Identifying Potential DNA Methylation Markers In Early-stage Colorectal Cancer

BackgroundColorectal cancer(CRC)is the second leading malignancy that accounts for 9.2%of mortality worldwide.Early detect and polypectomy can prevent cancer and reduce mortality.Therefore,screening for colorectal cancer is important for early diagnosis of colorectal cancer.Epigenetics is one of the most important ways to regulate the gene expression of the human.In recent years,with the advancement of epigenetics research,DNA methylation has become the most studied and clearest way of apparent modification.In the development of tumors,aberrant DNA methylation is an important inducement,which can occur in the early stage of tumorigenesis and it has tissue specificity,suggesting that methylation can be uesed as a new tumor molecular marker of tumor with unique advantages.Yet current screening modality mostly involves invasive colonoscopy and sigmoidoscopy while non-invasive tests have limited detection sensitivity in early stage CRC.Accurate biomarkers and noninvasive approach to detect early colorectal cancer becomes urgent clinical needs.In the preliminary work our group found a panel of genes related to early colorectal cancer.Our next aim is to verify whether the methylation level of this group of biomarkers can distinguish non-cancerous from colorectal cancer tissue samples,explore its methylation levels in different colorectal subtypes,and construct a predictive model for early colorectal cancer,It provides reference for early diagnosis,early screening and prognosis for colorectal cancer.Method1.We collected tissue samples of colorectal cancer patients enrolled in Southern Medical University successively:non-cancerous tissue(control group);colorectal cancer tissue,and subtype were divided into different groups;clinical data were collected.2.Preparation DNA sample:Genomic DNA(gDNA)and RNA were extracted from paraffin sections using Qiagen's ALLPrep DNA/RNA FFPE Kit.Genomic DNA was stored at-20° after quality detect.3.Methlight detect tissue DNA methylation:The extracted DNA was bisulfited,and the specific operation was carried out according to the protocol of Zymo DNA Methylation-Direct MagPrep kit.All the products of bisulfited were used for multiple methylation amplification.The specific methylated probe was used to determine the amount of multimethylated amplification products and the results were calculated.4.Data were analyzed by R software,The T-test was used for examined,the differences among different groups,p-value<0.05 is considered statistically significant.Model assessment was performed using the pROC and ggplot2 package.Results1.In the panel of genes,SFMBT2,ITGA4,THBD,ZNF304,KCNJ12,VAV3-AS1,EVC,TWIST1 make a significant difference between cancerous and non-cancerous tissue2.In colorectal cancer tissues,the combination of three methylation markers of KCNJ12 VAV3-ASI EVC can significantly distinguish between early colorectal cancer(stage0-stagel)and advanced colorectal cancer(stage?-stage?).3.We choosed the optimal gene combination:SFMBT2,ITGA4,THBD,ZNF304to establish the logistic regression mathematical model and plot the ROC curve to test its prediction results,the mean AUC value for trainset is 0.97,for testset is 0.96.ConclusionIn our work,Two panels of methylation markers for the purpose of CRC early detection and CRC stratification are identified on a tissue level with respective classification models showing promising clinical performances in current cohort The potential of these biomarkers in the application of systematic management of CRC,and precancerous lesions is proposed.