Methylation Status Of The T-cadherin Gene Promotor In Peripheral Blood Mononuclear Cells Is Associated With HBV-related Hepatocellular Carcinoma Progression

BackgroundPrimary liver cancer(PLC)is one of the most prevalent malignancies worldwide,and hepatocellular carcinoma(HCC)is the most common pathological type of it.Various causes are accounting for HCC cases,and hepatitis B virus(HBV)infection is the major one.Because of the limitation of existing technology to diagnosis and screen HCC including AFP level,ultrasound,computed tomography and magnetic resonance imaging,it is necessary to find a new noninvasive biomarker to detect hepatocellular carcinoma at an earlier.DNA methylation is one of the epigenetic mechanisms to regulate gene expression and frequently occurs in human cancer cells Some studies have reported the connection between the abnormal methylation of tumor-suppressor genes and carcinogenesis in liver.T-cadherin(CDH13)is a new member of the cadherin superfamily and possesses multiple functions.On the one hand,CDH13 can inhibit tumorigenicity and exert negative effects on the tumorigenic properties of parenchyma cells,on the other hand,CDH13 has positive roles in endothelial and vascular cells during the neointima formation in tumor neovascularization.HCC is typically a hyper-vascular tumor,few data are available on the total expression and methylation status of CDH13 in sera under the above co-operation.This study is aimed at detecting the methylation level of CDH13 promoter in peripheral blood mononuclear cells(PBMCs)of HCC patients,liver cirrhosis patients(LC),chronic hepatitis B(CHB)patients and normal controls(NCs),so as to explore the diagnostic and predicted value of CDH13 promoter methylation in HCCMethodsOur study included 26 NCs,65 CHB patients,14 LC patients and 157 HCC patients.We mainly focused on the mRNA expression and methylation status of CDH13 in peripheral blood mononuclear cells(PBMCs),which were detected by quantitative real-time polymerase chain reaction(RT-qPCR)and methylation-specific polymerase chain reaction(MSP),respectively.Results1.The expression levels of CDH13 were NOT the same across different groups.The mRNA average level was lowest in HCC group and highest in NCs group(p<0.001,Kruskal-Wallis H test).mRNA level in CHB group was higher than HCC(p<0.001)while lower than NCs(p<0.05).And mRNA level in early-stage of HCC was obviously lower than that in end-stage(p<0.05)2.Methylation frequency of the CDH13 promoter was significantly higher in HCC patients than that in the NCs(67.52%vs 0.00%,p<0.001)and CHB groups(67.52%vs 52.31%,p<0.05).And more hyper-methylation of CDH13 promoter existing in early-stage HCC than that in end-stage HCC(77.27%vs 44.68%;p<0.001).3.CDH13 mRNA level was significantly and relatively lower in methylated groups than that in unmethylated groups among the whole participants(p<0.001),HCC(p<0.001),CHB(p<0.001)and LC(p<0.05).We demonstrated that the CDH13 mRNA expression in control group was significantly higher than that of CHB and LC group,indicating that the results of MSP were consistent with the results of gene expression,and evidenced that hyper-methylation of CDH13 promoter down-regulated CDH13 mRNA expression indeed4.The methylation level of CDH13 promoter in HCC might be influenced or partly influenced by some critical factors such as TBil,ALB and AFP(p<0.05).However,it had no connection with age(p=0.489),gender(p=0.602),ALT(p=0.251),AST(p=0.065),PT(p=0.462)and HbeAg(p=0.507).5.HBV-related HCC patients had a higher methylation frequency of CDH13 than that in NCs,CHB and LC groups.As an important factor in signaling pathway to promote carcinogenesis regulating by CDH13,JNK level was also significantly higher in HCC(4.24±1.34 ng/mL)than that in NCs(2.50±0.47 ng/mL,p<0.001),CHB(2.92±0.53 ng/mL,p<0.05)and LC(2.46±2.11 ng/mL,p<0.001).6.The areas under ROC curve(AUC)evaluated by serum AFP level were 0.582 in male[standard error(SE)=0.039,95%confidence interval(CI)0.506-0.659;p<0.05]and 0.640 in female(SE=0.079,95%CI 0.486-0.794;p=0.086)respectively.The AUCs evaluated by the combination of AFP level and age were 0.637 in male(SE=0.038,95%CI 0.561-0.712;p<0.001)and 0.674 in female(SE=0.076,95%CI 0.524-0.824;p<0.05).The AUCs evaluated by the frequency of CDH13 methylation were 0.622 in male(SE=0.040,95%CI 0.543-0.701;p<0.05)and 0.705 in female(SE=0.074,95%CI 0.559-0.851;p<0.05).The total sensitivity was 67.95%(66.67%in male and 72%in female),the total specificity was 39.05%(36.71%in male and 46.15%in female),the positive likelihood ratio was 1.74(1.82 in male and 1.56 in female)and the negative likelihood ratio was 1.11(1.05 in male and 1.34 in female).The AUCs evaluated by the combination of CDH13 methylation and age were 0.723 in male(SE=0.035,95%CI 0.655-0.791;p<0.001)and 0.744(SE=0.070,95%CI 0.606-0.882;p<0.05)in female.While the combination of the age,methylated CDH13 level and AFP level showed a better score:AUC=0.796(SE=0.031,95%CI 0.735-0.857;p<0.001)in male and AUC=0.832(SE=0.057,95%CI 0.721-0.944;p<0.001)in female compared to any markers alone.7.The methylation of CDH13 promoter was an independent predictor for assessing the prognosis of HCC patients(r=-1.378,p<0.05,OR=0.284).Conclusions1.Compared with NCs and CHB group,there were aberrant hypermethylation of CDH13 promotor and a lower expression level of CDH13 mRNA in HBV-related HCC patients.And the expression level of JNK was higher than that of NCs and CHB,which suggesting that hypermethylation of CDH13 in PBMCs was associated with the down-expression of its mRNA2.Among HBV-related HCC patients,CDH13 in PBMCs was hyper-methylated and under-expressed compared with LC patients,CHB patients and normal controls in the progress of early HCC.But in the end-stage of HCC,CDH13 methylation level becomes lower than before under all the combined factors3.Combining age,AFP level and methylation level of CDH13 in PBMCs to make early diagnosis of HCC showed significant progress in diagnostic value.Thus,methylated CDH13 provides a new non-invasive approach for studying liver conditions4.For the significant reduction of CDH13 methylation level in the end-stage of HCC compared with the early-stage,CDH13 methylation status can be an independent factor of the poor prognosis in HCC patients.The methylation status of the CDH13 promoter in PBMCs was a potential noninvasive biomarker to predict the prognosis of HCC patients.