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Effect of IFNAR Promoter Methylation of PBMCs on MDM2 Expression and Its Relationship with Oxidative Stress in Patients with Chronic Hepatitis BBackgroundHepatitis B virus(HBV)infection is a global human health problem,at least 257 million people live with chronic HBV infection around the world.Chronic hepatitis B(CHB)infection may progress to liver cirrhosis and hepatocellular carcinoma.Therefore,timely and effective treatment of CHB is particularly important.Type I interferon is a first-line agent for CHB treatment.Type I Interferon(IFN)exerts its effects via binding to the type I interferon receptor(IFNAR)at cell surface,including IFNAR1 and IFNAR2,activating the Janus kinase/signal transducer and activator of transcription 1(JAK/STAT1)signaling pathway.STAT1 is a negative regulator of MDM2,which inhibition of p53-mediated transcriptional activity through binding with the p53 trans-activation domain and degradation of p53 through functioning as an E3 ubiquitin ligase.STAT1 could inhibit the expression of MDM2,the elevated p53 level was shown to inhibit replication of HBV via enhanced type I IFN antiviral signaling and apoptosis in infected cells.DNA methylation is one of the main epigenetic modulations involving chemical modification of DNA.DNA obtains a methyl group through DNA methyltransferase,which plays an important role in gene expression without changing the DNA sequence.Oxidative stress is caused when the balance between the oxidants and antioxidants is broken.Studies have shown that oxidative stress-induced epigenetic changes may be involved in the occurrence and progression of diseases.This study is mainly to detect the methylation status of IFNAR and MDM2 promoters in peripheral blood mononuclear cells(PBMCs),and the relationship between IFNAR promoter methylation and oxidative stress.Objective1.To study and clarify the methylation status of IFNAR and MDM2 promoters in PBMCs of CHB patients and healthy controls(HCs).2.To study and clarify the expression of IFNAR and MDM2 mRNA in CHB and HCs.3.To study the effect of INFAR promoter methylation on the expression of MDM2 mRNA in CHB patients.4.To study the relationship between IFNAR promoter methylation and oxidative stress in CHB patients.MethodsTotal of 169 participants were recruited from January 2014 to October 2016 in the Department of Hepatology,Qilu Hospital of Shandong University,including 148 CHB patients and 21 HCs.CHB patients fulfilled the 2009 AASLD Practice Guidelines and were hepatitis B surface antigen(HBsAg)positive for at least 6 months.We used methylation-specific polymerase chain reaction(MSP)to detect the methylation status of IFNAR and MDM2 in PBMCs from chronic hepatitis B(CHB)and HCs.In addition,quantitative real-time polymerase chain reaction(RT-qPCR)was performed to evaluate whether DNA methylation influences IFNAR and MDM2 expression in PBMCs.Enzyme-linked immunosorbent assay(ELISA)was used to determine the MDA and GSH in plasma from CHB and HCs.All statistical analyses were performed with SPSS 22.0 version(SPSS,Inc.,Chicago,IL)and Graphpad Prism 5.0(San Diego,CA,USA).The differences between variables were analyzed by Mann-Whitney U test.The Chi-square test was applied to categorical data.The correlation between variables was analyzed by the Spearman correlation.p<0.05 was considered statistically significant.Results1.The methylation frequency of IFNAR promoter in PBMCs of CHB patients was significantly lower than that of HCs(IFNAR1:p=0.03;IFNAR2:p<0.001).The methylation frequency of MDM2 promoter in PBMCs of CHB patients was not significantly different from that of HCs(p>0.05).2.The mRNA expression of IFNAR in PBMCs of CHB patients was significantly higher than that in HCs(IFNAR1:p=0.031;IFNAR2:p=0.027).There was no significant difference in the expression of MDM2 mRNA in patients with CHB compared with HCs(p>0.05).3.In patients with CHB,the expression of IFNAR mRNA in the IFNAR promoter methylated group was significantly lower than that in the unmethylated group(IFNAR1:p=0.023;IFNAR2:p=0.044).4.MDM2 mRNA expression in CHB patients with IFANR promoter methylation was significantly higher than that in CHB patients without IFNAR promoter methylation(IFNAR1:p=0.001;IFNAR2:p=0.008).5.The plasma levels of MDA in CHB patients were significantly higher than that