Circular RNA circC3P1 restrains kidney cancer cell activity by regulating miR-21/PTEN axis and inactivating PI3K/AKT and NF- kB pathways.

Kidney cancer (KC) seriously impacts public health. We detected the function and mechanism of circular RNA C3P1 (circC3P1) in KC cells. CCK-8, flow cytometry, migration, and invasion assay were respectively used to investigate the efficacies of circC3P1 and microRNA (miR)-21 on cell viability, apoptosis, migration, and invasion. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), circC3P1, and miR-21 expression were changed by cell transfection and detected by quantitative reverse-transcription polymerase chain reaction. Moreover, the apoptosis/pathways-related proteins and proteins were detected by western blot analysis. Besides, the relation between PTEN and miR-21 was detected by luciferase assay. circC3P1 and PTEN were downregulated while miR-21 was upregulated in KC tissues. circC3P1 declined cell viability, migration, and invasion and caused apoptosis. Furthermore, circC3P1 negatively regulated miR-21; miR-21 mimic could reverse the efficacies of circC3P1. Besides, circC3P1 restrained the PI3K/AKT and NF-κB pathways by downregulating miR-21. Finally, PTEN was authenticated as a target of miR-21. circC3P1 restrained KC cell growth, migration, and invasion by regulating miR-21/PTEN axis and inactivating PI3K/AKT and NF-κB signaling pathways.

Circular RNA circFNDC3B protects renal carcinoma by miR-99a downregulation.

Various circular RNAs (circRNAs) have been reported to involve in carcinoma. This study explored the role and mechanism of circRNA circFNDC3B (circFNDC3B) in renal carcinoma (RC). The detection indicators in this paper were viability, colony, and migration, which respectively investigated by Cell Counting Kit-8, colony formation, and migration assay. Reverse transcriptase quantitative polymerase chain reaction tested and cell transfection changed circFNDC3B and miR-99a expression. Moreover, western blot tested relate-proteins of proliferation, migration, and cell pathways were examined by western blot. circFNDC3B was upregulated at RC tissues. circFNDC3B enhanced cell viability, colony and migration, and miR-99a mimic played reverse impacts. Furthermore, circFNDC3B negatively regulated miR-99aand circFNDC3B restrained the janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3) and extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathways by miR-99a downregulation. Overexpression of circFNDC3B enhanced cell viability, colony formation and migration by miR-99a downregulation via JAK1/STAT3 and MEK/ERK pathways.

Clusterin promotes growth and invasion of clear cell renal carcinoma cell by upregulation of S100A4 expression.

BACKGROUND AND OBJECTIVE: Clusterin promotes cell proliferation, motility and invasiveness in human renal cell carcinoma (RCC) cells but the underlying molecular mechanisms of this action are largely unknown. The aim of this study was to investigate the effects of clusterin on cancer cell growth, invasion and S100A4 expression and to determine the effects of clusterin on in vitro cell proliferation and migration and in vivo tumour growth in RCC cells. METHODS: We have established stable transfectants of highly invasive Caki-1 human RCC cells with expression of clusterin shRNA targeting clusterin (Caki-1/clusterin shRNA). We also established stable transfectants of 786-O human RCC cells with expression of clusterin cDNA plaismid (786-O/clusterin cDNA). Clusterin and S100A4 expression was detected by reverse transcription (RT) PCR and western blot assay; Caki-1/clusterin shRNA and 786-O/clusterin cDNA clones were subjected to in vitro-invasion assays. Cell viability and cell growth was assessed in MTT and clonogenic assay. Specific small interfering RNA was employed to down-regulate S100A4. The expression plasmid for S100A4 (pCMV-S100A4) was used to upregulate S100A4. Caki-1/clusterin shRNA clones were injected subcutaneously in nude mice to determine tumour growth and cancer cell invasiveness in vivo. Xenograft tumour tissues were assessed by immunohistochemistry and frozen tissues were used for the detection of S100A4 and clusterin. RESULTS: Overexpression of clusterin increased cell invasiveness; and targeting clusterin reduced cell invasiveness in vitro. This increase in cell invasiveness was mediated by S100A4. Targeting clusterin decreased cell proliferation and down-regulated cellular S100A4 levels in Caki-1 cells; Overexpression of clusterin increased cell proliferation and up-regulated cellular S100A4 levels in 786-O cells; Stable Caki-1/clusterin shRNA transfectants produced smaller xenograft tumours containing reduced S100A4 protein levels in vivo. Stable 786-O/clusterin cDNA transfectants produced larger xenograft tumours containing increased S100A4 protein levels in vivo. CONCLUSION: Our results indicate that clusterin promotes growth and invasion in RCC cells in vitro and in vivo through upregulation of S100A4; And targeting clusterin confers growth inhibitory and anti-invasive properties in RCC cells in vitro and in vivo through a down-regulation of S100A4. These findings provide the rationale for future oncostatic strategies aimed at suppressing clusterin-mediated signal transduction pathways as a novel therapeutic approach in human RCC.

