Correlation Study Of High-risk Type HPV Detection, DNMT1 Expression And DAPK Methylation Status Detection In Cervical Lesions

Objective：1. To detect the expression of DNMT1 protein and the methylation status of suppressorgene DAPK promoter in cervical lesions, discuss their role in the development of cervical lesions2. To Study the relationship among infection of high-risk type HPV, expression of DNMT1and methylation status of DAPK promoter in cervical lesions, and to further understand thepathogenesis of the disease, to help early screening and monitoring of disease progression.Methods:1.Source of specimen We randomly collected the 150 patients were treated with TCT andcolposcopic examinations from 2011 January to 2011 December. According to the results ofcervical biopsy pathologic diagnosis under colposcopy, divided into cervical intraepithelialneoplasia grade I ( CIN I ) in 30 cases, cervical intraepithelial neoplasia grade II ( CINII )30cases, cervical intraepithelial neoplasia ( CIN III ) in 30 cases, squamous cell carcinoma( SCC ) in 30 cases as study group; the other with negative for intraepithelial lesion ormalignancy ( NILM ) as the control group of 30 cases.2. Detection of high-risk type HPV-DNA infection For patients with TCT residualpreservation solution, DNA was extracted, the SPR method for detection of HPV, HPV-DNAinfection was detected by the biosensor chip.3. Detection of the expression of DNMT1 protein state The selected the TCT residualpreservation solution of 150 patients (preservation period of not more than 3weeks), via theThinprep2000 system to produce 1cell slides. Then put the sliders under 95% alcohol fixation for24 hours at room temperature, then dry them in the air, store them in the refrigerator at 4 degreesbackup to DNMT1 protein was with immunocytochemistry. Cells picture were observed under amicroscope. The case with brown particles in cytoplasm and stromal cells was positive.4. Detection of tumor suppressor gene DAPK promoter methylation status Patients’TCTresidual preservation solution, with MSP technology，were detected the methylation status on thecervical exfoliated cells DAPK gene promoter region. Determination of results PCR productswere walking in 8% PAGE. Get methylated product, whether they appear non-methylatedproduct, both determinated to be methylated positive; only a non-methylated product were foundfor unmethylated; has not been methylated product and non-methylated product is the sign of thefailure of the experiment.5. Statistical analysis Using SPSS18.0 software package for the Statistical analysis,α= 0.05 of inspection standard.Result:1.150cases of cervical lesions in cervical exfoliated cells in each phase of high-risk typeHPV-DNA detection: Total high-risk type HPV positive rate was 80.67% (121/ 150), trend ofχ2= 30.658, P < 0.05,with the extent of the lesion progression, HPV infection rate is alsoshowing a trend of increasing.2. Expression of DNMT1protein in each group: In 6.67% of NILM group; CIN group56.67%;CIN II group 63.33% group66.67%; CIN; SCC76.67%DNMT1 protein expression. DNMT1protein in various types of cervical lesions in the positive expression rates have significantdifference (P < 0.05), and with the extent of the lesion progression, DNMT1 protein expressionrate showed increasing trend (P < 0.05).3. The experiment of promoter region of DAPK gene in CpG island methylation positive ratewas 26.67% (40/ 150), with the extent progression of the lesion, DAPK gene methylation rateswere 3.33%, 10.00%, 13.33%, 46.67%, 60.00%, there are significant differences among thegroups ( P < 0.05), and showed a trend of increasing( P < 0.05).4. In cervical lesions during the development of sick, fection of high-risk type HPV,DNMT1protein expression and DAPK gene methylation every two were positively correlated ( P< 0.05), The correlation coefficient is respectively: 0.340、0.214、0.444.Conclusion：With the rise of the level of cervical lesions, high-risk type HPV persistent infection,abnormal high expression of DNMT1protein and DAPK tumor suppressor gene abnormalhypermethylation, the promotion of relations may exist between between each Indicators. Newideas and new methods were proposed for the discovery and monitoring of the severity ofcervical lesions.