of HCs,GSH levels were significantly lower than HCs(MDA:p<0.001;GSH:p<0.001).MDA expressed in methylated group with CHB was significantly increased compared with unmethylated group(IFNAR1:p=0.018;IFNAR2:p=0.041),anti-oxidative stress parameters GSH was decreased in methylated group than that of unmethylated group with CHB(IFNAR1:p=0.030;IFNAR2:p=0.029).Conclusions1.In this study,we found that the IFNAR promoter of PBMCs in CHB patients had an abnormal methylation,the IFNAR promoter methylation frequency was lower than that of HCs.The methylation frequency of MDM2 promoter of PBMCs in CHB patients had no difference with HCs.2.The methylation of IFNAR promoter of PBMCs in CHB patients resulted in the expression of IFNAR decreased and the expression of MDM2 mRNA increased,which suggested IFNAR promoter methylation might affect MDM2 mRNA expression.IFNAR promoter methylation status and MDM2 expression may influence the anti-HBV effect of type I interferon.3.The methylation of the IFNAR promoter of PBMCs in CHB patients may be related to oxidative stress,which may provide novel insight into epigenetic regulation induced by oxidative stress in the process of CHB.PART ? Diagnostic Value of MDM2 Promoter Methylation of PBMCs in Patients with hepatitis B virus-related hepatocellular carcinomaBackgroundHepatocellular carcinoma(HCC)is the major type of primary liver cancer,accounting for 90%of liver cancer cases worldwide.HCC is the fifth most frequent malignant tumors,and the third leading cause of cancer-related mortality in the world.In China,HCC is also a malignant tumor with high morbidity and mortality,about 383,000 people die from HCC every year.The main risk factors for HCC include chronic hepatitis B virus(HBV)infection,alcoholic liver disease,chronic hepatitis C virus infection,non-alcoholic fatty liver disease.In China,hepatitis B virus-related HCC(HBV-related HCC)caused by chronic HBV is the most common.Even with recent advancements in medical technology,however,due to the insidious onset of the HCC,most patients are at middle-late stage when they are diagnosed.Hepatectomy and liver transplantation are potentially treatment options in the early stage of HCC.Alpha-fetoprotein(AFP)is the biomarker for the diagnosis of HCC,but about 40%of HCC patients without elevation in serum AFP levles.It is particularly important to find more sensitive and non-invasive biomarkers.Epigenetic modifications,including DNA methylation,play an important role in the development of tumors.DNA methylation is a heritable change that affects gene expression when the DNA sequence has not changed.DNA methylation is of great importance for gene expression and can have dire consequences for cells.DNA methylation includes DNA hypermethylation and hypomethylation.In most cases,DNA hypomethylation is defined as a reduction in methylation levels compared to"normal" methylation levels,relates to increased gene expression.DNA hypomethylation is widely studied in many tumors,such as liver cancer,gastric cancer and lung cancer.Several studies have shown that DNA hypomethylation of certain genes can be used as biomarkers for diagnosis.Murine double minute-2(MDM2)is an E3 ubiquitin ligase,and the gene encodes a negative regulator of the p53 tumor suppressor.MDM2 binds to the transcriptional activation domain of p53 to regulate the stabilization and activation of p53.Under normal physiological conditions,MDM2 and p53 form a self-regulating negative feedback loop to ensure a balance between them.However,the negative feedback loop is often observed to be broken in various tumors.MDM2 overexpression,in particular,has been implicated in several tumors,such as liver cancer,gastric cancer and sarcoma.Regardless of the state of p53,MDM2 not only binds to p53 and negatively regulates p53,but also contributes to the development of HCC.However,the methylation status of MDM2 and diagnostic value in HBV-related HCC patients has not yet been exploredObjectiveWe aimed to detect the methylation status of MDM2 promoter in PBMCs of HBV-related HCC patients and the value of MDM2 promoter methylation in the diagnosis of HBV-related HCC.MethodsOne hundred patients with HBV-related HCC,31 patients with liver cirrhosis(LC),and 37 patients with chronic hepatitis B(CHB)were recruited from June 2016 to February 2018 at the Department of Hepatology,Qilu