The Biologicai Function,Regulatory Mechanism And Clinical Significance Of MicroRNA-214 In Bladder Cancer

BackgroundAs one of the common urologic neoplasms, bladder cancer is the ninth most common cancer diagnosed worldwide and accounts for the second leading cause of death among male patients with genitourinary tract malignancies worldwide with 386,300 new cases and 150,200 death cases every year. Bladder cancer is also the fourth most common cancer diagnosed among male patients in developed countries and the seventh in developing countries. Although approximately 75% of cases are grouped as non-muscle-invasive bladder cancer (NMIBC) with a relatively high 5-year survival rate, other 25% of patients with muscle-invasive bladder cancer (MIBC) are subjected to subsequent metastatic disease after the first aggressive treatment. Investigating new therapeutic modalities and identifying prognostic biomarkers for bladder cancer based on novel molecular networks have become research hotspots recently.MicroRNAs (miRNAs) represent a class of small, endogenous, non-coding, 22-nucleotides RNA molecules that function as post-transcriptional regulator by partially pairing with the 3’untranslated regions (3’-UTR) of target mRNAs, leading to mRNA degradation or translational repression. It has been firmly established that miRNAs can regulate>60% of human protein-coding genes and play crucial roles in various physiopathologic processes. miRNAs have been identified either as tumor suppressors or oncogenes. In view of their disease- and tissue-specific expression profiles and amazing regulatory potential, miRNAs are being appraised as fascinating biomarkers for cancer diagnosis and prognosis.Some studies have reported that miR-214 was up-regulated and contributed to disease progression and distant metastases in malignant melanoma, whereas others have indicated that miR-214 was down-regulated and had a tumor-suppressive effect in cervical cancer, breast cancer and human hepatocellular carcinoma. The above evidence points out that miR-214 may play pivotal and diverse roles in oncogenesis of various tumor types, yet potential mechanisms of miR-214 are still not completely unraveled. So far, there have been little published papers involving the functional analysis and molecular network of miR-214 in bladder cancer, though a few miRNA profiling studies showed that miR-214 was dysregulated in bladder cancer.Currently, the diagnostic and surveillance strategies for bladder cancer mainly rely on cystoscopy and urine cytology. However, the former is rather invasive, costly and difficult to detect flat lesions or carcinoma in situ (CIS); the latter is more specific but fails to detect low-grade bladder cancer sensitively. Therefore, investigating novel noninvasive circulating biomarkers with high sensitivity and specificity is of utmost importance for diagnosis and prognosis of bladder cancer. Tumor-specific circulating miRNAs have been recognized as potential noninvasive biomarkers in cancer and can stably exist in various body fluids (including serum, plasm, urine, tears, bronchial lavage, colostrum, breast milk, as well as cerebrospinal, pleural, peritoneal, seminal and amniotic fluids) with extracellular vesicular structures called exosomes protecting them from degradation. In this paper, we aimed to investigate whether circulating miR-214 in urine could be detected and serve as a noninvasive biomarker, supposing that its expression pattern reflects that in the neoplastic tissues. It is critical to figure out whether expression profiles of circulating miRNAs mirror miRNA profiles of tumor tissues for improving the overall diagnostic specificity of the biomarkers.In view of these, by examining miR-214 expression in bladder cancer tissues and urine, handling its expression levels in vitro, identifying its downstream target gene and performing relative functional experiments, we aim to assess the value of miR-214 in early diagnosis and prognostic judgment of bladder cancer, investigate its functional role and molecular network in proliferation, apotosis, migration and invasion of bladder cancer and lay the basis for finding out novel diagnostic, prognostic and therapeutic targets for bladder cancer.Part 1 Down-regulation of miR-214 expression in bladder cancer tissues and its correlation with clinicopathological parameters and prognosisObjective:To investigate the miR-214 expression status in bladder