1.Clinical Staging Prediction Value And Biological Function Of MicroRNAs In Bladder Cancer 2.Value Of Serum MicroRNAs In Prediction And Prognosis For Lymph Node Metastasis In Gastric Cancer

BackgroundBladder cancer(BC)is one of the most prevalent malignancies in urinary system in China,which has seriously threatened human life and health.70-80%of patients are initially diagnosed with non-muscle-invasive BC(NMIBC),and 50-70%of these tumors will recur.The primary clinical treatment of NMIBC is transurethral resection of bladder tumor(TURBT).20-30%of patients are initially diagnosed with muscle-invasive BC(MIBC).The prognosis of MIBC is poor and the overall survival rate is only 60%.These patients may need systemic treatment such as radical cystectomy and radiotherapy.Therefore,accurate prediction of muscle-invasive tumor of BC has important significance for guiding on timely and effective treatment.Currently,the standard test used to measure the local stage of BC is transurethral resection.However,this approach is invasive and can not precisely determine the depth of invasion in some cases.The sensitivity and specificity of traditional urinary cytology are low,which limits its clinical application in noninvasive evaluation of MIBC.In addition,imaging exams such as ultrasound and computed tomography(CT)have limited ability to predict tumor stage.Therefore,sensitive and specific biomarkers are urgently needed to improve current strategies for preoperative prediction of tumor stage in BC.MicroRNAs(miRNAs)are a subset of small non-coding RNAs(typically 19-25 nucleotides in length)that play important roles in regulation of tumorigenesis,development and metastasis.Expression profiles of miRNAs are unique in various types of cancer.In different stages of tumor,miRNAs can also show specific expression signatures.Previous studies have demonstrated that numerous cell-free miRNAs exist stably in human serum and have potential roles in diagnosis,staging and prediction of outcomes in cancer.Recently,we have identified a diagnostic 6-miRNA panel(miR-152,miR-148b-3p,miR-3187-3p,miR-15b-5p,miR-27a-3p and miR-30a-5p)in serum of BC,which paved the way for finding novel noninvasive biomarkers for BC screening.Based on previous findings,the present study was designed to screen cell-free miRNA profiles in serum of MIBC patients and establish predictive miRNA panel for MIBC.Moreover,the value of serum miRNAs for prognosis of MIBC was further assessed.Finally,expression of miRNAs in tissues of BC patients was measured and systematic cell function experiments were performed to investigate biological functions of miRNAs in tumorigenesis and invasion of BC.This study would comprehensively evaluate values of miRNAs on clinical staging of BC through clinical and basic experiments,which could provide references for discovery biomarkers for staging and new therapeutic targets of BC.Chapter OneEstablishment of predictive panel based on serum cell-free microRNAs for muscle-invasive bladder cancerObjectiveThe aim of this study was to screen serum cell-free miRNA profiles in MIBC and establish cell-free miRNA panel for prediction of tumor stage in MIBC.Meanwhile,value of cell-free miRNAs in prognosis of MIBC patients was further accessed.Methods1.Serum samples form 207 MIBC patients,285 NMIBC patients and 193 controls were firstly collected in Qilu Hospital,Shandong University.2.In the discovery phase,serum samples from 6 MIBC patients,6 NMIBC patients and 6 healthy donors were respectively subjected to miSeq sequencing.MiRNAs with significant deregulations larger than two-fold change among three groups were selected as candidate biomarkers for MIBC.In addition,6 miRNAs were also selected because they had been shown to have diagnostic values for BC in our previous study.3.In the training phase,candidate miRNAs were tested with reverse transcription quantitative real-time PCR(RT-qPCR)assays in 111 MIBC patients,111 NMIBC patients and 187 controls.Based on differentially expressed miRNAs,the predictive 4-miRNA panel for MIBC was constructed using logistic regression model.Receiver operating characteristic(ROC)curves were established to discriminate the predictive ability of 4-miRNA panel.4.In the validation phase,serum from another independent cohort of 90 MIBC patients and 168 NMIBC patients were prospectively entered into the discriminatory model to validate predictive accuracy of the constructed algorithm.Meanwhile,predictive ability of 4-miRNA panel was compared with traditional urine cytology and pathological grade.5.MIBC patients in the validation phase were followed up for survival analysis.Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate prognostic values of serum cell-free miRNAs for MIBC patients.Results1.By miSeq sequencing,234,278 and 193 miRNAs were detectable(? 10 copies)in MIBC group,NMIBC group and control group,respectively.Expression of a miRNA was considered altered in MIBC only if at least 50 copies were detected by miSeq sequencing together with significant deregulations larger than two-fold change among three groups.Based on these criteria,23 miRNAs were selected as differentially expressed(miR-125b-5p,miR-130a-3p,miR-4732-3p,miR-3065-3p,miR-422a-3p,miR-378a-3p,miR-122-5p,miR-136-5p,miR-22-3p,miR-146a-5p,miR-205-5p,miR-769-5p,miR-19a-3p,miR-107,miR-320a,miR-423-5p,miR-486-3p,miR-574-3p,miR-4429,miR-205-5p,miR-103a-3p,miR-23a-3p and miR-362-5p).In addition,miR-152,miR-148b-3p,miR-3187-3p,miR-15b-5p,miR-27a-3p and miR-30a-5p were also selected because they had been shown to have diagnostic value for BC in our previous study.Thus,29 miRNAs were selected as candidates for further testing via RT-qPCR.2.29 candidate miRNAs were tested with RT-qPCR using an independent cohort of 111 MIBC patients,111 NMIBC patients and 187 controls.4 miRNAs(miR-422a-3p,miR-486-3p,miR-103a-3p and miR-27a-3p)showed differential expression levels in MIBC vs.Normal,NMIBC vs.Normal,and MIBC vs.NMIBC comparisons(all at p<0.05).ROC analysis revealed that the corresponding area under ROC curves(AUCs)of 4 miRNAs were 0.767(95%confidence interval[CI],0.706?0.821),0.718(95%CI,0.654 to 0.776),0.706(95%CI,0.641?0.765)and 0.641(95%CI,0.575?0.704),respectively.3.A stepwise logistic regression model to estimate the risk of being predicted with MIBC was applied on training data set.The predicted probability of being diagnosed with MIBC from the logit model based on 4-miRNA panel,logit(p=MIBC)=-1.5992 +(0.4938xmiR-422a-3p)+(0.4197×miR-486-3p)+(0.4168×miR-103a-3p)+(0.2335×miR-27a-3p)was used to construct the ROC curve.The AUC of the 4-miRNA panel was 0.894(95%Cl,0.846 to 0.931)and the optimal cut-off value was 0.0282,providing sensitivity of 90.99%and specificity of 72.97%.A threshold of 0.0282 was selected to ensure good predictive ability for MIBC.4.Using the classification threshold score of<0.0282 derived above,132 samples were identified as noninvasive tumors and 126 samples were identified as invasive tumors.After unblinding,121 of 168 noninvasive tumors[specificity,70.06%(95%CI,79.1 to 90.1)]and 79 of 90 invasive tumors[sensitivity,90.00%(95%CI,81.9 to 95.3)]were correctly identified resulting in an AUC of 0.880(95%Cl,0.834 to 0.917).The AUC of the 4-miRNA panel for MIBC was markedly higher than those of pathological grade(AUC=0.779,95%CI,0.724 to 0.828)and urine cytology(AUC=0.602,95%Cl,0.539 to 0.662).The predictive specificity of 4-miRNA panel for Ta and T1 was 61.82%and 71.68%,respectively.The predictive sensitivity of miRNA panel for T2,T3 and T4 was 78.57%,87.18%and 95.65%,respectively.5.In the validation phase,survival analysis was performed on 90 MIBC patients and the median follow-up time was 56(range 3-73)months.Kaplan-Meier survival analysis revealed that MIBC patients with low miR-486-3p and miR-103a-3p expression levels showed significantly reduced overall survival(OS)than those with high miR-486-3p and miR-103a-3p levels(p=0.002 and p=0.034,respectively).Multivariate Cox proportional hazards regression model analysis revealed that miR-486-3p(p=0.042),miR-103a-3p(p=0.021),tumor stage(p=0.030)and lymph node status(p=0.025)could act as independent prognostic factors for OS of MIBC.ConclusionsSerum cell-free miR-422a-3p,miR-486-3p,miR-103a-3p and miR-27a-3p were identified as potential biomarkers for MIBC prediction.The 4-miRNA panel demonstrated higher predictive accuracy than urinary cytology and pathological grade.In addition,miR-486-3p and miR-103a-3p