| Objective: The purpose is to discover a panel of DNA methylation sites associated with esophageal squamous cell carcinoma(ESCC)prognosis in ESCC with TCGA(The Cancer Genome Atlas)public database resources,and verify the reliability in the established ESCC cohort.By analyzing the correlation between methylation levels and pathological parameters or prognostic parameters,we aim to reveal the role of DNA methylation in ESCC.The combination of biomarker detection and clinical information can be used to construct a predictive model to evaluate the risk of tumor prognosis.The project intends to study the function of a novel ESCC related gene FAM178 B in cell level,observe the effect of demethylation drug on gene expression and the effect of FAM178 B overexpression on the biological behavior of ESCC cell lines,and preliminarily discuss the mechanism of FAM178 B gene in ESCC.Methods: 1.The data of Human Methylation 450 K BeadChip,Illumina HiSeq_RNA-Seq Version 2 and clinical information of 68 ESCC male patients were downloaded from the TCGA website and UCSC Cancer Browser website.R software was used to load the data and build a dataset.The methylation sites were screened according to the annotation.Spearman method was used to analyze the correlation between methylation levels and gene expression.After adjusting age and clinical stage,Cox multivariate regression model was used to analyze the hazard ratio(HR)and 95% confidence interval(CI)for each methylation site.2.Formalin-fixed paraffin-embedded(FFPE)tumor tissues from 135 ESCC patients were used to measure the methylation levels of 10 candidate genes by quantitative methylation specific polymerase chain reaction(qMSP).The percent of methylated reference(PMR)represented the methylation level.The relationship between methylation and clinical parameters was analyzed by Pearson Chi-square test or Fisher exact probability method.Kaplan-Meier method was used to draw the survival curves.Log-rank test was used to compare the differences.Univariate and multivariate Cox regression models were used to calculate HR value and 95% CI.3.ESCC cell lines TE-1 and Eca-109 were cultured in vitro.The treatment group was with 10μmol/L of 5-Aza-2’-deoxycytidine(5-Aza-CdR)culture medium,and the negative control group was with the same volume of dimethylsulfoxide(DMSO).Cells of each group,being cultured for 0h,48 h and 72 h respectively,were collected.The expression of FAM178 B gene was detected by reverse transcription polymerase chain reaction(RT-PCR).4.After constructing the lentiviral vector carrying FAM178 B overexpression,we detected the protein expression of target gene by Western blot.HK293 T cells were transfected and packaged to obtain high titer lentiviral vector.TE-1 and Eca-109 cell lines affected by lentiviral vector were divided into the test group(ESCC cell lines with FAM178 B overexpression lentiviral vector)and control group(ESCC cell lines with negative lentiviral vector).We used colony formation assay,Transwell assay and apoptosis test to evaluate the effects of FAM178 B overexpression on the ability of cell proliferation,invasion and apoptosis.Results: 1.In TCGA data,we got 867 methylation sites significantly correlated with gene expression and the prognosis,and all sites are located on CpG islands.48 methylation sites(Spearman r <-0.5,HR > 2.0)had the potential of tumor suppressor and reflecting the clinical application value.32 sites were located in the promoter region,referring to 15 genes which were used for subsequent sample test.Other 16 sites were located in the gene body region.2.With a PMR cutoff value of 4%,the methylation level of each gene was divided into hypermethylation and hypomethylation.The hypermethylation frequencies of FAM178 B,FAM83C,PDLIM4,PRSS27,KLHDC7 B,CALML5,MT1 L,HOXC11,EVC2 and OTOP3 were 94.81%,85.19%,42.22%,85.93%,82.96%,94.07%,83.70%,57.04%,44.44% and 84.44%.The risk of FAM83 C hypermethylation in poorly differentiated tumors was 63.1% lower than that in moderately and well differentiated tumors(OR = 0.369,95% CI = 0.140-0.972,P = 0.039).The risk of PRSS27 hypermethylation in poorly differentiated tumors was 3.305 times that in moderately differentiated and well-differentiated patients(OR = 4.305,95% CI = 0.946-19.585,P = 0.042).The risk of PDLIM4 hypermethylation in patients without lymph node metastasis was 51.1% lower than that in patients with