Circular RNA circARHGEF28 inhibited the progression of prostate cancer via the miR-671-5p/LGALS3BP/NF-κB axis.

Circular RNAs (circRNAs) play crucial roles in various biological processes, including prostate cancer (PCa). However, the precise roles and mechanism of circRNAs are complicated. Hence, we studied the function of a circRNA that might be involved in the progression of PCa. In this study, we found that circARHGEF28 was frequently downregulated in PCa tissues and cell lines. Furthermore, gain- and loss-of function experiments in vitro showed that circARHGEF28 inhibited proliferation, migration, and invasion of PCa. Additionally, circARHGEF28 suppressed PCa progression in vivo. Bioinformatics analysis and RNA pull-down and capture assay found that circARHGEF28 sponged miR-671-5p in PCa cells. Importantly, qRT-PCR and dual luciferase assays found that Lectin galactoside-binding soluble 3 binding protein (LGALS3BP) was downstream of miR-671-5p, and western blot analysis further confirmed that LGALS3BP negatively regulated the nuclear factor kappa-B (NF-κB) pathway. These results demonstrated that circARHGEF28 abolished the degradation of LGALS3BP by sponging miR-671-5p, thus blocking the activation of the NF-κB pathway. Our findings revealed that circARHGEF28/miR-671-5p/LGALS3BP/NF-κB may be an important axis that regulates PCa progression.

Integrated microbiome and metabolome analysis reveals novel urinary microenvironmental signatures in interstitial cystitis/bladder pain syndrome patients.

BACKGROUND: The pathogenesis of interstitial cystitis/bladder pain syndrome (IC/BPS) has not been elucidated, but urinary microorganisms and metabolites have been shown to be closely associated with the inflammatory response of IC/BPS. Nevertheless, the exact mechanisms related to this response have not been clarified. METHODS: 16S rRNA sequencing and untargeted metabolomics techniques were used to analyse the urinary microbial and metabolite profiles of 30 IC/BPS patients and 30 healthy controls, and correlation analyses were performed to explore the mechanisms by which they might be involved in the inflammatory response of IC/BPS. RESULTS: Twenty-eight differential genera, such as Lactobacillus and Sphingomonas, were identified. A total of 44 differential metabolites such as 1,3,7-trimethyluric acid and theophylline were screened. The abundance of Lactobacillus and Escherichia-Shigella was significantly higher in the urine of female IC/BPS patients and healthy controls compared to males, while Bacteroides and Acinetobacter were lower than in males. The results of the Pearson correlation analysis suggested that differential microorganisms may influence the composition of metabolites. The Lactobacillus genus may be a protective bacterium against IC/BPS, whereas Sphingomonas may be a pathogenic factor. The differential metabolite theophylline, as an anti-inflammatory substance, may downregulate the inflammatory response of IC/BPS. CONCLUSIONS: This study revealed microbial and metabolite profiles in the urine of IC/BPS patients versus healthy controls in both males and females. We also found some microorganisms and metabolites closely related to the inflammatory response of IC/BPS, which provided directions for future aetiological and therapeutic research.

HMMR promotes prostate cancer proliferation and metastasis via AURKA/mTORC2/E2F1 positive feedback loop.

Although dysregulated HMMR is linked to prostate cancer (PCa) prognosis, the precise mechanisms remain unclear. Here, we sought to elucidate the role of HMMR in PCa progression as well as underlying mechanism. Herein, we found that upregulation of HMMR frequently observed in PCa samples and was associated with poor prognosis. Additionally, HMMR significantly promoted PCa proliferation and metastasis through gain- and loss-of function approaches in vitro and in vivo. Mechanistically, HMMR may interact with AURKA and elevated AURKA protein level through inhibiting ubiquitination-mediated degradation, which subsequently activated mTORC2/AKT pathway to ensure the reinforcement of PCa progression. Moreover, upregulated E2F1 caused from sustained activation of mTORC2/AKT pathway in turn function as transcription factor to promote HMMR transcription, thereby forming a positive feedback loop to trigger PCa progression. Importantly, administration of the mTOR inhibitor partially antagonised HMMR-mediated PCa progression in vivo. In summary, we not only reveal a novel possible post-translation mechanism mediated by HMMR involved in AURKA regulation, but also describe a positive feedback loop that contributes to PCa deterioration, suggesting HMMR may serve as a potential promising therapeutic target in PCa.

