| DNA methylation is the most important epigenetic mechanism. The changes of it play role in abnomal genetic expression and unstablity of genome., which promote to tumor抯appearance and promotion. It is known that hypermethylation of CpG islands in promotor region have equal importance to genetic defects, and its status have ralations to depression of tumor-surpression-genes. We have used MSP(Methylation Specific PCR) to detect the methylation status of RASSF1A gene in bladder tumor. Though the results showed ralations between the status and the tumor, its clinical diagnostic values are not satifying. This reaech want to improve the clinical diagnostic values of this detection by the following two ways:1. The Influence to Clinical Values by Optimizing MSP Annealing朤emperatures. Objectives Optimize MSP by optimizing its annealing-temperature, so as to improve its results better coincidence to clinical diagnoses. Methods We set a standard sample as following: firstly, prepare full methylated sample authenticated by Bisulfite-Sequence(100% M), and the full unmethylated sample (0% M); secondly, mix them with volume ratio 1∶3; then run gradient PCR with the range of lower 5℃and upper 5℃just to the reference annealing temperature used by former research articles. Choose the annealing-temperatures that only occur one special desired bound after electrophoresis. After this, use these temperatures as optional annealing-temperatures to run MSP in all samples. Finally, compare their sensitivities,specificities,predictive values and likelihood ratios and ROC. Results we split samples into Group A(under 70 years old)and Group B (above 70 years old). 60.7℃(Group A) negative predictive values is 0, negative likelihood ratios is 1.00, sensitivity is 1.00, ROC area is 0.750; 64. 0℃(Group A) negative predictive values is 0.8, negative likelihood ratios is 0.56, sensitivity is 0.33, ROC area is 0.583;60.7℃(Group B) negative predictive values is 0.67, negative likelihood ratios is 0.60, ROC area is 0.529; while other data are no statistics difference. Conclusions there is evidence shows that we can detect bladder cancer affiliated by MSP, especially for the diagnoses to eliminate cancer. In clinical diagnoses, there are a lot of biology factors work on to the results, such as ages. We may optimizing MSP by adjusting annealing-temperatures to get better diagnostic values.2. Analysis RASSF1A methylation patterns by McMSP Objectives detect TSG RASSF1A promoter region methylation pattern based on real-time PCR, and compare McMSP and MSP in sensitivity and diagnostic values, and reduce cost and manipulation. Methods firstly, run MCA to the MSP products; secondly,add SYBR Green I to the pre-PCR mix; Finally, dilute the original DNA templates and compare the sensitivity and ROC areas between MSP and McMSP. Results McMSP can perform perfectly even the original DNA templates＜2pg, while, MSP become introducible even the original DNA templates＞100pg; unuse commercial agent lots, and delute SYBR Green I to 10000×, the resules are satisfying; McMSP ROC analysis, Group A, B and total samples抯Tm cut-off data are 62.525,63.535,62.935; ROC areas data are 0.846, 0.750,0.819. Conclusions analyzing promoter region methylation pattern by McMSP is quick, sensitive and advantage, as well as its better diagnostic values.Meanwhile, we investigate the methylation status of RASSF2A,NORE1A and the protein expression of RASSF1A in bladder carcinoma. Objectives screen methylation status of RASSF1A gene family, and reveal the ralation between methylation status of RASSF1A and gene expression in protein level. Methods the methylation status of promoter of RASSF2A and NORE1A was examined by MSP in 54 cases of bladder carcinoma tissues and adjacent normal tissues from 54 cases; Bladder TCC(n=38)and normal bladder tissues(n=18)were detected for RASSF1A expression by immunohistochemistry. Results 2/54 of bladder carcinoma tissues showed RASSF2A hypermethylation, and NORE1A showed 0/54; None of paired adjacent normal tissues and three normal bladder tissues showed RASSF2A and NORE1A hypermethylation; the positive rates of RASSF1A expression in bladder TCC were 39%, while in normal bladder tissues were 83%. The RASSF1A expression in TCC were obviously lower than those in normal tissues; compare the (-) samples by immunohistochemistry with co-ralation DNA methylation and mRNA expression status, the co-ralation samples are 83.3 % (20/24) in DNA methylation, and 79.2% (19/24) in mRNA depression. Conclusions the levels of RASSF1A methylation are low, which is unsuitable to clinical screen. There are significant ralations between RASSF1A expression status and TCC, as well as perfect ralations between its DNA methylation status and mRNA depression. |
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