| Background and objectives:Prostate cancer (PCa) represents the most common noncutaneous malignancy of the urinary system and a common cause of cancer-related mortality for men worldwide. Characteristically, it is a clinically heterogeneous, multifocal disease. Although PCa is apparently confined to the prostate, it encompasses a broad spectrum of diseases, some of which are featured with extremely indolent behavior and unfavorable clinical outcome. Thus, PCa has become a major public health challenge. In recent years, more and more clinically important tumors as well as other slow-growing cancers were detected due to the wide utilization of prostate-specific antigen (PSA) testing. Despite the considerable advances in diagnosis and adjuvant therapy, the clinical outcome of PCa patients has not been improved markedly. Various clinicopathological features, such as Gleason score, preoperation PSA level and pathological stage, are currently often used to predict prognosis in PCa patients. However, an increasing number of observations showed that PCa patients with the equivalent PSA level, Gleason score, and the pathologic stage could have different clinical outcomes, implying that it is a multi-step process of carcinogenesis and mechanisms influencing progression and prognosis of this malignancy. Therefore, there is an urgent necessary to identify new diagnostic and prognostic markers for better understanding of PCa pathogenesis, for improvement on therapeutic efficiency and clinical outcome of PCa patients.Accumulating evidence has demonstrated a pivotal role of microRNAs (miRNAs) in the development of various human cancers. miRNAs represent a novel class of small, non-coding RNAs which regulate expression levels of their target genes via completely or partially complementarily binding to the 3’-untranslated region (3’-UTR) of mRNA, leading to mRNA degradation or blocking translation [. Imperfect base pairing between miRNAs and their target mRNAs make them be involved into many essential cellular processes, such as development, proliferation, differentiation, cell fate determination and apoptosis. Emerging studies also reveal that miRNAs play crucial roles in several pathological pathways, such as host-viral interactions and tumorigenesis. Functionally, miRNAs have been demonstrated to act as either oncomiRNAs or tumor suppressors. Similar to other malignancies, PCa has a specific miRNA expression profile. In 2011, our research group reported characterization of miRNA profiles of PCa by miRNA microarray analyses (the data are deposited in the Gene Expression Omnibus (GEO) repository database (http://www.ncbi.nlm.nih.gov/geo/, accession number GSE34932). We found that 11 miRNAs were up-regulated and 17 miRNAs were down-regulated in PCa. Among them, miR-224, which has been implicated in controlling cell proliferation or apoptosis and contributing tumorigenesis of various human malignancies, is one of the downregulated miRNAs in PCa. Another study of our research group discovered that the down-expression of miR-224 is associated with poor prognosis of PCa patients, and further indicated that miR-224 could inhibit progression of human PCa by downregulating TRIB1. Since one miRNA can target multiple genes simultaneously, we believed that the underlying mechanisms of miR-224 have not been fully elucidated. Therefore, we carried out experiments to identify other target genes of miR-224 in PCa and to characterize the roles of miR-224-target mRNA axis in PCa tumorigenesis and progression.Materials:One human PCa cell line DU145 was purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibico, USA),2 mM L-glutamine, and antibiotics. Normal human prostate epithelial cells (PREC) were purchased from Lonza Company and were cultured in PrEGM Bullet kit (Lonza, USA) with antibiotics. All cell lines were maintained at 37 ℃ in a humidified chamber supplemented with 5% CO2.The study was approved by the Research Ethics Committee of Guangzhou First Municipal People’s Hospital, Guangzhou Medical College, China. Informed consent was obtained from all patients. All specimens were handled and made anonymous according to the ethical and legal standards.For miR microarray and quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis,4 and 20 pairs of primary PCa and adjacent non-tumorous frozen samples were obtained from the tissue bank at Guangzhou Medical College, respectively. All tissues for RNA extraction were obtained immediately after transurethral prostate resections or suprapubic prostatectomy. No patients recruited in this study received chemotherapy or radiotherapy before the surgery. The pathological diagnosis was performed preoperatively and confirmed postoperatively.For survival analysis, the Taylor dataset which is a large PCa dataset with microarray expression data for miRNAs and mRNAs, and information on survival for the patients including 149 primary PCa tissues and 29 adjacent non-cancerous prostate tissues were also collected Methods:Two online programs miRWalk (last upated on 15th March 2013) [15] and miRanda (August 2010 Release Last Update:2010-11-01) were used to predict possible target genes for miR-224.The miR microarray analysis was performed as our previously described. Data are available at the Gene Expression Omnibus (GEO) repository database [http://www.ncbi.nlm.nih.gov/geo/, accession number GSE34932]. miRNAs were extracted from 20 pairs of primary PCa tissues and adjacent normal prostate tissues with the miR Extraction Kit (Bioteke, China). The qRT-PCR analyses were carried out according to the protocol of All-in-OneTM miR qRT-PCR Detection Kit (GeneCopoeia, China). miRNA expression in each sample was normalized to that of the RNU6B housekeeping gene. The specificity of PCR amplification was confirmed by melting curve analysis and by agarose gels electrophoresis. Relative quantification of target miRNA expression was calculated using the comparative cycle threshold (CT) method. Mean± SE was calculated from independent experiment.The miR-224 coding sequence was cloned in the pMIRNAl lentivectors (SBI, USA) for expressing the miR-224 precursor. The scramble control hairpin pCDH-CMV-MCS-EF1-copGFP was purchased from the same vendor for negative controls. To package the construct,293TN cells were transfected with miR-224/miR-NC by pPACKH1 Packaging Plasmid Mix (CatNo:LV500A-1, SBI, USA), and then after 3 days, the virus particles are collected according the packaging protocol of SBI with the Lenti-Concentin Virus Precipitation Solution (CatNo: LV810A-1, SBI, USA). DU145 cells were infected with TransDux virus transduction reagent (CatNo:LV850A-1, SBI, USA). The infected cells were isolated with a flow cytometer and cultured in 96-well plates.The siRNA oligos for inhibiting CAMKK2 (si-CAMKK2) and the scramble oligos (si-NC) were purchased from GenePharma (Shanhai, China). The CAMKK2 coding sequence (without 3’-UTR) was cloned into pGCMV/EGFP/Neo-Vector (GenePharma, China). The blank vector was used as negative control. Fugene transfection reagents (Roche, USA) were used to transfect siRNA or plasmids.Proteins were extracted 48 hours post-transfection for Western blot analyses. Proteins (40μg) were fractioned on SDS-PAGE and transferred onto Hybond nitrocellulose membranes (GE Healthcare). The membranes were blocked with 5% skim milk in PBS-Tween 20 and probed with anti-CAMKK2 (rabbit polyclonal, ab96531, dilution 1:150, Cambridge, UK) or anti-p-actin antibody (rabbit polyclonal, sc-7210, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, USA). The results were visualized with the SuperSignal West PICO chemiluminescent detection system (Pierce Biotechnology). β-actin was used as an internal loading control.The expression of miR-224-targeted genes was evaluated by using a luciferase reporter assay in DU145 cells. The putative miR-224 complementary site in the 3’-UTR of CAMKK2 mRNA, or mutant sequence were cloned into the pGL3 luciferase reporter vector (Promega, USA). DU145 cells cultured in 24-well plates were co-transfected with 100 ng of luciferase reporter plasmid,10 ng miR-224 mimic or NC mimic, and 2 ng pRL-SV40 RLuci vector (Promega, USA). After 48 hours post the transfection, cells were washed with PBS and then lysed with the lysis buffer. The luciferase activities were detected with the dual-luciferase reporter assay system (Promega, USA) according to manufacturer’s instructions. The light output was quantitated by the bioluminescence detector Victor3V (PerkinElemer, USA).For cell viability assays,2×103 cells were seeded in 96-well plates and cultured for 24, 48, and 72 hours. Cells were then incubated with 20μl of CCK-8 solution (Cat No:C0038, Beyotime, China) for 4 hours at 37℃. The absorbance was measured at the wavelength of 495 nm with a spectrophotometer. Data were expressed as mean± SE of three independent experiments.For cell cycle assays, one 10 cm dish cell were collected and fixation by using 70% ethanol, and store at 40℃ for overnight. Before the staining, re-collected the cell and resuspend cells with PBS, then stain the cell with propidium Iodide (PI) solution and incubate at 37℃ for 15 minutes. Acquire data on flow cytometer (BD FACSVerse± System, BD Biosciences) and analysis the data with BD FACSuiteTM software. Data were expressed as mean ± SE of three independent experiments.The transwell inserts (8-μm pores) were filled with 50μlof a mixture of serum-free RPMI1640 medium and Matrigel (1:10; BD Biosciences, USA). The inserts were then placed in 24-well tissue culture plates (Transwell, Corning, USA) containing 10%FBS-medium. After solidification by incubation in at 37℃ for 4 hour,5×104 cells in 200μl medium were placed in upper chambers. Following 48 hours of incubation at 37℃ with 5% CO2 and in culture medium with mitomycin to stop the mitosis, the membranes were fixed with 10% formalin and stained with 0.05% Crystal Violet. The number of cells that migrated through the pores was assessed and the data were expressed as mean ± SE of three independent experiments.For the scratch wound-healing motility assay, a scratch was made with a pipette tip when the cells reach the confluence. After being cultured under standard conditions with mitomycin for 48 hours, plates were washed twice with fresh medium to remove non-adherent cells and then photographed. The cell migrated from the wound edge were counted and the data were expressed as mean±SE of three independent experiments.The versionl3.0 SPSS for Windows (SPSS Inc, IL, USA) and SAS 9.1 (SAS Institute, Cary, NC) softwares were used for statistical analysis. Continuous variables were expressed as mean±SE. Statistical