could provide prognostic information for MIBC patents.Chapter TwoExpression and biological function of miR-27a-3p in pathogenesis of bladder cancerObjectiveThe aim of this study was to analyze the expression levels of miRNAs in tissues of MIBC and NMIBC patients and to investigate the biological functions of miR-27a-3p in tumorgenesis and development of BC by experiments on cell lines in vitro.Methods1.Expression levels of miR-422a-3p,miR-486-3p,miR-103a-3p and miR-27a-3p in tissues of 40 MIBC patients and 40 NIMBC patients were measured by RT-qPCR assays.2.Differentially expressed miRNA(miR-27a-3p)was selected to further detect its expression in 40 pairs of muscle-invasive tumor and adjacent normal tissue.The relationship between miR-27a-3p and clinicopathological parameters in BC patients was analyzed.3.RT-qPCR assays were performed to measure expression levels of miR-27a-3p in BC cells(T24)and immortalized normal epithelial cells(SV-HUC-1).4.miR-27a-3p mimic and miR-27a-3p inhibitor were respectively transfected into T24 BC cells by LipofectamineTM 2000.At the same time,control groups including miR-NC group and mock group were established.The transfection efficiency of BC cells in each group was measured by RT-qPCR.5.After T24 BC cells were transfected with miR-27a-3p mimic and inhibitor,the effects of miRNAs on proliferation,migration and invasion ability of cells were analyzed by CCK8 assays,scratch assays,Transwell migration assays and Transwell invasion assays,respectively.Results1.Expression of miR-27a-3p was significantly down-regulated in tissues of MIBC compared with NMIBC(p<0.05).Expression miR-27a-3p was also down-regulated in tumor tissues compared with adjacent normal tissues in 70%MIBC patients(larger than 2 fold change).However,there were no significant differences in expression of miR-422a-3p,miR-486-3p and miR-103a-3p in tissues between MIBC group and NMIBC group(p>0.05).2.Analysis on correlation between miRNAs and clinicopathological characteristics revealed that expression of miR-27a-3p significantly correlated with tumor stage(p=0.03)and lymph nodes metastasis(p-0.01).No significant associations were found between expression of miR-27a-3p with sex(p=0.33),age(p=0.16),grade(p=0.92)and pathological type(p=0.42).3.RT-qPCR assays revealed that expression of miR-27a-3p was significantly down-regulated in T24 cells compared with immortalized normal epithelial cells(p<0.01).4.After transfected with miR-27a-3p mimic,growth rate of T24 BC cells in miR-27a-3p mimic transfected group was significantly reduced at 48h,72h and 96h compared with miR-NC group and mock group(all at p<0.05).After transfected with miR-27a-3p inhibitor,growth rate of T24 BC cells in miR-27a-3p inhibitor transfected group was significantly accelerated at 48h,72h and 96h compared with miR-NC group and mock group(all at p<0.05).5.In the scratch test,wound healing rates of miR-NC group,mock group and miR-27a-3p mimic group at 24h after transfection were(35.27 ± 2.01)%,(28.7±2.05)%and(15.57 ± 1.06)%,respectively.Wound healing rate of BC cells in miR-27a-3p mimic transfection group was significantly lower than that in miR-NC group and mock group(p<0.05).In contrast,wound healing rates of miR-NC group,mock group and miR-27a-3p inhibitor group at 24h after transfection were(24.57±1.60)%,(17.63 ± 2.91)%and(51.03±1.29)%,respectively.Wound healing rate of miR-27a-3p inhibitor transfection group was significantly higher than that in miR-NC group and the mock group(p<0.05).6.In Transwell migration assay,the number of transmembrane cells in miR-27a-3p mimic group was significantly lower than that in miR-NC group and mock group at 24h after transfection(p<0.05).The number of transmembrane cells in miR-27a-3p inhibitor group was significantly higher than that in miR-NC group and mock group at 24 h after transfection(p<0.05).7.In Transwell invasion assay,the number of transmembrane cells in miR-27a-3p mimic group was significantly lower than that in miR-NC group and mock group at 24h after transfection(p<0.05).The number of transmembrane cells in miR-27a-3p inhibitor group was significantly higher than that in miR-NC group and mock group at 24h after transfection(p<0.05).ConclusionsCompared with