T-cell immunoglobulin mucin-3 expression in bladder urothelial carcinoma: Clinicopathologic correlations and association with survival.

BACKGROUND: T cell immunoglobulin mucin-3 (Tim-3) was initially recognized as a pivotal immune checkpoint inhibitor that maintains immune homeostasis and tolerance. Recently, Tim-3 has been demonstrated to play an important role in tumor-associated immune suppression and aberrant Tim-3 expression has been reported in several human malignancies. However, the role of Tim-3 in bladder urothelial carcinoma (BUC) remains largely unknown. The present study aims to investigate Tim-3 expression in BUC and analyze correlations with clinicopathologic outcomes and postoperative survival. METHODS: Tim-3 protein expressions were detected in paraffin embedded sections from 100 patients with BUC by immunohistochemistry. Expressions were correlated with clinicopathologic outcomes and postoperative survival. RESULTS: Tim-3 protein was over-expressed in bladder cancer cells, tumor infiltrating lymphocytes and endothelial cells from patients with BUC. The expression levels of Tim-3 were significantly correlated with advanced pathological grade and T stage. Moreover, another immune checkpoint molecule programmed death receptor-1(PD-1) was also over- expressed in BUC tissues and had a significant correlation with Tim-3. Multivariate analysis showed that Tim-3 expression, as well as PD-1 expression was both independent predictors of disease-free survival and overall survival in patients with BUC. CONCLUSION: Tim-3 over-expression implies adverse clinical outcomes for BUC, which suggests it is a potential prognostic biomarker and a novel therapeutic target in BUC.

B7-H1/PD-1 blockade therapy in urological malignancies: current status and future prospects.

The stimulatory and inhibitory coreceptors expressed by T lymphocytes are known to play critical roles in regulating cancer immunity. An array of inhibitory coreceptors involved in the inhibition of T-cell functions and the blockade of immune activation have been discovered in recent years, the most important of which are cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmmed death-1 (PD-1), and B7 homolog 1 (B7-H1). Immunotherapies targeting T-cell coinhibitory molecules have proved to be effective in cancer treatment. Several kinds of monoclonal antibodies have been tested in preclinical studies, with better outcomes than conventional therapies in many malignancies. Common urological malignancies including renal cell carcinoma, bladder cancer and prostate cancer are supposed to be immunogenic cancer types and not so sensitive to conventional therapies as other malignancies. This review will focus on B7-H1/PD-1 blockade therapy in urological malignancies, summarizing the results of clinical trials as well as the challenges and prospects of this emerging immunotherapy.

Recombinant bacillus Calmette-Guérin in urothelial bladder cancer immunotherapy: current strategies.

Bacillus Calmette-Guérin (BCG) has been used in the intravesical treatment of urothelial bladder cancer (UBC) for three decades. Despite its efficacy, intravesical BCG therapy is associated with some limitations such as side effects and BCG failure, which have inspired multiple ways to improve it. Recent advances have focused on recombinant BCG (rBCG) which provides a novel tactic for modification of BCG. To date, a number of rBCG strains have been developed and demonstrated to encourage efficacy and safety in preclinical and clinical studies. This review summarizes current rBCG strategies, concerns and future directions in UBC immunotherapy with an intention to encourage further research and eventually to inform clinical decisions.

Clusterin plays an important role in clear renal cell cancer metastasis.

