Study On Estrogen Receptor 1 Promoter Methyl At Ion In Acute-on-Chronic Hepatitis B Liver Failure And Hepatitis B Virus-related Hepatocellular Carcinoma

PART I Methylation Status of Estrogen Receptor 1 Promoter is Associated with a High 28-day Mortality in Patients with Acute-on-chronic Hepatitis B Liver FailureBackgroundAcute-on chronic liver failure(ACLF)is an acute and severe hepatic insults based on an underlying chronic liver disease and is a complicated life-threatening syndrome characterized by jaundice,coagulation disorders.hepatic encephalopathy and ascites.In China,hepatitis B virus infection is the majority etiology of ACLF and HBV associated ACLF has been identified as acute-on-chronic hepatitis B liver failure(ACHBLF),accounting for 70%of ACLF.ACHBLF usually progresses rapidly into multiple organ failure and with a high mortality up to 50-90%in a short time.Early identification of ACHBLF patients with high mortality and properly intervention at accurate check time maybe ameliorate the ongoing liver injury and decrease the mortality rate.Several prognostic models has been generally used to evaluate the severity of liver failure.Although model for end-stage liver disease(MELD)is the common standard to prognosticate critically ill patients with liver failure,it is not designed to target the etiology of HBV infection and does not take the complications of' ACHBLF into consideration such as ascites and hepatic encephalopathy.Therefore,an objective and accurate predictive model,which could distinguish patients with high mortality at early stage,is required urgently.Regardless of pathophysiology.ACHBLF is a complex autoimmune disease with higher prevalence in men than women.This sexual disparity maybe due to lower production of estrogen and/or a reduced response to estrogen.The biological effects of estrogens are mediated by ESR,especially by ESR1 in the liver to attenuate hepatocyte apoptosis,suppress hepatic fibrosis and play an anti-carcinogenic role in patients with chronic hepatitis.None documents are ssoaiated with aberrant methylation of ESR1 promoter in patients with ACHBLF.ObjectiveIn this study,we determine and compare the methylation frequency of ESR1 promoter in PBMC from healthy controls,chronic hepatitis B,pre-ACHBLF patients and ACHBLF patients(early,mid-term andadvanced stages)in order to dynamically evaluate ESR1 promoter methylation changes with severe clinical condition.We also investigated the association of ESR1 promoter methylation with clinical characteristics and analyze the potential early predictive value of ESR1 promoter methylation for 28-day mortality in ACHBLF patients.Materials and MethodsA total of 131 ACHBLF patients,73 patients with CHB and 40 healthy controls(HCs)were recruited at Qilu Hospital of Shandong University from September 2009 to Jan 2016.Of all the 131 ACHBLF patients.113 patients hospitalized were disgnoses ACHBLF at the first(67 patients were at early stage,29 at mid-term with 29,17 at advanced).the rest 18 patients was initially pre-ACHBLF and progressed into ACHBLF finally during the inpatient time.All ACHBLF patients were followed up 28 days,and were recorded the outcomes.The diagnosis of ACHBLF was made according to the guideline in 2012.Methylation-specific polymerase chain reaction(MSP)was performed to determine the methylation frequency of ESR1 promoter and real-time fluorescent quantitative PCR performed to evaluate the expression of ESR1 mRNA.Cox proportional hazards regression analysis was used to determine the risk factors affecting survival time..Survival curves were drawn using Kaplan-Meier method and the statistical significance was determined using log-rank test.The diagnostic value of various clinical characteristics and associated criterion was assessed by receiver operating characteristic(ROC)curve.Results1.No significant difference associated with HBeAg,HBeAb and HBV DNA load between ACHBLF and CHB group.There were no significant difference among ACHBLF.CHB and HCs for age,gender and Cr.However,significant differences were found with respect to alanine aminotransferase,aspartate aminotransferase,total bilirubin,albumin(ALB),prothrombin time activity and international normalized?ratio(P<0.001,respectively)among these three groups.The incidence rate of liver cirrhosis(LC),ascites,hepatic