Roles Of DNA Methylation In T Regulatory Cells Differentiation From Peripheral Blood Of Patients With Chronic HBV Infection And Prognosis Of Hepatocellular Carcinoma

Over the past few decades,the number of liver diseases has been on the rise and has become one of the leading causes of human illness and death worldwide.The Global Burden of Disease project reported that in China alone,liver diseases,especially viral hepatitis(mainly viral hepatitis B),non-alcoholic fatty liver disease and alcoholic liver disease,affect approximately 300 million people.Despite medical and technological advances and breakthroughs in the development of vaccines and antiviral drugs,the socio-economic and medical burden and negative impact of liver diseases continue to increase globally as life expectancy increases and lifestyles and nutrition change.At the same time,China is also facing enormous pressure and challenges from liver-related diseases,and it is estimated that more than one fifth of the population in China is negatively affected by different forms of liver disease.Viral hepatitis,especially viral hepatitis B,plays an important role in causing liver disease in our large population of patients with liver disease.It is of concern that a large proportion of patients with liver disease with or without clinical treatment,will eventually and inevitably progress to liver cancer with the progress of diseases.The annual mortality rate associated with liver cancer has increased significantly over the past two decades and is largely concentrated in the Asia-Pacific region.Epigenetics is the science of altering gene function and gene expression in a reversible and heritable manner without altering the DNA sequence of the genome.Epigenetic modification systems including DNA methylation,RNA-associated silencing and histone modification can be used to initiate and maintain epigenetic silencing.As an important mode of epigenetic regulation mode,DNA methylation can regulate gene expression by,for example,altering protein-DNA interactions and ultimately changing the chromatin conformation.Methylation of CpG sites in the human genome is maintained by a series of DNA methyltransferases(DNMTs),which play different roles in silencing transposons,resisting viral sequences and inhibiting the transcription of specific genes.PART I:5-Aza-2’-deoxycytidine Induced Demethylation Affected the Frequency of T Regulatory Cells from CD4+naive T Cells Isolated from the Peripheral Blood of Patients with Chronic HBV InfectionBackgroundChronic Hepatitis B Virus(HBV)infection is a major contributor to liver diseases and a major challenge to global public health.Approximately 6%of the population is chronically infected with HBV,with a high risk of progressive into chronic hepatitis B(CHB),liver cirrhosis(LC),and hepatocellular carcinoma(HCC).In particular,patients with chronic HBV infection harbor an increased percentage of T regulatory cells(Tregs)in peripheral blood compared with controls,besides,Tregs are essential for the immune tolerance and play vital and complex roles in the immune balance in the progression of chronic HBV infection.However,the differentiation of naive CD4+T lymphocytes into Treg during chronic HBV infection remains an important question to be addressed.It was previously reported that HBV infection can induce epigenetic regulation in vivo.DNA methylation,as an important epigenetic regulatory mode,controls gene expression by altering protein-DNA interactions and ultimately chromatin conformation.Forkhead box P3(Foxp3)is the critical transcription factor in the differentiation of Tregs and the methylation region is reported to be located in the Treg-specific demethylated region(TSDR)of the Foxp3 gene.However,it is still unclear how methylation pattern affects Tregs differentiation tendency in the progression of chronic HBV infection,which is the focus of our study.Objective5-Aza-2’-deoxycytidine as a classic and power methylation inhibitor is widely used in scientific labs and clinical practices,in the present research,it was used to treat naive CD4+T cells isolated from patients with CHB,LC and HCC at different stages of chronic HBV infection.Then proceed to cell culture stimulation of CD4+naive T lymphocyte differentiation using cytokines such as TGF-β1 and IL-2.After 4 days of culture,cells were collected.The frequency of Tregs in peripheral blood was determined by flow cytometry from patients with CHB,LC,HCC and healthy controls(HCs).Gene expression of Foxp3,DNMTs,PD-1 were detected by Quantitative real-time polymerase chain reaction(qRT-PCR)and DNA methyltransferases(DNMTs)Activity was also determined by using a DNA methyltransferase activity assay.This study aimed to investigate the potential effects of demethylation treatment in the differentiation of Tregs from peripheral blood of patients with chronic HBV infection in vitro.Methods1.In our study,51 CHB patients,47 LC patients,40 HCC patients and 17 HCs were recruited from the Department