Identification of a potential structure-based GPCR drug for interstitial cystitis/bladder pain syndrome: in silico protein structure analysis and molecular docking.

INTRODUCTION AND HYPOTHESIS: There is currently no effective treatment for interstitial cystitis / bladder pain syndrome (IC/BPS) and thus seriously reduces the quality of life of patients. The purpose of this study is to analyze the structure and function of G protein coupled receptors related to IC/BPS by integrating bioinformatics and provide basis for the development of new drugs for IC/BPS. METHODS: We used ProtParam and DNAMAN to analyze the physical and chemical properties of GPR18 and GPR183 proteins. The secondary and tertiary structure, conservative domain, phosphorylation site of both proteins were predicted by ProtScale, PredictProtein, SWISS-MODEL and GPS5.0 respectively. Multiple sequence alignment of the proteins were carried out by DNAMAN and the phylogenetic tree was constructed by MEGA. Further, the molecular docking verification of cannabidiol and both proteins were carried out by using AutoDock Vin. RESULTS: GPR18 and GPR183 proteins were composed of 331 and 361 amino acids respectively. α-helix is the highest in the secondary structure of the two proteins. Both proteins contain seven transmembrane domains specific to G protein coupled receptors. And homology analysis showed that the two proteins had high homology. In terms of molecular docking, cannabidiol, a non psychoactive component extracted from the cannabis, can form effective molecular binding with GPR18 and GPR183 proteins. CONCLUSIONS: We identified the structures of GPR18 and GPR183 proteins and their highly homologous evolutionary properties. Furthermore, both proteins can form effective binding with cannabidiol which provides new insights for the development of IC/BPS drugs by targeting G protein coupled receptors.

Preliminary Exploration of a New Therapy for Interstitial Cystitis/Bladder Pain Syndrome: Botulinum Toxin A Combined with Sapylin.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is an intractable disease without long-term effective therapy. This study aims to evaluate the efficacy and safety of botulinum toxin A (BoNT/A) plus Sapylin, which might modulate the immune response of the bladder in the treatment of IC/BPS patients. We retrospectively investigated the clinical outcomes among 34 patients who accepted repeated Sapylin instillations after 200 U of BoNT/A submucosally injected into bladder walls (Mix group) and 28 patients who received BoNT/A alone (Control group). Each of the bladder walls (left, right, anterior and posterior) was injected six times with 8 U of BoNT/A per injection. The primary outcome measure was the global response assessment. The results showed that at 6 months post-injection, the response rate in the Mix group was remarkably higher than that in the Control group (58.8% vs. 28.6%, p < 0.05). The mean effective duration of the responders in the Mix group was apparently better than that in the Control group (27.5 (range 0-89) vs. 4.9 (range 0-11) months, p < 0.05). None of the patients experienced serious adverse events. In conclusion, repeated intravesical instillations of Sapylin after BoNT/A injection can produce significantly better clinical outcomes than BoNT/A alone in IC/PBS patients.

WGCNA and molecular docking reveal key hub genes and potential natural inhibitor in interstitial cystitis/bladder pain syndrome.

INTRODUCTION AND HYPOTHESIS: The etiology and treatment of interstitial cystitis/bladder pain syndrome are still controversial. The purpose of this study is to determine the key genes and specific regulatory pathways related to it and to find potential drug-active components through integrated bioinformatics. METHODS: The data set GSE11783 was downloaded from GEO database. The modules significantly related to interstitial cystitis/bladder pain syndrome were identified by weighted correlation network analysis. The genes in the key modules were analyzed by functional enrichment and protein interaction by Cytoscape software, and finally the core hub genes were screened. Furthermore, the molecular docking verification of active components and key proteins was carried out by using AutoDock Vin software. RESULTS: Among the 14 modules derived from WGCNA, turquoise module had the highest correlation with IC/BPS (r = 0.85, P < 0.001). The genes in the module were mainly enriched in the biological processes such as the interaction between cytokines and cytokine receptors and chemokine signaling pathway. The genes in the related modules of differentially expressed genes and WGCNA traits were intersected to obtain the core hub genes. Protein-protein interaction network analysis showed that the key genes were upregulated genes CCR7 and CCL19. In terms of molecular docking, triptolide, the active component in the traditional anti-inflammatory drug Tripterygium wilfordii, can form effective molecular binding with both core hub genes. CONCLUSIONS: Our study identified the core hub genes CCR7 and CCL19, which acted as essential components in interstitial cystitis/bladder pain syndrome. Furthermore, CCR7 and CCL19 can form effective binding with triptolide, which will provide new insights into the development of new therapies for interstitial cystitis/bladder pain syndrome.

