Integrative analysis and risk model construction for super­enhancer­related immune genes in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer associated with poor prognosis, and accounts for the majority of RCC-related deaths. The lack of comprehensive diagnostic and prognostic biomarkers has limited further understanding of the pathophysiology of ccRCC. Super-enhancers (SEs) are congregated enhancer clusters that have a key role in tumor processes such as epithelial-mesenchymal transition, metabolic reprogramming, immune escape and resistance to apoptosis. RCC may also be immunogenic and sensitive to immunotherapy. In the present study, an Arraystar human SE-long non-coding RNA (lncRNA) microarray was first employed to profile the differentially expressed SE-lncRNAs and mRNAs in 5 paired ccRCC and peritumoral tissues and to identify SE-related genes. The overlap of these genes with immune genes was then determined to identify SE-related immune genes. A model for predicting clinical prognosis and response to immunotherapy was built following the comprehensive analysis of a ccRCC gene expression dataset from The Cancer Genome Atlas (TCGA) database. The patients from TCGA were divided into high- and low-risk groups based on the median score derived from the risk model, and the Kaplan-Meier survival analysis showed that the low-risk group had a higher survival probability. In addition, according to the receiver operating characteristic curve analysis, the risk model had more advantages than other clinical factors in predicting the overall survival (OS) rate of patients with ccRCC. Using this model, it was demonstrated that the high-risk group had a more robust immune response. Furthermore, 61 potential drugs with half-maximal inhibitory concentration values that differed significantly between the two patient groups were screened to investigate potential drug treatment of ccRCC. In summary, the present study provided a novel index for predicting the survival probability of patients with ccRCC and may provide some insights into the mechanisms through which SE-related immune genes influence the diagnosis, prognosis and potential treatment drugs of ccRCC.

RAB3D, upregulated by aryl hydrocarbon receptor (AhR), promotes the progression of prostate cancer by activating the PI3K/AKT signaling pathway.

Many patients with prostate cancer (PCa) cannot be diagnosed until an advanced stage, which make PCa become a large threat to human health. It is an urgent need to explore novel biomarkers for accurate diagnosis and targets for the effective treatment of PCa. This study aimed to investigate the effects of RAB3D (which belongs to the secretory Rab GTPases) on the progression of PCa. The results showed that RAB3D was highly expressed in PCa tissues compared to normal tissues according to the gene expression omnibus dataset. Consistent with the bioinformatics results, RAB3D exhibited a higher expression in PCa cells. Overexpression of RAB3D promoted the proliferation, migration, and invasion of PCa cells, whereas the knockdown of RAB3D led to the opposite results. The procancer effects of RAB3D were further confirmed by the in vivo growth of xenograft model. Subsequently, RAB3D upregulated the PI3K/AKT signaling pathway both in vivo and in vitro. LY294002 (a PI3K inhibitor) rescued the RAB3D upregulation-induced promotion of malignant phenotypes of PCa cells. Furthermore, the transcription activity of RAB3D was found to be enhanced by aryl hydrocarbon receptor (AhR; a transcription factor). The AhR silencing-induced inhibition of the proliferation and migration of PCa cells was reversed by the overexpression of RAB3D. Taken together, RAB3D, upregulated by AhR, promotes the PCa progression by activating the PI3K/AKT signaling pathway.

OIP5 Is a Novel Prognostic Biomarker in Clear Cell Renal Cell Cancer Correlating With Immune Infiltrates.