OBJECTIVE: Clusterin (CLU) is implicated in regulating clear renal cell carcinoma (CRCC) progression and metastasis, yet the mechanisms are not elucidated. In the present study, we explored the potential role of CLU in CRCC metastasis. METHODS: Levels of CLU mRNA and CLU protein were measured by RT-PCR and immunohistochemistry analysis in 22 CRCC with metastasis and 22 without metastasis and 22 samples of normal kidney tissue. After CLU silencing and re-expression, the migration and invasion in vitro and in vivo of Caki-2 cells were determined by wound healing assay, transwell migration assay and pulmonary nodule assay, respectively. The expression of pERK1/2 and MMP-9 were detected by RT-PCR and Western blot assay. RESULTS: We found a significant increase of CLU and CLU mRNA expression in CRCC, and the expression of CLU is strongly correlated in patients with metastatic disease. We discovered that CLU-rich Caki-2 cells displayed higher invasive ability which prompted us to investigate if CLU silencing could reduce the migration and invasion in Caki-2 cells. Compared with the vector-transfected cells, CLU knocked-down (CLUi) cells showed reduced migration and invasion in vitro, as well as decreased metastatic potential in experimental metastasis. Re-expression of CLU in CLUi cells restored the invasive phenotypes. We found that MMP-9 was downregulated in CLUi cells. We also discovered that levels of activated ERK1/2 correlated with the rich expression of CLU and MMP-9. CONCLUSION: Our data suggest that CLU may regulate aggressive behavior of human CRCC cells through modulating ERK1/2 signaling and MMP-9 expression.

Expression of transcription factors snail, slug, and twist in human bladder carcinoma.

BACKGROUND: Slug, snail, and twist are transcription factors that regulate the expression of tumor suppressors such as E-cadherin. In this study, we aimed to examine the expression of these transcription factors in human bladder carcinoma. METHODS: We first investigated expression of slug, snail, twist and E-cadherin in five bladder Carcinoma cell lines by reverse transcription-polymerase chain reaction and western blotting. Furthermore, we investigated slug, snail, and twist and E-cadherin expression by immunohistochemistry with bladder carcinoma (tumor, n = 120; background, n = 42). RESULTS: Expression of slug mRNA and protein was detected in all cell lines, twist was clearly expressed in two out of five bladder carcinoma cell lines, snail was not expressed, and E-cadherin was detected in 3 cell lines. 44.2% (53/120) of human bladder carcinoma tissues and 38% (16/42) background tissue showed an expression of twist; 62.5% (75/120) of human bladder carcinoma tissues and 40% (17/42) background tissue showed an expression of slug, 15.8% (19/120) of human bladder carcinoma tissues and 76% (32/42) background tissue showed an expression of snail, and 25.8% (31/120) cases were negative for E-cadherin expression in carcinoma tissues. Expression of slug and twist shows increased levels in tumors, whereas snail seems reduced. Statistically significant correlations were found between twist, slug, and E-cadherin expression. Immunohistochemistry analysis showed that twist was elevated with increasing tumor stage (P = 0.001), the grade (P < 0.001), the progression (P = 0.035). Slug was elevated and snail was reduced with increasing nodal involvement (tumor-node-metastasis status) (P = 0.004, P = 0.01). E-cadherin was reduced in expression corresponding with tumor grade (P < 0.01). Positive twist, slug and E-cadherin expression clearly predicted poorer PFS (P = 0.042, P = 0.014, P = 0.001). In the multivariate analysis, only snail and E-cadherin expression were independent prognostic factors for OS (P = 0.002, P < 0.001). CONCLUSIONS: These data demonstrate that twist, snail and slug have inappropriate expression in bladder carcinoma and that this may play a part in the progression of human bladder carcinoma.

The E-cadherin repressor slug and progression of human extrahepatic hilar cholangiocarcinoma.

OBJECTIVES: This study explored the expression and function of Slug in human extrahepatic hilar cholangiocarcinoma (EHC) to identify its role in tumor progression. METHODS: The expression of Snail and Slug mRNA in 52 human tissue samples of EHC was investigated. The mRNA of Snail and Slug were quantified using reverse transcriptase-PCR, and correlations with E-cadherin expression and clinicopathological factors were investigated. We then investigated transfection of Slug cDNA in endogenous E-cadherin-positive human EHC FRH0201 cells, selectively induced the loss of E-cadherin protein expression, and then small interfering RNA (siRNA) for inhibition of Slug expression in endogenous Slug-positive human EHC QBC939 cells, selectively induced the loss of Slug protein expression. A Boyden chamber transwell assay was used for invasion. RESULTS: Slug mRNA was overexpressed in 18 cases (34.6%) of EHC compared with adjacent noncancerous tissue. E-Cadherin protein expression determined in the same 52 cases by immunohistochemistry was significantly down-regulated in those cases with Slug mRNA overexpression (P = 0.0001). The tumor and nontumor ratio of Slug mRNA was correlated with nodal metastasis(p = 0.0102), distant metastasis (p = 0.0001)and Survival time(p = 0.0443). However, Snail mRNA correlated with neither E-cadherin expression nor tumor invasiveness. By inhibiting Slug expression by RNA interference, we found that reduced Slug levels upregulated E-cadherin and decreased invasion in QBC939 cell. When the QBC939 cells was infected with Slug cDNA,, significant E-cadherin was downregulated and increased invasion in QBC939 cell. CONCLUSIONS: The results suggested that Slug expression plays an important role in both the regulation of E-cadherin expression and in the acquisition of invasive potential in human EHC. Slug is possibly a potential target for an antitumor therapy blocking the functions of invasion and metastasis in human EHCs.

