| Background and objectives:Prostate cancer (PCa) is one of the most common malignancies and the second leading cause of cancer-related death in men.25%-50%patients treated with the radical prostatectomy, a standard treatment for localized PCa, experience recurrence during follow up. Due to the heterogeneity and relative poor-understanding of PCa, current prognostic methods and biomarkers offer limited tools for determine the outcome of PCa. Several methods, including PSA measurement and Gleason score, have been widely applied to assess the diagnosis and prognosis of PCa patients in clinic. However, those methods are not without limitations. For instance, Gleason score is frequently underestimated and PSA levels can become elevated in many conditions. Moreover, patients with similar PSA levels, Gleason scores and pathological stages have different outcomes due to PCa heterogeneity. Therefore, new biomarkers and methods warrant to be identified for the accurate assessment of PCa and the in-depth design of therapeutic regimens for cancer patients.MicroRNAs (miRNAs) produced by the ribonuclease Ⅲ-enzyme Dicer are small, noncoding RNAs, which can regulate gene expression by modulating mRNA translation or cleavage. They are involved in major cellular processes, such as cell proliferation, invasion, differentiation and apoptosis. Accumulated evidence showed that dysregulations of miRNAs could play important roles in the initiation and progression of cancers with various tissue origins.MiR-15and miR-16were the first miRNAs identified in cancer development, which were reported to be deleted or down-regulated in the majority (approximately68%) of B cell chronic lymphocytic leukemias cases. Since then, many miRNAs have been demonstrated to play pivotal roles during tumorgenesis in different organs including prostate. Ozen et al evaluated miRNA expression profiles using a microarray of16specimens of prostate adenocarcinoma and found general miRNAs under expression, especially miR-125, miR-145and miR-let7c. In2011, we characterized miRNA profiles in PCa tissues 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]). Overall,11up-regulated and17down-regulated miRNAs were identified to differentially express in PCa. Among these, miR-30c is one of the down-regulated miRNAs in PCa.MiR-30family consists of5miRNAs (miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) that are present in humans. From our microarray results, miR-30c is the only differential expression miRNA from the family in PCa. Previous studies showed that the expression levels of miR-30c frequently increased in various human tumor types, including ovarian carcinoma, mesothelioma, and primary cutaneous anaplastic large cell lymphoma. However, miR-30c expression levels could decrease in breast carcinoma, renal carcinoma and bladder cancer. However, the role of miR-30c in PCa remains unclear. The aim of our study is to investigate miR-30c expression pattern in both PCa cell lines and patients, to analyze the potential association between miR-30c and its clinical significance, and to determine its effect on tumorigenesis for PCa.Materials:Three human PCa cell lines PC3, DU145, LNCaP and one normal prostate epithelial cell line RWPE-1(as control) were purchased from the American Type Culture Collection (Manassas, VA, USA).A total of103patients with pathologically confirmed PCa were treated at Guangzhou First People’s Hospital affiliated to Guangzhou Medical University, between January2003and July2011. The study was approved by Research Ethical Committee of Guangzhou First People’s Hospital affiliated Guangzhou Medical University. Informed consents were obtained from all patients. None of the patients recruited in this study had received either chemotherapy or radiotherapy before the surgery. Those patients with prior anti-hormonal treatment were also excluded. Prostate tissues were immediately collected from these patients after radical prostatectomy. Each case was classified by WHO criteria and staged according to the TNM classification and the Gleason grading system.Methods:The miRNA microarray analyses were performed as previous described. In this experiment, frozen samples of PCa tissues and benign prostate tissues were obtained from the tissue bank at Guangzhou Medical University. Data are available at the Gene Expression Omnibus (GEO) repository database [http://www.ncbi.nlm.nih.gov/geo/, accession number GSE34932].The qRT-PCR analyses were