Circular RNA DHX33 promotes malignant behavior in ccRCC by targeting miR-489-3p/MEK1 axis.

Mounting evidence indicates that circular RNAs modulate the initiation of clear cell renal cell carcinoma (ccRCC). However, their specific roles in the malignancy of ccRCC is understudied. Here, we present a novel circular RNA, circDHX33, that is up-regulated in ccRCC cell lines and tissues. Upregulated circDHX33 in ccRCC patients significantly correlates with advanced TNM stage and metastasis. Suppressing circDHX33 expression inhibits the proliferation and invasion of cultured cells, and suppresses tumor growth in vivo. Mechanistically, we show that circDHX33 promotes ccRCC progression by sponging miR-489-3p and modulating MEK1 expression. In conclusion, our findings suggest that circDHX33 plays a role in promoting ccRCC via the miR-489-3p/MEK1 axis and may serve as a novel therapeutic target for the treatment of ccRCC patients.

circPRRC2A promotes angiogenesis and metastasis through epithelial-mesenchymal transition and upregulates TRPM3 in renal cell carcinoma.

Background: Circular RNAs (circRNAs) have been identified as essential regulators in a plethora of cancers. Nonetheless, the mechanistic functions of circRNAs in Renal Cell Carcinoma (RCC) remain largely unknown. Methods: In this study, we aimed to identify novel circRNAs that regulate RCC epithelial-mesenchymal transition (EMT), and to subsequently determine their regulatory mechanisms and clinical significance. Results: circPRRC2A was identified by circRNA microarray and validated by qRT-PCR. The role of circPRRC2A in RCC metastasis was evaluated both in vitro and in vivo. We found that increased expression of circPRRC2A is positively associated with advanced clinical stage and worse survivorship in RCC patients. Mechanistically, our results indicate that circPRRC2A prevents the degradation of TRPM3, a tissue-specific oncogene, mRNA by sponging miR-514a-5p and miR-6776-5p. Moreover, circPRRC2A promotes tumor EMT and aggressiveness in patients with RCC. Conclusions: These findings infer the exciting possibility that circPRRC2A may be exploited as a therapeutic and prognostic target for RCC patients.

HOXA11-AS promotes the growth and invasion of renal cancer by sponging miR-146b-5p to upregulate MMP16 expression.

Recently, increasing studies showed that long noncoding RNAs (lncRNAs) play critical roles in tumor progression. However, the function and underlying mechanism of HOMEOBOX A11 antisense RNA (HOXA11-AS) on renal cancer remain unclear. In the current study, our data showed that the expression of HOXA11-AS was significantly upregulated in clear cell renal cell carcinoma (ccRCC) tissues and cell lines. High HOXA11-AS expression was associated with the advanced clinical stage, tumor stage, and lymph node metastasis. Function assays showed that HOXA11-AS inhibition significantly suppressed renal cancer cells growth, invasion, and ETM phenotype. In addition, underlying mechanism revealed that HOXA11-AS could act as a competing endogenous RNA (ceRNA) that repressed miR-146b-5p expression, which regulated its downstream target MMP16 in renal cancer. Taken together, our findings suggested that HOXA11-AS could promote renal cancer cells growth and invasion by modulating miR-146b-5p-MMP16 axis. Thus, our findings suggested that HOXA11-AS could serve as potential therapeutic target for the treatment of renal cancer.

Decreased expression of long non-coding RNA NBAT-1 is associated with poor prognosis in patients with clear cell renal cell carcinoma.

OBJECTIVE: accumulating evidence suggest that long non-coding RNAs (lncRNAs) may play important roles in human cancers. LncRNA neuroblastoma associated transcript-1 (NBAT-1) was initially identified to be involved in the progression of neuroblastoma. However, there is no report about the role of NBAT-1 in clear cell renal cell carcinoma (ccRCC). The purpose of this study is to investigate the clinical significant of NBAT-1 in ccRCC. METHODS: the expression pattern of NBAT-1 in ccRCC patients and renal cancer cell lines was detected by using quantitative real-time PCR (qRT-PCR), and its correlation with clinicopathologic features and prognosis of patients with ccRCC was assessed by Kaplan-Meier method and Cox proportional hazards model, respectively. Small interfering RNA (siRNA) was transfected into 786-O and ACHN cells to determine the effect of NBAT-1 knockdown on renal cancer cells. RESULT: NBAT-1 expression is significantly decreased in ccRCC tissues and renal cancer cells compared with adjacent normal tissues and normal human proximal tubule epithelial cell line HK-2, and its low level is associated with advanced features and poor prognosis. Also, multivariate analysis identified NBAT-1 expression as an independent prognostic factor for ccRCC. In vitro assays indicated that knockdown of NBAT-1 expression increased renal cancer cell proliferation, migration and invasion. CONCLUSIONS: NBAT-1 is a novel molecular correlated with ccRCC progression; and it may represent a prognostic biomarker and therapeutic target in renal cancer diagnosis and treatment.

