Clinical And Experimental Analysis Of MicroRNAs As Early Diagnosis And Stage Predictive Markers For Bladder Cancer

BackgroundBladder cancer (BC) is the most common malignancy in the urinary tract in China and is a serious threat to human health. BCcari be divided into nonmuscular-invasive BC (NMIBC) (pTis, pTa, and pTl) and muscular-invasive BC (MIBC)(pT2-pT4). The biological characteristics of the two tumors and the clinical treatment program are significantly different. NMIBC is also known as superficial BC. The minimally invasive treatment choice of NMIBC is transurethral resection of the bladder tumor (TUR-BT) and the prognosis is relatively good. MIBC features a high degree of malignancy, early metastasis and poor prognosis. The 5-year survival rate of MIBC is only 50-60%. For MIBC, the standard treatment program is radical cystectomy and pelvic lymph node dissection. About 30% patients are diagnosed with MIBC and 30% of NMIBC patients will progress to MIBC. Early diagnosis of BC and preoperative prediction of MIBC is the key to rational choice of treatment options.Currently, cystoscopy, urine cytology and image check are the screening methods of BC. Cystoscopy is the common method of diagnosis of BC, but it can cause varying degrees of urethral and bladder injury, infection, and other complications. Urinecytology is noninvasive and has the advantages of specificity. However, its sensitivity is lower than 50%, only 30% for early patients, and is easy subject to subjective factors. CT and ultrasonography are the most commonly used imaging methods for the diagnosis and preoperative staging prediction of BC. However, it is not easy to detect small lesions in the bladder, and the prediction accuracy is limited. TUR-BT is mainly used for the treatment of NMIBC, and also could provide a staging basis for MIBC. But because of the risk and compliance of surgery, it is limited in the application of BC staging prediction. The focus of attention is to find new biomarkers with high sensitivity and specificity for early detection of BC and preoperative prediction of MIBC.MicroRNAs (miRNAs) play a vital role in the development of tumors by regulating expression of genes at post-transcriptional levels by binding to completely or incompletely target mRNA molecules. The analysis of miRNAs in different types of tumors revealed that the miRNAs expression profile had significant tumor specificity and had a specific expression pattern in different stages of tumors,suggesting that it could be a potential marker for tumor diagnosis. Recent studies have shown that there are abundant and stable miRNAs in the urine supernatant, which are closely related to the occurrence and development of urinary system malignancy, this provides a new idea for noninvasive diagnosis of tumor. The selection of urinary cell-free miRNAs and the introduction of a model for the noninvasive diagnosis and prediction of MIBC will help early detection and clinical treatment regimens of BC.Quantitative real time polymerase chain reaction (RT-qPCR) is the most commonly used technique for quantitative detection of miRNAs. The standardization of detection data is mainly based on the correction of the reference gene. The use of an optimal reference gene for the detection of miRNAs can improve the reproducibility and sensitivity of RT-qPCR assays. However, there has been no study on searching for the reference gene for the screening of urinary cell-free miRNAs by RT-qPCR in BC.In this study, we first used high-throughput sequencing technology and RT-qPCR to screen and determine the optimal reference gene that can be used for the quantitative detection of urinary cell-free miRNAs. Using the identified reference genes, differentially expressed miRNAs were measured. A diagnostic model for BC using logistic regression analysis was constructed and evaluated. Also, the differential expression profiles of urinary miRNAs with predictive value of MIBC were screened by the above techniques. The predictive model of MIBC was established by logistic regression