The Role Of HBV-induced Epigenetic Alterations In Genes Expression Of Liver Cells With Chronic Hepatitis B

Background and aimsHepatitis B virus (HBV) infection, which can lead to acute hepatitis, chronic hepatitis,cirrhosis and hepatocellular carcinoma(HCC),is one of the most important reasons for theserious threat to human health.There are about350million people worldwide suffer fromchronic HBV infection, about120million in our country. Always, researchers pay closeattention to the immunological mechanism of HBV infection. In recent decades, theresearch about epigenetics pathogenic mechanism of HBV has become a research hotspot.Hepatitis B virus alters the expression of host cellular genes to support its replication andsurvival and to promote the liver cell injury. However, the underlying mechanism remainedincompletely understood. H3K4me3was reported as an active histone modification markand H3K27me3was a repressive mark. In our study, we investigated the role of H3K4me3and H3K27me3in the HBV-induced epigenetic changes through chromatinimmunoprecipitation-sequencing. Thus, our reseach will reveal the role of the alteredhistone modifications caused by HBV in the HBV-mediated liver pathogenesis.Furthermore,our research will provide new treatment strategy for clinical treatment of patients withchronic hepatitis B.MethodsAccording to relevant requirements, cultured the HepG2.2.15and HepG2cell lines. Inthe two cell lines, RNA-sequencing (RNA-Seq) and chromatin immunoprecipitation-sequencing (ChIP-Seq) were carried out to acquire the digital gene expression profiling(DGE) and the genome-wide’s histone methylation modification, respectively. Through theanalysis of bioinformatics, acquired differential expression gene profile related HBV. Inthese differentially expressed genes, we selected these closely associated with HBV diseasegenes and according to the function of the genes for classified analysis. Liver biopsyspecimens from CHB patients who were treatment naive (n=31)or who had been treatedwith a2year course of Telbivudine (600mg orally per day)(n=8) and individualsconfirmed as hepatitis virus negative (n=7,for use as healthy controls) were collected for analysis. HepG2cell and HepG2.2.15cells(treated and untreated whith Telbivudine) werecultureed as cell model for experiment. Quantitative real-time polymerase chain reaction(QPCR) was used to determine the expressions of HBV covalently closed circular DNA(cccDNA) in cells and liver tissues. QPCR and chromatin immunoprecipitation-quantitative real time polymerase chain reaction (ChIP-QPCR) were used to determine theexpressions and histone methylation modification of the specific genes, which were closelyassociated with HBV disease, in cells and liver tissues.The correlation among HBVcccDNA, the expressions level and the histone methylation modification level of thespecific genes were analyzed.All statistical analyses were performed using GraphPad Prismsoftware4.0(GraphPad Software). The two-tailed Student’s t-test was used to analyze thedifferences of messenger RNA (mRNA) expression and histone methylation amount in theHepG2and HepG2.2.15cells. Statistical significance was defined by a P value≤0.05.Results(1) First，digital gene expression profiling(DGE) were determined for both the HepG2and HepG2.2.15cells by RNA-Seq. The expression of vast genes have been changedbetween the two cell types. Among those genes, some were upregulated and the othersgenes were downregulated.(2) We detected the genome-wide distribution profile of H3K4me3and H3K27me3inthe HepG2and HBV-transformed HepG2.2.15cells by ChIP-Seq. Found that HBV inducesgenome-wide alteration of chromatin histone methylation in it’s host cells and thesechanges of locus-specific histone methylations are closely associated with differentialexpression of host genes induced by HBV.(3) A lot of significantly differentially expressed genes were identified in HepG2.2.15cells, as compared to HepG2cells by digital gene expression profiling (DGE) analysis.Many of these differentially expressed genes have a close relationship with HBV relateddisease, it imply that the expression of host genes changed induced by HBV plays animportant role in the HBV related disease.(4) We selected representative differentially expressed genes and according to thefunction of the genes for classified analysis. Indicate that a abundant of differentiallyexpressed genes are involved in virus entry into host cells, inflammation of host cells, hostcell fibrosis and carcinogenesis. So, it suggest that HBV cause disease may by changing theexpression of disease related genes. In our study, we also found that treatment of HepG2.2.15cells with the anti-HBV drug Telbivudine substantially restored the H3K4me3level to that of untransformed HepG2cells with HBV. Meanwhile, our analysis of the liversamples from health control and chronic hepatitis B patients revealed that treatment of thepatients with Telbivudine not only corrected the target gene expression but also theepigenetic modification of critical genes, especially the modification of H3K4me3. Thus itimply that epigenetic mechanisms may play a role in Telbivudine anti-HBV therapy.(5) In our results,we found that the expression of the histone methyltransferasesSMYD3and EZH2that regulate histone H3-specific methylation showed no difference inHepG2cell with or without HBV existence. So, it indicats that the changes of histonemethylations of host genes, induced by HBV, are not due to affects on expression of thehistone methyltransferases EZH2and SMYD3.ConclusionsIn the results of our reseach, suggest that HBV alter the histone methylations ofspecific host genes and the expression of these specific host genes take place correspondingchange. In these genes many are critically involved in HBV entry, inflammation, fibrosisand carcinogenesis of host cells. Interestingly, treatment the HepG2.2.15cells with theanti-HBV drug Telbivudine substantially restored the H3K4me3level to that ofuntransformed HepG2cells with HBV. More importantly, our analysis of liver samplesfrom health control and chronic hepatitis B patients revealed that treatment of the patientswith Telbivudine not only corrected the target gene expression but also the epigeneticmodification of critical genes. In addition, the expression of the histone methyltransferasesSMYD3and EZH2that regulate histone H3-specific methylation showed no difference inHepG2cell with or without HBV existence. Thus, our data suggest that abnormal histonemodifications might critically involved in HBV-mediated liver pathogenesis andTelbivudine therapy might benefit patients with HBV-related chronic infection, livercirrhosis and even hepatic carcinoma.