MiR-506 is down-regulated in clear cell renal cell carcinoma and inhibits cell growth and metastasis via targeting FLOT1.

BACKGROUND: Some microRNAs (miRNAs) are abnormally expressed in cancer and contribute to tumorigenesis. In the present study, we investigated the role of miR-506 in clear cell renal cell carcinoma (ccRCC). METHODS: miR-506 expression was detected in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 and ccRCC specimens by quantitative real-time-PCR. We assessed the association of miR-506 expression with pathology and prognosis in ccRCC patients. We over-expressed and knocked-down miR-506 expression in two renal cancer cell lines, 786-O and ACHN, and assessed the impact on cell proliferation, migration and invasion. A luciferase reporter assay was conducted to confirm the target gene of miR-506 in renal cancer cell lines. RESULTS: miR-506 was significantly down-regulated in renal cancer cell lines and ccRCC specimens. Low miR-506 expression in ccRCC specimens was associated with an advanced clinical stage and poor prognosis. miR-506 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Over-expression of miR-506 in renal cancer cells decreased cell growth and metastasis, In contrast, down-regulation of miR-506 expression promoted renal cancer cell growth and metastasis. FLOT1, a potential target gene of miR-506, was inversely correlated with miR-506 expression in ccRCC tissues. Consistent with the effect of miR-506, knockdown of FLOT1 by siRNA inhibited cell malignant behaviors. Rescue of FLOT1 expression partially restored the effects of miR-506. CONCLUSIONS: miR-506 exerts its anti-cancer function by directly targeting FLOT1 in renal cancer, indicating a potential novel therapeutic role in renal cancer treatment.

Combination of quercetin and hyperoside inhibits prostate cancer cell growth and metastasis via regulation of microRNA­21.

Previous studies have reported that hyperoside and quercetin in combination (QH; 1:1) inhibited the growth of human leukemia cells. The aim of the present study was to investigate the anti­cancer effect of QH on prostate cancer cells. The results demonstrated that QH decreased the production of reactive oxygen species (ROS) and increased antioxidant capacity in PC3 cells at various concentrations (2.5­60 µg/ml) with peak inhibition and augmentation changes of 3.22­ and 3.00­fold, respectively. Following treatment with QH for 48 and 72 h, the IC50-values on PC3 cells were 19.7 and 12.4 µg/ml, respectively. Western blot analysis revealed that QH induced apoptosis in human prostate cancer cells via activation of caspase­3 and cleavage of poly(adenosine diphosphate ribose) polymerase. In addition, QH significantly inhibited the invasion and migration of PC3 cells as well as reduced the expression of numerous prostate tumor­associated microRNAs (miRs), including miR­21, compared to that of untreated human prostate cancer cells. QH was also found to enhance the expression of tumor suppressor programmed cell death protein 4, which was negatively regulated by miR­21. Furthermore, induced overexpression of miR­21 using pre­miR­21 oligonucleotides attenuated the beneficial effect of QH on prostate cancer cells. In conclusion, the results of the present study indicated that QH exerted an anti­cancer effect on human prostate cancer cells, the mechanism of which proceeded, at least in part, via the inhibition of the miR­21 signaling pathway.

Upregulation of long non-coding RNA MALAT1 correlates with tumor progression and poor prognosis in clear cell renal cell carcinoma.

