长链非编码RNA LINC01006对前列腺癌细胞增殖和侵袭的影响

摘要 目的：探究长链非编码RNA LINC01006对前列腺癌（prostate cancer, PCa）细胞增殖和侵袭能力的影响。方法：体外培养人前列腺正常上皮细胞系RWPE-1,人PCa细胞系LNCaP、22Rv1、PC3、C4-2b,应用实时定量PCR（qRT-PCR）技术检测上述细胞LINC01006的表达;分别通过转染小干扰RNA（siRNA）或过表达LINC01006的慢病毒载体,在LNCaP和PC3细胞中敲减LINC01006或稳定过表达LINC01006;应用CCK8、克隆形成实验检测LINC01006对PCa细胞增殖能力的影响;应用Transwell侵袭实验检测LINC01006对PCa细胞侵袭能力的影响;通过网站预测LINC01006的转录调控因子及其结合位点。结果：相较于正常前列腺上皮细胞系RWPE-1,PCa细胞系LNCaP、22Rv1、C4-2b和PC3中LINC01006表达明显升高（P<0.05）。敲减LINC01006后的PCa细胞系LNCaP和PC3的增殖和侵袭能力被显著抑制（P<0.05）,过表达LINC1006则明显促进PCa细胞系LNCaP和PC3的增殖、侵袭能力（P<0.05）。通过PROMO网站预测可见AR是LINC01006的潜在转录调控因子,通过Cistrome DB数据库发现LINC01006上游启动子区域存在AR富集;敲减、抑制AR后LNCaP细胞中LINC01006表达明显升高（P<0.05）。结论：LINC01006在PCa细胞系中呈高表达,促进PCa细胞的增殖和侵袭,其受到AR负向调控,可能在PCa发生发展和去势抵抗形成过程中发挥作用。     

Prostate Cancer Associated Lipid Signatures in Serum Studied by ESI-Tandem Mass Spectrometryas Potential New Biomarkers

Prostate cancer (PCa) is one amongst the most common cancersin western men. Incidence rate ofPCa is on the rise worldwide. The present study deals with theserum lipidome profiling of patients diagnosed with PCa to identify potential new biomarkers. We employed ESI-MS/MS and GC-MS for identification of significantly altered lipids in cancer patient’s serum compared to controls. Lipidomic data revealed 24 lipids are significantly altered in cancer patinet’s serum (n = 18) compared to normal (n = 18) with no history of PCa. By using hierarchical clustering and principal component analysis (PCA) we could clearly separate cancer patients from control group. Correlation and partition analysis along with Formal Concept Analysis (FCA) have identified that PC (39:6) and FA (22:3) could classify samples with higher certainty. Both the lipids, PC (39:6) and FA (22:3) could influence the cataloging of patients with 100% sensitivity (all 18 control samples are classified correctly) and 77.7% specificity (of 18 tumor samples 4 samples are misclassified) with p-value of 1.612×10⁻⁶ in Fischer’s exact test. Further, we performed GC-MS to denote fatty acids altered in PCa patients and found that alpha-linolenic acid (ALA) levels are altered in PCa. We also performed an in vitro proliferation assay to determine the effect of ALA in survival of classical human PCa cell lines LNCaP and PC3. We hereby report that the altered lipids PC (39:6) and FA (22:3) offer a new set of biomarkers in addition to the existing diagnostic tests that could significantly improve sensitivity and specificity in PCa diagnosis.     

Nuclear Ep-ICD Expression Is a Predictor of Poor Prognosis in “Low Risk” Prostate Adenocarcinomas