Opa interacting protein 5 (OIP5), overexpressed in some types of human cancers, has been reported to be associated with the carcinogenesis of human cancer. However, its contribution to cancer immunity remains unknown. Furthermore, the relationship between OIP5 and cancer immunity remains uncertain. In our research, we explored the different expression of OIP5 between 539 ccRCC and 72 normal renal tissues base on TCGA data set. We analyzed the associations between OIP5 expression with ccRCC progression and survival. Next, we compared immune cell profiles in cancer tissues and normal tissues in the Cancer Genome Atlas (TCGA) ccRCC cohort. We found that the level of immune cell infiltration was correlated with the copy number of OIP5 gene in ccRCC. The effect of OIP5 on immune activity was verified by Gene Set Enrichment Analysis of RNA-seq data from 32 ccRCC cell lines in the public database. Moreover, a pathway enrichment analysis of 49 OIP5-associated immunomodulators demonstrated the involvement of the T cell receptor signaling pathway, the JAK-STAT signaling pathway, the NF-kappa B signaling pathway and the primary immunodeficiency pathway. In addition, using OIP5-associated immunomodulators, we constructed multiple-gene risk prediction signatures using the Cox regression model. Our results provided insights into the role of OIP5 in tumor immunity and revealed that OIP5 may be a potential immunotherapeutic target for ccRCC. Designated immune signature is a promising prognostic biomarker in ccRCC.

Enhanced Expression of CNTD2/CCNP Predicts Poor Prognosis in Bladder Cancer Based on the GSE13507.

Bladder cancer is one of the most common urogenital malignancies in the world, and there are no adequate prognostic indicators. CNTD2 is one of the atypical cyclins, which may be related to the cell cycle and even the development of cancers. Early studies have shown that CNTD2 is closely related to the occurrence and development of many malignant tumors. However, the mechanism of CNTD2 in bladder cancer has not been reported. In our research, we explored the different expressions of CNTD2 between 411 bladder cancers and 19 normal bladder tissues based on the TCGA dataset. CNTD2-related signaling pathways were identified through the GSEA. We analyzed the associations of CNTD2 expression and bladder cancer progression and survival using GSE13507. Compared with 19 cases of normal bladder tissue, CNTD2 gene expression was increased in 411 cases of bladder cancer. The high expression of CNTD2 strongly correlated with grade (P < 0.0001), T classification (P = 0.0001), N classification (P = 0.00011), M classification (P = 0.044), age (P = 0.027), and gender (P = 0.0012). Bladder cancer patients with high CNTD2 expression had shorter overall survival (P < 0.001). In the meantime, univariate and multivariate analyses showed that the increased expression of CNTD2 was an independent factor for poor prognosis in bladder cancer patients (P < 0.001 and P < 0.001, respectively). CNTD2 expression is closely related to bladder cancer progression, and the high expression of CNTD2 may be an adverse biomarker in bladder cancer patients.

H3 relaxin protects against calcium oxalate crystal-induced renal inflammatory pyroptosis.

OBJECTIVES: Calcium oxalate (CaOx) crystals can activate inflammatory cytokines by triggering inflammasomes, which cause damage to the adhered epithelium, a dysfunctional microenvironment and even renal failure. However, a comprehensive and in-depth understanding of the mechanisms underlying the effects of these crystals on damage and cytokine function in renal tubular epithelial cells (TECs) remains limited and to be explored. MATERIALS AND METHODS: We detected the pyroptosis of TECs induced after exposure to CaOx crystals and demonstrated the significance of cytokine activation in the subsequent inflammatory processes through a proteomic study. We then conducted animal and cell experiments to verify relevant mechanisms through morphological, protein, histological and biochemical approaches. Human serum samples were further tested to help explain the pathophysiological mechanism of H3 relaxin. RESULTS: We verified that crystal-induced extracellular adenosine triphosphate (ATP) upregulation via the membrane purinergic 2X7 receptor (P2X7 R) promotes ROS generation and thereby activates NLRP3 inflammasome-mediated interleukin-1ß/18 maturation and gasdermin D cleavage. Human recombinant relaxin-3 (H3 relaxin) can act on the transmembrane receptor RXFP1 to produce cAMP and subsequently improves crystal-derived damage via ATP consumption. Additionally, endogenous relaxin-3 was found to be elevated in patients with renal calculus and can thus serve as a biomarker. CONCLUSIONS: Our results provide previously unidentified mechanistic insights into CaOx crystal-induced inflammatory pyroptotic damage and H3 relaxin-mediated anti-inflammatory protection and thus suggest a series of potential therapeutic targets and methods for but not limited to nephrocalcinosis.