cancer tissues and its correlation with clinicopathological parameters and prognosis.Methods:1. Expression level of miR-214 was detected in 106 pairs of human bladder cancer frozen tissues (31 NMIBC and 75 MIBC) and matched adjacent normal specimens by qRT-PCR.2. The correlation between miR-214 expression and clinicopathological parameters in bladder cancer tissues were estimated using Mann-Whitney U test or Kruskal-Wallis test, as appropriate.3. Survival curves of miR-214 and other clinicopathological parameters in bladder cancer were delineated with the univariate Kaplan-Meier method and compared by log-rank test.4. Multivariate Cox proportional hazards regression model was applied to assess prognostic values of miR-214 and other clinicopathological parameters in bladder cancer.Results:1. miR-214 was significantly underexpressed in 74% of bladder cancer tissues (P<0.001) with the median expression level ten-fold lower than that of the noncancerous specimens (median expression=0.084 and 0.864, respectively).2. The attenuated miR-214 expression was significantly associated with history of NMIBC (P=0.033), multifocality (P=0.003), higher grade (P<0.001), higher tumor stage (P<0.001) and higher lymph node status (P<0.001).3. During the follow-up,54.7%(58 of 106) of patients with bladder cancer experienced recurrence and the 5-year overall survival rate was 61.3%(65 of 106). Kaplan-Meier survival curve revealed that bladder cancer patients with low miR-214 expression underwent significantly shorter RFS (log-rank test=26.207; P<0.001) and OS (log-rank test=45.174; P<0.001) than those with high expression, while multivariate Cox regression analysis showed that miR-214 was neither independent prognostic variable of RFS (P=0.269) nor OS (P=0.397) for bladder cancer patients.4. Then, separate analysis was performed for the NMIBC and MIBC tumors. In the NMIBC group,54.8% of NMIBC patients (17/31) presented recurrence, but miR-214 neither predicted RFS or OS when dysregulated by Kaplan-Meier analysis. In the MIBC group, local or metastatic relapse was observed for 54.7% of the 75 MIBC patients (41/75) and the 5-year overall survival rate was 45.3%(34/75). Kaplan-Meier survival analysis revealed that decreased expression of miR-214 was significantly associated with poorer prognosis in terms of both RFS (log-rank test=14.214; P<0.001)and OS (log-rank test=13.797; P<0.001). Multivariate Cox regression analysis showed that miRNA-214 was an independent prognostic predicator of RFS (RR=0.178; 95%CI,0.072-0.443; P<0.001) and OS (RR=0.230; 95%CI,0.097-0.547; P<0.001)for MIBC.Conclusion:miR-214 is attenuated in bladder cancer tissues and correlated with adverse clinical features, which manifest that it may be embroiled and play a role in the development and progression of bladder cancer. miR-214 is an independent prognostic predicator of RFS and OS for MIBC. associated with poorer prognosis in terms of both RFS (log-rank test=14.214; P<0.001)and OS (log-rank test=13.797; P<0.001). Multivariate Cox regression analysis showed that miRNA-214 was an independent prognostic predicator of RFS (RR=0.178; 95%CI,0.072-0.443; P<0.001) and OS (RR=0.230; 95%CI,0.097-0.547; P<0.001)for MIBC.Conclusion:miR-214 is attenuated in bladder cancer tissues and correlated with adverse clinical features, which manifest that it may be embroiled and play a role in the development and progression of bladder cancer. miR-214 is an independent prognostic predicator of RFS and OS for MIBC.Part 2 Biological function and regulatory mechanism of miR-214 in pathogenesis of bladder cancerObjective:To handle miR-214 expression levels in vitro, identify its downstream target gene and perform relative functional experiments, and investigate functional role and molecular network of miR-214 in bladder cancer.Methods:1. miR-214 expression level was detected in the nonmalignant SV-40 immortalized bladder epithelial cell line (SV-HUC-1) and two bladder cancer cell lines (T24,5637) by using qRT-PCR.2. Bladder cancer cells (T24 and 5637) were transiently transfected with miR-214 mimic using Lipofectamine 2000 as experiment groups, transfected with miR-negative control mimic as negative control group (miR-NC group), and merely transfected with Lipofectamine 2000 as blank group (mock group). To ensure actual transfection, qRT-PCR was carried out to detect transfection efficiencies in every experiment 24 hours later. CCK8 assay, wound-healing