carried out as previous described. MiRNA was extracted from3PCa cell lines,1normal prostate cell lines,103PCa tissues and20benign prostate tissues using miRNA extraction kit (Bioteke, China). The qRT-PCR analyses were performed according to the protocol of All-in-OneTM miRNA qRT-PCR Detection Kit (GeneCopoeia, China). MiRNA expression in each sample was normalized to U6B housekeeping gene as internal control. Relative quantification of miR-30c expression was calculated using the comparative cycle threshold (Ct) method. Each sample was examined in triplicate and date was expressed as mean±SD.We purchased commercial pGCMV/EGFP/Neo-Vector with an overexpression of miR-30c, and the corresponding blank-vector as control from Genepharma (Shanghai, China). LNCaP and DU145were plated in medium without antibiotics approximately24h before transfection. A final concentration of100nM of miR-30c overexpression vector and their respective negative control were transfected using Lipofectamine2000(Invitrogen) according to the manufacturer’s instructions. After48h, cells were harvested for further experiments.An MTT assay was used to evaluate the proliferation ability of transfected DU145and LNCaP cell lines. Briefly,1000-10000cells were seeded into96-well culture plates for12,24,48,72and96h. Cells were then incubated with20ul of3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT,5mg/ml, Sigma, St Louis, MO) for4h at37℃. The supernatant was then discarded and150ml of dimethyl sulfoxide (DMSO) was added to solubilize the crystals for20min at room temperature. Absorbance was measured at the wavelength of495nM using a spectrophotometer (Multiskan MK3, Thermo Scientific).Scratch wound-healing motility assay was performed to evaluate the migration ability of transfected DU145and LNCaP cell lines. A scratch was set with a pipette tip running through the dish when the cells were seeded into6-well plate and grown to confluence. Wound monolayers were washed twice with PBS to remove cell debris. Then, cells were transfected with miR-30c vector and control for48hours. After washing, representative pictures were then photographed. The cells migrated from the wound edge was counted. Data were represented as mean±SD of three independent experiments.We used a transwell invasion assay to evaluate the invasion ability of transfected DU145and LNCaP cells. CytoSelect Cell Invasion Kit (Cell Biolabs, Inc.) was used for invasion assay according to manufacture’s instructions. Briefly, after cells were transfected for48hours,5×104cells were harvested by trypsinization, washed with serum-free medium to25×104/ml and placed in the upper chamber. The lower chamber contained10%fetal bovine serum used as a chemo-attractant. Following24h of incubation at37℃with5%CO2, cells were then removed from the top of the inserts.Then, cells that migrated through the membrane were fixed, stained and quantified by counting16independent symmetrical visual fields under the microscope. Data was represented as mean±SD of three independent experiments.Statistical treatment:The software of SPSS version17.0for Windows (SPSS Inc, IL, USA) was used for statistical analysis. Statistical analyses of two groups were conducted using student’s t test. One-way ANOVA was used for comparisons of three independent groups. The results of MTT assay were analyzed by factorial design analysis of variance. Fisher’s exact test was performed to evaluate the association of miR-30c expression with clinicopathological features. Moreover, Kaplan-Meier method and log-rank test was then used to determine the relationship between miR-30c expression and survival. For multivariate analysis, Cox proportional regression model was also conducted. P value less than0.05was considered statistically significant.Results:1. From our microarray results, miR-30c was one of the most down-regulated miRNAs in PCa tissues compared with that in normal tissues (2.09-fold, P=0.016). QRT-PCR was also conducted to examine the expression levels of miR-30c in3human PCa cell lines (PC-3, DU145, and LNCaP) and a normal epithelial prostate cell line (RWPE-1). The data showed that miR-30expression levels were down-regulated in all3PCa cell lines compared with RWPE-1(PC-3,1.85-fold, P=0.025; DU145,2.04-fold, P=0.013; LNCaP,3.12-fold, P<0.001), which further confirmed the result from microarray analyses. Moreover, we further detected the expression levels of miR-30c in103PCa tissues and20benign prostate tissues. A