Research Of P16 Gene Methylation, Expression Of P16 Protein And DNMT1 In Cervical Lesions

ObjectiveCervical cancer is a common reproductive system cancer of women, persistent infection with high-risk HPV is recognized as the main cause of cervical cancer. DNA methylation, as a new subject, is payed widely attention in the research of molecular mechanism of cervical cancer. Hypermethylation of promoter region CpG islands of tumor suppressor gene is considered to be the important mechanism in inactivation of tumor suppressor genes. p16 gene CpG island s methylation is a high occurrence event in kinds of tumors,and it is closely related with cervical cancer. It is still controversial about the interaction of p16 CpG islands methylation and HPV16 infection in cervical lesions.p16 protein shows abnormal overexpression in cervical cancer.DNMT1,as a key enzyme of DNA methylation,whose dysfunction can lead to hypermethylation of tumor suppressor genes,shows abnormal expression in a variety of tumors,but there is less relevant reports about the role of DNMT1 in cervical cancer. This study aimed to explore the role of p16 gene CpG islands methylation,p16 protein,DNMT1,HPV16 and interaction of last factors in the development of cervical cancer.MethodsA case-control study was used. The study population comprised 53 cervical inflammation patients of Maternal and Child Health Center in Taiyuan ,105 cases of cervical intraepithelial neoplasia(CIN)(including52 cases of CINⅠand 53cases of CINⅡ/Ⅲ) of the Second Hospital affiliated to Shanxi Medical University and 80 new cases of cervical squamous cell carcinoma diagmosed by pathology of Shanxi Tumor Hosiptal during september 2009 to may 2010. Demographic characteristics,sexual health , reproductive factors and other related information of the study population were collected using structured questionnaire. Cervical tissue was collected by surgery of colposcopy biopsy. HPV16 was detected by PCR.p16 gene methylation was detected by methylation specific PCR(MSP).Western blotting was used to detected the expression of p16 protein and DNMT1 protein.Real-time quantitative PCR(real time PCR) was used to examine the level of mRNA of HPV16 E2, E6, p16 and DNMT1.Using SPSS16.0, Measurement data was analyzed by ANOVA and T-test; count data was analyzed byχ2 test andχ2 trend test; related factors were analyzed by Logistic regression analysis;the qualitative and quantitative measurements of interaction effects were analyzed by additive model and RERI,AP,S. Results(1)The rate of HPV16 infection in CINⅠ(71.2%),CINⅡ/Ⅲ(71.7%) (81.3%) were both significantly higher than that in cervical inflammation(49.1%)(χ2=5.340,P=0.021;χ2=5.679,P =0.017;χ2 =15.292,P﹤0.001). The level of mRNA of HPV16 E2 and E6 was significantly higher in CINⅡ/Ⅲand cervical cancer than that in cervical inflammation. The difference of statistically significance was not found in CINⅠ.(2) The expression of DNMT1 protein was significantly higher in CINⅠ(1.328±0.379), CINⅡ/Ⅲ(1.840±0.369) and cervical cancer(2.275±0.553) than in cervilities(0.908±0.287),and the differences were all statistically significant. The level of DNMT1 mRNA in CINⅡ/Ⅲand cervical caner was both higher than that in cervilities and the differences were all statistically significant. The difference of level of DNMT1 mRNA in CINⅠand cervicitis was not found. The expression of DNMT1 protein and its mRNA increased with the progress of cervical lesions.(3) The rate of p16 gene methylation was significantly higher in CINⅠ(13.5%), CINⅡ/Ⅲ(17.0%) and cervical cancer(32.5%) than that in cervicitis(0%)(χ2=7.771,P =0.005;χ2=5.634,P =0.018;χ2=21.411,P﹤0.001).The expression of p16 protein was significantly different in cervicitis(0.679±0.287), CINΙ(1.364±0.385), CINⅡ/Ⅲ(1.901±0.444) and cervical cancer(2.228±0.578)( F=135.434,P﹤0.001). The level of p16 mRNA in cervical caner was both higher than that in cervicitis and the differences were all statistically significant. The difference of level of DNMT1 mRNA in CINⅠ, CINⅡ/Ⅲand cervicitis was not found. The ratio of p16 CpG islands methylation and expression of p16 protein and its mRNA increased with the progress of cervical lesions.(4) It indicated that there were positive addictive interactions between HPV16 and p16 gene CpG island methylation, HPV16 and p16 protein overexpression, DNMT1 protein overexpression and p16 gene CpG island methylation ,DNMT1 protein overexpression and p16 protein overexpression in CINΙ, CINⅡ/Ⅲand cervical cancer.(5) HPV16, p16 protein, DNMT1 protein, cleaning after sex, pregnancies and menopause were involved in multinomial Logistic regression model in CINⅠ.p16 protein, p16 gene CpG island methylation, DNMT1 protein, HPV16, frequency of cleaning pudendum, cleaning after sex and menopause were involved in multinomial Logistic regression model in CINⅡ/Ⅲ. p16 protein, p16 gene CpG island methylation, DNMT1 protein, farmers, cleaning after sex, pregnancies, menopause, gynecological diseases history and education were involved in multinomial Logistic regression model in cervical cancer.Conclusion(1) p16 CpG island methylation,p16 protein overexpression and its mRNA overexpression may increase the risk of cervical lesions; The ratio of p16 CpG islands methylation and expression of p16 protein and its mRNA increased with the progress of cervical lesions. p16 CpG island methylation and HPV16 or p16 protein overexpression and HPV16 may exist in synergy in cervical lesions.(2) The high expression of DNMT1 protein and mRNA may be related to cervical lesions; The expression of DNMT1 protein and its mRNA increased with the progress of cervical lesions.DNMT1 protein overexpression and p16 CpG island methylation ,DNMT1 protein overexpression and p16 protein overexpression may exist a synergistic effect in cervical lesions. (3) HPV16 and multi-parity pregnancies are the common risk factors of cervical intraepithelial neoplasia and cervical cancer. Cleaning after sex and menopause are the common protective factors of cervical intraepithelial neoplasia and cervical cancer. Being a farmer is the common risk factor of CINⅠand cervical cancer. Gynecological diseases history is a risk factor of cervical cancer. High education level has a protective effect in cervical cancer.