Methylation Profile Of Tumour-Associated Genes Promoter In Urine Sediments And Diagnosis Research For Bladder Cancer

Objectives: The aim of the study is to set up a profile of bladder cancer. We use methylation specific polymerase chain reaction(Msp) method to detect the tumor tissue of the patients which clinically final diagnosis by pathology for bladder transitional cell cancer and non bladder cancer patients (control group), the related gene methylated status about the urine sediments; get the message about bladder transitional cell cancer in related gene methylated profile about cast-off cells from urine of our nation., To approach the application value to use related gene methylation detect in the diagnosis of bladder transitional cell cancer, and the same time which is a preliminary research to judgement the prognosis of bladder transitional cell cancer.The research's experiment divide into four bulks:1.Detecting the promoter status of a panel of tumor-related genes in bladder cancer cell lines.2. Setup a methylation profile of bladder transitional cell carcinoma; Evaluate the role of detecting methylation markers in urin in diagnosis of bladder cancer.3. Evaluate the reliability of of detecting methylation markers in urin in diagnosis of bladder cancer.4. Evalute the role of methylaion markers in urine as prognostic factors in bladder cancre. Experiment 1: Detecting promoter methylation status of CpG islands in in bladder cancer cell lines.Methods: Methytlation specific PCR were used to detect promoter region CpG island methylation status of 20 genes (ABCC6, ALX4, BCL2, BMP3B, BRCA1,CCNA1, CDH13, CFTR, DRM, HPP1, ITGA4, PTCHD2, MT1A, MTIN 1, MYOD1, RASSF1A, RPRM, RUNX3, SALL3,TMS1)in 3 bladder cell lines. The bladder cancer cell lines (T24, 5637 and SCaBER).Results: 19 of 20 genes (ABCC6, ALX4, BCL2, BMP3B, CCNA1, CDH13, CFTR, DRM, HPP1, ITGA4, MT1A, MTIN 1, MYOD1, PTCHD2, RASSF1A, RPRM, RUNX3, SALL3 and TMS1) tested in bladder cancer cell lines (T24, 5637 and SCaBER) have at least one present allele methylated, Except BRCA1. They display heterozygosis methylated or homozygosis methylated.Experiment 2: Setup a DNA methylation profile of bladder transitional cell carcinoma and explore its diagnostic useMethods: 66 patients which clinically final diagnosis by pathology for bladder transitional cell cancer were included in this study. 12 candidate gen (SALL3,MT1A,HPP1, MYOD1,BRCA1,ABCC6,ALX4,CDH13,CFTR,MINT1,Rassf1a,TMS1) were chosen form results in experiment 1. Methylation status of 12 candidate genes in urine DNA samples were detected by MSP. Get the message about bladder transitional cell cancer in related gene methylated spectra about cast-off cells from urine of our nation. To compare and statistics analysis with the result of control group(non-cancerous urinary diseases:33 cases,the healthy volunteers: 7 cases, and the neurological diseases: 8 cases).Diagnostic value of bladder cancer were evaluated by analyze the sensitivity and specificity by statistic methods. Results: The 12 gene promoter region CpG island methylation profile of 66 patients which clinically final diagnosis by pathology for bladder transitional cell cancer is as below: SALL3 65.1% (43/66), MT1A 43.9% (29/66), HPP1 46.9 (31/66), MYOD1 33.3% (22/66), BRCA1 19.7 %(13/66), ABCC6 51.5% (34/66), ALX4 22.7% (15/66), CDH13 21.2 % (14/66),CFTR 54.5% (36/66), MINT 1 13.6% (9/66), Rassf1a 40.9% (27/66), TMS1 13.6% (9/66).Within 7 healthy volunteers and 8 neurological patients, 12 genes were unmethylated. In terms of 33 non-tumor patients, only 4 of them were MTA1 methylated12.1 %(4/33), 3 of them were RASSF1A methylated 9.0 %(3/33), the others 10 genes were unmethylated. The methylation frequency of 12 genes in bladder cancer group were significantly higher than that in non-tumor group. According to the standard which we set to diagnose bladder cancer by utilize DNA methylation, when combination detect 12 genes of 66 patients which clinically final diagnosis by pathology for bladder transitional cell cancer, the result: masculine 63 case, Negative 3 case, masculine rate 95.5%, false negative rate 4.5%. Detect non-cancerous urinary diseases 33case, false masculine 4 case, Negative 29 case, false masculine rate 12.1%, false negative rate 87.9%.Through statistics analysis theb sensitivity is 95.5%, specificity 87.9%, Youden's index 87.9%, masculine likelihood ratio 0.83, Negative likelihood ratio 0.05, detect concordance 93%, the result were highly goodness of fit with clinical pathologic diagnosis, Indicated that detecting the DNA methylation status of urine