Shotgun proteome expression profile of microdissected superficial transitional cell carcinoma and biomarker discovery from urine.

The aim of this study was to globally characterize the expression profile of superficial transitional cell carcinoma using shotgun proteome strategy and to discuss the biomarker panel identified from urine. We identified 440 commonly expressed proteins from four samples of superficial transitional cell carcinoma using laser capture microdissection coupled with two-dimensional liquid chromatography tandem mass spectrometry. The identified proteins were further analyzed using bioinformatics tools and compared with the published literature. Compared with the entire list of the International Protein Index, there were 41/22 Gene ontology (GO) terms found to be enriched/depleted within the biological process annotation. GO biological process enrichment/depletion analysis was consistent with the results of urine proteome analysis. Proteins classified under the terms cell adhesion, cell proliferation and cell differentiation are good candidate biomarkers for cancer detection from urine. In conclusion, the present study identified an extensive expression profile in superficial transitional cell carcinoma of the bladder, providing information for the understanding of cancer cell biology and the discovery of a biomarker panel from urine.

[Effect of astrgaloside IV on the long-term consequences of renal ischemia-reperfusion injury in rat].

OBJECTIVE: To observe the effect of astrgaloside IV (Astr) on the long-term consequences of renal ischemia-reperfusion injury (IRI) in rat. METHODS: Fifty-four male Sprague-Dawley rats were randomized into 3 equal groups: IRI group, Astr group, and sham operation group. All rats underwent right nephrectomy and isolation of the left renal artery. The left renal arteries of the IRI group and Astr group were gripped by vascular clamp for 60 minutes and that of the sham operation group was only isolated without gripping. Two milliliters of Astr solution (0.1 g/L) was perfused into the stomach of the rats in the Astr group three days before and after the operation respectively. The rats in the IRI and sham operation groups were perfused with normal saline of the same volume. Four, twelve, and twenty-four weeks after the operation 24-hour urine specimens of the rats were collected to detect the urine protein. At each time point 6 rats from each group were anesthetized and blood was collected from the abdominal aorta to measure the level of serum creatinine (Cr), their left kidneys were taken out to undergo pathological examination and extraction of mRNA. Histochemistry was used to detect the expression of tumor growth factor (TGF)-beta1 protein in the renal tissues. RT-PCR was used to detect the expression of TGF-beta1 mRNA. Collagen staining and immunohistochemistry were used to measure the proportion of collagen positive material to the total area. RESULTS: The level of urine protein was increased progressively, those 12 and 24 weeks after the operation in the IRI group were significantly higher than those in the Astr and sham operation groups (all P < 0.05). The serum Cr 4 weeks after the operation was 36 micromol/L +/- 4 micromol/L, significantly higher than those in the Astr and sham operation groups (31 micromol/L +/- 8 micromol/L and 31 micromol/L +/- 5 micromol/L), and the serum Cr levels 4 weeks 12 and 24 weeks after the operation in the IRI group remained significantly higher than those in the Astr and sham operation groups (all P < 0.05). Collagen staining showed that the glomerular basement membrane, tunica adventitia vasorum, and adventitia of renal tubule were remarkably redder in the IRI than in the Astr and sham operation groups. The expression of TGF-beta1 protein was progressively increased since 12 weeks after the operation in the IRI group, significantly stronger in the Astr and sham operation groups. The expression of TGF-beta1 mRNA was progressively increased since 12 weeks after the operation in the IRI and Astr groups, significantly stronger than that in the sham operation group (P < 0.05). However, the expression of TGF-beta1 mRNA 24 weeks after the operation was significantly stronger in the IRI group than in the Astr group (P < 0.05). CONCLUSION: After renal IRI the probability of development of renal fibrosis increases. Astrgaloside IV markedly ameliorates renal injury by downregulating the TGF-beta1 expression.