LncRNA ADAMTS9-AS2 inhibits cell proliferation and decreases chemoresistance in clear cell renal cell carcinoma via the miR-27a-3p/FOXO1 axis.

Accumulating evidence reveals the principal role of long noncoding RNAs in the progression of clear cell renal cell carcinoma (ccRCC). However, little is known about the underlying mechanism of ADAM metallopeptidase with thrombospondin type 1 motif, 9 antisense RNA 2 (ADAMTS9-AS2) in ccRCC. Here, bioinformatics analyses verified ADAMTS9-AS2 is a long noncoding RNA and its high expression was associated with better prognosis of ccRCC. ADAMTS9-AS2 was clearly downregulated in ccRCC clinical samples and cell lines. Clinical data showed low-expressed ADAMTS9-AS2 was correlated with worse overall survival in ccRCC patients. Next, miR-27a-3p was identified as an inhibitory target of ADAMTS9-AS2 by dual-luciferase reporter and RNA immunoprecipitation assays. Both overexpressed ADAMTS9-AS2 and underexpressed miR-27a-3p in ccRCC cell lines led to the inhibition of cell proliferation and the reduction of chemoresistance. Additionally, Forkhead Box Protein O1 (FOXO1) was confirmed as the inhibitory target of miR-27a-3p. Induced by ADAMTS9-AS2 overexpression, cell proliferation and chemoresistance exhibited an obvious reduction, FOXO1 expression showed an evident increase, but all were reversed after miR-27a-3p was simultaneously overexpressed. Collectively, these results suggest ADAMTS9-AS2 inhibits the progression and impairs the chemoresistance of ccRCC via miR-27a-3p-mediated regulation of FOXO1 and may serve as a prognostic biomarker and therapeutic target for ccRCC.

Bone marrow-derived mesenchymal stem cells attenuate tubulointerstitial injury through multiple mechanisms in UUO model.

Current evidence supports the use of bone marrow-derived mesenchymal stem cells (MSCs) for a diverse range of clinical applications, and many studies have shown that MSCs have renal-protective effects, but the mechanism is not well understood. Therefore, in this study, we aim to further identify whether MSCs can attenuate renal fibrosis by decreasing tubulointerstitial injury in a unilateral ureteral obstruction (UUO) model. In this study, we cultured MSCs and then transplanted them into a UUO model through the tail vein. Histology, cell proliferation, peritubular capillary (PTC) loss and myofibroblast markers were examined on days 3, 7 and 14 after surgery. We demonstrated that renal interstitial fibrosis in the MSC group was significantly attenuated compared with the UUO and DMEM groups. Moreover, MSC treatment inhibited the loss of PTCs and increased parenchymal cell proliferation. In addition, UUO-induced activation and proliferation of myofibroblasts were suppressed by MSC infusion. Furthermore, MSCs attenuated tubulointerstitial infiltration of macrophages in UUO mice. Tubulointerstitial damage plays a very important role in the progression of chronic kidney disease (CKD). PTC loss, macrophage recruitment, and myofibroblast activation are directly correlated with the development of renal tubulointerstitial fibrosis. Our results suggest that MSC infusion in the UUO model is a promising therapeutic strategy for promoting kidney repair.