of Hepatology,Qilu Hospital of Shandong University from June 2018 to December 2018.Peripheral venous blood was retrieved from each subject,Naive CD4+T Cell Isolation Kit Ⅱ was used for naive CD4+T cell magnetic sorting.A fraction of cells stained with PE-conjugated Anti-Human CD45RA antibody were analyzed by flow cytometry to perform a quality control isolation procedure.2.Freshly isolated naive CD4+T cells were cultured in serum-free hematopoietic cell medium with 10%fetal bovine serum,all the cells were treated with 5 ng/mL recombinant human TGF-β1 and 20 ng/mL recombinant human IL-2 for the differentiation of Tregs.Cells in the experimental groups were also treated with 0.055μM 5-Aza-2’-deoxycytidine to inhibit the gene methylation.Control groups were treated with the same concentration of the vehicle(acetic acid)used to dilute 5-Aza-2’-deoxycytidine.Cells were harvested at 4 days after 5-Aza-2’-deoxycytidine treatment.3.Nuclear proteins generated from cultured cells were used to determine relative levels of DNMTs activity using DNMT Activity Quantification Kit.4.Flow cytometry was used to assess cell differentiation after 5-Aza-2’-deoxycytidine treatment.5.Total RNA was extracted from cultured cells of both 5-Aza-2’-deoxycytidine-treated groups and control groups.Expression of Foxp3,DNMT1,DNMT3a,DNMT3b and PD-1 was detected by qRT-PCR,to determine the effect of 5-Aza-2’-deoxycytidine on the expression of the above genes compared to the acetate acid-treated control.6.Clinical parameters collected for statistical analysis.Results1.The activity of DNMTs was significantly decreased after 5-Aza-2’-deoxycytidine-treatment than vehicle in all the subjects.There is no significant difference in the reduction of DNMTs activity between chronic HBV-infected patients and HCs.5-Aza-2’-deoxycytidine down-regulated the expression of DNMT1 but up-regulated the expression of DNMT3a and DNMT3b in samples from all subjects.The decreased DNMT1 mRNA expression was significant in samples from patients with HCC compared to HCs.There was no significant difference between chronic HBV-infected patients and HCs on the increase of DNMT3a and DNMT3b mRNA expression.2.The frequency of Tregs derived from naive CD4+T cells in polarizing conditions from peripheral blood of chronic HBV infected patients was significantly higher than that from HCs.In samples from patients with chronic HBV infection,the increase of Tregs percentage was significantly higher in LC and HCC than that in CHB,but there was no significant difference between LC and HCC.3.Compared with control groups which treated with acetic acid,5-Aza-2’-deoxycytidine-treatment up-regulated the Foxp3 mRNA level in induced Tregs in vitro.The increase of Foxp3 mRNA from 5-Aza-2’-deoxycytidine treated groups was particularly significant higher in samples from patients with HCC and LC compared with HCs.In samples from peripheral blood of CHB patient,we did not find any significant correlations between the increase of Foxp3 mRNA and a series of clinical parameters,including HBV DNA load.In samples from peripheral blood of LC patients,the increase of Foxp3 mRNA was significantly correlated with portal hypertension,aspartate aminotransferase(AST),and platelet(PLT),no significant correlation was found between the increase of Foxp3 mRNA and HBV DNA load.In samples from peripheral blood of HCC patients,there were significant positive correlations between the increase of Foxp3 mRNA and alkaline phosphatase(ALP),alanine aminotransferase(ALT),age and ascites.There was a significant negative correlation between the increase of Foxp3 mRNA and haemoglobin(HGB),no significant correlation was found between the increase of Foxp3 mRNA and HBV DNA load.4.As a cell-surface receptor belonging to CD28/CTLA-4 family,PD-1(Programmed death-1,PD-1)inhibits activation of T cells.Foxp3 cooperates with CTLA-4 for the proper function of Tregs,and they need to be co-expressed in T cells to prevent lymphopoiesis.The expression of PD-1 mRNA in induced Tregs was significantly increased after 5-Aza-2’-deoxycytidine-treatment in all the subjects.This increase of PD-1 mRNA was more noticeable in patients with LC and HCC compared to HCsConclusion1.5-Aza-2’-deoxycytidine causes methylation status change.Compared to those from HCs,5-Aza-2’-deoxycytidine might have the ability to enhance the frequency of Tregs of peripheral blood from patients with Chronic HBV Infection.2.Compared with acetic acid-treated group,5-Aza-2’-deoxycytidine showed the potential ability to enhance expression of Foxp3 mRNA and PD-1,especially in patients with LC and HCC.3.The altered methylation status and increased frequency of Tregs brought by 5-Aza-2’-deoxycytidine might have a variety of different clinical consequences in patients from different stages of chronic HBV infection,for which a comprehensive analysis is required.Our findings may provide new ideas for clinical remission of chronic HBV infection and provide new targets for future