Exploring The Potential Biomarkers In Bladder Transitional Cell Carcinoma

Bladder cancer is the most common urinary cancer in China.More than 90%of bladder cancers are bladder transitional cell carcinomas(BTCC).Bladder cancer diagnosis is mainly dependent of cystoscope examination combined with histopathologic evaluation now.However,cystoscope examination is invasive and conventional histopathologic evaluation,encompassing tumor grade and stage,is inadequate to accurately predict the behavior of most bladder cancers.To establish which non-muscle-invasive cancers will recur or progress and which invasive cancers will metastasize,new potential prognostic biomarkers for bladder cancer patients remain to the identified.It was reported recently that epigenetics status and the expression of microRNA(miRNA) always change in bladder cancer,which provide potential targets for bladder cancer diagnosis and treatment.Therefore,we explored the changes of epigenetics status and the expression of microRNA in bladder cancer in this thesis.Epigenetics refers to changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence,including DNA methylation, histone methylation and acetylation and DNA footprinting et al.DNA methylation is the most clear epigenetic pattern and is widely studied.It is reported that the promoter region of tumor repressor genes was hypermethylated in bladder cancers,which provides potential targets for bladder cancer diagnosis.P16 gene is an important tumor suppressor gene.Hypermethylation was also found in p16 gene in some bladder cancer tissues.However,no systematic studies have been carried out in p16 gene methylation status among Chinese bladder cancer patients.Therefore,in the first part of the thesis, a methylation-specific polymerase chain reaction(MSP) was performed to detect p16 gene CpG island methylation in the matched samples of tumor tissue and urine exfoliated cells from 35 cases of bladder transitional cell carcinoma patients.Recent studies have shown that microRNA plays an important role in tumorigenesis and tumor progression.MicroRNAs are～22-nt small regulatory RNAs that are thought to control gene expression at the posttranscriptional level by targeting cognate target mRNAs for either degradation or translational repression.It was reported that the expression of single microRNA or microRNA profiles changed in many tumor tissues or cancer cells.The differential expressed microRNAs may serve as potential targets for cancer diagnosis and treatment.Exploring the role of microRNAs in bladder cancer will help us to understand the mechanisms of bladder cancer development and progression,as well as develop new biomarkers for bladder cancer diagnosis.However, the differential expressed microRNAs in bladder cancer remain to be identified. Therefore,in the second part of the thesis,the changes in microRNA expression in bladder transitional cell carcinoma were studied.PartⅠ:Studying the p16 gene methylation status in bladder transitional cell carcinoma.To investigate the role of p16 gene methylation in bladder cancer development,a methylation-specific polymerase chain reaction(MSP) was performed to detect p16 gene CpG island methylation in tumor tissue samples from 35 cases of bladder transitional cell carcinoma patients.The mucous membrane of urinary bladder in opposite side of the pathological changes was taken as the control in 9 of 35 cases.To investigate the clinical significance of p16 gene methylation in urjnary exfol iated cells for early diagnosis of bladder transitional cell carcinoma,p16 gene methylation in the matched urine exfoliated cells from the 35 BTCC patients was studied.The urine samples from normal people and from the patients without urinary tumor were taken as the control.Data showed that the methylation of p16 gene CpG island was detected in 51.4%(18/35) of the tumor tissues,45.7%(16/35) of the urine exfoliated cells, respectively.Logistic regression revealed that the methylation was not related to the age,gender,tumor pathologic grade and clinical stage(P＞0.05).The p16 gene methylation of urinary samples was detected in 10 out of 20 superficial BTCC patients. And the methylation also existed in the tumor tissues of the matched urine samples. The urine samples from the patients without urinary tumor or disorders showed no methylation.Both the positive predictive value and specificity of the p16 gene methylation in the urinary exfoliated cells were 100.0%.And the false positive was 0%. No p16 CpG island methylation was found in normal urothelium(9 cases).The differences were statistically significant(P＜0.001).Taken together,our results demonstrated that p16 gene CpG island was hypermethylated in bladder transitional cell carcinoma and that the methylation of p16 gene CpG island in