| Objective:To quantitatively measure the methylation levels of SLIT2,SOX1 and JAM3 genes of exfoliated cells from different cervical lesions that infected by highrisk human papillomavirus(confirmed by method of HC-II).comparing the genes methylation levels between groups.To analyze its role in cervical cancer occurrence and development,and to explore their application value in the early cervical cancer screening.Methods: Collected exfoliated cells of different cervical lesions that infected by high-risk human papillomavirus.According to histopathologic diagnosis results,there were45 cases with normal cervix,47 cases with cervical intraepithelial lesion I(CINI),70 cases with CINII/III,80 cases with cervical cancer.DNA methylation of SLIT2,SOX1 and JAM3 genes were quantified using by bisulfite and pyrosequencing in all specimens,and to evaluated their diagnostic value for cervical cancer and advanced precancerous lesions.Statistical analysis was performed by SPSS Version20.0,measurement results were expressed by mean±standard deviation.using the One-way ANOVA to analyze the methylation level,and multiple comparision between different groups were analyzed by the LSD.Receiver operating characteristic(ROC)curves were made and the area under the curve(AUC)were determined for the detection of the cervical(pre)-cancerous lesions.Results:1.The mean methylation value of SLIT2 were(5.29±1.29)%,(5.75±2.57)%,(11.65±6.74)% and(25.22±14.63)% in the normal cervix,CINI,CINII/III and cervical cancer groups,respectively,there were statistical significance between groups(F=66.72,P<0.001).The mean methylation value of SOX1 were(5.78±1.17)%,(5.35±1.36)%,(13.17±8.42)% and(32.30±16.38)% in the normal cervix,CINI,CINII/III and cervical cancer groups,respectively,comparision among groups(F=96.81,P<0.001).The mean methylation value of JAM3 were(3.84±1.84)%,(3.96±1.79)%,(13.78±7.06)% and(25.58±13.09)% in the normal cervix,CINI,CINII/III and cervical cancer groups,respectively,had a obvious differences between groups(F= 92.77,P<0.001).Showed that each gene methylation level in cervical cancer group was obviously higher than that of other groups(P<0.001),and methylation increased with the degree of cervical lesions.2.The area under the ROC curve(AUC)was 0.914 for SLIT2 gene methylation,0.953 for SOX1 gene and was 0.925 for JAM3 gene,to discriminate cervical cancer from CINII/III,CINI,and normal.The optimal cut-off value for SLIT2 gene methylation was set at 14.08%,with the sensitivity of 74.0% and with specificity of 90.0%.The cut-off value for SOX1 was set at 17.25%,with the sensitivity of 85.0%and with specificity of 90.0%.If the cut-off value was set at 16.13%,the sensitivity and the specificity of JAM3 gene methylation were 81.0% and 90.0%respectively.Smilarly,the area under the ROC curve(AUC)was 0.940 for SLIT2 gene methylation,0.933 for SOX1 gene and was 0.982 for JAM3 gene,to discriminate CINII/III and worse lesions from CINI and normal.The optimal cut-off value for SLIT2 gene methylation was set at 7.08%,with the sensitivity of 86.5% and with specificity of 88.0%.The cut-off value for SOX1 was set at 9.58%,with the sensitivity of 83.2% and with specificity of 100.0%.If the cut-off value was set at 10.37%,the sensitivity and the specificity of JAM3 gene methylation were 85.8% and 100.0%respectively.3.The combination of SLIT2,SOX1 and JAM3 genes methylation for detection of cervical cancer,we found that the panel of SLIT2/SOX1 with the sensitivity of 82.9%and specificity of 88.0%,with sensitivity of 85.8% and specificity of 90.8% for CINII/III.The panel of SLIT2/JAM3 with the sensitivity of 87.1% and specificity of86.7% for cervical cancer,with the sensitivity of 90.0% and with specificity of 89.7%for CINII/III.The panel of SOX1/JAM3 with the sensitivity of 82.4% and specificity of 91.0% for cervical cancer,with the sensitivity of 90.0% and with specificity of92.9% for CINII/III.Conclusions:1.The hypermethylation of tumor suppressor SLIT2,SOX1 and JAM3 exsit in cervical cancer,and the methylation level increased with the degree of cervical lesions.2.Quantitative measurement the methylation level of SLIT2,SOX1 and JAM3 genes,with high sensitivity and specificity,for the detection of cervical cancer and advanced precancerous lesions.3.The combination of gene detection for cervical cancer and advanced precancerous lesion have better dignostic efficacy.4.Pyrocequencing technology was suitable for early cervical cancer screening due to its advantages of operating easily,high accuracy,good reproducibility,high flux,and low cost. |
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