A Novel Risk Factor Model Based on Glycolysis-Associated Genes for Predicting the Prognosis of Patients With Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is one of the most prevalent cancers among males, and its mortality rate is increasing due to biochemical recurrence (BCR). Glycolysis has been proven to play an important regulatory role in tumorigenesis. Although several key regulators or predictors involved in PCa progression have been found, the relationship between glycolysis and PCa is unclear; we aimed to develop a novel glycolysis-associated multifactor prediction model for better predicting the prognosis of PCa. METHODS: Differential mRNA expression profiles derived from the Cancer Genome Atlas (TCGA) PCa cohort were generated through the "edgeR" package. Glycolysis-related genes were obtained from the GSEA database. Univariate Cox and LASSO regression analyses were used to identify genes significantly associated with disease-free survival. ROC curves were applied to evaluate the predictive value of the model. An external dataset derived from Gene Expression Omnibus (GEO) was used to verify the predictive ability. Glucose consumption and lactic production assays were used to assess changes in metabolic capacity, and Transwell assays were used to assess the invasion and migration of PC3 cells. RESULTS: Five glycolysis-related genes were applied to construct a risk score prediction model. Patients with PCa derived from TCGA and GEO (GSE70770) were divided into high-risk and low-risk groups according to the median. In the TCGA cohort, the high-risk group had a poorer prognosis than the low-risk group, and the results were further verified in the GSE70770 cohort. In vitro experiments demonstrated that knocking down HMMR, KIF20A, PGM2L1, and ANKZF1 separately led to less glucose consumption, less lactic production, and inhibition of cell migration and invasion, and the results were the opposite with GPR87 knockdown. CONCLUSION: The risk score based on five glycolysis-related genes may serve as an accurate prognostic marker for PCa patients with BCR.

A robust signature of immune-related long non-coding RNA to predict the prognosis of bladder cancer.

BACKGROUND: Bladder cancer is the second most common malignant tumor in the urogenital system. The research investigated the prognostic role of immune-related long non-coding RNA (lncRNA) in bladder cancer. METHODS: We extracted 411 bladder cancer samples from The Cancer Genome Atlas database. Single-sample gene set enrichment analysis was employed to assess the immune cell infiltration of these samples. We recognized differentially expressed lncRNAs between tumors and paracancerous tissues, and differentially expressed lncRNAs between the high and low immune cell infiltration groups. Venn diagram analysis detected differentially expressed lncRNAs that intersected the above groups. LncRNAs with prognostic significance were identified by regression analysis. Multivariate Cox analysis was used to establish the risk score model. Then we established and evaluated the nomogram. Additionally, we performed gene set enrichment analysis to explore the potential functions of the screened lncRNAs in tumor pathogenesis. RESULTS: Three hundred and twenty differentially expressed lncRNAs were recognized. We randomly divided patients into the training data set and the testing data set at a 2: 1 ratio. In the training data set, 9 immune-related lncRNAs with prognostic significance were identified. The risk score model was constructed to classify patients as high- and low-risk cohorts. Patients in the low-risk cohort had better survival outcomes than those in the high-risk cohort. The nomogram was established based on the indicators including age, gender, tumor-node-metastases stage, and risk score. The model's predictive performance was confirmed by the receiver operating characteristic curve analysis, concordance index method, calibration curve, and decision curve analysis. The testing data set also achieved similar results. Bioinformatics analysis suggested that the 9-lncRNA signature was involved in the modulation of various immune responses, antigen processing and presentation, and T cell receptor signaling pathway. CONCLUSIONS: Our study uncovered the prognostic value of immune-related lncRNAs for bladder cancer and showed that they may regulate tumor pathogenesis in various ways.