Hospital of Shandong University.All HBV-related HCC patients were selected according to the 2010 update of the American Association for the Study of Liver Diseases Practice Guidelines for Management of hepatocellular carcinoma,hepatitis B surface antigen positivity for>6 months.The inclusion criteria for LC patients followed the evidence-based clinical practice guidelines for liver cirrhosis 2015.CHB patients were selected according to the practice guidelines for managing CHB established in the 2018 update of the American Association for the Study of Liver Diseases.We detected the methylation status of MDM2 by methylation-specific polymerase chain reaction(MSP),and we quantitatively analyzed the mRNA levels of MDM2 in peripheral blood mononuclear cells(PBMCs)by real-time quantitative PCR(RT-qPCR).Statistical analysis was performed using SPSS version 19.0 software(SPSS,Chicago,IL,USA),GraphPad Prism 6.0(San Diego,CA,USA)and MedCalc software(MedCalc,Ostend,Belgium).The quantitative variables differences between groups were compared using Mann-Whitney U test or Kruskal-Wallis H test.The categorical variables were compared with Chi-squared tests.We explored risk factors for MDM2 promoter methylation using binary logistic regression analysis.Associations of the MDM2 expression levels with the clinicopathological characteristics of patients were analyzed by the Spearman correlation test.In order to assess the diagnostic value of MDM2 promoter methylation,we combined MDM2 promoter methylation and the AFP to measure the area under the receiver operating characteristic(ROC)curve.The results were considered statistically significant when p<0.05.Results1.The methylation frequency of MDM2 detected in PBMCs was 72.97%(27/37)in the CHB patients,64.52%(20/31)in the LC patients,30.00%(30/100)in the HBV-related HCC patients.The MDM2 methylation frequency in the HBV-related HCC patients was significantly lower than that in CHB patients(p=0.000)and LC(p=0.001).However,no difference in the MDM2 methylation frequency was detected between the CHB and LC patients(p>0.05).2.MDM2 promoter methylation was significantly correlated with distant metastasis(p=0.040),TNM stage(p=0.035)and BCLC stage(p=0.044).There was no correlation between MDM2 promoter methylation and other clinicopathological characteristics,such as gender,age,AFP,vascular invasion,lymph node metastasis,number of tumors,tumor size(p>0.05).The statistical logistic regression analysis of risk factor showed none significantly different results(p>0.05).3.MDM2 mRNA levels were obviously higher in the HBV-related HCC patients than in the CHB patients(p=0.003)and LC patients(p=0.010).There were no significant differences in MDM2 mRNA levels between the CHB and LC patients(p>0.05).In the HBV-related HCC patients,the MDM2 mRNA levels in the unmethylated group were significantly higher than those in the methylated group(p=0.024).4.We found a positive correlation between MDM2 mRNA levels and HBV DNA(p=0.009),alanine aminotransferase(ALT)(p=0.016),aminotransferase aspartate(AST)(p=0.024).However,there was no correlation between MDM2 mRNA levels and total bilirubin(TBIL),albumin(ALB)or prothrombin time(PT).5.The ROC curve was established,including AFP,methylation status of MDM2 and combined determination,to identify the HBV-related HCC,CHB and LC.We compared the diagnostic value of AFP(cut off point:20 ng/ml),MDM2 methylation,and combined determination to distinguish HBV-related HCC from CHB.For the combination determination,the sensitivity was 89.00%,the specificity was 62.16%,the positive predictive value(PPV)was 87.25%,and the negative predictive value(NPV)was 62.07%,which was better than AFP(sensitivity:52%,specificity:62.16%,PPV:78.79%,NPV:32.39%)and MDM2 promoter methylation(sensitivity:70.00%,specificity:72.97%,PPV:87.5%,NPV:43.37%).Moreover,the area under curve(AUC)of the combined determination was 0.756[standard error(SE)=0.0434,95%CI=0.675-0.825)],which was significantly higher than AFP levels(AUC=0.639,SE=0.0471,95%CI=0.553-0.720;p=0.0056)and slightly higher than MDM2 promoter methylation(AUC=0.715,SE=0.0436,95%CI=0.632-0.789;p>0.05).Meanwhile,AFP(cut off point:20 ng/ml)combined with MDM2 methylation in distinguishing the HBV-related HCC patients from LC patients showed a sensitivity of 89.00%,specificity of 58.06%,PPV of 87.25%,and NPV of 62.07%,which were