analysis and validated. Finally, the biological function of the related miRNAs in BC was studied by using BC tissue samples and in vitro cell experiments.This study established new biomarkers of urinary cell-free miRNAs for early diagnosis and staging prediction in BC, in order to provide new ways for noninvasive screening and staging prediction and provide new targets for the effective treatment of BC.Part One. Identification and validation of reference genes for RT-qPCR detection of urinary microRNAs in bladder cancerPurposesQuantitative real time polymerase chain reaction (RT-qPCR) is the most commonly used method for the detection of cell-free microRNAs (miRNAs) in urine.It is important to obtain reliable RT-qPCR detection data by selecting a more stable reference gene. The aim of this study was to screen and validate the reference genes in RT-qPCR detection of urinary cell-free miRNAs in bladder cancer (BC).Methods1 . Urine samples from 6 BC patients and 6 healthy donors were sequenced on a Miseq sequencing platform (Illumina). In order to identify potential reference miRNAs for BC, we selected miRNAs showed that lower than 1.2-fold changes with no significant differences between the two groups.2. Candidate reference miRNAs and U6 were subjected to RT-qPCR assays using a cohort of 80 BC patients and 80 controls, because U6 is commonly used for expression normalization.3. Variable stabilies of selected reference miRNAs was evaluated using geNorm and NormFinder, in order to determine the optimal reference gene.4. To further validate the stability of the identified reference genes, we performed RT-qPCR assays on another cohort (63 BC patients and 63 controls).Results1. To identify potential reference miRNAs for BC, we selected miRNAs showed lower than 1.2-fold changes with no significant differences between the two groups. Using the above criterion, 13 miRNAs (let-7b-5p, miR-532-5p, miR-23b-5p,miR-100-5p, miR-20a-5p, miR-28-3p, miR-34a-5p, miR-17-5p, miR-133a-3p,miR-194-5p, miR-30b-5p, miR-98-5p, and miR-99b-5p) were determined as candidate reference gene. U6 was also selected because U6 is commonly used for expression normalization.2. Among the 13 candidate reference miRNAs and U6, four miRNAs(miR-17-5p, miR-133a-3p, miR-30b-5p, and miR-98-5p) were not detectable and the Cq values of two miRNAs (miR-194-5pand miR-99b-5p) were above 35. Seven reference miRNAs (miR-100-5p, miR-20a-5p, miR-23b-5p, miR-28-3p, miR-34a-5p,miR-532-5p, and let-7b-5p) and U6passed the quality control process and there was no evidence of differential expression of these genes between BCs and controls.3. Variable stabilies of selected reference miRNAs and U6 were evaluated using geNorm and NormFinder. The two algorithms both identified let-7b-5p as the most stably expressed reference gene, and let-7b-5p and miR-532-5p were selected as the most stable pair of reference genes.4. To further validate the stability of the identified reference genes, we used another cohort (63 BC patients and 63 controls). Based on the Cq value of each validated reference gene, there was no evidence of differential miRNA expression between BCs and controls and among different stages of BC.ConclusionWe identified the combination of let-7b-5p and miR-532-5p as the most suitable reference genes for urine miRNA detection by RT-qPCR.Part Two. Cell-free microRNA expression signatures in urine serve as novel noninvasive biomarkers for the .diagnosis and recurrence prediction of bladder cancerPurposesBladder cancer (BC) is one of the most common and lethal urological malignances worldwide. The incidence of BC has substantially increased over the last years. Currently, cystoscopy, urine cytology and image check are the screening methods of BC. Despite their advantage, they are invasive or lack sensitivity and specificity and cannot meet clinical needs. Previous studies have shown the existence of a large amount of stable microRNAs (miRNAs) in human urine, and laid the foundation for studying the role of urinary miRNAs in the diagnosis of BC. In