The study on the function and cell source of interleukin-6 in interstitial cystitis/bladder painful syndrome rat model.

OBJECTIVE: The elevated expression of interleukin-6 (IL-6) in patients with interstitial cystitis/bladder painful syndrome (IC/BPS) has been demonstrated, but the role of IL-6 in IC/BPS and its source remain to be explored. METHODS: IC/BPS rat model was created in female rats by using long-term intermittent intravesical hyaluronidase (0.5 ml, 4 mg/ml). After modeling, IL-6 stimulation group, and anti-IL-6R group were treated with recombinant rat IL-6 and tocilizumab, respectively. Symptomatic changes were detected by Vonfrey pain score and urodynamics, and hematoxylin-eosin (HE) staining, mast cell staining and Masson staining were used to evaluate the changes of inflammation in the bladder tissue of rats. Cell sources of IL-6 was explored through enzyme linked immunosorbent assay (ELISA) test, reverse transcription polymerase chain reaction (RT-PCR), and western-blot test on the supernatant of coculturing rat bladder epithelial cells and rat macrophages. RESULTS: The Vonfrey pain scores of the model group and IL-6 stimulation group were significantly higher than those of the control group, while the anti-IL-6R group were significantly lower (p < .05). Compared with the blank control group, urodynamic results showed that the urination interval of the model group and IL-6 stimulation group was significantly shortened, and the maximum bladder capacity was significantly reduced (p < .05), and anti-IL-6R treatment significantly alleviated the inflammatory response of bladder tissue. The results of HE, Mast cell staining, and Masson staining showed that the inflammatory response of bladder tissue after anti-IL-6R treatment was significantly reduced. Through cells coculture, the relative expression of IL-6 from model group was found significantly higher than blank control group by RT-PCR, ELISA, and western blot test (p < .05). CONCLUSIONS: IL-6 played an essential role in the development of IC/BPS rat model as a proinflammation cytokine. Further evidence from coculture proved that macrophages are the cell resource of IL-6 in IC/BPS.

Clinicopathological significance and prognostic value of cancer-associated fibroblasts in prostate cancer patients.

INTRODUCTION: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment were considered to play an essential role in tumor growth and development. However, few studies have assessed the prognostic and clinicopathological significance of CAFs in prostate cancer (PCa) patients. METHODS: One hundred thirty pairs of PCa tissues and normal adjacent tissues (NATs) were immunostained with fibroblast activation protein and α-smooth muscle actin to quantify CAFs. Bioinformatics analysis was used to uncover the possible biological functions of CAFs. RESULTS: More CAFs were identified in PCa tissues than in NATs. High density of CAFs may be associated with advanced-stage disease, higher Gleason scores, lymphatic metastases, higher PSA, and poor biochemical recurrence-free survival in PCa. Bioinformatics analysis showed that CAFs may regulate tumor progression and recurrence through ECM modification, PI3K-Akt signaling pathway and regulation of cytoskeleton. CONCLUSION: In summary, our study uncovered the clinicopathological significance and potential mechanism of CAFs and indicated that CAFs may be a useful prognostic biomarker in PCa.

Robot-Assisted Nephrectomy Using the Newly Developed EDGE SP1000 Single-Port Robotic Surgical System: A Feasibility Study in Porcine Model.

Objectives: To evaluate the feasibility and safety of the EDGE SP1000 single-port robotic platform by performing nephrectomy in live porcine model. Materials and Methods: Robotic nephrectomy was performed on sample group of five gilts using the EDGE SP1000 single-port robotic system. The continuous vital signs of all gilts were monitored throughout the operation to examine the safety of the operation. Data regarding surgical complications and technical difficulties throughout the operation were recorded for future evaluation. Finally, the survival of the sample gilts 2 weeks after the operation were recorded. Results: The robot-assisted nephrectomy yielded an impressive result that all sample gilts survived after 2 weeks. Furthermore, neither surgical complications nor technical difficulties were encountered during the operation. The average duration to establish the operation channel was 12.4 ± 1.52 minutes, the average time taken to install EDGE SP1000 single-port robotic system was 2.8 ± 0.84 minutes, the average time to dissociate and remove the kidney was 47 ± 5.61 minutes, and average total operational duration was 71 ± 5.24 minutes. Most importantly, the surgeon and assistant who were using the system found it convenient and performed excellently during the operation. Conclusions: This study shows that the EDGE SP1000 single-port robotic surgical system is safe and feasible for the use of nephrectomy in gilts. Our further research will expand the application of the EDGE SP1000 system to other urologic procedures and accumulate more preclinical data for further clinical trial.

