of human urothelial carcinoma (UC) cells as well as its mRNA and protein expression, which were detected by RT-PCR and western blot, respectively. UC cell proliferation was analyzed by WST-1 assay and Hoechst 33258 staining. The effect of TGFBI on UC cell metastasis was analyzed using adhesion, migration and invasion assays. We found that TGFBI increased the proliferation of UC cells. Moreover, TGFBI enhanced the adhesion, migration and invasion of UC cells by upregulating MMP-2, MMP-9 and calpain-2 expression. We evaluated the effect of Epirubicin (EPI) on the regulation of TGFBI expression and found that TGFBI acts as a downstream target of EPI and is suppressed by EPI in UC cells. EPI is more effective in inhibiting the proliferation and metastasis of UC cells with high TGFBI expression. This study demonstrates that TGFBI might lead to tumorigenesis and progression of UC and those cells with high TGFBI expression may be vulnerable to relapse. EPI could prove to be a therapeutic option in patients with high TGFBI expressing UC cells.

FOXJ1 promotes bladder cancer cell growth and regulates Warburg effect.

Forkhead Box J1 (FOXJ1) which belongs to Fox gene family, plays complex and crucial roles in processes of development, organogenesis, regulation of the immune system, as well as progression of several malignancies. However, how FOXJ1 functions in bladder cancer remains unclear. Here, we report that FOXJ1 is upregulated in most bladder cancer patients, and predicts poor clinical outcomes. FOXJ1 facilitates bladder cancer cell proliferation and colony formation. FOXJ1 knockdown suppresses bladder tumor growth in nude mice. Mechanistically, FOXJ1 enhances glycolysis by increasing glucose uptake, lactate production and extracellular acidification rate (ECAR), and decreasing ATP generation and oxygen consumption rate (OCR) in bladder cancer cells. Our findings provide clues regarding the role of FOXJ1 as a tumor inducer in bladder cancer and an enhancer in glycolysis. Targeting FOXJ1 could be a potential therapeutic strategy in bladder cancer.

VHL-TGFBI signaling is involved in the synergy between 5-aza-2'-deoxycytidine and paclitaxel against human renal cell carcinoma.

PURPOSE: To analyse the role of von Hippel-Lindau (VHL) and transforming growth factor ß-induced (TGFBI) in synergistic mechanisms of 5-aza-2'-deoxycytidine (DAC) and paclitaxel (PTX) against renal cell carcinoma (RCC). METHODS: To elucidate the role in the synergy between DAC and PTX against RCC cells, TGFBI expression was regulated using siRNA technology and an expression vector containing the full-length cDNA for TGFBI was also transfected into RCC cells. The proliferation of RCC cells was evaluated using the WST-1 assay and TGFBI expression was detected by real-time PCR (RT-PCR), and Western blot. RESULTS: The results indicated that the expression of TGFBI was significantly decreased by DAC or PTX alone in vitro and in vivo. Moreover, the combination of DAC and PTX caused a synergistic decrease in the expression of TGFBI in RCC cells. We also investigated the effect of VHL-TGFBI signaling on the synergy between DAC and PTX, although the synergy between the two medications was not abolished by interfering with VHL activity or TGFBI expression. RCC cells without VHL activity and RCC cells expressing high levels of TGFBI displayed an increased synergistic effect compared to control cells. CONCLUSION: Our study suggests that VHL-TGFBI signaling is involved in the synergy between DAC and PTX against RCC cells. In addition, the synergy between DAC and PTX is more effective in VHL inactive RCC cells.

VHL-TGFBI signaling is involved in the synergy between 5-aza-2'-deoxycytidine and paclitaxel against human renal cell carcinoma.

PURPOSE: To analyse the role of von Hippel-Lindau (VHL) and transforming growth factor ß-induced (TGFBI) in synergistic mechanisms of 5-aza-2'-deoxycytidine (DAC) and paclitaxel (PTX) against renal cell carcinoma (RCC). METHODS: To elucidate the role in the synergy between DAC and PTX against RCC cells, TGFBI expression was regulated using siRNA technology and an expression vector containing the full-length cDNA for TGFBI was also transfected into RCC cells. The proliferation of RCC cells was evaluated using the WST-1 assay and TGFBI expression was detected by real-time PCR (RT-PCR), and Western blot. RESULTS: The results indicated that the expression of TGFBI was significantly decreased by DAC or PTX alone in vitro and in vivo. Moreover, the combination of DAC and PTX caused a synergistic decrease in the expression of TGFBI in RCC cells. We also investigated the effect of VHL-TGFBI signaling on the synergy between DAC and PTX, although the synergy between the two medications was not abolished by interfering with VHL activity or TGFBI expression. RCC cells without VHL activity and RCC cells expressing high levels of TGFBI displayed an increased synergistic effect compared to control cells. CONCLUSIONS: Our study suggests that VHL-TGFBI signaling is involved in the synergy between DAC and PTX against RCC cells. In addition, the synergy between DAC and PTX is more effective in VHL inactive RCC cells.

Profiling of mRNA and long non-coding RNA of urothelial cancer in recipients after renal transplantation.