research.PART II:A New Prognostic Model Identified from Integrating analysis of Methylation and Expression Profiles to Predict Overall Survival in Hepatocellular CarcinomaBackgroundHepatocellular carcinoma(HCC)as a serious end-stage liver disease,represents the main histological type of liver cancer(LC),is not only one of the malignant tumors with the highest morbidity and mortality in the world,but also one of the leading causes of cancer death in China.In our country,approximately 383,000 people die from HCC each year,accounting for 51%of the world’s deaths from hepatocellular carcinoma.Epidemiological surveys conducted in China have shown that chronic hepatitis B virus(HBV)infection affects approximately 90 million people in China and is a major cause of liver cancer,with approximately 80%of HCC cases in China originating from HBV infection.Epigenetics is the science of altering gene function and gene expression in a reversible and heritable manner without altering the DNA sequence of the genome.Epigenetic alterations have been demonstrated to play a crucial role in the development,progression,and invasion of many cancers including HCC.As an important and common epigenetic mechanism,DNA methylation can lead to activation of multiple oncogenes or silence tumor suppressor genes.Studies have shown that abnormal de novo methylation of CpG islands is a hallmark of human cancer and that alterations in DNA methylation levels may precede those of other tumor markers,making it a potentially better marker for predicting tumourigenesis or prognosis.In the first part of our study,we found that altered methylation status mediated by 5-Aza-2’-deoxycytidine may cause abnormal differentiation of Treg cells and abnormal expression of some genes such as Foxp3,DNMTs and PD-1 in HCC patients.These genes are involved in immune regulation and immune processes such as virus clearance and tumor cell clearance in HCC patients,and their altered expression may affect the immune status of the patient’s organism,leading to the prolongation or progression of the disease.All of the above studies highlight altered DNA methylation as an important molecular event leading to hepatocarcinogenesis.The Cancer Genome Atlas(TCGA)is a large-scale cancer genome project jointly supervised by National Cancer Institute and National Human Genome Research Institute,It molecularly characterized over 20,000 primary cancer and matched normal samples spanning 33 cancer types.An obvious advantage of TCGA is the integration of data from different independent studies to obtain a greater number of samples for more reliable analysis.At present,there still are some pathways and factors that regulates the carcinogenesis of HCC remains unclear,also,there is a lack of effective molecular prognostic model for HCC patients in clinical practice.Therefore,integrated bioinformatics methods used to analyze cross-platform high-throughput data on a large scale is still important.ObjectiveThis study aims to identify methylation differential genes(MDGs)and methylation-driven genes of HCC from the TCGA database.Gene ontology(GO)enrichment analysis,pathway analysis as well as survival analysis need to be performed on these genes,ultimately to identify a set of robust prognostic signatures from MDGs and create a molecular model which can predict the overall survival of HCC through DNA methylation levels.Methods1.The original datasets comparing DNA methylation,clinical information and transcriptome profiling data between 418 HCC patients and 58 normal controls were downloaded from the TCGA database.2.The original TCGA data were processed into a methylation expression matrix file using affy R package.limma package was used to screen MDGs,edgeR package was used to screen differentially expressed genes(DEGs),MethylMix package was used for identifying methylation-driven genes.3.Gene-functional annotation enrichment analysis was performed by DAVID Bioinformatics Resources 6.8 and Cytoscape software.The enrichment information to analyze MDGs at the functional level was obtained from Biological processes(BP),Cellular component(CC)and Molecular function(MF)respectively.4.ConsensusPathDB(CPDB)was performed to identify the pathways associated with methylation-driven genes.5.STRING online database was used for analyzing the protein-protein interaction(PPI)and visualizing PPI network of MDGs.6.Univariate COX analysis was performed to assess the association between MDGs and overall survival of HCC patients using survival package in R.In addition,survival package was also used for analyzing the relationship between gene methylation levels combined with gene expression data and the survival time of HCC patients.A set of prognosis-related signatures were identified and Hazard ratios(HRs)were used for identifying protective(HR<1)and risky(HR>1)genes.Least absolute shrinkage and selection operator(LASSO)analysis using glmnet package was applied to these selected signatures to generate prognostic model.Risk