Identification of potential crucial genes associated with carcinogenesis of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a common genitourinary malignancy with high mortality. However, the molecular pathogenesis of ccRCC remains unclear and effective biomarkers for daily practice are still limited. Thus, we aimed to identify the potential crucial genes and pathways associated with carcinogenesis of ccRCC and further analyze the molecular mechanisms implicated in tumorigenesis. In the present study, expression profiles GSE 66270, GSE 53757, GSE 36895, and GSE 76351 were downloaded from GEO database, including 244 matched primary and adjacent normal tissues, furthermore, the level 3 RNAseq dataset (RNAseqV2 RSEM) of KIRC was also downloaded from The Cancer Genome Atlas (TCGA), which consist of 529 ccRCC tumors and 72 normal tissues. Then, differentially expressed genes (DEGs) and pathway enrichment were analyzed by using R software. A total of 129 up- and 123 down-regulated genes were identified, which were aberrantly expressed both in GEO and TCGA data. Second, Gene ontology (GO) analyses revealed that most of the DEGs were significantly enriched in integral component of membrane, extracellular exosome, plasma membrane, cell adhesion, and receptor binding. Signaling pathway analyses indicated that DEGs had common pathways in signal transduction, metabolism, and immune system. Third, hub genes were identified with protein-protein interaction (PPI) network, including PTPRC, TGFB1, EGF, MYC, ITGB2, CTSS, FN1, CCL5, KNG1, and CD86. Additionally, sub-networks analyse was also performed by using MCODE plugin. In conclusion, the novel DEGs and pathways in ccRCC identified in this study may provide new insight into the underlying molecular mechanisms that facilitates RCC carcinogenesis.

808nm light triggered black TiO2 nanoparticles for killing of bladder cancer cells.

The black TiO2 nanoparticles are synthesized via a facile calcination method combined with an in-situ controllable solid-state reaction approach. The results indicate that the photocatalyst with a narrow band gap of ~2.32 eV extends the photoresponse to visible light and near infrared region. And thus more reactive oxygen species can be obtained to induce the cell-killing under 808 nm light triggering. The as-obtained black TiO2 nanoparticles exhibiting low toxicity, good biocompatibility and high anticancer effect in vitro, is demonstrated as efficient photosensitizers for phototherapy to kill the bladder cancer cells. These findings suggest that the facile synthetic black TiO2 nanomaterials will have broad application in biomedicine.

Downregulation of lncRNA SDPR-AS is associated with poor prognosis in renal cell carcinoma.

Renal cell carcinoma (RCC) is a common type of kidney cancer. Normally, surgical treatment can prolong life, but only for patients with early stage tumors. However, it is difficult for early detection strategies to distinguish between benign and malignant kidney tumors. Therefore, potential biomarkers for early diagnosis and prognosis of RCC are needed. Intriguingly, mounting evidence has demonstrated that many long noncoding RNAs (lncRNAs) are strongly linked to cancers. Indeed, promising RCC-associated lncRNA biomarkers have also been identified. However, the functional and prognostic roles of the antisense (AS) serum deprivation response (SDPR) lncRNA (SDPR-AS) in RCC remain largely unknown. The aims of this study were to investigate the expression and prognostic relevance of SDPR-AS in RCC. We uncovered the downregulated expressions of both lncRNA SDPR-AS and its protein-coding gene, SDPR, in RCC tissues compared to the matched normal tissues. Furthermore, SDPR-AS and SDPR expressions were positively correlated. Overexpression and knockdown experiments suggested that SDPR-AS and SDPR were coregulated in RCC cell lines. In addition, overexpression of SDPR-AS suppressed cell migration and invasion, but not cell growth. Furthermore, expression of SDPR-AS was associated with tumor differentiation and lymphatic metastasis. Kaplan-Meier survival and log-rank tests demonstrated the association of elevated expression of SDPR-AS with increased overall survival. In conclusion, our results suggest that the SDPR-AS may serve as a prognostic biomarker and therapeutic target of RCC.

Retroperitoneal Laparoscopic Partial Nephrectomy for Tumors Larger than 7 cm in Renal Cell Carcinoma: Initial Experience of Single-Institution.