lymph node metastasis(OR = 0.488,95% CI = 0.242-0.985,P = 0.044).And the risk of PDLIM4 hypermethylation was reduced by 62.3% in patients without perineural and lymphvascular invasion than that in patients with perineural and lymphvascular invasion(OR =0.377,95% CI = 0.156-0.909,P = 0.026).3.The 5-year overall survival rate of 135 ESCC patients was 18.3%.Univariate Cox analysis showed that the hypermethylation of FAM178B(HR = 1.881,95% CI = 1.334-2.652,P < 0.001),PRSS27(HR = 4.789,95% CI = 1.195-19.184,P = 0.027),PDLIM4(HR = 7.646,95% CI = 1.595-36.662,P = 0.011),EVC2(HR = 2.313,95% CI = 1.364-3.924,P = 0.002),OTOP3(HR = 1.379,95% CI = 1.045-1.822,P = 0.023),CALML5(HR = 3.416,95% CI = 1.426-8.181,P = 0.006)and MT1L(HR = 1.559,95% CI = 1.086-2.240,P = 0.016)could affect the OS rate of ESCC patients and act as risk factors.Further subgroup analyses revealed that many clinical parameters were considered as confounding factors causing the correlation between methylation and prognosis.After adjusting these confounding factors with multivariate Cox regression model,we have found that increased methylation levels of FAM178B(HR = 1.684,95% CI = 1.160-2.445,P = 0.006),PRSS27(HR = 5.116,95% CI = 1.320-19.826,P = 0.018),PDLIM4(HR = 10.264,95% CI = 2.297-45.857,P = 0.002),EVC2(HR = 2.412,95% CI = 1.396-4.169,P = 0.002)and CALML5(HR = 3.173,95% CI = 1.327-7.586,P = 0.009)could predict the poor prognosis of ESCC independently.The prognostic index(PI)was obtained by stepwise forward method,and the formula was PI = 1.109 × X5 + 0.557 × X9 + 0.650 × FAM178 B + 1.440 × CALML5 + 0.701 × EVC2.X5 referred to the depth of invasion and X9 referred to the status of perineural and lymphvascular invasion.Taking the upper and lower quartiles of PI as the boundary,all patients were divided into low-risk group(PI < 2.474),middle-risk group(2.474 ≤ PI < 3.197)and highrisk group(PI ≥ 3.197).The OS rate of these three groups had a statistical difference(χ2 = 29.716,P < 0.001).4.After 48 hours and 72 hours treatment with 5-Aza-CdR,the expression changes of FAM178 B were not significantly different(F = 2.987,P = 0.126).Interestingly,the expression levels of FAM178 B in TE-1 cell were downregulated,and the difference was statistically significant(F = 576.460,P < 0.001).5.Colony formation assay showed that the clone forming number of Eca-109 cell line in the experimental group was higher than that in the control group(268 ± 4 vs.212 ± 3,P < 0.001).The clone forming number of TE-1 cell line in the experimental group was higher than that in the control group(67 ± 2 vs.58 ± 2,P = 0.005).The result of Transwell assay showed that the invasion cells per field in the Eca-109 group was more than that in the control group(77 ± 1.52 vs.66 ± 1.49,P = 0.048).No positive result was found in TE-1 cell line.The result of apoptosis test showed that the apoptosis percentage of Eca-109 cell line in the experimental group was higher than that in the control group [(6.56 ± 0.04)% vs.(5.18 ± 0.08)%,P < 0.001].No positive result was found in TE-1 cell line.Conclusions: 1.The integration analysis of methylation chip and transcriptome sequencing data is helpful to discover the potentially prognosis-related markers from massive methylation sites.2.The phenomenon of simultaneous methylation of multiple genes exists in ESCC.Multivariate Cox regression analysis shows that methylated FAM178 B,PRSS27,PDLIM4,EVC2 and CALML5 genes are independent risk factors for ESCC postoperative prognosis.3.The prognostic index of five factors(the depth of invasion,the status of perineural and lymphvascular invasion,FAM178 B methylation,CALML5 methylation and EVC2 methylation)can be effectively used to predict the long-term survival of ESCC patients.4.The effects of demethylation drug on the expression of FAM178 B gene in ESCC cell lines are different,suggesting tumor heterogeneity when CpG island methylation in the promoter region regulates gene expression.There may be other molecular mechanisms involved in gene transcription process during carcinogenesis.5.Overexpression of FAM178 B can promote the proliferation and enhance the ability of ESCC cell invasion;meanwhile,it accelerates the apoptosis of tumor cells.Taken together,FAM178 B may act as an oncogene in ESCC pathogenesis,and it is worthwhile to research the exact mechanism in ESCC prognosis. |
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