higher than the results of AFP(sensitivity:52.00%,specificity:58.06%,PPV:80.00%,NPV:27.27%)and MDM2 methylation(sensitivity:70%,specificity:64.52%,PPV:86.42%,NPV:40.00%).The combined determination AUC was 0.735(SE=0.0477,95%CI=0.651-0.809],which was higher than that of AFP alone(AUC=0.634,SE=0.0533,95%CI=0.545-0.716;p=0.0128)and MDM2 methylation(AUC=0.673,SE=0.0494,95%CI=0.585-0.752;p=0.0356).ConclusionsIn conclusion,this study showed MDM2 promoter hypomethylation in PBMCs of HBV-related HCC patients.Moreover,the combination of MDM2 promoter methylation status and serum AFP might improve the diagnostic efficiency of HBV-related HCC.PART ? Relationship between MDM2 Promoter Methylation and Oxidative Stress in PBMCs of Patients with Hepatitis B Virus-Associated Hepatocellular CarcinomaBackgroundHepatocellular carcinoma(HCC)is most common primary liver cancer and the third leading cause of cancer deaths worldwide.Main risk factors include hepatitis virus infection(hepatitis B virus,hepatitis C virus),alcohol consumption,metabolic liver disease(especially nonalcoholic fatty liver disease).Especially in China,HCC induced by chronic hepatitis B virus(HBV)infection is more universal.DNA hypomethylation leads to the activation of oncogenes,which contributes to cancer development and progression.DNA methylation can change gene expression without altering the DNA sequence.The murine double minute 2(MDM2)level was increased in a variety of cancers,such as liver cancer,gastric cancer.In our previous study,we have found that MDM2 promoter hypomethylation in peripheral blood mononuclear cells(PBMCs)of HCC patients.However,the mechanism leading to DNA hypomethylation is not clear.Several studies have shown that oxidative stress could lead to DNA methylation including hypermethylation and hypomethylation.Oxidative stress is an imbalance between oxidants and antioxidants.Oxidative stress was assessed by measuring oxidants,antioxidants and related metabolites in plasma.Metabolomics is defined as "the detailed analysis of endogenous metabolites" and has been widely used in finding disease biomarkers and exploring the mechanism of cancer occurrence and development.Metabolomics,as a supplement to genomics and proteomics,can better reflect the phenotype of cells.Even though MDM2 methylation status and increased oxidative stress are all play a role in diseases,no relationship has yet been explored between them.Objective1.We aimed to detect the methylation status of MDM2 promoter in PBMCs of HBV-related HCC patients and HCs.2.Our study aimed to explore the relationship between MDM2 promoter methylation and oxidative stress in HBV-related HCC patients.MethodsWe enrolled in 117 patients with HBV-related HCC from the Department of Hepatology,Qilu Hospital of Shandong University,between June 2016 to August 2019.In addition,we recruited 26 healthy controls.The HBV-related HCC patients fulfilled the 2010 update of the American Association for the Study of Liver Diseases Practice Guidelines for Management of hepatocellular carcinoma,hepatitis B surface antigen positivity for>6 months.We detected the methylation status of MDM2 in PBMCs from the hepatitis B virus-related HCC(HBV-related HCC)patients and the healthy controls(HCs)by methylation-specific polymerase chain reaction(MSP).Real-time quantitative PCR(RT-qPCR)was used to detected the MDM2 mRNA level.We also determined the oxidative stress parameter levels in plasma of the patients with HBV-related HCC and HCs by enzyme-linked immunosorbent assay(ELISA).The differences between continuous variables were analyzed by Mann-Whitney U-test.Categorical values were presented by relative frequencies.The Chi-square test was applied to categorical data.The correlation between difference variables were analyzed by the Spearman correlation.Statistical analysis was performed using SPSS version 19.0 software(SPSS,Chicago,IL,USA)and GraphPad Prism 6.0(San Diego,CA,USA).Thirty-six patients with HBV-related HCC and 11 HCs were selected for detection of metabolites in plasma by ultra high performance liquid chromatography-mass spectrometry(UHPLC-MS).Data obtained from metabolomics were imported into the SIMCA software(V16.0.2,Umea,Sweden)for principal component analysis(PCA),orthogonal partial least squares discrimination analysis(OPLS-DA).Metabolites with p<0.5,variable importance for the