encephalopathy(HE)and spontaneous bacterial peritonitis(SBP)in ACHBLF group was 55.75%,66.37%,30.97%and 27.43%,respectively and the mortality was 67.26%.The patients who died had a higher bilirubinemia(P = 0.003)and MELD score(P<0.001),had an increased incidence of HE(P 0 0.001)than survivors in patients with ACHBLF.2.A highly significant increase of methylation frequency of ESRI was found in PBMCs of ACHBLF compared to CHB(P<0.001)and NC(P<0.001).However,no differences of DNA methylation frequency were detected between NC and CHB.Non-survivors showed a significantly higher methylation frequency than survivors(P<0.001).And the patients with ESR1 methylation had a lower expression of ESR1 mRNA than without methylation(P<0.001).3.ESR1 promoter methylation was significantly associated with ALB(P = 0.027)and MELD scores(P = 0.044).However.there were no significant correlations with other clinic-pathological parameters.4.ESR1 promoter methylation was varied with patient's severe condition.The more severe condition,the higher frequency of ESR1 promoter methylation was.5.HE,MFLD score and ESR1 promoter methylation was the independent risk factors associated with 28-day mortality of ACHBLF.6.ESRI promoter methylation showed a significantly higher value to predict 28-day mortality of early ACHBLF than MELD score>24.6(P = 0.015).ConclusionsAbnormal ESR1 methylation occurred in ACHBLF patients and varied with patient's severe condition.ESR1 methylation might potentially serve as a early prognostic biomarker for 28-day mortality of ACHBLF in a noninvasive approach.PART ? Sera DNA Methylation of CDH1,DNMT3b and ESR1 Promoters as Biomarker for the Early Diagnosis of Hepatitis B Virus-Related Hepatocellular CarcinomaBackgroundHepatocellular carcinoma(HCC)is the most common malignant liver tumor and is the second leading cause of cancer related death in China.Hepatitis B virus(HBV)infection is the majority risk factor of HCC,which caused HCC has been called HBV-related HCC.At the initial stage,HCC is absent with pathognomonic symptoms,but progresses quickly in a short time,HCC cases have already developed locally advanced disease or distant metastasis by the time of diagnosis.Although recent advances in combined therapy of HCC,the 5-year survival rate is about 30%-40%.And the 5-year survival rate in HBV-related HCC is much worse with only 12%.The excellent prognosis is depend on the early diagnosis.Alpha-fetoprotein(AFP)and imageological examination is widely used but it has inadequate sensitivity and specificity restricted by self to identify HCC.especially at the early stage of HCC and with smaller volume.Therefore,new biomarkers are needed for the early diagnosis of HBV-related HCC from chronic hepatitis B(CHB)and HBV-related liver cirrhosis.Abnormal methylation of epigenetic events has been developed as biomarker in the development,progression and response to treatments.HBx protein encoded by HBV genome increases the expression of DNA methyltransferases(DNMT)to induce the aberrant methylation to lead oncogene activation and anti-oncogene deactivation.disturb the program of cell growth,proliferation and apoptosis and finally progress into HBV-related HCC development,invasion and metastasis.Sole serum gene methylation as biomarker for the early diagnosis of HCC is limited in clinical application,it could not reflect the complex procedure of multiple genes contributed to the carcinogenesis.Combined methylation of a series genes associated with different signal pathways may increase the accuracy of early diagnosis of HCC.HBV-related HCC is a complex autoimmune disease with higher prevalence in men than women.This sexual disparity maybe due to lower production of estrogen and/or a reduced response to estrogen.Estrogens exert their effect primarily via estrogen receptor(ESR).especially with ESR1 in liver.Aberrant ESR1 expression in liver has been implicated in stimulating hepatocyte injury and may act as HCC inducers or promoters.DNMT3h is the key enzyme to catalyze DNA methylation at the 5'cytosine of CpG sites.E-cadherin protein encoded by CDH1 is a Ca2+-dependent homophilic interaction in epithelial tissues,and decreased expression or loss of E-cadherin due to hypermethylation of CDH1 can promote invasive and metastatic behavior in many epithelial tumors.However,no