IntroductionMolecular markers for predicting prostate cancer (PCa) that would have poor prognosis are urgently needed for a more personalized treatment for patients. Regulated intramembrane proteolysis of Epithelial cell adhesion molecule results in shedding of the extracellular domain (EpEx) and release of its intracellular domain (Ep-ICD) which triggers oncogenic signaling and might correlate to tumor aggressiveness. This study aimed to explore the potential of Ep-ICD and EpEx to identify PCa that have poor prognosis.MethodsImmunohistochemical analysis of Ep-ICD and EpEx was carried out in normal prostate tissues (n = 100), benign prostate hyperplasia (BPH, n = 83), and prostate cancer (n = 249) using domain specific antibodies. The expression of Ep-ICD and EpEx was correlated with clinico- pathological parameters and disease free survival (DFS).ResultsReduced expression of nuclear Ep-ICD and membrane EpEx was observed in PCa in comparison with BPH and normal prostate tissues (p = 0.006, p < 0.001 respectively). For patients who had PCa with Gleason Score less than 7, preserved nuclear Ep-ICD emerged as the most significant marker in multivariate analysis for prolonged DFS, where these patients did not have recurrence during follow up of up to 12 years (p = 0.001).ConclusionReduced expression of nuclear Ep-ICD was associated with shorter disease free survival in patients with a Gleason Score less than 7 and may be useful in identifying patients likely to have aggressive tumors with poor prognosis. Furthermore, nuclear Ep-ICD can differentiate between normal and prostate cancer tissues for ambiguous cases.     

The prognostic impact of tumour NSD2 expression in advanced prostate cancer

Abstract

Purpose: To assess the prognostic significance of the nuclear receptor binding SET protein 2 (NSD2), a co-activator of the NFkB-pathway, on tumour progression in patients with advanced prostate cancer (PCa).

Methods: We retrospectively assessed NSD2 expression in 53 patients with metastatic and castration-resistant PCa. Immunohistochemical staining for NSD2 was carried out on specimen obtained from palliative resection of the prostate. Univariable and multivariable analyses were performed to assess the association between NSD2 expression and PCa progression.

Results: Of the 53 patients, 41 had castration-resistant PCa and 48 men had metastases at time of tissue acquisition. NSD2 expression was increased in tumour specimen from 42 patients (79.2%). In univariable Cox regression analyses, NSD2 expression was associated with PSA progression, progression on imaging and overall survival (p?=?0.04, respectively). In multivariable analyses, NSD2 expression did not retain its association with these endpoints.

Conclusions: NSD2 expression is abnormal in almost 80% of patients with advanced PCa. Expression levels of this epigenetic regulator are easily detected by immunohistochemistry while this biomarker exhibited prognostic value for PCa progression and death in univariable analysis. Further studies on NSD2 involvement in PCa proliferation, progression, metastasis and resistance mechanisms are needed.     

The clinical significance of circulating miR-21, miR-142, miR-143, and miR-146a in patients with prostate cancer

BackgroundProstate cancer (PCa) is the most common type of solid tissue cancer among men in western countries. In this study, we determined the levels of circulating miR-21, miR-142, miR-143, miR-146a, and RNU 44 levels as controls for early diagnosis of PCa.MethodsThe circulating miRNA levels in peripheral blood samples from 43 localized PCa patients, 12 metastatic PCa (MET) patients, and a control group of, 42 benign prostate hyperplasia (BPH) patients with a total of 97 volunteers were determined the by PCR method.ResultsNo differences in the DCT values were found among the groups. In PCa and PCaMet groups the expression of miR21 and miR142 were higher compared to the BHP group. No other differences were observed among the other groups. miR21 expression in the PCa group was 6.29 folds upregulated whereas in the PCaMet group 10.84 folds up-regulated. When the total expression of miR142 is evaluated, it showed a positive correlation with mir21 and mir 146 (both p<0.001). Also, the expression of miR146 shows a positive correlation with both miR21 and miR143 (both p<0.001). Expression of miRNAs was found to be an independent diagnostic factor in patients with Gleason score, PSA, and free PSA levels.ConclusionsOur study showed that co-expression of miR21, miR-142, miR-143, and miR-146a and the upregulation of miR-21 resulted in increased prostate carcinoma cell growth. In the PCaMet group, miR21 is the most upregulated of all miRNAs. These markers may provide a novel diagnostic tool to help diagnose PCa with aggressive behavior.     

