The Epigenetic Drift Of Patients With HBV-related Acute-on-chronic Liver Failure

Chronic HBV infection is the major cause of acute-on-chronic liver failure (ACLF) in China. ACLF is an important clinical syndrome characterized by severe liver dysfunction and hepatic encephalopathy that may result in a high rate of morbidity and mortality. Although advances in intensive care and medical management have improved the outcome, the prognosis of severe hepatitis B remains poor. This may be related to the fact that the pathophysiology of this complex event is still incompletely understood. Improved understanding of the pathophysiology of this complex event is warranted. Our previous study found that although the same infection of HBV, monozygotic twins (MZ twins) showed different clinical phenotype, which indicated the host epigenetics factors may play an important role in influencing the clinical phenotype after infection with HBV. Recently, accumulating evidence suggests that DNA methylation, gene imprinting and microRNAs (miRNAs) involved in the pathogenesis of viral replication, host-viral interactions, inflammation and immune regulation. Given the importance of epigenetics factors in the pathophysiology of the HBV-related liver diseases, we hypothesized that epigenetics factors are important in the development of ACLF.Recognition the epigenetic drift between patients with ACLF and the control samples may help to identify those that are involved in progression of ACLF and establish the basis to unravel their pathogenic role. Here, we firstly addressed this hypothesis by using a microarray-based screening the DNA methylation, gene imprinting and miRNAs expression profiling in PBMCs of a pair of MZ twins with ACLF and asymptomatic carrier (AsC) separately.Method:1. Agilent Human CpG Islands arrays were used to analyze the genome-wide DNA methylation profiling between MZ twins. At the same time, the gene expression profiles between MZ twins were identified by the Roche Nimblegen cDNA microarray. Potential DNA methylation genes which related to the pathogenesis of ACLF were identified by analysis the expression and known function of methylated gene.2. The different expression imprinted genes were screened by Roche Nimblegen cDNA microarray. Quantitative real-time RT-PCR (qRT-PCR) was used to validate the microarray results between52patients with ACLF and48asymptomatic carriers. Heterozygotes of IGF2were identified by PCR-restriction fragment length polymorphism (RFLP). Allelic expression of IGF2was detected by RT-PCR and RFLP. The difference of loss of imprinting (LOI) between ACLF patients and asymptomatic carriers was analyzed by χ2test.3. Genome-wide miRNA expression profiling between MZ twins was screened by Exiqon miRNA arrays. qRT-PCR was used to validate the microarray results between104patients with ACLF and96asymptomatic carriers. Potential target genes for differentially regulated miRNAs were identified by monitoring the miRNA and mRNA expression on a genome-wide basis used different algorithms such as TargetScan, miRanda and PicTar.Results:1.47methylated genes down-regulated and88unmethylated genes up-regulated in the patient with ACLF in comparison with his twin brother with asymptomatic HBsAg carrier. These genes were related to the inflammatory signaling pathway, the expression of inflammatory mediators, microcirculation disturbance and etc.7methylated genes such as ZEBk、USP47、 BTF3L4and PAK6were related to the the pathogenesis of ACLF.2.13imprinted genes such as IGF2, IGF2AS, KCNQ1DN and CDKN1C up-regulated and8imprinted genes such as SGCE, PEG10, DLX5and L3MBTL2down-regulated in the patient with ACLF. The expressions of IGF2, DKN1C and TFPI2were significantly increased in patients with ACLF (P<0.001). The LOI of IGF2in patients with ACLF was higher than asymptomatic carriers (55.56%vs.21.05%, P=0.045).3. We identified53differentially expressed miRNAs between MZ twins, of which45miRNAs up-regulated and8miRNAs down-regulated in ACLF patient, which were validated by qRT-PCR. The expressions of hsa-mir-16and hsa-let-7a were significantly increased in ACLF patients with8.5-fold and8.6-fold separately (P<0.001). BCL2, CARD8, EDA, IL1RAPL1, LTB and FZD10were predicted as the target genes of hsa-mir-16. CERCAM, IGF2BP1, OPRM1and MAP4K3were predicted as the target genes of hsa-let-7a.Conclusion:1. The expression of imprinted gene and miRNAs, and the statue of DNA methylation were difference between the patients with ACLF and AsC, which indicated epigenetics factors probably involved in the pathophysiology of the HBV-related ACLF.2. The expressions of IGF2were significantly increased in patients with ACLF which was related to the LOI of IGF2in patients with ACLF.3. The expressions of hsa-mir-16and hsa-let-7a were significantly increased in ACLF patients. BCL2, CARD8, EDA, IL1RAPL1, LTB and FZD10were predicted as the target genes of hsa-mir-16. CERCAM, IGF2BP1,OPRM1and MAP4K3were predicted as the target genes of hsa-let-7a.