this study, we aimed to develop a urinary miRNAs panel for diagnosing and predicting recurrence of BC.Methods1. Urine samples from 6 BC patients and 6 healthy donors were sequenced on a Miseq sequencing platform (Illumina). MiRNAs with larger than 2-fold change between the two groups were selected as candidate miRNAs.2. In the training phase, expression of the selected miRNAs was measured by RT-qPCR assays in a cohort of 150 BC patients and 150 controls. Receiver operating characteristic (ROC) curves were established to discriminate BCs from controls.3. A diagnostic miRNA panel was constructed based on a logistic regression model for the differentiation between the BC group and the control group. Then it was validated by RT-qPCR assays in a cohort of 120 BC patients and 120 controls.4. Urine cytology was used to compare the diagnostic performance of the miRNA panel in a cohort of 120 BC patients and 120 controls in the validation set.5. The Mann-Whitney U test or Kruskal-Wallis test was used to test the correlation between expression levels of miRNAs and clinicopathological characteristics.6. Kaplan-Meier analysis with the log-rank test was used for survival curves.The Cox proportional hazard regression model was used to determine the independent prognostic factors.Results1. Miseq sequencing revealed 256 and 308 miRNAs with at least 50 copies in BC patients and controls, respectively. The miRNAs were considered as altered only if the absolute fold change was significantly larger than 2-fold between BC and control groups. 23 miRNAs were differentially expressed between the two groups,among which 16 miRNAs were highly expressed (let-7e-5p, let-7i-5p, miR-7-5p,miR-22-3p, miR-23a-3p, miR-29a-3p, miR-125b-5p, miR-126-5p, miR-148a-3p,miR-184-5p, miR-181b-5p, miR-186-5p, miR-221-3p, miR-375, miR-574-5p, and miR-941) and 7 miRNAs were down-regulated (miR-10a-3p, miR106b-5p,miR-200a-3p, miR-423-5p, miR-429, miR-455-5p, and miR-505-3p).2. The RT-qPCR assay was used to confirm expressions of 23 candidate miRNAs which were selected from the previous step. RT-qPCR analysis revealed that five miRNAs (miR-7-5p, miR-22-3p, miR-29a-3p,miR-126-5p, and miR-375) were up-regulated and two (miR-200a-3pand miR-423-5p) were down-regulated in BCs.The diagnostic performance of the seven miRNAs was evaluated by ROC analysis.The AUCs of these miRNAs were 0.639, 0.803, 0.67, 0.705, 0.748, 0.692, and 0.72,respectively.3. In the training set, a stepwise logistic regression model was constructed for diagnosis of BC as follows: logit(p=BC)=0.7792-(0.0823×miR-7-5p)-(0.2015×miR-22-3p)-(0.0223×miR-29a-3p)-(0.0793×miR-126-5p)+(0.1522xmiR-20 Oa-3p)-(0.1545×miR-375)+(0.2234xmiR-423-5p). ROC analysis revealed that the AUC of the miRNA panel was 0.923 (95%CI, 0.886 to 0.950). The sensitivity of the miRNA panel was 82.00% and the specificity was 96.00%.4. In the validation set, the ability to predict BC by the constructed seven-miRNA panel was further assessed. The AUC of the miRNA panel was 0.916(95% Cl, 0.873 to 0.948, sensitivity=85.00%, specificity=86.67%). The AUCs of the panel for Ta, T1 and T2-T4 were 0.864 (sensitivity=81.25%, specificity=85.00%),0.930 (sensitivity=89.74%, specificity=86.67%) and 0.978 (sensitivity=97.78%,specificity=90.83%), respectively. The AUCs of the panel for low grade BC and high grade BC were 0.911 (sensitivity=80.88%, specificity=91.67%) and 0.958(sensitivity=94.23%, specificity=89.17%), respectively. In addition, urine cytology was used to compare the diagnostic performance of the seven-miRNA panel. The AUC of urine cytology was 0.642 (95% Cl, 0.577 to 0.702, sensitivity=30.83%,specificity=97.50%). The corresponding AUCs of this panel for Ta, T1 and T2-T4 were significantly higher than those of urine cytology, which were 0.531,0.628 and 0.724, respectively.5. The correlation between expression levels of the seven miRNAs and clinicopathological characteristics: high level of miR-22-3p and miR-375 expression,along with the low level of miR-423-5p expression significantly correlated with advanced tumor