assay, Transwell assay and Annexin V-FITC/PI double labeling flow cytometry were applied to assess the changes in cell proliferation, migration, invasion and apoptosis posttransfection, respectively. Western blot was used to detect caspase-3 and cleaved caspase-3 after transfection.3. To identify potential target genes of miR-214, we took advantage of an integrated bioinformatics analysis, starBase v 2.0, which can jointly use five public available algorithms (including targetScan, picTar, PITA, RNA22 and miRanda). The pmiR-ReportTM vectors were constructed with wild type (WT)-target gene sequences or mutant (MUT)-target gene sequences. Bladder cancer cells (T24 and 5637) were co-transfected with miR-NC/miR-214 mimics and WT-target gene 3’-UTR vector/MUT-target gene 3’-UTR vector using Lipofectamine 2000. The activities of Renilla and firefly luciferases in cell lysates were measured using the Dual-Luciferase assay kit. Expression of target gene in both mRNA and protein levels were detected by qRT-PCR and western blot in miR-214 reexpressed bladder cancer cells.4. Expression level of target gene was detected in bladder cancer frozen tissues and matched adjacent normal specimens by qRT-PCR. The relationship between target gene expression and clinicopathological features were analyzed using Mann-Whitney U test or Kruskal-Wallis test, as appropriate. Survival curves of target gene in bladder cancer were depicted with the Kaplan-Meier method and compared by log-rank test. Cox proportional hazards regression model was used to estimate prognostic value of target gene in bladder cancer.5. siRNAs against target gene were transfected into bladder cancer cells (T24 and 5637). qRT-PCR and western blot were applied to detect transfection efficiencies. CCK8 assay, wound-healing assay, Transwell assay and Annexin V-FITC/PI double labeling flow cytometry were applied to assess the changes in cell proliferation, migration, invasion and apoptosis posttransfection, respectively. Western blot was used to assess caspase-3 and cleaved caspase-3 posttransfection.Results:1. Expression level of mature miR-214 was found to be significantly down-regulated in bladder cancer cells (T24,5637) compared with that in HUC cells by using qRT-PCR.2. Relative miR-214 expression in T24 and 5637 cells transfected with miR-214 mimic was 22319±2284 (P<0.001) and 10276±1836 (P<0.001) times higher than that transfected with mock or miR-NC mimic confirmed by way of real-time PCR 24 hours posttransfection. Compared with mock group and miR-NC group, the proliferation ability of cells in miR-214 mimic group was significantly decreased 48h, 72h and 96h posttransfection (P<0.001). In wound-healing assay, the healing rate of miR-214 mimic group (T24,15.17%±2.86%; 5637,43.33%±3.33%) were significantly lower than those in mock group (T24,82.00%±1.41%;5637, 75.67%±3.08%) and miR-NC group (T24,80.83%±3.60%; 5637,75.00%±3.52%) 24h after transfection (P<0.001).In Transwell migration assay, the number of penetrating T24 cells in miR-214 mimic group was (49.46±4.36)% of that in mock group (P<0.001) and the number of penetrating 5637 cells in miR-214 mimic group was (25.42±2.36)% of that in mock group (P<0.001). In Transwell invasion experiment, the penetrating rate of cells in miR-214 mimic group (T24, 11.67%±3.72%; 5637,3.40%±0.32%) were significantly lower than those in mock group (T24,41.17%±6.21%; 5637,26.17%±3.19%) and miR-NC group (T24, 40.50%±5.79%; 5637,25.17%±4.07%) (P<0.001). Apoptosis rate of cells in miR-214 mimic group (T24,57.10%±2.85%; 5637,41.94%±2.84%) were significantly higher than those in mock group (T24,18.61%±1.56%; 5637,12.72%±3.26%) and miR-NC group (T24,18.81%±1.57%; 5637,13.05%±3.17%) 24h after transfection (P<0.001). Protein level of caspase-3 and cleaved caspase-3 in miR-214 mimic group was significantly higher than those in miR-NC group 48h posttransfection.3. Integrated bioinformatics analysis (targetScan, picTar, PITA and miRanda) demonstrates that PDRG1 contains one potential complimentary miR-214 binding site on its 3’-UTR. PDRG1 was verified as a direct downstream target for miR-214 by Dual-Luciferase reporter assay in bladder cancer (P<0.001). Depressed endogenous expression of PDRG1 in both mRNA (P<0.001) and protein levels were observed in miR-214 reexpressed bladder cancer cells.4. Compared to matched normal tissues (median expression=0.00024), PDRG1 was significantly overexpressed in 81% of