adjacent normal tissues and NMIBC tissues,expression of miR-27a-3p was significantly down-regulated in MIBC tissues.Down-regulated miR-27a-3p could significantly promote proliferation,migration and invasion of BC cells,and has important biological functions in tumorigenesis and progression of BC.This study may lay theoretical foundation for clarifying roles of miRNAs in pathogenesis of BC and finding new therapeutic targets for BC.BackgroundGastric cancer(GC)is one of the most common malignant tumors in digestive tract,and its morbidity and mortality have been increasing in recent years.Lymph node metastasis is the main path of GC metastasis and could serve as one of the most important prognosis factors for GC.Preoperative determination of lymph node status is critical in providing guide on tumor staging and planning optimal treatment for GC.Currently,postoperative histopathological biopsy is the "gold standard" for diagnosing lymph node metastasis of GC,but this method is invasive and is difficult to accurately obtain the range of lymph node metastasis.Imaging techniques such as endoscopic ultrasonography,computed tomography(CT),magnetic resonance imaging and positron emission tomography(PET)-CT are commonly used for preoperative assessment of lymph node status.However,the accuracy of endoscopic ultrasonography for lymph node metastasis is still limited.Although PET-CT can distinguish whether lymph nodes are infiltrated by cancer cells,it is not conducive to the detection of patients with lymph nodes micrometastasis.Limitations of these imaging techniques make them difficult to be reliable methods of diagnosing lymph node metastasis in GC.Meanwhile,traditional serum biomarkers for GC,including carcinoembryonic antigen(CEA),carbohydrate antigen 19-9(CA19-9)and carbohydrate antigen 72-4(CA72-4)have shown low sensitivity for lymph node metastasis prediction.Therefore,looking for noninvasive serological tumor biomarkers for lymph node metastasis prediction of GC is one of the focuses in academic community.MicroRNAs(miRNAs)are ubiquitous non-coding RNAs in eukaryotes and play important roles in regulation of crucial pathological processes in lymph node metastasis of tumors.Accumulating evidence indicates that numerous stable cell-free miRNAs exist in human serum and expression profiles of these miRNAs are tissue-specific and phase-specific.Previous studies demonstrated that distinctive patterns of circulating cell-free miRNAs have been reported to predict lymph node metastasis in various types of cancers,such as colorectal cancer,cholangiocarcinoma,and cervical squamous cell carcinoma.In fact,differential expression of several circulating miRNAs including miR-1207-5p,miR-146a and miR-148a have been reported in GC patients with lymph node metastasis.However,the predictive efficacy and prognosis value of serum cell-free miRNAs in GC patients with lymph node metastasis positive(LNPs)is still not clear.In the present study,we performed miSeq sequencing and two phases of reverse transcription quantitative real-time PCR(RT-qPCR)assays to systematically investigate serum cell-free miRNA expression profiles in LNPs.4-miRNA panel was constructed by multivariate logistic regression model and the predictive value of the established panel was further analyzed in the validation set.In addition,correlation between expression of serum cell-free miRNAs and prognosis of LNPs was further assessed.ObjectiveThe aim of this study was to identify miRNA expression profiles in LNPs and to establish serum cell-free miRNAs panel for predicting lymph node metastasis in GC.Moreover,the value of serum cell-free miRNAs in predicting the prognosis of LNPs was further evaluated.Methods1.Serum samples from 203 LNPs,203 GC patients with lymph node metastasis negative(LNNs)from General Surgery Department and 83 healthy control individuals from Healthy Physical Examination Centre of Qilu Hospital,Shandong University were recruited.2.In the screening phase,serum samples from 10 LNPs,10 LNNs and 10 healthy controls were respectively subjected to miSeq sequencing and candidate miRNAs with significant differences in expression among three groups were firstly identified.3.In the training phase,candidate miRNAs