documents reports sera DNA methylation of ESR1,DNMT3b and CDH1 as the biomarker for the early diagnosis of HBV-related HCC with noninvasive detection.ObjectiveWe detected and compared methylation frequency of ESR1,DNMT3b and CDH1 from HBV-related HCC,HBV-related liver cirrhosis(HBV-related LC).CHB cases and normal control(NC),and analyze the association of abnormal methylation with clinic-pathological characteristics at the progression of HBV-related HCC,explore the predictive value of sera cfDNA methylation as a noninvasive biopsy of the tumor to diagnose HBV-related HCC.Materials and MethodsA total of 406 subjects including 183 HCC(153 HBV-related HCC and 30 non-HBV-related HCC),47 HBV-related LC,126 CHB and 50 NC were randomly recruited at Qilu Hospital of Shandong University,from January 2011 to May 2013.The diagnosis of HCC was made according to the practice guidelines on the management of primary carcinoma of the liver in 2005.A total of 406 sera were collected before any treatments.Methylation-specific PCR(MSP)was applied to determine the methylation frequency of ESR1,DNMT3b and CDH1.Chi-square test or Fisher's exact test was used to compare methylation frequency of ESR1,DNMT3b and CDH1 genes among different groups.The diagnostic value of combined methylation and serum AFP was assessed by receiver operating characteristic(ROC)curve.Results1.A highly significant increase of methylation frequency of the three genes(30.07%of ESR1,41.18%of DNMT3b and 27.45%of CDH1)was found in the sera of HBV-related HCC compared to HBV-related LC(P<0.001,P<0.001,P = 0.007;respectively),CHB(P<0.001,P<0.001,P<0.001;respectively)and NC(P<0.001,P<0.001,P<0.001;respectively).However,no differences of DNA methylation frequency were detected between NC and CHB,NC and HBV-related LC,CHB and HBV-related LC.2.CDH1 exhibited a significantly higher level of methylation in non-HBV-related HCC(14/30,46.67%)compared with HBV related HCC(42/153,27.45%)(P = 0.037).No significant differences of methylation frequency of DNMT3b(P = 0.905)and ESR1(P =0.709)was found between these two groups.3.In the patients with HBV-related HCC,ESR1 methylation in the cirrhosis and resection groups was significantly higher than without cirrhosis group and other treatments groups,respectively(P=0.005.P?0.027):There was a significantly higher level of DNMT3b methylation frequency in patients with tumor size>10 cm than patients with tumor size 3-10 cm(P = 0.028)and DNMT3b methylation was significantly higher in solitary nodules than in the multiple nodule group(P = 0.045);A significantly higher methylation level of CDH1 was found in males than females(P?0.026).No significant differences of methylation frequency were found between groups stratified by AFP,HBeAg,histological differentiation,macrovascular invasion and micrometastases.4.In the patients with non-HBV-related HCC,ESR1 promoter methylation was significantly higher in the group with AFP>400 ng/ml than the group with AFP<400 ng/ml(P = 0.048);a significantly higher level of DNMT3b methylation frequency in patients with tumor size<3 cm than patients with tumor size 3-10 cm(P = 0.010).No significant associations of methylation frequency of CDH1 wass found with these clinic-pathological characteristics.5.When considering the different combination of methylation between HBV-related HCC and non-HBV-related HCC,methylation of CDH1 and LESR1 genes occurred in 21.62%of HBV-related HCC.whereas in 0%of non-HBV-related HCC considering two genes methylation.6.Combined methylation showed a sensitivity of 84.48%,specificity of 66.26%,positive likelihood ratio of 2.50 and negative likelihood ratio of 0.23.The area under the ROC curve(AUC)was significantly higher than serum AFP levels(P = 0.003).Moreover,the AUC of combined methylation was also significantly higher than serum AFP levels to discriminate HBV-related HCC from HBV-related-LC patients(P? 0.045).Conclusions1.Aberrant methylation of ESR1,DNMT3b and CDHI frequently occurred in the sera of HBV-related HCC,indicating that serum combined methylation might a potential biomarker as a noninvasive detection for the early diagnosis of HBV-related HCC.2.The difference in methylation frequency of the same genes and gene combinations provides novel information that HBV might have important influences on genes methylation.