The molecular mechanism and signal transduction pathways involved in urothelial cancer (UC) after renal transplantation (RTx) remain unknown. In this study, we investigated the profiling of messenger RNA (mRNA) and long non-coding RNA (lncRNA) in RTx recipients with UC. The mRNA and lncRNA of six pairs of UC and corresponding normal urothelial tissues in RTx recipients were profiled using Arraystar Human lncRNA Microarray V3.0, which is designed for the global profiling of 26,109 coding transcripts and 30,586 lncRNAs. Quantitative real-time PCR (qRT-PCR) was used to validate the differentially expressed mRNAs and lncRNAs. Molecular function classification and biological process classification for the differentially expressed mRNAs were analyzed with Gene Ontology. The key pathways that were associated with UC after RTx were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Compared to normal urothelial tissues, 1597 mRNAs were upregulated and 1032 mRNAs were downregulated in UC; 2107 lncRNAs were upregulated and 1794 lncRNAs were downregulated (greater than twofold). Further qRT-PCR analysis of mRNA and lncRNA expression showed well consistency with the data of microarray analysis. The expression of matrix metalloprotease (MMP)-3, MMP-10, MMP-12, and MMP-13 was significantly increased, while the expression of CD36 was decreased in UC after RTx. Co-expression analysis of lncRNAs and their nearby coding genes showed that lncRNAs may play critical roles in regulating nearby genes in the carcinogenesis of UC. Our results also suggest that peroxisome proliferator-activated receptor (PPAR) signaling may be involved in UC after RTx. Moreover, several cytokines and their receptors were also significantly upregulated in UC after RTx, suggesting that cytokines might be modulated and participated in the carcinogenesis of UC after RTx. We analyzed the potential molecular mechanism and pathways involved in the UC of RTx recipients. Our results revealed that several key regulatory pathways and lncRNAs play critical roles in the carcinogenesis of UC, and suggest that UC in RTx recipients may be more likely to invade and metastasis. However, the detailed functional analysis of these mechanisms should be further performed in the future.

Synergistic roles of p53 and HIF1α in human renal cell carcinoma-cell apoptosis responding to the inhibition of mTOR and MDM2 signaling pathways.

INTRODUCTION: mTOR and MDM2 signaling pathways are frequently deregulated in cancer development, and inhibition of mTOR or MDM2 independently enhances carcinoma-cell apoptosis. However, responses to mTOR and MDM2 antagonists in renal cell carcinoma (RCC) remain unknown. MATERIALS AND METHODS: A498 cells treated with MDM2 antagonist MI-319 and/or mTOR inhibitor rapamycin were employed in the present study. Cell apoptosis and Western blot analysis were performed. RESULTS AND CONCLUSION: We found that the MDM2 inhibitor MI-319 induced RCC cell apoptosis mainly dependent on p53 overexpression, while the mTOR antagonist rapamycin promoted RCC cell apoptosis primarily through upregulation of HIF1α expression. Importantly, strong synergistic effects of MI-319 and rapamycin combinations at relatively low concentrations on RCC cell apoptosis were observed. Depletion of p53 or HIF1α impaired both antagonist-elicited apoptoses to differential extents, corresponding to their expression changes responding to chemical treatments, and double knockdown of p53 and HIF1α remarkably hindered MI-319- or rapamycin-induced apoptosis, suggesting that both p53 and HIF1α are involved in MDM2 or mTOR antagonist-induced apoptosis. Collectively, we propose that concurrent activation of p53 and HIF1α may effectively result in cancer-cell apoptosis, and that combined MDM2 antagonists and mTOR inhibitors may be useful in RCC therapy.

Decitabine induces G2/M cell cycle arrest by suppressing p38/NF-κB signaling in human renal clear cell carcinoma.

OBJECTIVE: The anti-neoplastic effects of decitabine, an inhibitor of DNA promoter methylation, are beneficial for the treatment of renal cell carcinoma (RCC); however, the mechanism of action of decitabine is unclear. We analyzed gene expression profiling and identified specific pathways altered by decitabine in RCC cells. METHODS: Four human RCC cell lines (ACHN, Caki-1, Caki-1, and A498) were used in this study; growth suppression of RCC cells by decitabine was analyzed using the WST-1 assay. Apoptosis and cell cycle arrest were examined using flow cytometric analysis. Gene expression of RCC cells induced by decitabine was evaluated with cDNA microarray, and potential biological pathways were selected using Ingenuity Pathway Analysis. The activity of the p38-NF-κB pathway regulated by decitabine was confirmed by Western blotting. RESULTS: Decitabine suppresses the proliferation of RCC cells in vitro. Although decitabine did not significantly induce apoptosis, decitabine caused cell cycle arrest at G2/M in a dose-dependent manner. Gene expression regulated by decitabine in RCC cells was investigated using microarray analysis. Ubiquitin carboxyl terminal hydrolase 1 (UCHL1), interferon inducible protein 27 (IFI27), and cell division cycle-associated 2 (CDCA2) may be involved in growth suppression of RCC cells by decitabine. The phosphorylation of p38-NF-κB pathway was suppressed by decitabine in RCC cells. CONCLUSIONS: We investigated gene expression profiling and pathways modulated by decitabine in RCC cells. Decitabine was shown to suppress the growth of RCC cells via G2/M cell cycle arrest and the p38-NF-κB signaling pathway may play a role in the anti-neoplastic effect of decitabine in RCC cells.