significant decrease in miR-30c transcript levels was detected in PCa tissues, compared to that observed in benign tissues (3.27±0.62vs.6.85±0.33, P<0.001).2. We further analyzed the possible correlations between miR-30c expression levels and various clinicopathological parameters from103PCa samples. For clinical analysis, the patients were firstly classified into a high expression group(≥3.36) and a low expression group (<3.36) according to median relative expression (3.36) of miR-30c from qRT-PCR. The decreased expression levels of miR-30c was frequently observed in PCa patients with high Gleason score (P=0.009), advanced pathological stage (P=0.016), and biochemical recurrence (P=0.034). However, there was no statistically significant association between miR-30c expression and other features, including preoperative PSA levels and surgical margin (all P>0.05).3. We then further investigated the prognostic implication of miR-30c in PCa. We have conducted Kaplan-Meier survival and log-rank test analyses and found the reduced expression of miR-30c was associated with shorter biochemical recurrence-free survival time (P=0.023). Estimated5-year biochemical recurrence-free survival probability for the miR-30c high expression vs low expression group were85.1%vs60.4%, respectively. 4. Univariate analysis indicated that miR-30c (P<0.001), Gleason score (P<0.001), preoperative PSA (P=0.011), pathological tumor stage (P<0.001) and surgical margin (P=0.043) could be prognostic factors for biochemical recurrence-free survival. We then used the Cox proportional hazards multivariate model to examine the correlation of clinicopathological factors and miR-30c expression with biochemical recurrence-free survival. Indeed, multivariate analysis indicated that miR-30c (P=0.002), Gleason score (P=0.001), and pathological tumor stage (P=0.010) demonstrated statistically significant correlations in proportional hazard regression model. These results suggest that miR-30c expression level can serve as an independent predictor of biochemical recurrence-free survival in PCa patients with PCa.5. To determine whether miR-30c has effects on proliferation, migration and invasion of PCa cells, we have conducted several assays in vitro. Then, we transiently transfected LNCaP and DU145cells with has-miR-30c vector, respectively. Cells transfected with blank vector were used as negative control (miR-NC). The miR-30c expression levels were measured at48hours after transfection by qRT-PCR. Data showed that miR-30c expression level in miR-30c-transfected cells was significantly increased compared that in control cells (LNCaP, fold=3.87, P<0.01; Du145, fold=4.32, P<0.01).6. An MTT assay was used to evaluate the proliferation ability of miR-30c-transfected PCa cells. The result showed that the viability of miR-30c-transfected LNCaP and DU145cells was significantly lower than that of NC-transfected cells at24h,48h and72h after transfection (all P<0.05), indicating that overexpression of miR-30c has significant negative effects on cell proliferation.7. We further conducted wound healing assay to investigate the effect of miR-30c on cell migration. The results indicated that miR-30c markedly reduced the migration of LNCaP and DU145cells (LNCaP:240±19vs.520±43cells, P<0.01; DU145:490±67vs.660±52cells, P<0.01).8. In addition, the trans well assay with Matrigel was also performed to determine the effect of miR-30c on the invasive ability of PCa cells. The results clearly revealed that overexpression of miR-30c significantly reduced the migrated miR-30c-transfected LNCaP and DU145cells compared with control cells (LNCaP:67±6vs.120±13cells/field, P<0.01; DU145:130±15vs.220±18cells/field, P<0.01;). Taken together, these results suggested that miR-30c could suppress proliferation, migration and invasion of PCa cells..Conclusions:1. miR-30c can distinguish patients with prostate cancer from patients with BPH, indicating it may serve as turmor suppressor.2. miR-30c expression levels are associated with clinical progression for PCa.3. Although a larger cohort study in studying miR-30c expression is still necessary, our results indicate miR-30c may serve as prognostic factor for patients with PCa.4. miR-30c can serve as tumor suppressor, in which KRAS-MAPK pathway may be involved.5. miR-30c functions as a tumor suppressor for PCa cells by inhibiting cell proliferation, invasion and migration. |
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