Impct On Methylation Of CpG Island In The Promoter Region Of P14ARF Cin Period Cell After Treated By Folid Acid And 5-Aza-CdR

OBJECTIVE:(1) To test the promoter methylation status of tumor suppressor gene P14ARF (P14alternative reading frame) in the CIN tissues. (2) To study the role of folic acid and 5-Aza-2'-deoxycytidine (5-Aza-CdR) in the treatment of cervical intraepithelial neoplasia role in terms of the influence in P14ARF, and to provide the theoretical basis of drug treatment in stage of the cervical precancerous lesions.METHODS:(1) Source of specimen Collect 89 thecervical tissues including cervical intraepithelial neoplasia I,II,III of outpatients and inpatients who were diagnosed without lesions and any therapy from September 2010 to February 2011 in the Second Hospital of Shanxi Medical University. Take the lesion point of cervix, and divided it into two pieces immediately, one was fixed by routine pathological examination of formaldehyde, and the other was put into the cold phosphate buffer saline solution and was transferred to the lab for culturing.(2) Detection the methylation status of tumor suppressor gene P14ARF promoter. use the methylation specific PCR (methylation-specific PCR, MSP) technology to detect the P14ARF promoter methylation status in 59 cases of cervical intraepithelial neoplasia after drug intervention in vitro. Depending on the differences of drug intervention, the tissues were divided into the 5-Aza-CdR group, the folic acid group,the folic acid and the 5-Aza-CdR combination group and control group (that is, before the inter- vention group).(3) Results determined 10μL PCR products were obtained in 2% agarosegel electro- phoresis. Ethidium Brmide were used for dyeing. Use earners to observe and take pictures. 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, 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 SPSS 17.0 software was used to make Chi-square test, P<0.05 was considered statistically significant.RESULTS:(1) The probability of cultureing cervical epithelial tissues were CIN I/10.6 (5/47),CIN II 37.5%(9/24) CIN 11138.9%(7/18);(2) The probability of P14ARF gene methylation was increasing with the severity of CIN (CINⅠ14.8%, CINⅡ52.6%, CINⅢ70.0%); (3) After 48 hours" interfer to the 59 cases CIN explants samples, the methylation rates in the control group (that is, before intervention group), the Folid acid group, the 5-Aza-CdR group, and the two drugs combination group were 35.6% (21/59),22.0%(13/59),16.9%(10/59) and 3.4%(2/59) seperately. And the de-methylation effects in the Two-drug combination group were significantly better than the single-agent group. X2 test was used to compare with each other group.There were significant differences between the Two-drug combination group and other groups (p<0.05). Compared the control group with 5-Aza-CdR group X2=5.295,P=0.035; Compare the control group with the two drugs group, the difference was statistically significant(P<0.01);Compared folid acid group with Two-drug combination group (X2=7.265,p=0.004)the statistic is significantly difference;There is significantly statistic difference between 5-Aza-CdR group and Two-drug group (X2=4.256,p=0.029).CONCLUSION:(1) The methylation rate of tumor suppressor gene P14ARF was increasing with the development of the CIN grade,which indicates the hyper of P14ARF gene methylation may be related to the occurrence of cervical lesions in the development of important events. (2) Folic acid combined with 5-Aza-CdR could partially reverse the high methylation status of P14ARF gene, and it can be expected as one effective drug to the treatment of cervical intra- epithelial neoplasia.