scores were calculated based on gene methylation levels and coefficients.7.A key gene,TNFRSF12A,with the dual characteristics of MDG and methylation driven gene,was identified from the prognostic model and shown to be significantly associated with survival in HCC patients in a univariate COX regression analysis.Survival analysis was performed in terms of TNFRSF12A methylation levels and integration of TNFRSF12A methylation levels with self-expression levels,respectively.A mixed model of this gene was constructed using MethylMix R package.Integration analysis of TNFRSF12A methylation level and its expression level was performed to determine the correlation between them.Results1.167(57 Hypomethylated and 110 Hypermethylated)MDGs with significant differences in methylation levels between normal controls and HCC patients were screened from the TCGA database.285 genes whose methylation level was significantly associated with their own expression were identified as methylation driven genes.2.For BP,MDGs showed enrichment mainly in the positive regulation of transcription from RNA polymerase Ⅱ promoter,transcription from RNA polymerase II promoter,negative regulation of transcription from RNA polymerase II promoter and nucleosome assembly;For CC,MDGs showed enrichment mainly in the nuclear nucleosome,nucleosome,transcription factor complex and nuclear chromatin;For MF,MDGs showed enrichment mainly in the sequence-specific DNA binding,transcription factor activity,transcriptional activator activity.CPDB was performed to identify the pathways associated with the methylation-driven genes.The top five pathways with the most significant correlations were Regulation of Insulin-like Growth Factor(IGF)transport and uptake by Insulin-like Growth Factor Binding Proteins(IGFBPs),Plasma lipoprotein remodeling,Statin Pathway,Vitamin K Metabolism and TYROBP Causal Network;the top five pathways that contained the most methylation driven genes were Metabolism,Pathways in cancer-Homo sapiens(human),Nuclear Receptors Meta-Pathway,Focal Adhesion-PI3K-Akt-mTOR-signaling pathway and Cytokine-cytokine receptor interaction-Homo sapiens(human).3.For MDGs,Genes with 10 or more connections/interactions were as follows:SOX1,POU4F1 and SEX3,they can be considered as central node genes in MDGs.4.Univariate Cox regression analysis screened a set of prognosis-related signatures from MDGs:HIST1H1D,RP11-476B1.1,OR2AK2,TNFRSF12A,CTD-2313N18.8,AC133644.2,RP11-467L13.4,LINC00989,RP11-395114.2,RP4-575N6.4,LRRIQ1,RNA5SP75,RP11-713D19.1,HRs were used for identifying protective(HR<1)and risky(HR>1)genes.High-risk signatures mean that the risk of HCC patient increases as the degree of gene methylation increases;on the contrary,low-risk signatures mean that as the degree of gene methylation increases,patient risk decreases.LASSO analysis was then applied to these selected signatures to generate prognostic model.Risk scores were calculated based on gene methylation levels and coefficients.Risk score=(0.458)*HIST11H1D+(-3.723)*RP11-476B1.1+(-0.341)*OR2AK2+(-2.163)*TNFRSF12A+(-0.405)*CTD-2313N18.8+(-0.394)*AC133644.2+(-1.008)*RP11-467L13.4+(-3.355)*INC00989.This model was compared with other clinical data for univariate and multivariate survival analysis,demonstrating its utility as an independent prognostic factor.The area under the receiver operating characteristic(ROC)curve(AUC)for the 8-gene model was 0.725,Youden index was 0.326,and the corresponding risk score was-1.299.According to this,HCC patients were divided into high-risk group and low-risk group.HCC patients from high-risk group had statistically significantly worse overall survival compare to those from low-risk group.5.A key gene,TNFRSF12A was found in the prognostic model,which belongs to both MDGs and methylation-driven genes.TNFRSF12A is generally hypomethylated in HCC patients and hypermethylated in normal controls.In addition,TNFRSF12A expression decreased with the increase of its methylation level.We performed survival analysis and found that among HCC patients,the methylation degree of TNFRSF12A was significantly correlated with survival time.Conclusion1.Our research has identified a registry of methylation-related genes and pathways that are important for regulating the carcinogenesis of HCC.2.Combined with gene methylation levels and expression profiles,a powerful new molecular model that can predict the prognostic of patients with HCC has been established.These findings will set us targets for further research on the molecular mechanism of HCC.3.A key gene,TNFRSF12A,was extracted from the prognostic model,which possesses the dual characteristics of HCC methylation differential genes and HCC methylation driven genes.Its methylation level and expression level were shown to be significantly associated with survival of HCC patients in univariate COX regression analysis and survival analysis.It may be a potential target for the treatment of HCC or for determining the prognosis of HCC patients.