| Object: Detecting the levels of circulating tumor DNA(ctDNA)methylation in the ESCC patients to screen ctDNA methylation biomarkers for clinical diagnosis of esophageal squamous cell carcinoma.Methods:(1)Using methylated DNA immunoprecipitation sequencing(Me DIP-seq)to analyze the differences of ctDNA methylation levels in plasma of 8 ESCC patients and 8 normal controls and screen the differential genes.(2)Detecting Cp G methylation levels in promoter regions of BMP7,YTHDF2,SLC3A2,VIRMA and PLCE1 genes in ESCC tissues and adjacent normal tissues by Mass ARRAY DNA assay.(3)Using immunohistochemistry to explore the expression of BMP7,YTHDF2,SLC3A2,VIRMA and PLCE1 proteins in 113 cases of ESCC and 106 cases of adjacent normal tissues and the relationship between survival prognosis and expression was analyzed by Kaplan-Meier method.(4)Using ELISA to further verify the expression of BMP7,SLC3A2 and PLCE1 in serum.(5)Using ROC curve and AUC to analyze the methylation level of promoter regions of BMP7,YTHDF2,SLC3A2,VIRMA and PLCE1,as well as the protein expression levels and serum for the diagnosis of ESCC.(6)ECA9706 and ECA109 were transfected with YTHDF2 si RNA,cell proliferation was detected by CCK8 and plate cloning assay,cell migration was detected by Transwell assay and cell cycle change was detected by flow cytometry.(7)Statistical analysis was performed with Graph Pad Prism 8 and SPSS25.0.Results: We found 17,913 and 22,145 ctDNA methylated genes in ESCC and normal samples by Me DIP-seq.Through differential analysis,we found 7672 differential ctDNA methylated genes were found,including 690 differential genes in the promoter region.The length was 200-800 bp and distributed on all chromosomes.According to the differential multiple of ctDNA methylation differential genes,enrichment function and location in KEGG pathway,we initially selected 15 ctDNA methylation differential genes for immunohistochemical verification,among which the protein expression levels of 5 genes(BMP7,YTHDF2,SLC3A2,VIRMA and PLCE1)were significantly different between ESCC patients and adjacent normal tissues.In ESCC,the methylation level of Cp G＿24.25 in BMP7 promoter region was significantly higher than that in normal control(P=0.021).The methylation levels of Cp G＿10.11 in YTHDF2 promoter region,Cp G＿23.24.25.26.27 in SLC3A2 promoter region and Cp G＿4 in PLCE1 promoter region were significantly lower than those in normal control(P=0.021,P=0.042 and P=0.014).The differences among Cp G units in VIRMA promoter region were not statistically significant(P > 0.05).The areas under ROC curve of Cp G＿18 and Cp G＿24.25 methylation levels in BMP7 promoter regions for ESCC diagnosis were0.771 and 0.833,respectively,corresponding to sensitivity of 66.7% and 75.0%,and corresponding specificity of 87.5% and 87.5%.The combined diagnosis value of the two Cp G units in the BMP7 promoter region was higher,the area under ROC curve was 0.896,and the corresponding sensitivity and specificity were 83.3% and 87.5%,respectively.The area under ROC curve of SLC3A2 promoter cp G＿23.25.26.27 for ESCC diagnosis was 0.631,and the corresponding sensitivity and specificity were93.1% and 33.3%,respectively.The area under ROC curve of Cp G＿4 in PLCE1 promoter region for the diagnosis of esophageal squamous cell carcinoma was 0.612,corresponding sensitivity and specificity were50.0% and 70.7%,respectively.BMP7,YTHDF2 and PLCE1 proteins were mainly expressed in the cytoplasm,and the expression of BMP7 protein in ESCC tissues was significantly lower than that in adjacent normal tissues(P<0.001);The expression of YTHDF2 and PLCE1 in ESCC tissues was significantly higher than that in adjacent normal tissues(P<0.001);SLC3A2 protein was mainly expressed in cell membrane,and its expression in ESCC tissues was significantly higher than that in normal adjacent tissues(P< 0.001);VIRMA protein was mainly expressed in the nucleus,and its expression in ESCC tissues was significantly higher than that in adjacent normal tissues(P< 0.001).The value of BMP7,YTHDF2,SLC3A2,VIRMA and PLCE1 scores in diagnosing ESCC was analyzed by ROC curve.It was found that the protein expression levels of these five molecules had high diagnostic value,and the areas under ROC curve were 0.911,0.974,0.983,0.842 and 0.659,respectively.The corresponding sensitivity was 78.0%,93.8%,96.3%,77.2% and 33.6%,and the corresponding specificity was 85.7%,88.4%,97.2%,86.1% and 93.0%.Kaplan-meier analysis showed that high expression of YTHDF2 was associated with poor survival prognosis of ESCC patients,while the expression of BMP7,SLC3A2,VIRMA and PLCE1 were not statistically different from the survival prognosis of ESCC patients.ELISA showed that the level of BMP7 in ESCC in serum was lower than that in normal control group,the difference was statistically significant(P=0.043).The level of PLCE1 in ESCC was higher than that in normal control group,the difference was statistically significant(P=0.008).The level of SLC3A2 in ESCC was not significantly different from that of the normal control group(P< 0.05).The serum levels of BMP7 and PLCE1 were helpful for the diagnosis of ESCC.The area under ROC curve was 0.661 and 0.749,respectively.The sensitivity was 68.0% and 63.0%,and the specificity was 65.5% and 99.3%.To further verify the biological function of YTHDF2,it was found that knocking down YTHDF2 in ESCC cell lines,the proliferation and migration ability of were significantly reduced and the cell cycle also stagnated in S phase.Conclusion:(1)The differential ctDNA methylation genes BMP7,YTHDF2,SLC3A2,VIRMA and PLCE1 have high sensitivity and specificity for the diagnosis of ESCC in promoter methylation level,protein expression level or serum level.(2)The high expression of YTHDF2 is an indicator of poor prognosis in ESCC patients,and knocking down YTHDF2 can inhibit the proliferation and migration of esophageal cancer cells and affect the cell cycle. |
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