projection(VIP)>1,fold change>2 or<0.5 were screened as significant different metabolits.The two-sided p<0.05 was considered statistically significant.Results1.The methylation frequency was significantly decreased in the HBV-related HCC patients compared with HCs(p<0.001).We observed that the MDM2 methylation was associated with TNM stage(p=0.037).However,there were no significantly differences among gender,age,AFP,number of tumors,tumor size and vascular invasion.2.The mRNA levels of MDM2 were increased in the HBV-related HCC patients compared with HCs(p<0.001).The MDM2 mRNA levels showed a significant positive correlation with HBV DNA(p=0.017),ALT(p=0.018),AST(p=0.034),respectively,and no obvious correlation between MDM2 mRNA levels and TBIL(p=0.072),ALB(p=0.596)or PT(p=0.762).We also observed that the mRNA levels of MDM2 were significantly decreased in the methylated group compared to the unmethylated group in the HBV-related HCC patients(p=0.027).3.Our results showed that oxidative stress parameters,MDA,were significantly increased in the HBV-related HCC patients compared to HCs(p<0.001).Furthermore,the expression levels of antioxidant proteins,SOD and GSH,in plasma were lower in the HBV-related HCC patients than that in the HCs(SOD:p=0.009;GSH:p=0.040)We found that the plasma MDA levels were significantly higher in the MDM2 unmethylated group than in the MDM2 methylated group in the HBV-related HCC patients(p=0.027).Moreover,the levels of SOD and GSH were decreased in the MDM2 unmethylated group compared with the MDM2 methylated group in the plasma of the HBV-related HCC patients(SOD:p<0.001;GSH:p=0.017).4.Plasma metabolites were significantly different between HBV-related HCC and HCs,with a total of 216 differential metabolites.In the positive ion mode,a total of 144 differential metabolites were identified,including 88 up-regulated and 56 down-regulated metabolites.In the negative ion mode,a total of 72 differential metabolites were identified,including 51 up-regulated and 21 down-regulated metabolites.These differential metabolites belong to amino acids,bile acids,fatty acids,phospholipids,carbohydrates,organic acids,organic heterocyclic compounds and other compounds.5.There were significant differences in differential metabolites between the MDM2 methylated group and unmethylated group in HBV-related HCC patients,with a total of 81 differential metabolites.There were 52 differential metabolites(33 up-regulated and 19 down-regulated)in the positive ion mode;29 differential metabolites(19 up-regulated and 10 down-regulated)in the negative ion mode.These differential metabolites belong to amino acids,fatty acids,phospholipids,bile acids,nucleosides,organic acids and other compounds.In addition,we found that the metabolites with antioxidant effects,reductin D1 and leucine,were significantly higher in the MDM2 methylated group than in the MDM2 unmethylated group(p<0.05),and the levels of S-adenosine methionine,which can provide methyl groups,were significantly higher in the MDM2 methylated group.The metabolites indoxyl sulfate and p-cresol sulfate related to oxidative damage are significantly lower than those of the MDM2 unmethylated group(p<0.05).6.The metabolites were identified in HBV-related HCC patients involving several critical metabolic pathways including arginine and proline metabolism,arginine and ornithine metabolism,alanine and aspartate and glutamate metabolism,nitrogen metabolism,compared with HCs.The metabolites were identified in MDM2 promoter methylated group of HBV-related HCC patients involving critical metabolic pathways including cysteine and methionine metabolism,histidine metabolism,pentose and glucuronide interconversion,alanine and aspartate and glutamate metabolism compared to the unmethylated group.The metabolic pathway most significantly associated with differential metabolites in the MDM2 promoter methylated group and unmethylated group of HBV-related HCC patients was cysteine and methionine metabolism.Conclusions1.The methylation frequency of MDM2 promoter in PBMCs of HBV-related HCC patients was significantly lower than in HCs.2.The MDM2 promoter hypomethylation in PBMCs of HBV-related HCC patients was associated with oxidative stress,in which cysteine and methionine metabolism may play an important role. |
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