BACKGROUND: To report our initial experience dealing with retroperitoneal laparoscopic partial nephrectomy (LPN) for tumors larger than 7 cm in renal cell carcinoma (RCC). MATERIALS AND METHODS: A total of 15 patients with malignant tumors larger than 7 cm underwent retroperitoneal LPN at our institution. Patient baseline demographics, perioperative outcomes, pathological characteristics, and estimated glomerular filtration rate (eGFR) were analyzed retrospectively in our collected database. RESULTS: The tumor size is 7.5 (7.1-9.0) cm. Nine (60.00%) patients, 4 (26.67%) patients, and 2 (13.33%) patients suffered from preoperative chronic kidney disease (CKD) at stage I, II, and III, respectively. The median operating time was 121 (90-330) minutes and the warm ischemia time (WIT) was 29 (12-45) minutes, with the estimated blood loss of 50 (10-1200) mL. The preoperative eGFR was 81.26 mL/min per 1.73 m2 (ranging from 56.15 to 140.47), eGFR on 1 and 30 days postoperative was 70.49 mL/min per 1.73 m2 (ranging from 50.32 to 137.73) and 75.13 mL/min per 1.73 m2 (ranging from 54.07 to 142.99), respectively. At last follow-up, the eGFR was 72.78 mL/min per 1.73 m2 (ranging from 51.28 to 137.86); no stage migration for CKD was observed. Major complications included 2 patients requiring blood transfusions and 1 patient performing renal vein suture as well as single leak. CONCLUSIONS: Our initial experience suggests that retroperitoneal LPN maybe a feasible, safe, and effective procedure for selected tumors larger than 7 cm in RCC, with the advantage of renal function preservation and acceptable major surgical complications. Tumor size might not be the contraindication of LPN in the treatment of selected tumors.

Expression of microRNA-30c via lentivirus vector inhibits the proliferation and enhances the sensitivity of highly aggressive ccRCC Caki-1 cells to anticancer agents.

The clear cell renal cell carcinoma (ccRCC) is one of the most fatal urologic tumors, and the prognosis remains very poor for advanced or metastatic ccRCC. This study reveals the roles of microRNA (miR)-30c in regulating a highly aggressive ccRCC cell line proliferation by targeting MTA-1, which is a key mediator for human cancer metastasis. Results from quantitative real-time polymerase chain reaction showed that the expression of MTA-1, the target of miR-30c, was significantly higher in metastatic ccRCC specimens than in nonmetastatic ccRCC or nontumor specimens. Accordingly, endogenous miR-30c is at a much lower level in highly aggressive ccRCC Caki-1 cells than nontumor or ccRCC cell lines. Expression of miR-30c via lentivirus vector inhibits the proliferation, anchorage-independent growth, in vitro invasion or migration, or in vivo growth of Caki-1 cells by repressing MTA-1 protein expression. miR-30c also enhances the sensitivity of Caki-1 cells to anticancer agents, including sorafenib and paclitaxel. These data reveal the potential application of miR-30c and that its targeting gene, MTA-1, would be a potential target in metastatic ccRCC treatment.

KLF6 Suppresses Metastasis of Clear Cell Renal Cell Carcinoma via Transcriptional Repression of E2F1.

The transcription factor KLF6 has an essential role in the development and metastasis of multiple human cancers. Paradoxically, KLF6 expression was found to be attenuated in primary metastatic clear cell renal cell carcinoma (ccRCC), such that it is unclear how KLF6 affects malignant progression in this setting. In this study, we demonstrate that KLF6 attenuation in renal cells is sufficient to promote E2F1-mediated epithelial-mesenchymal transition and metastatic prowess. In a mouse xenograft model of human ccRCC, silencing KLF6 increased tumor cell proliferation and malignant character, whereas E2F1 silencing reversed these properties. These effects were corroborated in a metastatic model system, where we observed a greater number of pulmonary metastatic lesions formed by ccRCC cells where KLF6 was silenced and E2F1 enforced. Analysis of clinical specimens of ccRCC revealed that low levels of KLF6 and high levels of E2F1 correlated closely with ccRCC development. Overall, our results established the significance of activating the KLF6-E2F1 axis in aggressive ccRCC, defining a novel critical signaling mechanism that drives human ccRCC invasion and metastasis. Cancer Res; 77(2); 330-42. ©2016 AACR.

Bilateral Synchronous Sporadic Renal Cell Carcinoma: Retroperitoneoscopic Strategies and Intermediate Outcomes of 60 Patients.