Comparative analysis of retrograde intrarenal surgery and modified ultra-mini percutaneous nephrolithotomy in management of lower pole renal stones (1.5-3.5 cm).

BACKGROUND: To compare the safety and efficacy of retrograde intrarenal surgery (RIRS) and modified Ultra-mini percutaneous nephrolithotomy (UMP) in semi-supine combined lithotomy position for the management of 1.5-3.5 cm lower pole renal stones (LPSs). METHODS: A total of 63 patients with 1.5-3.5 cm LPSs who underwent RIRS (n = 33) or modified UMP (n = 30) in diameter between January 2017 and January 2019 were analyzed retrospectively. Modified UMP was performed in semi-supine combined lithotomy position and a 9.5/11.5 F ureteral access sheath (UAS) was inserted during the procedure in order to maintain low pelvic pressure and to facilitate the removal of stone fragments. Base-line parameters, stone characteristics, illness condition, operation time, postoperative hemoglobin (Hb) drop, postoperative creatinine (Cr) elevation, length of hospital stay, length of postoperative hospital stay, stone-free rate (SFR) and complications were compared between the two groups. RESULTS: There were no significant differences between the two groups in base-line parameters, stone characteristics and illness condition. The mean operating time of RIRS group was longer than UMP group (95.61 ± 21.9 vs. 55.0 ± 16.1 min, p < 0.001). The mean postoperative Hb drop was less in RIRS group (7.42 ± 4.7 vs. 15.70 ± 9.8 g/L, p < 0.001). The length of hospital stay and postoperative hospital stay for RIRS were shorter than UMP (4.76 ± 1.1 vs. 5.83 ± 0.8 d, p < 0.001, 2.97 ± 0.9 vs. 4.07 ± 0.9 d, p < 0.001). The Early SFR was higher in UMP group (54.5 vs. 80.0%, p < 0.050) while SFR at 1-month and 3-months postoperatively was similar in both groups (p = 0.504, p = 0.675). There were no significant differences between the two groups in complications (p = 0.228). CONCLUSION: For patients with 1.5-3.5 cm LPSs, both modified UMP and RIRS are safe and viable. The modified UMP technique was used in this study, application semi-supine combined lithotomy position and the retention of UAS can improve the surgical efficiency and maintain low pressure perfusion in the kidney, which resulted in superior treatment efficacy. Therefore, we highly recommend this technique for LPSs with heavy stone burdens.

Nox4 has a crucial role in uric acid­induced oxidative stress and apoptosis in renal tubular cells.

The purpose of the present study was to evaluate the effects of uric acid in promoting tubular cell apoptosis and verify the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4)­induced oxidative stress in this process. HK­2 cells were used as a human proximal tubular cell model and co­cultured with various concentrations of uric acid with or without pre­treatment with the Nox4 inhibitor diphenylene iodonium (DPI). The apoptotic rate and the amount of reactive oxygen species (ROS) were examined by flow cytometry. Furthermore, levels of Nox4, phosphorylated (p)­P38, p­extracellular signal­regulated kinase (ERK), B­cell lymphoma 2 (Bcl­2) and Bcl­2­extra large (Bax) were detected by western blot analysis. The results showed that treatment with uric acid decreased HK­2 cell viability and promoted apoptosis in a dose­dependent manner. This was paralleled with an upregulation of Nox4 as well as ROS overproduction, which activated the phosphorylation of P38/ERK and caused an imbalance of Bax/Bcl­2 in HK­2 cells. Of note, inhibition of Nox4 with DPI prevented uric acid­induced cell injury by suppressing ROS generation and P38/ERK activation. In conclusion, it was demonstrated that elevated uric acid promoted ROS­induced tubular cell apoptosis by upregulating Nox4 expression. The present study therefore provided possible mechanisms and a potential therapeutic target of uric acid­induced chronic kidney disease.