urinary exfoliated cells may be used as a biomarker for early diagnostic of superficial bladder transitional cell carcinoma.PartⅡ:Studying the differential expressed microRNAs in bladder transitional cell carcinoma.Instead of examining the global miRNA expression profile in bladder tumor tissues from one group of patients as compared with others as controls,the changes in miRNA expression in the tumor from 7 each individual patient were profiled by comparison with that in their own healthy bladder tissue.Overall,expression of a range from 40 to 111 miRNAs had over 1.5-fold change in a total of 464 miRNAs in bladder tumors compared with those in normal tissue in each patient;from 18 to 50 miRNAs were up-regulated whereas from 9 to 89 miRNAs were down-regulated.The tumor tissues collected in this study were classified into either the T1 or T2 group. In the T1 group,the differentiated grade was 1,1,and 1-2.A total of nine miRNAs (miR-129,miR-141,miR-494,miR-498,miR-500,miR-513 and three unknown miRNAs) were found to be up-regulated in every patient,whereas 13 miRNAs (miR-let-7a,7b,7c,7d,miR-143,miR-199a~*,miR-21,miR-24,miR-26a,miR-29c, miR-30a-5p,miR-30c,and miR-30e-5p) were down-regulated.In the up-regulated group,most of miRNAs had a mean twofold increase except for miR-141,which was increased more than fivefold.In the down-regulated group,the mean decrease was 2 to 3-fold for most miRNAs,and a sixfold increase was seen for miR-143.In the T2 group, the differentiated grade ranged from 1,1-2,2,and 3,which was not significantly different from the T1 group.But as all the changes in miRNA expression were screened in the tumor tissue relative to healthy tissue in this group of patients,no any up-regulated miRNA was commonly seen in every patient.However,four miRNAs (miR-26a,miR-29c,miR-30c,and miR-30e-5p) were commonly downregulated and were also found to be downregulated in the T1 group,suggesting that no specific miRNA expression change was detected in the T2 group.Overall,the common change in miRNA expression in the bladder tumor over that in normal tissue in these seven patients was a decrease in the expression of these four miRNA,as indicated by the 1.5-fold cutoff line.It was interesting to note that there was one patient,whose tumor was graded 3 with a poorly differentiated phenotype.Although it shared the down-regulation of four miRNAs as described above,the miRNA expression change profile in this tumor was remarkably different from those in the other six patients;there were fewer down-regulated miRNAs and the presence of its specific up-regulated miRNAs differed.In particular,a group of miRNAs was increased in this grade 3 tumor tissue; miR-21 was extremely elevated,close to tenfold,but either down-regulation or no significant change in the expression of this miRNA was noted in others.The miR-let-7 group(hsa-let-7a,hsa-let-7c,and hsa-let-7d) was significantly increased(2.07,1.71, and 4.20-fold) whereas others displayed a common decrease in these miRNAs.To evaluate the reliability of quantitation by the miRNA array assay in this study,the levels of three miRNAs(miR-let-7a,miR-30c,and miR-129) in all seven patients were examined by real-time RT-PCR.Data showed that the change pattern of of each miRNA expression measured by real-time RT-PCR was the same as miRNA array in each individual patient,although the change value was not exactly matched,which suggested that the miRNA array results were reliable.Therefore,innovative conclusions could be drawn base on the data:1.P16 gene CpG island was hypermethylated(51.4%) in bladder transitional cell carcinoma and the methylation was not related to the age,gender,tumor pathologic grade or clinical stage.The methylation of the promoter may induce p16 gene silence and therefore promote bladder transitional cell carcinoma tumorigenesis.2.P16 gene was hypermethylated(50%) in the urine exfoliated cells from superficial bladder transitional cell carcinoma patients,which might serve as the biomarker for early diagnosis of superficial bladder transitional cell carcinoma.3.In the T1 group of bladder transitional cell carcinoma,9 miRNAs(miR-129, miR-141,miR-494,miR-498,miR-500,miR-513 and three unknown miRNAs) were found to be up-regulated in every patient,whereas 13 miRNAs(miR-let-7a, 7b,7c,7d,miR-143,miR-199a~*,miR-21,miR-24,miR- 26a,miR-29c, miR-30a-5p,miR-30c,and miR-30e-5p) were down-regulated.While in T2 group, only 4 miRNAs(miR-26a,miR-29c,miR-30c,and miR-30e-5p) were downregulated.Overall,miR-26a,miR-29c,miR-30c,and miR-30e-5p were commonly downregulated in bladder transitional cell carcinoma,which might play an important role in bladder transitional cell carcinoma tumorgenesis.The data also suggested that microRNAs can be used as biomarkers for bladder cancer diagnosis and staging.