OBJECTIVE: To evaluate the presentation, management, pathology, and functional and oncological outcomes of patients undergoing retroperitoneoscopic treatment of bilateral synchronous sporadic RCC at our institution. METHODS: We retrospectively evaluated the records of 60 patients with bilateral synchronous sporadic RCC who underwent retroperitoneoscopic treatment at the General Hospital of People's Liberation Army from 2008 to 2014. The estimated glomerular filtration rate was calculated and compared among different surgical procedures. The overall survival and recurrence free survival were assessed based on information from recent follow-up. RESULTS: Fifty-six patients underwent bilateral retroperitoneoscopic surgeries in staged procedures, and four patients underwent bilateral retroperitoneoscopic surgeries in simultaneous procedures. Among the former group of patients, 34 underwent bilateral partial nephrectomy, 12 underwent radical nephrectomy followed by partial nephrectomy, and 10 underwent partial nephrectomy followed by radical nephrectomy. Bilateral partial nephrectomy can better preserve renal function (p = 0.040) and the sequence of partial nephrectomy and radical nephrectomy did not affect functional outcomes (p = 0.790). One patient undergoing simultaneous procedures developed acute renal failure and required temporary hemodialysis. At 3 and 5 years, overall survival rates were 93.0% and 89.4%, and recurrence free survival rates were 90.5% and 81.6%. High nuclear grade (p = 0.014) was related to disease recurrence. CONCLUSIONS: Staged bilateral partial nephrectomy was efficient in preserving renal function. The survival of patients with bilateral synchronous sporadic renal tumors was similar to that of patients with unilateral nonmetastatic tumors. Nuclear grade was an independent prognostic factor of disease recurrence.

Cross-talk between EPAS-1/HIF-2α and PXR signaling pathway regulates multi-drug resistance of stomach cancer cell.

EPAS-1/HIF-2α (Endothelial PAS domain-containing protein 1/hypoxia-inducible transcription factors 2α) is a transcription factor expressed in a wide range of human cancers, including stomach cancer. Although EPAS-1 has been studied for years, its function in oncogenic transformation processes needs to be further investigated. In this study, we found that EPAS-1 would promote the growth of stomach cancer cell line BGC-823. Our results revealed that EPAS-1 interacts with Pregnane X Receptor (PXR), a nuclear receptor that regulates multiple genes' transcription involved in multi-drugs resistance (MDR) process. Protein-protein interaction between EPAS-1 and PXR was identified by co-immunoprecipitation and GST-pull down assays. By this interaction, EPAS-1 recruited PXR to its response elements in promoter/enhancer regions of CYP3A4, a PXR target gene. Over-expression of EPAS-1 increased the expression of PXR responsive genes, enhanced the proliferation of BGC-823 cells and boosted the resistance of BGC-823 cells against the cytotoxicity of chemotherapeutic drugs, e.g. Mitomycin C and Paclitaxel. Reduction of EPAS-1 level via its siRNA disrupted the proliferation, and enhanced the susceptibility of BGC-823 cells to those chemotherapeutic drugs. Our findings suggested that EPAS-1 and PXR may cooperatively participate in development and especially MDR process of stomach cancer. These findings may contribute to more effective targeted drugs discovery for the stomach cancer therapy.

Estrogen receptor α enhances the transcriptional activity of ETS-1 and promotes the proliferation, migration and invasion of neuroblastoma cell in a ligand dependent manner.