bladder cancer tissues (median expression=0.00632) detected by qRT-PCR (P<0.001). Moreover, there was an inverse correlation between miR-214 and the PDRGl expression in bladder cancer tissues (r=-0.367, P<0.001). Up-regulated PDRG1 expression was significantly associated with gender (P=0.012), higher tumor grade (P=0.001), higher tumor stage (P<0.001) and higher lymph node status (P<0.001). Kaplan-Meier survival curve showed that bladder cancer patients with high PDRGl expression suffered significantly shorter RFS (log-rank test=6.578; P=0.010) and OS (log-rank test=8.990; P=0.003) than those with low expression, while Cox regression analysis demonstrated that PDRGl was neither independent prognostic variable of RFS (P=0.457) nor OS (P=0.145) for bladder cancer patients. Performing separate Kaplan-Meier analyses for the NMIBC and MIBC tumors, PDRG1 neither predicted RFS nor OS when dysregulated in both groups.5. Relative PDRG1 mRNA expression in T24 and 5637 cells transfected with si-PDRG1 was 0.33 ±0.09 (P<0.001) and 0.57±0.05 (P<0.001) times of that in mock group 24h after transfection. Compared with si-NC group and mock group, the proliferation ability of cells in si-PDRG1 group was significantly decreased 48h,72h and 96h posttransfection (P<0.001). In wound-healing assay, the healing rate of si-PDRG1 group (T24,20.67%±2.42%; 5637,52.00%±5.97%) were significantly lower than those in mock group (T24,81.00%±1.55%; 5637,73.83%±2.93%) and miR-NC group (T24,79.00%±3.52%; 5637,71.83%±1.94%) 24h after transfection (P<0.001). In Transwell migration assay, the number of penetrating T24 cells in si-PDRG1 group was (55.78±11.23)% of that in mock group (P<0.001) and the number of penetrating 5637 cells in si-PDRG1 group was (35.59±2.28)% of that in mock group (P<0.001). In Transwell invasion experiment, the penetrating rate of cells in si-PDRG1 group (T24,14.33%±2.94%; 5637,7.00%±2.61%) were significantly lower than those in mock group (T24,44.50%±2.35%; 5637,31.00%±2.61%) and si-NC group (T24,43.83%±2.99%; 5637,29.67%±4.89%) (P<0.001). Apoptosis rate of cells in si-PDRG1 group (T24,52.83%±4.23%; 5637,34.75%±3.39%) were significantly higher than those in mock group (T24,16.89%±1.69%; 5637, 11.16%±2.51%) and si-NC group (T24,17.07%±1.82%; 5637,11.41%±2.50%) 24h after transfection (P<0.001). Protein level of PDRG1 was significantly lower while protein level of caspase-3 and cleaved caspase-3 in si-PDRG1 group was significantly higher than those in si-NC group 48h posttransfection.Conclusion:Our findings manifest for the first time that miR-214 could exert tumor-suppressive effects in bladder cancer by directly down-regulating oncogene PDRG1 to inhibit proliferation, migration and invasion while prompting apotosis of bladder cancer cells. Our study also supplies experimental evidences for gene target therapy of bladder cancer. proliferation ability of cells in si-PDRG1 group was significantly decreased 48h,72h and 96h posttransfection (P<0.001). In wound-healing assay, the healing rate of si-PDRG1 group (T24,20.67%±2.42%; 5637,52.00%±5.97%) were significantly lower than those in mock group (T24,81.00%±1.55%; 5637,73.83%±2.93%) and miR-NC group (T24,79.00%±3.52%; 5637,71.83%±1.94%) 24h after transfection (P<0.001). In Transwell migration assay, the number of penetrating T24 cells in si-PDRG1 group was (55.78±11.23)% of that in mock group (P<0.001) and the number of penetrating 5637 cells in si-PDRG1 group was (35.59±2.28)% of that in mock group (P<0.001). In Transwell invasion experiment, the penetrating rate of cells in si-PDRG1 group (T24,14.33%±2.94%; 5637,7.00%±2.61%) were significantly lower than those in mock group (T24,44.50%±2.35%; 5637,31.00%±2.61%) and si-NC group (T24,43.83%±2.99%; 5637,29.67%±4.89%) (P<0.001). Apoptosis rate of cells in si-PDRG1 group (T24,52.83%±4.23%; 5637,34.75%±3.39%) were significantly higher than those in mock group (T24,16.89%±1.69%; 5637, 11.16%±2.51%) and si-NC group (T24,17.07%±1.82%; 5637,11.41%±2.50%) 24h after transfection (P<0.001). Protein level of PDRG1 was significantly lower while protein level of caspase-3 and cleaved caspase-3 in si-PDRG1 group was significantly higher than those in si-NC group 48h posttransfection.Conclusion:Our findings manifest for the first time that miR-214 could exert tumor-suppressive effects in bladder cancer by directly down-regulating oncogene PDRG1 to inhibit proliferation, migration and invasion while prompting