Long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of cellular processes, such as cell growth, apoptosis, and carcinogenesis. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has recently been identified to be involved in tumorigenesis of several cancers such as lung cancer, pancreatic cancer, and cervical cancer. However, the role of lncRNA MALAT1 in clear cell renal cell carcinoma (ccRCC) remains unclear. Expression levels of lncRNA MALAT1 in ccRCC tissues and renal cancer cell lines were evaluated by quantitative real-time PCR (qRT-PCR), and its association with overall survival of patients was analyzed by statistical analysis. Small interfering RNA (siRNA) was used to suppress MALAT1 expression in renal cancer cells. In vitro assays were conducted to further explore its role in tumor progression. The expression level of MALAT1 was higher in ccRCC tissues and renal cancer cells compared to adjacent non-tumor tissues and normal human proximal tubule epithelial cells HK-2. The ccRCC patients with higher MALAT1 expression had an advanced clinical features and a shorter overall survival time than those with lower MALAT1 expression. And multivariate analysis showed that the status of MALAT1 expression was an independent predictor of overall survival in ccRCC. Additionally, our data indicated that knockdown expression of MALAT1 decreased renal cancer cell proliferation, migration, and invasion. Our data suggested that lncRNA MALAT1 was a novel molecule involved in ccRCC progression, which provided a potential prognostic biomarker and therapeutic target.

Metformin inhibits cell growth by upregulating microRNA-26a in renal cancer cells.

Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties and may reduce cancer risk and improve prognosis. However, the mechanism by which metformin affects various cancers, including renal cancer still unknown. MiR-26a induces cell growth, cell cycle and cell apoptosis progression via direct targeting of Bcl-2, clyclin D1 and PTEN in cancer cells. In the present study, we used 786-O human renal cancer cell lines to study the effects and mechanisms of metformin. Metformin treatment inhibited RCC cells proliferation by increasing expression of miR-26a in 786-O cells (P < 0.05). As a result, protein abundance of Bcl-2 and cyclin D1 was decreased and PTEN was increased in cells exposed to metformin. Also over-expression of miR-26a can inhibited cell proliferation by down-regulating Bcl-2, cyclin D1 and up-regulating PTEN expression. Therefore, these data for the first time provide novel evidence for a mechanism that the anticancer activities of metformin are due to upregulation of miR-26a and affect its downstream target gene.

High expression of long non-coding RNA SPRY4-IT1 predicts poor prognosis of clear cell renal cell carcinoma.

INTRODUCTION: Long non-coding RNAs (lncRNAs) play a key role in cellular processes, such as cell growth, apoptosis, and carcinogenesis. lncRNAs SPRY4-IT1 has recently been identified to be involved in tumorigenesis of several cancers such as non-small cell lung cancer and esophageal squamous cell carcinoma. However, the role of SPRY4-IT1 in clear cell renal cell carcinoma (ccRCC) remains unclear. METHODS: The expression of SPRY4-IT1 was examined in ccRCC patients and renal cancer cell lines by using quantitative real-time PCR (qRT-PCR). The relationship between SPRY4-IT1 level and clinicopathological parameters of ccRCC was analyzed with the Kaplan-Meier method and Cox proportional hazards model. Small interfering RNA (siRNA) was used to suppress SPRY4-IT1 expression in renal cancer cell line 786-O. In vitro assays were performed to further explore its role in renal cancer progressio. RESULTS: The relative level of SPRY4-IT1 was significantly higher in ccRCC tissues compared to the adjacent normal renal tissues. And higher expression of SPRY4-IT1 was found in renal cancer cell lines compared with the normal human proximal tubule epithelial cell line HK-2. The ccRCC patients with higher SPRY4-IT1 expression had an advanced clinical stage and poorer prognosis than those with lower SPRY4-IT1 expression. Multivariate analyses by Cox's proportional hazard model revealed that expression of SPRY4-IT1 was an independent prognostic factor in ccRCC. In vitro assays, our results indicated that knockdown of SPRY4-IT1 reduced renal cancer cell proliferation, migration, and invasion. CONCLUSIONS: Our data suggested that lncRNA SPRY4-IT1 might be considered as a potential prognostic indicator and a potential target for therapeutic intervention in RC.

Bromodomain 4 protein is a predictor of survival for urothelial carcinoma of bladder.