BACKGROUND: It is well known that estrogen receptor α (ERα) participates in the pathogenic progress of breast cancer, hepatocellular carcinoma and head and neck squamous cell carcinoma. In neuroblastoma cells and related cancer clinical specimens, moreover, the ectopic expression of ERα has been identified. However, the detailed function of ERα in the proliferation of neuroblastoma cell is yet unclear. METHODS: The transcriptional activity of ETS-1 (E26 transformation specific sequence 1) was measured by luciferase analysis. Western blot assays and Real-time RT-PCR were used to examine the expression of ERα, ETS-1 and its targeted genes. The protein-protein interaction between ERα and ETS-1 was determined by co-IP and GST-Pull down assays. The accumulation of ETS-1 in nuclear was detected by western blot assays, and the recruitment of ETS-1 to its targeted gene's promoter was tested by ChIP assays. Moreover, SH-SY5Y cells' proliferation, anchor-independent growth, migration and invasion were quantified using the MTT, soft agar or Trans-well assay, respectively. RESULTS: The transcriptional activity of ETS-1 was significantly increased following estrogen treatment, and this effect was related to ligand-mediated activation of ERα. The interaction between the ERα and ETS-1 was identified, and enhancement of ERα activation would up-regulate the ETS-1 transcription factor activity via modulating its cytoplasm/nucleus translocation and the recruitment of ETS-1 to its target gene's promoter. Furthermore, treatment of estrogen increased proliferation, migration and invasion of neuroblastoma cells, whereas the antagonist of ERα reduced those effects. CONCLUSIONS: In this study, we provided evidences that activation of ERα promoted neuroblastoma cells proliferation and up-regulated the transcriptional activity of ETS-1. By investigating the role of ERα in the ETS-1 activity regulation, we demonstrated that ERα may be a novel ETS-1 co-activator and thus a potential therapeutic target in human neuroblastoma treatment.

Downregulation of FOXO3a promotes tumor metastasis and is associated with metastasis-free survival of patients with clear cell renal cell carcinoma.

PURPOSE: To explore the mechanisms underlying clear-cell renal cell carcinoma (ccRCC) metastasis using transcriptional profiling and bioinformatics analysis of ccRCC samples, and to elucidate the role of FOXO3a in ccRCC metastasis. EXPERIMENTAL DESIGN: Gene expression profiling was performed using four primary metastatic and five primary nonmetastatic ccRCC samples. The mRNA and protein levels of FOXO3a in ccRCC samples were investigated by real-time reverse transcription PCR and immunohistochemistry, respectively. The association between metastasis-free survival of patients with ccRCC and FOXO3a mRNA levels was analyzed. Biologic functions of FOXO3a in renal cancer cell lines were investigated. The influence of FOXO3a on tumor metastasis was also studied in vivo orthotopic xenograft tumor model. Finally, the mechanism by which FOXO3a attenuation could increase invasion and migration of tumor cells was explored. RESULTS: Bioinformatics analysis of the profiling data identified FOXO3a as a key factor in ccRCC metastasis. FOXO3a expression was decreased in primary metastatic ccRCC samples. Patients with low FOXO3a mRNA levels had poor metastasis-free survival (P = 0.003). Knocking down FOXO3a induced tumor cell invasion and migration in the nonmetastatic ccRCC cells. Induced FOXO3a overexpression in SN12-PM6 cells could inhibit tumor metastasis in vivo. Downregulation of FOXO3a increased SNAIL1 expression, thereby activating the epithelial-mesenchymal transition (EMT) of RCC cell lines. CONCLUSIONS: The loss of FOXO3a induced EMT of tumor cells by upregulating SNAIL1, which promoted tumor cells metastasis in vitro and in vivo. Thus, FOXO3a could be considered as an independent prognostic factor in ccRCC metastasis and could be a marker of occult metastases.

Dicer is down-regulated in clear cell renal cell carcinoma and in vitro Dicer knockdown enhances malignant phenotype transformation.

