| Objective: To test DNA methylation levels by pyrosequencing,and to further explore the relationship between DNA methylation level and natural history of cervical intraepithelial neoplasia 1(CIN1),and the relationship between methylation levels of DNA in CIN2/3 group and prognosis of CIN2/3 after treatment,evaluating whether it is valuable for prognosis of CIN by follow-up.Methods: 1.Exfoliated cells of different degrees of cervical lesions were collected,from whom infected by HPV and the cervical lesions were confirmed by histopathology.The methylation levels of genes SLIT2,SOX1,JAM3,TERT and C13ORF18 in cervical exfoliated cells were tested by bisulfite modification combined with pyrosequencing to find the relationship between DNA methylation and CIN and cervical cancer.2.142 patients with CIN diagnosed by histological pathology were followed up for 24 months.According to the results of follow-up,the patients were grouped,and the methylation levels of JAM3,SLIT2,TERT,SOX1 and C13ORF18 in each group were compared.ROC curve analysis was used to evaluate the efficacy of CIN prognosis by the methylation level of each gene.Results: 1.The methylation levels of SLIT2,SOX1,JAM3,TERT and C13ORF18 increase with the severity of cervical lesions.2.After follow-up,of the 50 patients with CIN1,58%(29/50)were reversal,34%(17/50)were persistent,and 8% progressed(4/50).In the cases of progression,3 cases were diagnosed for CIN2 and 1 case was diagnosed for CIN3.Of the 92 patients with CIN2/3,2 were lost to follow-up and 3 were pregnant.Rate of recurrence accounted for 8%(7/87),and rate of cure accounted for 92%(80/87).3.The methylation levels of genes JAM3,SOX1,SLIT2,TERT and C13ORF18 were compared between groups of regression,persistence and progression of CIN1.The methylation levels of four genes between three groups,except for TERT,were statistically significant,and the DNA methylation level increased with the reversal,persistence and progression of the disease gradually.The regression group was significantly lower than the persistence or progression group.The methylation levels of the five genes of case of CIN2/3 after treatment were compared between group of recurrence and cure,which were statistically significant(P<0.001).4.Under the ROC curve(AUC)of genes JAM3,SOX1,SLIT2,and C13ORF18 for predicting CIN1 persistence or progression were 0.984,0.919,0.913 and 0.744,respectively.When methylation level of JAM3 had the optimal cut-off value(Ct)of 3.94%,the sensitivity was 95.2%,specificity was 93.1%;when the optimal Ct of SOX1 was 5.58%,the sensitivity was 90.5% and the specificity was 75.9%;when the optimal Ct of SLIT2 was 6.49%,the sensitivity was 95.2% and the specificity was 86.2%;when the C13ORF18 gene had the best Ct of 1.31%,the sensitivity was 66.7%,and the specificity was 72.4%.5.The AUC of genes JAM3,SOX1,SLIT2,TERT,C13ORF18 for predicting CIN2/3 after treatment recurrence were 0.971,0.966,0.946,0.927,0.955.When the optimal Ct of JAM3 was 15.94%,the sensitivity was 94.8% and the specificity was 93.7%;when the optimal Ct of SOX1 was 17.99%,the sensitivity was 88.3%,and the specificity was 93.7%;when the optimal Ct of SLIT2 was 14.92%,the sensitivity was 78.6% and the specificity was 91.0%.When the optimal Ct of TERT was 8.97%,the sensitivity was 91.4% and the specificity was 87.5%.When the optimal Ct of C13ORF18 was 14.92%,the sensitivity was 83.4% and the specificity was 92.5%.Conclusions: 1.The hypermethylation of genes SLIT2,SOX1,C13ORF18 and JAM3 is associated with the natural history of CIN1,and the level of methylation increases with the regression,persistence and progression of cervical lesions.2.The hypermethylation of the genes SLIT2,SOX1,C13ORF18,TERT and JAM3 was associated with the prognosis of CN2/3 after treatment,and the methylation level of recurrence group was significantly higher than the regression group.3.DNA methylation level has higher sensitivity and specificity for predicting prognosis of CIN1 and CIN2/3 after treatment,especially JAM3.4.DNA hypermethylation can be a prognostic marker of CIN. |
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