Methylight-based Analysis For Performance Of DNA Methylation Markers In Esophageal Carcinoma

BACKGROUNDEsophageal carcinoma(EC)is one of the most common gastrointestinal malignancies,mainly squamous cell carcinoma(SCC)in developing countries.Esophageal cancer has no definite characteristic symptoms in early stage,it is difficult to treat in the advanced stage and has a poor prognosis,whereas effective prevention and treatment strategies at the present stage was cornered because of accessibility for large-scale screening.Invasive screening methods have poor group compliance and accessibility.Therefore,finding reliable biomarkers to solve the problem of esophageal cancer screening and diagnosis will be of great significance for the adjustment and optimization of esophageal cancer prevention and treatment strategies.OBJECTIVEThis experiment is in the current international and domestic research progress of,on the basis of extensive literature research,then pick some genes of interest,which some relevant studies with reliable research methods and sufficient sample sizes have been screened out for genes with relatively good effects:STK3,ARHGEF4,NDRG4,SHANK2,TBX15,and some other areas,a total of 18 different methylated regions.Designing a sets of specific oligonucleotide primers,so that we could measure these MDMs performance in esophageal cancer tissue samples using Methylight technique.Comparing differences of performance between normal tissue,early stage tumor tissues,and progressed stage esophageal phase,also the levels of methylation,with these selected DMRs;The performance differences of these MDMs in different technological methods were analyzed by comparing with the research results in relevant fields all over the world,the role of epigenetic changes in the occurrence and development of tumors was explored based on this connection.At the same time,this study is expected to build a risk prediction model through the analysis of sample data,and explore the differences in methylation levels between tumor tissues and non-tumor tissues in the differentiation of esophageal cancer by single marker and multiple markers combined classification.The MDM with good performance verified in this experiment will give us some inspiration in the research of tumor markers for non-invasive diagnosis of esophageal cancer.MATERIALS AND METHODSThis research collected a total of 54 cases of ESCC FFPE samples from the Nanfang hospital.Tissue DNA was extracted by purification column extraction method,sample DNA was transformed by magnetic beads.By modifying design fluorescent methylation site specific oligonucleotide sequence,based on the Methylight technique to detect methylation level of samples.Then,biostatistical analysis was performed to compare whether the methylation levels of these DMRs were different in different stages and groups.Logical regression was used to build a diagnostic prediction model to evaluate the prediction ability of MDMS with better performance under the experimental technology conditions.ROC curve was drawn and the area under the curve was calculated.The performance differences between the marker combination classifier and the single methylation marker was compared.RESULTSAbout 1-2 CpG-islands were selected from the 14 differentially methylated regions included in the experimental design for the experiment,and 4 MDMs have good performance in differentiating tumor tissues from distal paracancer tissues group,also different staging groups.The top four markers are listed as following:Chr(19KN196484V1),STK3,OLPLAH,and ZNF542,AUC can reach 0.720-0.908 at 95%confidence interval with P.less than 0.001.Taking the boundary value of the maximum Yoden index,the MDM with the highest sensitivity is CHR19KN196484V1,which can reach 0.971.MDM with the highest specificity is STK3,which has a specificity of 0.857.CONCLUSION1.All the 14 MDMs included in this research were able to form gradient differences in the detection of human genome methylation standards,and had the ability to distinguish the methylation levels of DNA in different samples.2.95%confidence interval(P<0.001),AUC of top 4 markers coμLd reach 0.720-0.908.Taking the bound value of the maximum Yoden index,the MDM with the highest sensitivity was CHR19KN196484V1,which could reach 0.971;MDM with the highest specificity was STK3,with the specificity of 0.857.3.After constructing the prediction model,it can be concluded that the AUC of the double-labeled classifier combining CHR19K196484V1 and STK3 can reach 0.916,the specificity can reach 0.895 and the sensitivity is about 0.829 when the Youden index is the maximum.Eventhouge the specificity and sensitivity of the double-marker classifier was not significantly better than that of single MDM,the AUC was improved a lot.4.CHR19K196484V1and STK3 have the potential to be used as tumor markers for non-invasive examination of esophageal cancer.