OBJECTIVES: Although emerging evidence has shown that the deregulation of micro-ribonucleic acid (RNA) biogenesis machinery is involved in various human malignancies, this role has not been investigated in clear cell renal cell carcinoma (ccRCC). This study aims to determine whether Dicer, a key enzyme responsible for biogenesis of microRNA, is deregulated in ccRCC. The biological roles of Dicer in vitro are also determined. MATERIALS AND METHODS: The expression of Dicer at messenger RNA and protein levels was detected by real-time quantitative polymerase chain reaction and western blot, respectively, in human kidney tubule epithelial cell line, nonmetastatic 786-O ccRCC cell line, and metastatic ACHN ccRCC cell line, as well as in 42 cases of ccRCC surgical specimens including 14 cases with distant metastasis and their corresponding adjacent normal renal tissues. Dicer expression levels in specimens were also measured by immunohistochemical staining. Knockdown of Dicer expression in 786-O and ACHN ccRCC cell lines was achieved by transfecting short interfering RNA against Dicer. The effects of Dicer on cell proliferation, migration, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, flow cytometric analyses, and Boyden chamber Transwell assay, respectively. RESULTS: Compared with human kidney tubule epithelial cell line, Dicer expression levels were significantly down-regulated in 786-O and ACHN ccRCC cell lines, with the metastatic ACHN ccRCC cell line having even lower levels. Meanwhile, Dicer expression levels were significantly down-regulated in ccRCC surgical specimens compared with adjacent normal renal tissues, with the metastatic ones further reduced, and Dicer messenger RNA levels were significantly correlated with overall tumor-node-metastasis stage of ccRCC. In vitro, the knockdown of Dicer significantly promoted cell proliferation, migration, and invasion. CONCLUSIONS: Reduced expression of Dicer may play a role in the tumorigenesis of ccRCC and further decline may be associated with distant metastasis of ccRCC.

Laparoendoscopic single-site urethrovesical anastomosis training in an economical porcine model.

PURPOSE: To develop an economical animal model for laparoendoscopic single-site surgery (LESS) urethrovesical anastomosis (UVA) training. MATERIALS AND METHODS: A homemade single-port device was used and the uterus cervix and the ileum were chosen to simulate UVA to reduce costs. Ten trainees were randomly divided into two groups: the conventional LESS UVA (CLUVA) group and the transurethral assistant LESS UVA (TALUVA) group. In TALUVA, a laparoscopic forceps was inserted through the urethra to assist operation after the bladder neck was disconnected, whereas CLUVA followed the conventional steps. Anastomosis time and knotting time were recorded, and the learning curves of both groups were analyzed. After training, questionnaires were given to the trainees to assess the difficulties and the satisfaction of the training. RESULTS: The final mean operating time significantly declined in both groups. Except for the first lesson, the trainees in the TALUVA group operated faster than those in the other group. The results from the questionnaires show that all trainees were satisfied with the training, and LESS UVA was considered more difficult in the CLUVA group than in the TALUVA group. CONCLUSIONS: The female porcine model for LESS UVA was feasible and cost-effective. TALVUA could effectively reduce the difficulties involved in LESS UVA.

Overexpression of E2F1 promotes tumor malignancy and correlates with TNM stages in clear cell renal cell carcinoma.

BACKGROUND: Transcription factor E2F1 exerts effects on many types of cancers. As an upstream regulator of a host of genes, E2F1 can trigger diverse aberrant transcription processes that may dominate malignancy. Clear cell renal cell carcinoma (ccRCC) is the most common subtype in renal cell carcinoma which displays high malignancy and has a shortage of biomarkers in clinics. Our study aimed to explore the function of E2F1 in ccRCC and its correlation with clinicopathological parameters. METHODOLOGY/PRINCIPLE FINDINGS: Transcription factor E2F1 was mainly distributed in cancer cell nucleus and mRNA expression significantly increased in 72 cases of clear cell renal cell carcinoma (ccRCC) tissues compared with adjacent non-cancerous kidney tissues (p<0.001). The protein expression was consistent with mRNA expression. Further analysis in 92 cases indicated that E2F1 mRNA level expression was associated with the tumor pathologic parameters embracing diameter, Fuhrman tumor grade, pT stage, TNM stage grouping and macrovascular infiltration (MAVI). These surgical specimens had high grade tumors accompanied with an elevated E2F1 expression. Moreover, E2F1 transfection was found to contribute significantly to cancer cell proliferation, migration and invasion in vitro. CONCLUSIONS/SIGNIFICANCE: Overexpression of E2F1 may be a key event in the local and vascular infiltration of ccRCC indicated by the activation of matrix metalloproteinase (MMP) 2 and MMP9. These findings highlighted the implication of E2F1's function in the metastatic process. Furthermore, the clinical relevance of E2F1 in ccRCC pointed to a potential new therapeutic target.