Mehthylaion-based Biomarkers Of Colorectal Cancer:identification,differential Diagnosis And Prognostic Evluation

Background and ObjectivesColorectal cancer(CRC)is one of the common malignancy of the digestive tract.Worldwide,the incidence and mortality of colorectal cancer ranks third and fourth in common malignancies.With the advancment of living standards and changes in diet,the incidence and mortality of colorectal cancer are increasing continuously,which has become a major public health problem in China.As early diagnosis and prompt treatment are favorable for patients’ prognosis,it is particularly important to identify and find biomarkers to detect CRC at an early stage.The occurrence and progression of CRC is considered as a complex progress involving multiple factos,such as lifestyle,diet habits and other environmental factors.Except them,its main pathogenesis involves a series of genetic and epigenetic changes.DNA methylation as one of the important epigenetic regulation mechanisms,has been found to play an important role in CRC.Abnormal methylation in gene promoter regions can lead to transcriptional silencing of tumor suppressors or transcriptional activation of proto-oncogenes.Therefore,studying on DNA methylation may give hints to discover new CRC-realated genes,and elucidate the molecular mechanism of CRC.Moreover,it can provide new ideas for the diagnosis,treatment and prognosis of CRC.In present study,we focused on DNA methylation biomarkers and explored their impact on the development of CRC and applications in early diagnosis asmolecular markers.Materials and MethodsIn discovery stage,6 cases of CRC patients with tumor tissue and adjacent normal tissues were collected from the Sir Run Run Shaw Hospital of School of Medicine from 2006 to 2012.Methylated immunoprecipitation sequencing(MedIP-seq)and transcriptome sequencing(RNA-seq)were used to detect gene methylation levels and transcription levels.The differential genes were then validated using the expression and methylation data of 45 pairs of tumor tissues and matched para-cancer normal tissues in the Cancer and Tumor Gene Map(TCGA).Afterward,we used multiple target region methylation enrichment sequencing(MethylTarget)to test the methylation level of specific CpG sites in 122 subjects with a diagnosis of CRC in Zhejiang Taizhou Hospital from 2002 to 2011.Finally,genes and CpG sites with significant differences in methylation levels were selected for follow-up studies.Fluorescence quantitative PCR and MethylTarget were used to detect the changes in gene expression and methylation status in colorectal cancer cell lines before and after treatment with 5-aza-2’-deoxycytidine(5-Aza-dC).CCK8 was used to detec cell proliferation and transwell assay was performed to measure migration and invasion.LASSO Logistic regression was used to dimension reduction and construct a model for discriminating CRC based on methylation levels.The methylation risk score(MRS)was calculated according to the meaningful predictor β value in the model,and was further validated in GEO data.Area under the curve(AUC),sensitivity,specificity and Youden index were computed based on the receiver operating characteristic(ROC)curve analysis.The survival curve was fitted by Kaplan-Meier method,and the difference in survival rate was compared by Log-Rank test.Multivariate survival analysis was performed using Cox proportional hazard regression model.The prognostic index(PI)of each patient was calculated according to the regression coefficient given by Cox proportional hazard regression model.The model prediction effect was evaluated using the area under the time-dependent ROC curve(AUC(t)),overall discrimination index(IDI),and net weight classification index(NRI).ResultsPart I Genome-wide methylation and expression profiling identify methylation-associated genes in colorectal cancerAfter genome-wide sequencing and analyzing,we screened 53 genes with differential methylation and mRNA expression.According to the common phenomenon that levels of methylation in the promoter usually inversely associated with the gene expression at mRNA level,21 genes were selected for the further analyses.We then performed the validation on differential levels of regional methylation and expression of these 21 screened genes between the tumor and normal tissues with TCGA data,and 13 genes were identified as candidate genes.Next,we performed the validation at specific CpG sites of these 13 candidate genes in additional 122 Chinese CRC patients.We found that the expression of C14orf159,CADM3,CNRIP1,GRIA4,GSTM2,NRXN1,PRKG2 and RSP02 were high in tumor tissues,while low methylation in their promoters.CEACAM6,GRHL2 and SRPRB were down-regulated in tumor tissues with high methylation levels.After treatment with 5-Aza-dC,the methylation levels of CADM3,CEACAM6,GRIA4,NRXN1,PRKG2 and RSP02 were decreased,while CEACAM6,GRIA4 and NRXN1 exhibited higher expression in the four CRC cell lines.Finally,we selected CNRIP1 and GSTM2 to explore their biological function in CRC cell lines and found that CNRIP1-deficient and GSTM2-deficient could promote cell proliferation.While,we did not find their impact on cell migration and invation.Part II A methylation-based assay for differentiating normal and tumor tissues of colorectal cancerAfter LASSO regression,nine independent CRC-related CpG sites(cg1 5442728,cg06907680,cg26813458,cg08157672,cg20342184,cg23559689,cg12647497,cg01508023 and cg14733048)were screened out.Based on these nine CpGs,a discriminating model was constructed for distinguishing tumor tissue from normal tissue.Subsequently,it was verified in the GEO database(GSE48684 and GSE42752).Using this model,we obtained AUC values of 0.966 and 0.993 for GSE48684 and GSE42752,respectively.Part Ⅲ A methylation-based predictor for prognosis of colorectal cancerUsing the Cox proportional hazard regression model,methylation levels of four CpG sites were found to be associated with CRC prognosis,after adjusting for age,gender,TNM stage,and chemoradiotherapy.Then based on the stepwise Cox regression analysis,a prognostic index model based on cg25639415 and cg12754421 methylation level was constructed,and the efficacy of the model was further evaluated and verified.We found that prognostic index(PI)has a better ability to distinguish between 1-,3-,and 5-year survival of colorectal cancer patients,with AUC(t)of 0.613,0.659,and 0.770,respectively.The IDI and NRI of the model were significantly improved compared with the TNM stage(P=0.013).A combined predictive predictor(CP)was construced by combining PI and TNM stage to predict patient’s survival.It was found that CP can increase the AUC(t)by 11.26%than the TNM stage alone when predicting patient’s 5-year survival.ConclusionAfter multiple validation,we identified 11 genes which were aberrantly regulated by DNA methylation in tumors and showed as promising biomarkers for CRC.The promoter region methylation may be one of the mechanisms of CEACAM6,GRIA4,NRXN1 silence in CRC.GSTM2 and CNRIP1,which exhibited high methylation levels in cancerous tissues,were mainly responsible for cell proliferation and acted as potential tumor suppressor genes in CRC.Using methylation levels of nine CpG sites,we developed a discriminating CRC model,which had high sensitivity and specificity for discriminating normal and tumor tissues and might have potential practical value.The prognostic index we established has a high accuracy in predicting the 5-year survival of patients with colorectal cancer.The combined prognosis predictor is more comprehensive and accurate to predict patients’ survival.