Impct On Methylation Of CpG Island In The Promoter Region Of Dapk Gene In GIn Period After Treated By Folid Acid And 5-Aza-CdR

OBJECTIVE: 1. Testing the promoter methylation status of samples that the CIN tissues of tumor suppressor gene DAPK (death-associated protein kinase), Occurrence of changes in the role of cervical cancer;2. To study the role of folic acid and 5-Aza-2'-deoxycytidine (5-Aza-CdR) in the reversal of cervical intraepithelial neoplasia, and to provide the theoretical basis of drug treatment in stage of the cervical precancerous lesions.METHODS: 1. Source of specimen We collected the specimen of patients treated for cervical intraepithelial neoplasia lesions in patients from January 2010 to January 2011 in the Second Hospital of Shanxi Medical University, We take the same point of cervix in vitro lesions, and divided it into two pieces Immediately, the one was fixed by routine pathological examination of formaldehyde, and the other was put into the cold phosphate buffer saline solution used in the experiment.2. Detection of tumor suppressor gene DAPK promoter methylation status We select the tissues of CIN patients, Depending on the situation of drug intervention, were divided the tissues into the single 5-Aza-CdR treatment group, the folic acid and 5-Aza-CdR two drugs combination treatment group and control group (that is, before the intervention group).using the methylation specific PCR (methylation-specific PCR, MSP) technology, To detect the DAPK gene promoter methylation status in 60 cases of cervical intraepithelial neoplasia after in vitro drug intervention.3. Results determined 10μL PCR products were obtained in 2% agarose gel electro- phoresis, Ethidium Brmide dyes, imager and camera observation. If methylation products were obtained, no matter whether non-methylated products were obtained or not, they were judged to be methylation-positive; If the products appear non-methylated only, when the result was non-methylated; If obtained neither the methylation products nor the non-methylated products, it marks the experiment was failed.4. Statistical analysis using SPSS13.0 software to make Chi-square test, P<0.05 was considered statistically significant.RESULTS: 1.In the all 60 cases of the CIN tissues, the whole methylation rate was 21.7%(13∕60),with the advance of the CIN disease, the methylation rates of gene DAPK showed an increasing trend.2. The 60 cases of tissue samples were reaped after 24 hours which were interfered by different drugs, 5-Aza-CdR group, the two drugs combination group ,the methylation rates dropped to 8.3%% (5/60), 1.7% (1/60 ). Two-drug combination group de-methylation effects were significantly better than single-agent group, Compared with other groups there were significant differences (P<0.05).CONCLUSION: 1. DAPK hyper-methylation of tumor suppressor genes is the occurrence of cervical lesions in the development of important events, Reversal of DAPK gene hypermethylation status may be the prevention of cervical cancer, and containing the progress of cervical intraepithelial neoplasia as a new therapy.2. Folic acid combined with 5-Aza-CdR may reverse the high DAPK gene methylation status, and it was expected to be the effective drugs treatment of cervical intra- epithelial neoplasia.