Study On The Methylation Status Of Tace In Chronic Hepatitis B And Acute-On-Chronic Hepatitis B Liver Failure

PART I DEMETHYLATION OF TUMOR NECROSIS FACTOR-a CONVERTING ENZYME PROMOTER ASSOCIATED WITH HIGH HBEAG LEVEL IN CHRONIC HEPATITIS BBackgroundHepatitis B viral hepatitis is a worldwide disease, developing countries have high incidence. According to the world health organization report, about 2 billion people had been infected with HBV in the world, including 350 million people with chronic HBV infection. About 100 million people died of HBV associated liver dieases, including liver failure, hepatic cirrhosis and hepatocellular carcinoma. HBV infection caused great threat to human health. How to control disease progression and remove viruses has been a research hot spot. Hepatitis B e antigen (HBeAg) seroconversion is a crucial step during the progression of HBV infection. In patients with chronic hepatitis B, HBeAg seroconversion means clinical improvement, liver disease into the stationary phase, reducing liver fibrosis, liver cirrhosis, lower the incidence of hepatocellular carcinoma. However, there has been no sensitive and effective model for predicting the occurrence of HBeAg seroconversion until now.DNA methylation is one of the DNA modification of transcription level in the CpG island. The aberrant DNA hypermethylation of gene promoter regions is an important epigenetic mechanism that regulates gene expression leading to down-regulation and silencing of several genes. It plays an important role in maintaining normal cell differentiation. Tumor necrosis factor-a-converting enzyme (TACE) belongs to a disintegrin and metallopriteinases (ADAMs) family, cracking release active soluble extracellular domain structure. The methylation of TACE gene promoter may be associated with gene silencing. Therefore, aberrant methylation of TACE gene promoter may occur in patients with chronic hepatitis B (CHB) and may be associated with its disease progression. There is no study of the promoter methylation of TACE on CHB, especially on HBeAg positive CHB.ObjectiveTo evaluate the tumor necrosis factor-α converting enzyme (TACE) methylation status in patients with CHB and its association with clinical features.MethodsEighty patients with HbeAg-positive CHB,80 with HbeAg-negative CHB, and 40 healthy controls (HCs) were randomly enrolled in this study. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) and methylation status of TACE promoter was determined by methylation-specific polymerase chain reaction (MSP-PCR). The clinical and laboratory parameters were collected.Quantitative variables were expressed as the mean (centile 25; centile 75). Categorical variables were expressed as number (percentage). Statistical analyses of the data were performed with SPSS version 16.0 (Chicago, IL, USA). Categorical variables were compared by chi-squared test. Quantitative variables were compared by Student’s t test or Mann-Whitney U test. Spearman correlation coefficients were calculated to evaluate correlations between TACE methylation status and clinicopathological parameters. All statistical analyses were two-sided and P< 0.05 was considered statistically significant.Results1. One hundred and thirty of 160 patients with CHB (81.25%) and 38 of 40 HCs (95%) displayed TACE promoter methylation. The difference was significant (χ2= 4.501, P< 0.05).2. TACE promoter methylation frequency in HBeAg-positive CHB (58/80, 72.5%) was significantly lower than in HBeAg-negative CHB (72/80,90%; χ2= 8.041, P< 0.01) and HCs (χ2= 8.438, P< 0.01). However, no significant difference was observed in the methylation frequency between HBeAg-negative CHB and HCs (χ2=0.873, P> 0.05).3. In the HBeAg-positive group, TACE methylation frequency was significantly negatively correlated with HBeAg (r=-0.602, P< 0.01), alanine aminotransferase (r =-0.461, P< 0.01) and aspartate aminotransferase (r=-0.329, P< 0.01). In HBeAg negative CHB group, no significant correlation between TACE promoter methylation and Sex (r=-0.009, P=0.934), Age (r=0.023, P=0.842), HBeAg (r=0.076, P=0.500), Log10 [HBV DNA] (r=0.060, P=0.596), ALT (r=-0.111, P=0.327), AST (r=-0.073, P=0.519), ALB (r=0.016, P=0.886), TBIL (r=-0.144,P=0.201), PTA (r=0.156, P=0.167) and Cr (r=-0.023, P=0.836) could be observed.4. In the HBeAg-positive group, TACE promoter methylation frequency in ALT <2ULN group (18/20,90%) was significantly higher than in ALT≥2ULN group (40/60,66.7%;χ2= 4.096, P=0.043). In the HBeAg-negative group, there was no defferece between ALT<2ULN group(35/38,92.1%) and ALT≥2ULN group(37/42, 88.1%;χ2= 0.356, P=0.55)。ConclusionPatients with HBeAg-positive CHB have aberrant demethylation of TACE promoter, which might potentially serve as a biomarker for HBeAg seroconversion.PART II DEMETHYLATION OF TUMOR NECROSIS FACTOR-a CONVERTING ENZYME PROMOTER PREDICTS POOR PROGNOSIS IN ACUTE-ON-CHRONIC HEPATITIS B LIVER FAILUREBackgroundLiver failure is a common severe disease clinical syndrome, the case fatality rate is extremely high. It is of serious liver damage caused by a variety of factors, lead to liver function such as synthesis, detoxification, excretion and biogenesis happen serious obstacle even decompensation, jaundice, blood coagulation dysfunction, ascites and various clinical manifestations of hepatic encephalopathy, According to the histologic features. and illness development speed, liver failure were divided into four categories:acute liver failure, subacute liver failure, acute-on-chronic liver failure, chronic liver failure. Acute-on-chronic liver failure (ACLF) is an acute hepatic deterioration of chronic liver