BACKGROUND: Bromodomain 4 (BRD4) protein is a double bromodomain-containing protein that binds preferentially to acetylated chromatins. BRD4 is essential for cellular growth and has been implicated in cell cycle control, DNA replication and carcinogenesis. However, its expression profile and prognostic value in urothelial carcinoma of the bladder (UCB) have not been investigated. METHODS: Real-time quantitative PCR (qRT-PCR) and Western blot were used to explore BRD4 expression in UCBs and normal bladder tissues. Moreover immunohistochemistry (ICH) was used to detect the expression of BRD4 in UCBs. Spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data. RESULTS: Up-regulated expression of BRD4 mRNA and protein was observed in the majority of UCBs by qRT-PCR and Western blot when compared with their paired normal bladder tissues. Clinicopathological analysis was showed a significant correlation existed between the higher expression of BRD4 protein with the histological grade, lymph node metastasis and distant metastasis (P < 0.05); Survival analysis by Kaplan-Meier survival curve and log-rank test demonstrated that elevated BRD4 expression in bladder cancer tissue predicted poorer overall survival (OS) compared with group in lower expression. Notably, multivariate analyses by Cox's proportional hazard model revealed that expression of BRD4 was an independent prognostic factor in UCB. CONCLUSIONS: These results suggest that the aberrant expression of BRD4 in human UCB is possibly involved in the tumorigenesis and development, and the BRD4 protein could act as a potential biomarker for prognosis assessment of bladder cancer. Further studies on the cellular functions of BRD4 need to address these issues.

Up-regulation of flotillin-2 is associated with renal cell carcinoma progression.

Flotillin-2 (FLOT2) is a highly conserved protein isolated from caveolae/lipid raft domains that tether growth factor receptors linked to signal transduction pathway. FLOT2 has recently been identified to be involved in tumorigenesis of several cancers such as breast cancer, melanoma, and gastric cancer. However, the role of FLOT2 in renal cell carcinoma (RCC) remains unclear. The expression levels of FLOT2 in RCC patients and renal cancer cell lines were evaluated by quantitative real-time PCR (qRT-PCR) and Western blot. FLOT2 protein expression was also analyzed in archived paraffin-embedded RCC tissues using immunohistochemistry (IHC), and its association with overall survival of patients was analyzed by statistical analysis. Small-interfering RNA (siRNA) was used to suppress FLOT2 expression in RCC cell lines. In vitro assays were performed to further explore its role in tumor progression. The expression level of FLOT2 was higher in RCC tissues and cell lines than in corresponding adjacent normal tissues and normal human proximal tubule epithelial cell line HK-2. IHC analysis revealed high expression levels of FLOT2 in RCC specimens. The RCC patients with higher FLOT2 expression had an advanced clinical stage and poorer prognosis than those with lower FLOT2 expression. FLOT2 expression was an independent prognostic marker of overall RCC patient survival in a multivariate analysis. In vitro assays indicated that knockdown of FLOT2 reduced cell proliferation, migration, and invasion. Our data suggest that FLOT2 is a novel molecule involved in RCC progression, which provide a potential prognostic biomarker and therapeutic target.

VPA inhibits renal cancer cell migration by targeting HDAC2 and down-regulating HIF-1α.

Cell migration plays major roles in human renal cancer-related death, but the molecular mechanisms remain unclear. Valproic acid (VPA) is a broad-spectrum inhibitor of class I and II histone deacetylases and shows great anticancer activity in a variety of human cancers. In this study, we found that VPA significantly inhibited cell migration but not proliferation of human renal cancer ACHN cells. Mechanistic studies found that VPA significantly inhibited the expression of HIF-1α. Knockdown of HIF-1α could obviously inhibited cell migration, while over-expression of HIF-1α markedly rescued the inhibition of VPA on cell migration. Further studies found that knockdown of HDAC2 completely mimicked the effects of VPA on HIF-1α and cell migration, and over-expression of HIF-1α could also rescue the effects of HDAC2 knockdown on cell migration. Collectively, these results indicated that the potential of specific inhibition of HDAC2 by small molecular chemicals may lead to future therapeutic agents in human renal cancer treatment.

Foxl1 inhibits tumor invasion and predicts outcome in human renal cancer.

