Study On The Correlation Between Methylation Status Of HPV-16 E2 Binding Sites And Cervical Lesions

Cervical cancer is one of the most common malignancies of gynecologic reproductive system in the world,which brings serious physiological and psychological burden to the health of women.The incidence of cervical cancer in different countries is different.According to statistics,the number of new cases of cervical cancer in China is about 98,900,and the deaths is about 30,500,all of which are higher than those of the developed countries in the world.Persistent infection with high-risk human papillomaviruses?HR-HPVs?is the definitive etiological agent of cervical cancer.Among the HR-HPVs,HPV-16 and HPV-18 are the most prevalent types observed in cervical carcinoma.Currently,cervical cancer screening mainly depends on cytology and HPV DNA testing,but both methods have limitations.The sensitivity of cytology is low and the false negative rate is high.However,HPV DNA testing has increased sensitivity,but its specificity is poor.In addition,it is unable to determine whether there is a risk of further progression of cervical lesions in HPV infected individuals.Therefore,it is an urgent to find early indicators that can predict the development trend of cervical lesions,so that high-risk patients can be accurately detected from women with persistent HPV infection.As a result,the goal of precise prevention and treatment of cervical cancer can be achieved.With the further study of the pathogenesis of cervical cancer,the role of DNA methylation has gradually been recognized.Previous studies have confirmed that HPV-16 L1 gene methylation is closely associated with cervical lesions.However,it is highly controversial in describing the association between the methylation status of HPV-16 long control region?LCR?and the level of cervical lesions.The HPV-16LCR contains four E2 binding sites?E2BSs?with the same palindrome.Related studies reported binding of the E2 protein to E2BSs as transcription factors that may exert either activation or inhibition of E6 and E7 oncoproteins.However,the methylation status of four E2BSs in HPV-16 LCR may alter binding affinity to the E2protein,which affects transcriptional regulation of E2 on E6 and E7 oncoproteins.In this study,the methylation status of four E2BSs in HPV-16 LCR in clinical samples was quantitatively evaluated by pyrosequencing.To study the relationship between the methylation status of E2BSs and cervical lesions so as to search for new molecular markers for the early diagnosis of cervical cancer.Materials and methods1 ObjectsA total of 578 cervical cell specimens?median age 37 years,range 18-81 years?were collected from patients undergoing conventional liquid-based cytology tests from July 2016 to March 2017 in Henan Maternal and Child Health Hospital.Cytology were classified using the 2014 Bethesda Reporting System?TBS?by three professional cytopathologists.Hybridization typing was used to genotype HPV in specimens,and only HPV-16 single infection was included in the study.Finally,59specimens?median age 37 years,range 25-65 years?were included in the study.Among them,18 asymptomatic HPV-16 infection as the control group,LSIL?18?,HSIL?17?,SCC?6?.The study was approved by the institutional Ethics Committee of the Bioscience.Informed consent was obtained from all participants prior to inclusion.2 Methods2.1 Primer designThe sequence of four E2BSs in the HPV-16 LCR is querying in genebank?NC₀01526.4?.A total of three pairs of amplification and sequencing primers were designed using PyroMark Design 2.0 software.One of the primers in each amplification reaction was labeled with biotin at its 5'end.2.2 PyrosequencingThe sample DNA is converted using sulfite solution.The methylation level of each CpG site in the sample is quantified by pyrosequencing and Pyro Q-CpG software.2.3 bDNA assayThe levels of HPV E6/E7 mRNA in residual samples were assayed using Quantivirus^?HPV E6/E7 RNA 3.0 assay?bDNA?.3 Statistics analysisSPSS 21.0 software was used for statistical analyses.The correlation among the HPV-16 E2BSs methylation,E6/E7 mRNA level and the severity of cervical lesions were evaluated by Spearman rank correlation.Kruskal-Wallis and Mann-Whitney tests were used for comparison of non-parametric data.ROC curve was used to evaluate the sensitivity and specificity of methylation levels of E2BSs in the diagnosis of HSIL+?HSIL and SCC?.P-values<0.05 were considered significant.Results1.The methylation status of 8 CpG sites in HPV-16 LCR was successfully detected in each sample.The methylation levels of 8 CpG sites in HSIL+were higher than those in asymptomatic HPV-16 infection group?all P<0.05?.Compared with other CpG sites,the methylation status at 7100 locus were higher in the three groups,whereas the methylation status at 7073 locus was lowest.2.Spearman correlation analysis showed that E2BSs methylation levels were positively correlated with cervical lesions?r_s=0.614,0.599,0.289 and 0.502,respectively;all P<0.05?.The methylation levels of E2BS1,E2BS2 and E2BS4 were significantly higher in HSIL+than other two groups?all P<0.05?.However,in addition to E2BS4,the difference was significant between the LSIL and asymptomatic HPV-16 infection?both P<0.05?.3.The methylation level of E2BS3 were the lowest in HSIL+and LSIL groups?all P<0.05?.And the E2BS1 showed higher methylation compared to E2BS4?both P<0.05?.However,no significant difference was observed among the four E2BSs in asymptomatic HPV-16 infection?P>0.05?.4.The HPV E6/E7 mRNA level significantly increased with the increase of methylation levels of E2BSs?r_s=0.480,0.467,0.290 and 0.302,respectively;all P<0.05?.And the E6/E7 mRNA level has a moderate correlation with E2BS1 and E2BS2.5.The methylation levels of E2BS1 and E2BS2 had the best diagnostic value for predicting HSIL+?AUC=0.791,95%CI=0.668-0.914;AUC=0.806,95%CI=0.680-0.931?,which respectively achieved 73.9%sensitivity with 72.2%specificity and 73.9%sensitivity with 80.6%specificity.Conclusions1.The methylation status of E2BSs may affect the regulation of E2 protein on E6/E7 oncogene.2.HPV-16 E2BSs methylation levels are related to the severity of cervical lesions.Methylation may lead to the occurrence of cervical cancer.3.The methylation status of E2BS1 and E2BS2 may be used as an early molecular diagnostic marker for the severity of cervical lesions.