| Objective:The aim of this project was to detect the continuous methylation status of PAX1,p16 and miR-124-2 in normal cervical tissue cells as well as different levels of cervical lesions so that we could analyze the relationship between methylation and cervical lesions.We tried to explore the significance of methylation in cervical lesions’ occurrence and development.Furthermore,we preliminarily evaluated the clinical value of methylation during cervical lesion screening as well as early diagnosis.Methods:1.From December 2018 to November 2019,we collected 100 Liquid-Based Pap(LBP)samples for 4 groups that distinguished by pathology,which included: negative for intraepithelial lesion or malignance(NILM),low-grade squamous intraepithelial lesion(LSIL),high squamous intraepithelial lesion(LSIL)and squamous cell carcinoma(SCC).All patients were admitted to Shenzhen People’s Hospital and tested positive for HPV.2.According to case-control study process,we used Next-generation Sequencing,which based on the bisulfite sequencing,to quantitate the samples’ continuous methylation level of PAX1,p16 and miR-124-2 genes.3.Then we analyzed the sequencing results by mechanical algorithms and evaluated the relationship between the methylation of those genes and cervical lesions.Result:1.The mean age of samples was 41.22±11.59 years old,there were statistically significant differences between the groups,and the mean age of SCC was significantly higher than others.2.The infection rate of HPV 16/18 increased with the severity of the cervical lesions.Except 6 uncategorized samples,there were 76.6% samples had been infected HPV 16/18 in groups of HSIL and SCC while only 25.5% samples in groups of LSIL and NILM.3.Thirty-nine significant methylation sites were detected in PAX1 which included sites 1 to3,4 to 32 and 34 to 36,all of them had a similar performance in ROC curve.Take site(1,3)as an example: to use its mean methylation to diagnose HSIL+,the AUC was 0.79(95%CI,70-87%),the best cut of value was 0.04 and Youden index was 0.52,the corresponding sensitivity and specificity were respectively 70% and 82%.To use the mean methylation of site(1,3)to diagnose SCC,the AUC was 0.91(95%CI,84-96%),the best cut of value was 0.11 and Youden index is 0.75,the corresponding sensitivity and specificity were respectively 84% and 90.67%.4.Eleven significant methylation sites were detected in p16 which included sites 0 to 1 and3 to 9.To use the mean methylation of site(0,1)to diagnose HSIL+,the AUC was 0.88(95%CI,80-94%),the best cut of value was 0.15 and Youden index was 0.68,the corresponding sensitivity and specificity were respectively 68% and 100%.When use the mean methylation of site(0,1)to diagnose SCC,the AUC was 0.98(95%CI,93-100%),the best cut of value was 0.20 and Youden index was 0.84,the sensitivity and specificity were both 92%.To use the mean methylation of site(3,9)to diagnose HSIL+,the AUC was 0.89(95%CI,81-94%),the best cut of value was 0.09 and Youden index was 0.68,the corresponding sensitivity and specificity were respectively 72% and 96%.When use the mean methylation of site(3,9)to diagnose SCC,the AUC was 0.97(95%CI,92-100%),the best cut of value and Youden index as well as sensitivity and specificity were same as site(0,1).5.Ten significant methylation sites were detected in miR-124-2 which included sites 2 to 3and 5 to 9.To use the mean methylation of site(2,3)to diagnose HSIL+,the AUC was 0.84(95%CI,75-91%),the best cut of value was 0.13 and Youden index was 0.58,the corresponding sensitivity and specificity were respectively 72% and 86%.When use the mean methylation of site(2,3)to diagnose SCC,the AUC was 0.96(95%CI,90-99%),the best cut of value was 0.13 and Youden index was 0.80,the corresponding sensitivity and specificity were respectively 100% and 80%.To use the mean methylation of site(5,9)to diagnose HSIL+,the AUC was 0.85(95%CI,76-91%),the best cut of value was 0.08 and Youden index was 0.62,the corresponding sensitivity and specificity were respectively 72% and 90%.When use the mean methylation of site(5,9)to diagnose SCC,the AUC was 0.97(95%CI,91-99%),the best cut of value was 0.13 and Youden index was 0.84,the sensitivity and specificity are both 92%.Conclusion:Our study has confirmed that continuous methylation status in entire section gene of PAX1,p16 and miR-124-2 could be a reference for screening and early diagnosis of cervical lesions and complement the traditional HPV detection and cytology. |
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