stage (P=0.02, P=0.03, and P=0.03, respectively). High level of miR-29a-3p and miR-375 expressions correlated with positive lymph node metastasis,and higher level of miR-7-5p expression correlated with a higher tumor grade (P=0.03,P=0.04,and P=0.02, respectively).6. The identification of potential prognostic factors for the recurrence of BC:survival analysis was performed in the nonmuscular-invasive BC (NMIBC) group and the muscular-invasive BC (MIBC) group. In the NMIBC group, according to the Kaplan-Meier curve, patients with high miR-22-3p levels and low miR-200a-3p levels had dramatically lower recurrence-free survival (RFS) than those with low miR-22-3p levels and high miR-200a-3p levels (P=0.002, and P=0.040, respectively). Univariate Cox proportional hazards regression model analysis revealed a significant correlation between recurrence and miR-22-3p (P=0.004), miR-200a-3p (P=0.045) and tumor stage (P=0.006). Parameters significantly related to RFS in the univariate analysis were then put into the multivariate analysis to identify independent factors for prognoses of NMIBC. Multivariate analysis showed that miR-22-3p, miR-200a-3p and tumor stage retained their significance for recurrence of NMIBC (P=0.024,P=0.008, and P=0.008, respectively). In the MIBC group, there were no miRNAs that influenced patient predicted recurrence (all at P> 0.05).ConclusionA seven-miRNA panel (miR-22-3p, miR-29a-3p, miR-375, miR-7-5p,miR-126-5p, miR-423-5p, and miR-200a-3p) was designed as a novel diagnostic biomarker for BC based on a multivariate logistic regression model. Compared with traditional urine cytology, this panel was significantly superior based on its higher diagnostic accuracy. Furthermore, out of the seven miRNAs, miR-22-3p and miR-200a-3p were identified as independent factors for tumor recurrence in NMIBC.These findings suggest that urinary miRNAs obtained in a noninvasive manner may play important roles in the diagnosis and recurrence prediction of BC.Part Three. Prediction of muscular-invasive bladder cancer using a urinary cell-free microRNA panelPurposesBladder cancer (BC) can be divided into non-muscular invasive BC (NMIBC)(pTis, pTa, and pTl) and muscular-invasive BC (MIBC) (pT2-pT4).The biological characteristics of the two and the clinical treatment programsare significantly different.Non-muscular invasive BC also is known as superficial BC. The preferred transurethral resection of the bladder for minimally invasive treatment, and the prognosis is relatively good. However, muscular-invasive BC features a high degree of malignancy and poor prognosis. The 5-year survival rate was only 50-60%. The standard treatment program is the use of radical cystectomy at the same time as pelvic lymph node dissection. Preoperative treatment of muscular-invasive BC and non-muscular invasive BC is the key to the choice of treatment options, but also the current clinical problems need to be solved. The present study was designed to identify unique urinary cell-free microRNA (miRNA) signatures for prediction and prognosis of MIBC by Miseq sequencing and logistic regression model analysis.Methods1. In the discovery phase, pooled urine samples from 6 NMIBC patients, 6 MIBC patients and 6 healthy donors were subjected to Miseq sequencing in order to identify differentially expressed miRNAs among the three groups.2. In the training phase, potential miRNAs were tested with RT-qPCR in an independent cohort of 113 NMIBC patients, 113 MIBC patients and 156 controls to identify potential miRNAs. Receiver operating characteristic (ROC) curves were used to differentiate MIBC from NMIBC.3. Based on the logistic regression model, a MIBC miRNA panel was constructed. Based on the logistic regression model, the MIBC miRNA panel was constructed.In the validation phase, urine samples from another cohort of 102 NMIBC patients and 83 MIBC patients were used to validate the diagnostic accuracy of the constructed miRNA panel.4. Traditional urine cytology was performed on the 102 NMIBC patients and 83 MIBC patients. Comparisions