apotosis of bladder cancer cells. Our study also supplies experimental evidences for gene target therapy of bladder cancer.Part 3 Detection of cell-free miR-214 in urine and its potential diagnostic and prognostic value for bladder cancerObjective:To investigate potential of urinary cell-free miR-214 as a noninvasive biomarker for bladder cancer in this report.Methods:1. At screening stage, miR-214 expression in medium from 2 bladder cancer cell lines (T24 and 5637) was detected by qRT-PCR to determine whether it is secretory, and cell-free miR-214 expression was also determined in urine samples from 20 bladder cancer patients and 20 healthy controls.2. At validation stage, expression levels of miR-214 were assayed by qRT-PCR in urine samples from an independent set of 192 preoperative bladder cancer patients,80 matching postoperative patients and 169 healthy controls.79 matched tumor tissues were also analyzed to determine whether urinary cell-free miR-214 mirrored expression of miR-214 in tumors.3. Mann-Whitney U test or Kruskal-Wallis test was applied to estimate the correlation between urinary cell-free miR-214 expression and clinicopathological parameters as appropriate.4. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic efficiency of urinary extracellular miR-214.5. Survival curves of urinary cell-free miR-214 in bladder cancer were delineated with the univariate Kaplan-Meier method and compared by log-rank test.6. Multivariate Cox proportional hazards regression model was applied to assess prognostic value of urinary cell-free miR-214 in bladder cancer.Results:1. miR-214 was secreted from bladder cancer cell lines and down-regulated in urine supernatant of bladder cancer patients compared with healthy controls (P<0.001) during screening stage.2. At the validation stage, cell-free miR-214 levels were significantly attenuated in preoperative urine from bladder cancer patients (P<0.001), whereas its expression significantly increased in matched postoperative urine from patients who underwent tumor resection (P<0.001). There was a significantly positive correlation between miR-214 level in preoperative urine supernatant and matching tumor tissues (r= 0.6539,95%CI=0.5010 to 0.7673,P<0.001).3. Underexpressed extracellular miR-214 in urine was significantly associated with age (P=0.028), history of NMIBC (P=0.044), higher tumor stage (P<0.001), higher lymph node status (P<0.001) and higher grade (P<0.001).4. ROC curve analyses indicated that urinary cell-free miR-214 could forcefully differentiate bladder cancer (area under the curve; AUC= 0.838; 95% CI= 0.796 to 0.875) patients from healthy controls. At a cutoff value of 0.1551, the optimal sensitivity, specificity, positive and negative predictive values were 90.53%,65.62%, 69.90% and 88.70% in identifying a patient with bladder cancer.5. During the follow-up,58.9%(113/192) of patients with bladder cancer experienced recurrence and the 5-year overall survival rate was 57.3%(110/192). Kaplan-Meier survival curve revealed that bladder cancer patients with low urinary cell-free miR-214 expression underwent significantly shorter RFS (log-rank test=29.734; P<0.001) and OS (log-rank test=61.711; P<0.001) than those with high expression, while multivariate Cox regression analysis showed that urinary cell-free miR-214 was neither independent prognostic variable of RFS (P=0.277) nor OS (P=0.667) for bladder cancer patients.4. Then, separate analysis was performed for the NMIBC and MIBC tumors. In the NMIBC group,49.2% of NMIBC patients (31/63) presented recurrence, but urinary cell-free miR-214 neither predicted RFS or OS when dysregulated by Kaplan-Meier analysis.In the MIBC group, local or metastatic relapse was observed for 63.6% of the 129 MIBC patients (82/129) and the 5-year overall survival rate was 36.4% (47/129). Kaplan-Meier survival analysis revealed that decreased expression of urinary cell-free miR-214 was significantly associated with poorer prognosis in terms of both RFS (log-rank test=37.381, P<0.001) and OS (log-rank test=37.376,P<0.001). Multivariate Cox regression analysis showed that urinary cell-free miRNA-214 was an independent prognostic predicator of RFS (RR=0.264; 95%CI,0.149-0.468; P<0.001) and OS (RR=0.282; 95%CI,0.160-0.495; P<0.001) for MIBC.Conclusions:Urinary cell-free miR-214 is a hopeful non-invasive biomarker for tumor stratification, early diagnosis and prognostic assessment of bladder cancer.