UNLABELLED: The Forkhead Box L1 (Foxl1) transcription factor regulates epithelial proliferation and development of gastrointestinal tract, and has been implicated in gastrointestinal and pancreatic tumorigenesis. However, the role of Foxl1 in renal cancer development and progression remains to be elucidated. The study was conducted to investigate the expression of Foxl1 and its prognostic significance in clear cell renal cell carcinoma (ccRCC). Meanwhile, the function of Foxl1 in human ccRCC was further investigated in cell culture models. METHODS: Real-time quantitative PCR, western-blot, immunohistochemistry were used to explore Foxl1 expression in primary ccRCC clinical specimens and ccRCC cell lines. Foxl1 expression was up-regulated by over-expression vector in 786-O and ACHN cells, proliferation, cell cycle, migration and invasion were assayed. RESULTS: Foxl1 expression was down-regulated in the majority of the ccRCC clinical tissue specimens at both mRNA and protein levels. Clinic pathological analysis showed that Foxl1 expression was significantly correlated with tumor stage, lymph node metastasis, distant metastasis, clinical TNM stage (cTNM) and histological grade of renal cancer. The Kaplan-Meier survival curves revealed that low Foxl1 expression was associated with poor prognosis in ccRCC patients. Foxl1 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Mechanistic analyses demonstrated that over-expression of Foxl1 inhibits tumor cell growth, migration and invasion in renal cancer cells. CONCLUSIONS: These results suggest that Foxl1 expression is a candidate predictor of clinical outcome in patients with resected ccRCC and it plays an inhibitory role in renal tumor progression.

Argonaute 2 is up-regulated in tissues of urothelial carcinoma of bladder.

UNLABELLED: Argonaute 2 proteins (Ago2) have been demonstrated to be widely expressed and involved in post-transcriptional gene silencing and play key roles in carcinogenesis. However, its expression profile and prognostic value in urothelial carcinoma of the bladder (UCB) have not been investigated. METHODS: Real-time quantitative PCR (qRT-PCR) and Western blot were used to explore Ago2 expression in UCBs and normal bladder tissues. Moreover immunohistochemistry (ICH) was used to detect the expression of Ago2 in UCBs. Spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data. RESULTS: Up-regulated expression of Ago2 mRNA and protein was observed in the majority of UCBs by qRT-PCR and Western blot when compared with their paired normal bladder tissues. Clinic pathological analysis was showed a significant correlation existed between the higher expression of Ago2 protein with the Histological grade, lymph node metastasis and Distant metastasis (P<0.05); Survival analysis by Kaplan-Meier survival curve and log-rank test demonstrated that elevated Ago2 expression in cancer tissue predicted poorer overall survival (OS) compared with group in lower expression (62.2% VS 86.3%, P<0.05). Notably, multivariate analyses by Cox's proportional hazard model revealed that expression of Ago2 was an independent prognostic factor in UCB. CONCLUSIONS: These results suggest that the aberrant expression of Ago2 in human UCB is possibly involved with tumorigenesis and development, and the Ago2 protein could act as a potential biomarker for prognosis assessment of bladder cancer. Further studies on the cellular functions of Ago2 need to address these issues.

Epigallocatechin gallate attenuates interstitial cystitis in human bladder urothelium cells by modulating purinergic receptors.

BACKGROUND: Epigallocatechin gallate (EGCG) has exhibited antitumor properties against bladder cancer. However, its effects in interstitial cystitis (IC) have not been investigated. METHODS: Here, we performed repeated cystoscopy and re-biopsy of bladder mucosa before and after intravesical irrigation of EGCG in eight patients diagnosed with IC based on clinical and histopathologic assessments. Six normal bladder tissue samples were obtained from age-, race-, and sex-matched asymptomatic control subjects. IC symptom index was used to compare the therapeutic effect in IC patients. Patient-derived bladder epithelial cells were cultured and cell stretch experiments and ATP assays were performed. The expression of purinergic receptors X1, X2, and X3, and Y1, Y2, and Y11, in biopsied samples was detected by Western blotting and real-time polymerase chain reaction, respectively. Moreover, the expression of inducible NO synthase, phosphorylated Akt, and phosphorylated NF-κB was also assessed. RESULTS: All EGCG-treated patients demonstrated different extents of remission of symptoms. We found a significant upregulation in P2X1, P2X2, and P2X3 receptor proteins and P2Y1, P2Y2, and P2Y11 receptor transcripts in IC patients. However, EGCG therapy attenuated the expression of all purinergic receptors. In addition, EGCG demonstrated prominent antioxidative and antiinflammatory effects via inhibition of the upregulation of iNOS and phosphorylated NF-κB. Furthermore, the stretch-activated release of ATP in cultured bladder urothelial cells was greater in cells derived from IC patients, compared with those from the control patients, but EGCG, at all concentrations tested, effectively abolished the increase in ATP release from stretched IC patient-derived cells. CONCLUSIONS: Our study suggests that inhibition of the expression of purinergic receptors and ATP release in urothelial cells by EGCG supports further development of EGCG as a novel therapeutic option for IC.