were performed among the miRNA panel, urine cytology, and grade for MIBC prediction.5. The Kaplan-Meier method was used to estimate survival curves with the log-rank test. The Cox proportional hazards regression model was used to identify the independent prognostic factors of MIBC.Results1. The Miseq sequencing method was used to screen miRNAs with significant differential expression levels among MIBC, NMIBC and control groups. Among 478 urine miRNAs scanned by Miseq sequencing, 235, 188 and 276 miRNAs were detectable in MIBC, N-MIBC and control group, respectively. The differential expression of an miRNA was considered when at least 50 copies were detected and >2-fold change was observed among the three groups. According to these criteria, 24 miRNAs (let-7a-5p, let-7b-5p, miR-1, miR-100-3p, miR-126-5p, miR-133a,miR-143-3p, miR-146a-5p, miR-148a-3p, miR-17-5p, miR-181a-5p, miR-186-5p,miR-194-5p, miR-200a-5p, miR-22-3p, miR-23b-3p, miR-24-3p, miR-28-3p,miR-29a-3p, miR-30b-5p, miR-320a, miR-34a-5p, miR-375, and miR-1246) were differentially expressed among MIBCs, NMIBCs and controls.2. The 24 candidate miRNAs were first tested by RT-qPCR in an independent cohort of 113 MIBC patients, 113 NMIBC patients and 156 controls. Two miRNAs(miR-24-3p and miR-1246) showed differential expression levels in all three comparisons (MIBC vs. Normal, NMIBC vs. Normal, and MIBC vs.NMIBC). The potential of two miRNAs to discriminate non-muscle-invasive from muscle-invasive tumors was evaluated by ROC analysis. The corresponding AUCs of miR-24-3p and miR-1246 were 0.791 and 0.675, respectively.3. The stepwise logistic regression model was used to estimate the risk for prediction of MIBC in the training data set. Based on the two-miRNA panel, the prediction probability of MIBC was calculated from the logit model, logit(P=MIBC)=0.8575-0.2156xmiR-24-3p-0.2133xmiR-1246, which was used to construct the ROC curve. The performance of the established miRNA panel was also evaluated by ROC analysis. The AUC of the two-miRNA panel was 0.825 (95% CI,0.769 to 0.872) with a sensitivity of 76.11% and a specificity of 74.34%.4. The parameters estimated from the training set were used in a blind fashion to predict the probability of MIBC in an independent validation data set. The AUC of the predictive panel was 0.808 (95% CI, 0.744 to 0.862), with a sensitivity of 75.9%and a specificity of 75.49%. The AUC of the two-miRNA panel for MIBC was markedly higher than that of the tumor grade (AUC of 0.709,95% CI, 0.638 to 0.773)and urine cytology(AUC of 0.561, 95% CI, 0.487 to 0.634).5. Correlations between miRNA expression levels and patient survival:Kaplan-Meier survival analysis revealed that MIBC patients with high miR-24-3p expression levels showed significantly reduced overall survival (OS) than those with low miR-24-3p (P< 0.05). However, no significant correlation was found between the miR-1246 expression level and overall survival rate. Univariate Cox proportional hazards regression model analysis revealed that OS was significantly correlated with the miR-24-3p level (P=0.008) and tumor stage (P=0.008). Parameters significantly related to OS in the univariate analysis were applied to the multivariate analysis to identify the independent factors for prognosis. The results showed that miR-24-3p level (P=0.004) and tumor stage (P=0.014) could be usedas independent prognostic factors for OS of MIBC.ConclusionThis study revealed that urine miR-24-3p and miR-1246 were potential biomarkers for the diagnosis of MIBC. The two-miRNA panel generated from the multivariate logistic regression model demonstrated high prediction accuracy for MIBC. Compared with traditional urinary cytology and grade, this panel was significantly superior in terms of accuracy. Moreover, of the two miRNAs in the panel,miR-24-3p was identified as an independent factor for OS in MIBC patients.Part Four.Expression and biological function of microRNAs in bladder cancerPurposesThe aim of this study was to investigate expression of microRNAs (miRNAs) in staging