ARLTS1 and Prostate Cancer Risk - Analysis of Expression and Regulation

Prostate cancer (PCa) is a heterogeneous trait for which several susceptibility loci have been implicated by genome-wide linkage and association studies. The genomic region 13q14 is frequently deleted in tumour tissues of both sporadic and familial PCa patients and is consequently recognised as a possible locus of tumour suppressor gene(s). Deletions of this region have been found in many other cancers. Recently, we showed that homozygous carriers for the T442C variant of the ARLTS1 gene (ADP-ribosylation factor-like tumour suppressor protein 1 or ARL11, located at 13q14) are associated with an increased risk for both unselected and familial PCa. Furthermore, the variant T442C was observed in greater frequency among malignant tissue samples, PCa cell lines and xenografts, supporting its role in PCa tumourigenesis. In this study, 84 PCa cases and 15 controls were analysed for ARLTS1 expression status in blood-derived RNA. A statistically significant (p = 0.0037) decrease of ARLTS1 expression in PCa cases was detected. Regulation of ARLTS1 expression was analysed with eQTL (expression quantitative trait loci) methods. Altogether fourteen significant cis-eQTLs affecting the ARLTS1 expression level were found. In addition, epistatic interactions of ARLTS1 genomic variants with genes involved in immune system processes were predicted with the MDR program. In conclusion, this study further supports the role of ARLTS1 as a tumour suppressor gene and reveals that the expression is regulated through variants localised in regulatory regions.     

The Effect of Quercetin Nanosuspension on Prostate Cancer Cell Line LNCaP via Hedgehog Signaling Pathway

Background:Prostate cancer (PCa) is the second leading cause of cancer death in American population. In this manner, novel therapeutic approaches for identification of therapeutic targets for PCa has significant clinical implications. Quercetin is a potent cancer therapeutic agent and dietary antioxidant present in fruit and vegetables.Methods:To investigate the underlying mechanism by which the PCa was regulated, nanoparticles of quercetin were administrated to cells. For in vitro experiments, human PCa cell line LNCaP were involved. Cell viability assay and quantitative RT-PCR (qRT-PCR) for hedgehog signaling pathway genes were used to determine the key signaling pathway regulated for PCa progression.Results:The cell viability gradually decreased with increased concentration of quercetin nanoparticles. At 48 h, 40 mM concentration of quercetin treatment showed near 50% of viable cells. Quercetin nanoparticles upregulates Su(Fu) mRNA expressions and downregulates gli mRNA expressions in the LNCaP cells.Conclusion:The results showed that the hedgehog signaling targeted inhibition may have important implications of PCa therapeutics. Additionally, the outcomes provided new mechanistic basis for further examination of quercetin nanoparticles to discover potential treatment strategies and new targets for PCa inhibition.Key Words: Hedgehog, Prostate cancer, Proliferation, Quercetin nanoparticles, Signaling pathway     

Expression profile of WNT molecules in prostate cancer and its regulation by aminobisphosphonates

Skeletal metastases represent a frequent complication in patients with advanced prostate cancer (PCa) and often require bisphosphonate treatment to limit skeletal‐related events. Metastasized PCa cells disturb bone remodeling. Since the WNT signaling pathway regulates bone remodeling and has been implicated in tumor progression and osteomimicry, we analyzed the WNT profile of primary PCa tissues and PCa cell lines and assessed its regulation by bisphosphonates. Prostate tissue (n = 18) was obtained from patients with benign prostate hyperplasia (BPH) and PCa patients with different disease stages. Serum samples were collected from 62 patients. Skeletal metastases were present in 17 patients of whom 6 had been treated with zoledronic acid. The WNT profile and its regulation by bisphoshonates were analyzed in tissue RNA extracts and serum samples as well as in osteotropic (PC3) and non‐osteotropic (DU145, LNCaP) PCa cell lines. Several members of the WNT pathway, including WNT5A, FZD5, and DKK1 were highly up‐regulated in PCa tissue from patients with advanced PCa. Interestingly, osteotropic cells showed a distinct WNT profile compared to non‐osteotropic cells. While WNT5A, FZD5, and DKK1 were highly expressed in PC3 cells, WNT1 and SFRP1 mRNA levels were higher in DU145 cells. Moreover, zoledronic acid down‐regulated mRNA levels of WNT5A (?34%), FZD5 (?60%), and DKK1 (?46%) in PC3 cells. Interestingly, patients with skeletal metastases who received zoledronic acid had twofold higher DKK1 serum levels compared to bisphosphonate‐naive patients. The WNT signaling pathway is up‐regulated in advanced PCa, differentially expressed in osteotropic versus non‐osteotropic cells, and is regulated by zoledronic acid. J. Cell. Biochem. 112: 1593–1600, 2011. © 2011 Wiley‐Liss, Inc.     

Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa). However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR) as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight^™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.     

Explication of the roles of prostate health index (PHI) and urokinase plasminogen activator (uPA) as diagnostic and predictor tools for prostate cancer in equivocal PSA range of 4–10 ng/mL

BackgroundProstate cancer (PCa) is one of the most commonly encountered cancers and the leading cause of death worldwide. Currently used biomarkers accounts difficulties in discriminating benign from malignant cases or predicting outcome, so investigating new biomarkers performance is needed.ObjectivesAssessment of diagnostic and predictor roles of prostate health index (PHI) and urokinase plasminogen activator (uPA) in PCa.Methods194 males with initial tPSA of 4–10 ng/mL were categorized into three groups: PCa, benign prostatic hyperplasia (BPH) and healthy control. Serum levels of tPSA, fPSA, p2PSA, and uPA were performed by ELISA with calculation of PHI as (p2PSA/fPSA) × √PSA.ResultsPHI and uPA were significantly higher in PCa patients relevant to BPH and healthy control (p ≤ 0.001). Both markers outperformed all assessed biomarkers and showed the highest area under the curve (AUC) in ROC curve analysis. Both were significantly higher in PCa patients with {Gleason score ≥ 7, late stages (cT2b,c; T3), LN extension and distant metastasis}relative to their counterparts. Additionally, PHI and uPA and were independent predictors of distant metastasis and Gleason score ≥ 7, while PHI was predictor of LN invasion (β = 0.25, p = 0.004).ConclusionPHI and uPA would be of potential value in discriminating between PCa, BPH and healthy men in addition, both are promising as independent predictors of adverse pathological features.     

前列腺癌细胞系中环状RNA circRNA-1565的表达及鉴定

目的:探讨前列腺癌细胞系中的环状RNA circRNA-1565的表达及鉴定。方法:培养前列腺正常上皮细胞(RWPE-1)、4种前列腺癌细胞(22RV1、LNCap、PC-3、DU145),提取总RNA,采用RT-PCR检测不同细胞系中circRNA-1565的表达,并对其进行成环性验证及细胞内亚定位实验,进行差异比较。结果:circRNA-1565在正常前列腺细胞系(RWPE-1)中表达量极低,在转移性前列腺癌细胞系中高表达,在非转移性前列腺癌细胞系中低表达。且circRNA-1565是一条主要定位于细胞质内的具有有效环状结构的RNA分子。结论:circRNA-1565在不同前列腺癌细胞系中存在差异表达,可能会通过mi RNA sponge途径发挥生物学作用。     

Targeting lactate production and efflux in prostate cancer

Prostate cancer (PCa) is the most commonly diagnosed cancer in men worldwide. Screening and management of PCa remain controversial and, therefore, the discovery of novel molecular biomarkers is urgently needed. Alteration in cancer cell metabolism is a recognized hallmark of cancer, whereby cancer cells exhibit high glycolytic rates with subsequent lactate production, regardless of oxygen availability. To maintain the hyperglycolytic phenotype, cancer cells efficiently export lactate through the monocarboxylate transporters MCT1 and MCT4. The impact of inhibiting lactate production/extrusion on PCa cell survival and aggressiveness was investigated in vitro and ex vivo using primary tumor and metastatic PCa cell lines and the chicken embryo chorioallantoic membrane (CAM) model. In this study, we showed the metastatic PCa cell line (DU125) displayed higher expression levels of MCT1/4 isoforms and glycolysis-related markers than the localized prostate tumor-derived cell line (22RV1), indicating these proteins are differentially expressed throughout prostate malignant transformation. Moreover, disruption of lactate export by MCT1/4 silencing resulted in a decrease in PCa cell growth and motility. To support these results, we pharmacological inhibited lactate production (via inhibition of LDH) and release (via inhibition of MCTs) and a reduction in cancer cell growth in vitro and in vivo was observed. In summary, our data provide evidence that MCT1 and MCT4 are important players in prostate neoplastic progression and that inhibition of lactate production/export can be explored as a strategy for PCa treatment.