Gene expression profiling of WDR5 regulated genes in bladder cancer.

WD repeat domain 5 (WDR5) plays an important role in various biological functions through the epigenetic regulation of gene transcription (Wysocka et al., 2005 [1]; Sandstrom et al., 2014[2]; Ang et al., 2011[3]). Recently, our study found that WDR5 was upregulated in bladder cancer tissues, promoted bladder cancer cell proliferation, self-renewal and chemoresistance to cisplatin in bladder cancer cells in vitro, and tumor growth in vivo (Chen et al., 2015). To gain a molecular understanding of the role of WDR5 in promoting bladder cancer, we performed a genome-wide analysis on WDR5 knockdown by microarray gene expression profiling. Here we provide detailed experimental methods and analysis for the microarray data, which have been deposited into Gene Expression Omnibus (GEO): GSE59132.

Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells.

Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients.

NOTCH1 signaling promotes chemoresistance via regulating ABCC1 expression in prostate cancer stem cells.

Chemotherapy is a strategy for patients with advanced prostate cancer, especially those with castration-resistant prostate cancer. Prostate cancer stem cells (PCSCs) are believed to be the origin of cancer recurrence following therapy intervention, including chemotherapy. The mechanisms underlying the chemoresistance of PCSCs are still poorly understood. In the present study, fluorescence-activated cell sorting was used to isolate PCSCs from LNCaP and PC3 cell lines. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide was used to measure the cell viability. Quantitative real-time PCR and western blotting were utilized to evaluate the mRNA and protein levels. ShRNA was employed to knock down target gene expression. Chromatin immunoprecipitation (ChIP) was performed to explore the detailed mechanism underlying ABCC1 expression. Our results revealed that the sorted PCSCs showed enhanced chemoresistance ability than matched non-PCSCs. Protein level of activated form of NOTCH1(ICN1) was significantly higher in PCSCs. Inhibition of NOTCH1 with shRNA could decrease ABCC1 expression, and improve chemosensitivity in PCSCs. Finally, ChIP-PCR showed ICN1 could directly bind to the promoter region of ABCC1. In conclusion, NOTCH1 signaling could transactivate ABCC1, resulting in higher chemoresistance ability of PCSCs, which might be one of the important mechanisms underlying the chemoresistance of PCSCs.

Role of EZH2 in the growth of prostate cancer stem cells isolated from LNCaP cells.

Enhancer of zeste homolog 2 (EZH2) plays a crucial role in embryonic and somatic stem cells for their proliferation and differentiation. However, the roles and underlying mechanisms of EZH2 in prostate cancer stem cells (PCSCs) remain unknown. This study aimed to investigate the effects of EZH2 on PCSCs. PCSCs were isolated from the human prostate cancer cell line LNcap by fluorescence activated cell sorting (FACS). EZH2 expression was compared between PCSCs and non-PCSCs. The association between EZH2 function and PCSC growth was investigated using siRNA-mediated knock-down of EZH2. Cell growth was investigated by MTT, cell cycle and apoptosis of PCSCs were explored by flow cytometric analysis. Finally, the upstream pathway miRNA level was determined via a luciferase reporter assay, and the downstream pathway cycle regulators were examined via reverse transcriptase-polymerase chain reaction. The results showed that LNcap cell line comprised a greater proportion of CD44+/CD133+ cells by comparison to the PC-3 cell line. EZH2 was up-regulated in PCSCs compared with non-PCSCs. Silence of EZH2 inhibited cell growth and the cell cycle and promoted the progression of apoptosis. Furthermore, EZH2 was a direct target of miR-101 in PCSCs and EZH2's mRNA levels were inversely correlated with miR-101 expression and cyclin E2 (a cell-cycle regulator) was suppressed by siEZH2. In conclusion, EZH2 is essential for PCSC growth, partly through a negative regulation by miR-101 and positively regulating cyclin E2.