analyses of miRNA microarray and qRT-PCR were conducted using Wilcoxon signed-rank test. The Spearman correlation was calculated between the expression levels of miR-224 and CAMKK2 in PCa tissues. Kaplan-Meier method was used for the survival analysis and Cox regression analysis was used for the univariate and multivariate analysis. Differences were considered statistically significant when the p value was less than 0.05.Results:1. Our previous study reported that miR-224 may function as a tumor suppressor in human PCa. To determine the underlying regulatory mechanisms of miR-224 in PCa cells, two miRNA target predicting programs (miRWalk and miRanda) were employed to identify possible targets of miR-224. Both miRWalk and miRanda predicted CAMKK2 as a potential target of miR-224. To verify this prediction, DU145 cells were forced to overexpress miR-224 by transfection. Western blot analysis showed that the endogenous CAMKK2 expression in miR-224 transfected cells was significantly reduced at the protein level.To confirm CAMKK2 being a target of miR-224, the luciferase reporter containing the complimentary seed sequence of miR-224 at the 3’-UTR region of CAMKK2 mRNA was constructed. Luciferase activity assay showed that expression of the luciferase reporter was significantly reduced by co-transfection with hes-miR-224 plasmid. In contrast, expression of the reporter containing the mutated sequence of the same fragment was not affected by cotransfection with hes-miR224 plasmid. The results indicated that the fragment at the 3’-UTR of the CAMKK2 mRNA was the complementary site for the miRNA-224 seed region, and therefore, that CAMKK2 was a direct target of miR-224.2. Our previous study indicated that enforced expression of miR-224 could inhibit cell proliferation, invasion and migration of DU145 cells. To further verify whether the role of miR-224 in PCa was mediated via targeting CAMKK2, the expression of CAMKK2 in DU145 cells was knockdown via siRNA. As shown in Figure 2, CAMKK2 overexpression abolished effects of miR-224 in cell proliferation of DU145 cells, further cell cycle assay showed that CAMKK2 could promote the cell proliferation by increase the ratio of S+G2/M phase cells. However CAMKK2 didn’t show the significance affect on cell invasion and migration. Therefore, the data indicated that miR-224 could inhibit PCa cell proliferation partially mediated by targeting CAMKK2.3. Expression levels of miR-224 and CAMKK2 mRNA in 20 pairs of primary PCa and adjacent non-tumorous frozen samples were detected by qRT-PCR, and respectively normalized to RNU6B and P-actin. As shown in Figure 3, the expression levels of miR-224 and CAMKK2 mRNA were respectively lower and higher in PCa tissues than those in adjacent non-tumorous prostate tissues significantly (both P<0.001). Notably, the downregulation of miR-224 was negatively correlated with the upregulation of CAMKK2 mRNA in PCa tissues (Spearman’s correlation:r=-0.66, P=0.004), which was consistent with the statistical results based on Taylor dataset (Pearson’s correlation:r=-0.428, P<0.001).4. According to the data of Taylor dataset, the median values of miR-224 and CAMKK2 mRNA expression levels in PCa tissues were used as cutoff points to classified all 104 PCa patients into miR-224-low (n=52), miR-224-high (n=52), CAMKK2-low (n=52) and CAMKK2-high (n=52) expression groups. Of 104 patients, 38 (36.54%) cases were miR-224-low and CAMKK2-high expression, and 14 (13.46%) cases were miR-224-high and CAMKK2-low expression, and the other 52 (50.00%) cases were both low expression of miR-224 and CAMKK2 or both high expression of miR-224 and CAMKK2. Since the expression levels of miR-224 in PCa tissues were significantly correlated with those of CAMKK2, and our previous study indicated that miR-224 downregulation was significantly associated with PCa metastasis, high PSA level, and high Glesson scores (all P<0.05), we here investigated the associations of combined miR-224 downregulation and CAMKK2 upregulation (miR-224-low/CAMKK2-high) with clinicopathological characteristics of PCa patients. As show in Table 2, miR-224-low/CAMKK2-high was significantly associated with advanced clinical stage (P=0.028).5. Using the Taylor dataset, the association of combined miR-224 and CAMKK2 expression (miR-224/CAMKK2) with the overall survival of PCa patients was analyzed by Kaplan-Meier method. PCa patients in miR-224-low/CAMKK2-high group more frequently had shorter overall survival than those in groups with other expression patterns of two molecules (P=0.028). In addition, the univariate analysis showed that there were significant differences in the overall survival between patients with miR-224-low/CAMKK2-high expression and other expression patterns of two molecules (P=0.046). Furthermore, the multivariate analyses showed that miR-224/CAMKK2 (P=0.037) and Gleason score (P=0.021) were independent predictors for unfavorable overall survival.Conclusions:1. miR-224 and its target gene CAMKK2 may synergistically contribute to the malignant progression of PCa.2. Combined detection of miR-224 and CAMKK2 expression represents an efficient predictor of patient prognosis and may be a novel marker which can provide additional prognostic information in PCa. |
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