disease. Systemic hemodynamic changes generally accompany the development of ACLF. In China, hepatitis B virus (HBV) infection is one of the major causes of ACLF. Acute-on-chronic hepatitis B liver failure (ACHBLF) is the most common liver failure accounting for 80%～90% of all the ACLF patients. Because ACHBLF progresses critically and lack of specific clinical treatment measure, most of the patients’prognosis are poor.At present, the underlying molecular mechanism for the pathogenesis of ACHBLF is still unclear. Previous studies show that TNF-α, soluble tumor necrosis factor-a receptor I (sTNFRI) and sTNFRII play important roles in the pathogenesis of ACHBLF. In most inflammatory conditions, the main function of TNF-a is to augment cellular activation, cytokine production, and tissue destruction. TACE, a disintegrin and metalloproteinase, mediates the release of growth factors, receptors, and adhesion molecules. It is responsible for the shedding of cell-surface TNF-a. So far, there are fewer studies of TACE and ACHBLF.Abnormal DNA methylation has become one of the most promising molecular markers and a hot topic at present in the early diagnosis, risk assessment, early recurrence forecast, response monitoring to treatment and prognosis judgement of malignant disease. The research of the methylation of TACE may help to better understand its function in liver failure. That maybe supplies some useful clues in the further research on the pathogenesis mechanism of ACHBLF. Therefore, we investigate the methylation status of TACE in the patients with ACHBLF in this study.ObjectiveThis study aims to analyze TACE methylation status in ACHBLF and its potential role with prognosis.Methods45 ACHBLF patients,80 chronic hepatitis B (CHB) patients and 54 healthy controls (HCs) were enrolled. Basic characteristics and clinical data such as age, sex, liver function, were collected from each subject. TACE promoter methylation was measured by methylation specific polymerase chain reaction (MSP). The TACE mRNA level was determined by quantitative real time-polymerase chain reaction (RT-qPCR). The plasma levels of TACE, TNF-a, sTNFRI, sTNFRII were measured by Enzyme-linked immunosorbent assay (ELISA). The start date of the follow-up was the date of the diagnosis of ACHBLF. All the ACHBLF patients were followed up for at least 3 months and the outcome (death or survival) of each ACHBLF patient was recorded.Quantitative variables were expressed as the mean (centile 25; centile 75). Categorical variables were expressed as number (percentage). Statistical analyses of the data were performed with SPSS version 16.0 (Chicago, IL, USA). Difference in the methylation rate of TACE among patients with ACHBLF, CHB and HCs was analyzed by chi-squared test, while laboratory parameter difference among the three groups was analyzed by the Mann-Whitney U-test. TACE-methylated and TACE-unmethylated group of patients with ACHBLF was analyzed by Rank Sum Test. Survival curves were drawn using the Kaplan-Meier method and the statistical significance was determined using the log-rank test. Diagnostic values of TACE methylation and MELD score in predicting 3 month mortality for ACHBLF patients were assessed by the area under the Receiver operating characteristic curves (AUC). All statistical analyses were two sided, and P value <0.05 was considered to be statistically significant.Results1. There was no significant difference in ACHBLF, CHB and HC groups with respect to sex, and age (P>0.05), while significantly different ALT, AST, TBIL, PTA and HBV DNA levels were found in ACHBLF group compared to the other two groups (P< 0.05).2. Methylation of TACE was detected in 16 cases of 45 (35.56%) patients with ACHBLF,64 cases in 80 (80.00%) patients with CHB and 50 cases in 54 (92.59%) HCs. The rate of methylation in patients with liver failure was significantly lower than that in CHB (χ2=24.69, P<0.01) and HCs (χ2= 35.93, P<0.01). Significant difference was also found between CHB and HCs (χ2=4.03, P-0.045).3. There was no statistical difference between methylated and unmethylated group of ACHBLF patients with respect to Age, Sex, HBV DNA, ALT and AST (P>0.05). However, significant higher PTA was found in methylated than in unmethylated group of ACHBLF (P< 0.01). Meanwhile, lower TBIL、MELD levels and Mortality differences could be found in methylated group than unmethylated group of ACHBLF (P<0.01).4. TACE mRNA level was significantly higher in ACHBLF patients than CHB patients (t=2.41, P=0.02) and HCs (t=2.95, P<0.01). There was no significant difference between mRNA levels in CHB patients and HCs (t=-0.87, P=0.39). The methylation status of TACE was significantly inversely associated with its mRNA expression (r=-0.68; P<0.01). TACE mRNA level was significantly lower (t=8.11, P<0.01) in participants with aberrant methylation than those without.5. The levels of TACE, TNF-a, sTNFRI and sTNFRII were significantly higher in ACHBLF patients than CHB patients and HCs. There was no significant difference between mRNA levels in CHB patients and HCs. Meanwhile, the TACE level was significantly correlated with its mRNA expression (r=0.35; P<0.01).6. The three month mortality of ACHBLF patients was 55.56%(25/45). The mean survival time (from hospital admission) was 50.93 (SE 5.48,95% CI 40.19-61.68) days. We found that 3 month mortality was significantly associated with TBIL, PTA, MELD and Methylation.ConclusionTherefore, this study showed that the methylation of TACE in line with the severity of the ACHBLF, hypomethylation of TACE promoter occurred in ACHBLF and might serve as a potential prognostic marker.