model in the tissues of muscular invasive bladder cancer (MIBC) and non-muscular invasive bladder cancer (NMIBC). Expression levels of miRNAs in BC cells were measured, and functional experiments explored the predictive miRNAs in the process of biological functions, such as proliferation, migration, invasion and apoptosis, in order to clarify its role in the development and progression of BC and find a theoretical basis for the potential treatment of BC.Methods1. Expression of miR-24-3p and miR-1246 in frozen tissues of 35 MIBC and 35 NMIBC, and T24 cells were detected by RT-qPCR. In addition, expression of miRNAs, which were differentially expressed in MIBC, was detected in cancer tissues and matched normal tissues.2. The miR-1246 mimics, miR-1246 inhibitor and the corresponding negative control sequence were transferred into the T24 BC cell line with liposome 2000. The RT-qPCR method was used to detect the transfection efficiency.3. The proliferation, migration, invasion, and apoptosis of T24 BC cells were detected by CCK8 assay, wound-healing assay, Transwell migration experiment,Transwell invasion assay, and Annexin V-FITC / PI double labeling flow cytometry.Results1. The results showed that the expression level of miR-1246 in MIBC was significantly higher than that in NMIBC (P <0.05), while there was no significant difference of miR-24-3p between MIBC and NMIBC tissues (P> 0.05). The expression level of miR-1246 was significantly up-regulated in 77% of MIBC compared with normal tissues.2. After transfection with miR-1246 mimics at 24 hours, miR-1246 was significantly upregulated in T24 cells compared with the mock and miR-NC groups,(P <0.001). Compared with the mock and miR-NC groups, miR-1246 was significantly down-regulated (P <0.05) in the T24 cells after transfection of the miR-1246 antagonist group at 24 hours.3. Compared with the mock group and the miR-NC group, the growth rate of the transfected miR-1246 mimetic group T24 cells was significantly increased at 48 hours,72 hours,and 96 hours after transfection,and the difference was statistically significant (P <0.05). Compared with the mock group and the miR-NC group, the growth rate of the transfected miR-1246 antagonist group T24 cells was significantly decreased at 48 hours, 72 hours, and 96 hours after transfection, and the difference was statistically significant (P <0.05).4. The wound healing rate of the miR-1246 mimetic group was significantly higher than that of the mock group and the miR-NC group after 24 hours transfection(P <0.05). The wound healing rate of the miR-1246 antagonist group was significantly lower than that of the mock group and the miR-NC group (P <0.05). In the Transwell migration experiment, the number of transmembrane cells in the miR-1246 mimetic group of T24 cells was significantly higher than that of the mock group and the miR-NC group (P <0.05). The number of transmembrane cells in the miR-1246 antagonist group of T24 cells was significantly lower than that of the mock group and the miR-NC group (P <0.05). In the Transwell invasion experiment, the number of transmembrane cells in the miR-1246 mimetic group of T24 cells was significantly higher than that of the mock group and the miR-NC group (P <0.05). The number of transmembrane cells in miR-1246 antagonist group of T24 cells was significantly lower than that of the mock group and the miR-NC group (P <0.05).5. Apoptosis of the T24 cell line in the miR-1246 mimics group was not statistically different from the mock group and the miR-NC group (P> 0.05). In addition, the apoptotic rate of the miR-1246 antagonist group in T24 cells was not significantly different from that in the mock group and the miR-NC group (P> 0.05).ConclusionCompared with NMIBC, the expression levels of miR-24-3p and miR-1246 in MIBC were significantly up-regulated. Further study found that miR-1246 played an important role in invasion and metastasis and could promote cell migration and invasion in BC. This study provides a theoretical basis for finding new therapeutic targets for BC.