Identification And Evaluation Of A Novel Methylation Marker To Detect Cervical Neoplasia Based On Real Time PCR

Cervical cancer is the fourth most common female malignant tumor worldwide.In 2018,An estimated 570 thousand new cases of cervical cancer were diagnosed and 31 thousand women died from the disease all over the world.China is the high-risk country with 75 per 100 thousand incidence rate.Over 130 thousand new cases diagnosed each year and the disease results in 57 thousand women died in 2018.Cervical cancer is not only serious threat to women's health but also cause heavy economic and social burden on the country.Cervical cancer is the only tumor with a clear etiology so far.Persistent infection of high-risk type human papilloma virus(HPV)is the main cause of the disease,and high-risk HPV DNA is found to be present in 99.7%of cervical cancer specimens.Women have an 80%chance of get HPV infection in their lifetime,and fortunately most of the HPV infection are cleared by human immune system.Infections by the high-risk HPV types persist which then increase the risk of progression to cervical dysplasia and cancer.The development of cervical cancer is typically slow.The progression to cervical cancer begins with the development of precancerous changes in normal cells and it usually takes 10-30 years to develop cancer.This provide a perfect time-windows to screen for high grade neoplasia and prevent the cancer occur.Cervical cancer is probably the first cancer to be eliminated.HPV vaccination and screen program are the major precaution for prevention of cervical cancer.HPV vaccination is the primary prevention,the HPV type coverage of current nine-valent vaccine is limited and protection efficiency is not 100%,and most importantly HPV vaccination is not widely implemented.Cervical cancer screen program plays a key role according to the national clinical practice guideline even the vaccination program has been taken.Precancerous and early stage of invasive cancer can be 100%cured.The purpose of screen program is to discover the high grade neoplasia and get clinical intervene to prevent cervical cancer.There is no national's screening strategy across China,two main screen technology are used include liquid-based cytology(TCT)and HPV DNA detection.But the sensitivity of TCT is only about 60%.HPV test has been accepted as the best triage testing,however the specificity of HPV test is low,40-60%of the women with ASCUS significance are HPV positive,among which less than 1%are confirmed to have cancer.The missing diagnosed and over diagnosed problems always disturb the clinician due to the insufficient of the two strategies.There are urgent needs for novel tools to discover the precancerous cancer precisely during the cancer screen program.DNA methylation is common epigenetic mechanism that involves the transfer of a methyl group to the C5 carbon residues of cytosines.DNA methylation plays important roles in various disease.Aberrant methylation is related to the progression of the cervical cancer,thus the cancer specific methylation markers have great potential to be used in diagnose and treatment of cervical cancer in clinics.However,none of the cervical cancer-related methylation loci reported in current studies meet clinical needs.In this study,we integrate humanmethylation450k methylation data and RNA-seq gene expression data from The Cancer Genome Atlas(TCGA)to identify cervical cancer specific DNA methylation markers.By a serial criteria of selection we identified 10 methylation marks,then use bisulfite sanger sequencing to verify these methylation markers by 80 normal exfoliated cell and 20 cervical cancer specimens.At last,we identify a CpG methylation marker in CLEC14A gene which are highly methylated in cervical cancer specimens and unmethylated in normal specimens.The RNA expression level of the CLEC14 A is much higher in normal specimen than the cervical cancer specimen,which indicate DNA methylation influence the gene function and this gene may involve the progression of the cervical cancer.We find no study to be publish about CLEC41A gene relate to cervical cancer.we than developed a new method to detect the methylation of CLEC14A based on a bisulfite duplex real time PCR.A universe primer were designed to amplify the methylation and unmethylation DNA and differentiated by two different fluorescence labeled Taqman MGB probe specific to methylated and unmethylated DNA.The methylation level were calculated by using the formulas methylation%=methylated DNA/(methylated DNA+unmethylated DNA).Then we evaluate the analytical sensitivity,specificity and reproducibility performance of the method using the Siha and HE cell line.This method shows that methylated DNA detection sensitivity is 5%in the background of unmethylated DNA.Finally we verify the accuracy of the detection system by using 20 cervical cancer specimen and 50 normal specimen compare the bisulfite Sanger sequencing method.Then we explore the methylation status of the CLEC14A gene in a spectrum of cervical lesions including 90 normal?62 low-grade squamous intraepithelial lesion(LSIL)?51 high-grade squamous intraepithelial lesion(HSIL)and 20 cervical cancer.The average percentage of methylation of normal,LSIL,HSIL and cancer were 0.2%?4.6%?30.7%and 47.8%respectively.The level of methylation correlates with progression of cervical cancer.Receiver operating characteristic(ROC)curves were generated to calculate the sensitivity and specificity of each parameter and cut off values of the percentage of methylation reference for differentiation diagnosis.ROC curve analysis demonstrated that the sensitivity,specificity and accuracy for detection of cervical cancer were 100%,84.2%and 0.916 respectively;and 84.5%,90.8%and 0.924 for detection of HSIL+respectively.Compare to the TCT and HPV detection method,it greatly improves the accuracy of the test and can effectively reduce the problems such as overdiagnosis.In summary,we identified a new methylation marker associated with cervical cancer,and the function of this gene may be related to development of cervical cancer.These results indicated that quantitative CLEC14A methylation demonstrates great potential for cervical cancer screen,and also provided a new target for the study of cervical cancer occurrence and development.