sediments can serve as a useful tool for early diagnosis in bladder cancer.Experiment 3: Authenticity research about tumor tissue gene DNA methylated profile and urine sediments of bladder transitional cell cancer:Methods: Take 15 cases patients tumor tissue from cystoscopy biopsyed or exairesis from experiment 2, select 11 genes: ABCC6. BRCA1. ALX4. CDH13. CFTR. HPP1. MTNT1. Mt1a. Rassf1a. RPRM. Sall3, detect gene DNA methylated status by MSP to gain methylated profile, 9 case among the total were detected with coupled cast-off cells of urine sample, cast-off cells of urine DNA methylated spectra were compare with bladder cancer DNA methylated profile, to study correlativity between them, approach the authenticity to inspect urine sediments methylated Profile.Results: In 15 case bladder cancer tissue related gene methylated profile detection, 11 genes gene methylated profile as follow: ABCC6: 60%. BRCA1: 20%. ALX4: 30%. CDH13: 26.6%. CFTR: 30%. HPP1: 73.4%, MTNT1: 30%. Mt1a: 13.3%. Rassf1a: 26.6%. RPRM: 30%. Sall3: 30%. Of the total 9 case detected with coupled cast-off cells of urine sample, bladder cancer tissue with cast-off cells of urine methylated Profile coincidence of 11 genes in each patient, from 72.7% (8/11) to 100% (11/11) , average 86.8%, among the total 6 case methylated spectra coincidence exceed 90%, moreover the coincidence of each gene in the 11 detected genes was extremely highly, from 66.6% to 100%. X2 test of paired comparison of enumeration data analysis P>0.05, demonstration there have distinguished concordance between them.Experiment 4: 66 patients which clinically final diagnosis by pathology for bladder transitional cell cancer were refer to China uropoiesis disease diagnosis guide to undertake TNM stages, compare methylated profile among clinical I,II,III stages; palpated primarily and recrudescence patients were divided into groups to compare methylated profile; compare 23 case patient methylated profile before operation and after operation, to compare and analysis the DNA methylated profile change before before operation and after operation,and after operation. Results: 66 cases bladder transitional cell cancer patients compare among clinical I,II,III stages , major difference genes include: BRCA1 13.3%,19.4%,26.6%; MYOD1 26.6%,33.3%/40%; HPP1 53.3%/50.0%/33.3%; ABCC6 40% /47.2% /73% (11/15); CDH13 20% /27.8% 16.1%; CFTR53.3% /50%/67%; Rassfla26.6%/44.4%/47%; RPRM6.7%/16.7%/13%; MINT1 20% /44.4% /47%. MT1A and ALX4 have significance difference compare among clinical I,II,III stages (P≤0.05) .primarily and recrudescence group DNA methylated profile difference have follow genes: BRCA118.9% /23.1%; HPP1 66% /46.2%; CDH13为18.9/30.8%; MINT1 15.1%/7.7%; Rassf1a为35.8% (19/53): 61.5% (8/13); RPRM为15.1% /7.7%. X2 test of paired comparison of enumeration data analysis only Rassf1a has significance difference(P<0.05).Detect 12 genes methylated profile methylation rate change information before operation and after operation in 23 patients. Among 12 genes, BRCA1 unmethylated before operation and after operation, 5 genes include CDH13,CFTR,MTNT1,Myod,RPRM methylated before operation ,and unmethylated after operation, Their methylation transformation efficiency were 100%. Mtla has 9 case methylated before operation, and only 1case methylated after operation, methylation transformation efficiency was 87.5%. Sall3 has 15 case methylated before operation, and only 2case methylated after operation, methylation transformation efficiency was86.6%. ALX4 has 7 case methylated before operation, and only 1case methylated after operation, methylation transformation efficiency was 85.7%. Rassfla has 4 case methylated before operation, and only lease methylated after operation, methylation transformation efficiency was 75%. All genes we detect the methylation transformation efficiency were at higher level. Conclusion: To detect methylation status among can essential reflect cancer tissue methylation status, the result of detection demonstrate related gene DNA methylation change in bladder cancer are earlier period and frequently happen event. If association detect 12 genes DNA methylation status have hope effective detect bladder cancer. Urine sediments related gene in bladder transitional cell cancer methylated profile have no definite correlated relation among clinical TNM stages and primarily or recurrence patients, but be use as target to evaluate for exairesis clean and thorough whether or not. Nevertheless the MSP method detection steps are complex, time consuming fairly long, it will has along time before clinical application.