were verified by RT-qPCR in serum samples from 103 LNPs,103 LNNs and 73 healthy controls.Subsequently,miRNAs differentially expressed in LNPs were identified.4.By logistic regression model analysis,the predictive 4-miRNA panel for lymph node metastasis in GC was established based on differentially expressed miRNAs.Receiver operating characteristic(ROC)curve was used to evaluate predictive value of the constructed panel.5.In the validation phase,serum samples from another cohort of 90 LNPs and 90 LNNs were prospectively entered into the discriminatory model to validate the predictive accuracy of the constructed algorithm.Moreover,ROC analysis was performed on the predictive value of 4-miRNA panel for LNPs with different lymph node status(N1,N2 and N3).6.In the validation phase,90 LNPs were followed up for overall survival(OS)analysis.Survival curves were estimated with Kaplan-Meier method and log-rank test was used to compare the distributions of survival times.Cox proportional hazards regression model was employed to evaluate independent factors of OS.Results1.By miSeq sequencing,181,225 and 158 cell-free miRNAs were detected in control group,LNNs group and LNPs group,respectively.Expression of miRNA was considered "significantly altered" only if at least 20 copies were detected by miSeq sequencing,together with a larger than two-fold change in its expression level in LNPs vs.LNNs,LNPs vs.controls and LNNs vs.controls.19 candidate miRNAs were identified to be differentially expressed in LNPs.2.In the training phase,19 candidate miRNAs were tested with RT-qPCR and 4 miRNAs(miR-501-3p,miR-143-3p,miR-451a,and miR-146a)showed differential expression levels among three groups(all at p<0.05).Predictive accuracy of miR-501-3p,miR-143-3p,miR-451a,and miR-146a for lymph node metastasis measured by area under ROC curve(AUC)was 0.790(95%confidence interval[CI],0.728 to 0.844),0.774(95%CI,0.711 to 0.830),0.710(95%CI,0.642 to 0.771)and 0.675(95%CI,0.606 to 0.738),respectively.3.In the validation phase,the predicted probability of being predicted with lymph node metastasis from the logit model based on 4-miRNA panel,logit(p=LNPs)=-1.2020 +(0.3963×miR-501-3p)+(0.3768xmiR-143-3p)+(0.2494×miR-451a)+(0.2106×miR-146a)was used to construct the ROC curve.The observed AUC of the 4-miRNA panel was 0.891(95%CI,0.840 to 0.930)and the optimal cut-off value was-0.2398,providing a sensitivity of 75.73%and a specificity of 87.38%.To ensure good predictive ability for lymph node metastasis,a threshold of cut-off value-0.2398 was identified.4.Based on the classification threshold score of-0.2398,116 samples were identified as LNNs and 64 samples were identified as LNPs in the validation phase.Subsequent unblinding of the results showed that 79 out of 90 LNNs(specificity=87.78%)and 53 of 90 LNPs(sensitivity=63.33%)were correctly identified,resulting in an AUC of 0.822(95%CI,0.758 to 0.875).The AUCs of the panel for LNPs with N1,N2,and N3 were 0.709(95%CI,0.619 to 0.788),0.860(95%CI,0.787 to 0.915),and 0.914(95%CI,0.846 to 0.958),respectively.5.In the validation phase,Kaplan-Meier survival analysis revealed that LNPs with lower miR-451a and miR-146a expression levels showed significantly reduced OS than those with high miR-451a and miR-146a expression levels(both at p<0.05).The univariate Cox proportional hazards regression model analysis revealed that expression of miR-451a(p=0.001)and miR-146a(p=0.016)along with tumor stage(p<0.001)were significantly correlated with OS of LNPs.The multivariate Cox proportional hazards regression model analysis showed that miR-451a expression(p=0.028)and tumor stage(p=0.011)maintained their significance as independent prognostic factors for OS of LNPs.Conclusions1.This study revealed that serum cell-free miR-501-3p,miR-143-3p,miR-451a,and miR-146a could serve as potential biomarkers for predicting lymph node metastasis in GC.The established serum 4-miRNA panel can effectively discriminate LNPs from LNNs,which could provide a new detection approach for preoperative prediction of lymph node metastasis in GC.2.Serum miR-451a can be used to independently evaluate the prognosis of LNPs and is expected to become a new prognostic biomarker of lymph node metastasis in GC.