| Hepatocellular carcinoma (HCC) is one of the common malignancies and the third most ordinary cause of cancer-related mortality worldwide. The cause of primary hepatocelluar carcinoma is complicated, in Asia, which is one of the highest HCC incidence areas, most closely related to virus infection, such as hepatitis B virus (HBV) and hepatitis C virus (HCV). It is deduced that most of HCC patients in China were injuries from hepatitis infection and the long term chronic inflammation, accumulated necrosis and regeneration of hepatocytes, finally leaded to cell malignant transformation.The mechanism of hepatocarcinogenesis was still unclear recently, and the judgment on the diagnosis, treatment and prognosis of HCC were still highly depends on pathology and radiology. However, radiological examination is limited to its resolution when dealing with the small HCC and early HCC, and clinicopathologic features could be ambiguous due to the heterogeneous nature of cancer. The high rates of recurrence and metastasis of HCC was still a major obstacle for patient's long-term survival, even after liver resection and liver transplantation. It's an urgent need to identify the optimal targets and develop a reliable system for HCC diagnosis and evaluation of early recurrenceAberrant DNA methylation is believed to play an important role in hepatocarcinogenesis as a major epigenetic change, which is associated with global hypomethylation and specific promoter hypermethylation in cancer. Promoter hypermethylation of GSTP1, RASSF1A, RIZ1, APC, SOCS1and E-cadherin were considered as potential biomarkers in HCC. With the development of genome-wide screening, the global methylation profiling of cancer become clearer than ever before. However, in previous studies, although a number of genes were identified as biomarkers of tumor, only a few were confirmed by independent researches. After ruling out the difference between gene array platforms, different gene methylation profiles were found in HCC cases with different etiological backgrounds, pathophysiological processes, and histological characteristics were found with different gene methylation profiles.In this study, we focused on HBV-related HCC(HBHC) secondary to liver cirrhosis. In the first part, we analyzed the aberrant promoter methylation of cancer samples and adjacent liver samples using Methylation BeadChips, to seek for the potential novel biomarkers in HBHC. In the second part, we compared the global methylation profiles of HBHC subclasses with high-risk and low-risk, explored the putative biomarker for the prediction of early recurrence. Part I Methylation Profile of Single Hepatocytes Derived from Hepatitis B Virus-Related Hepatocellular CarcinomaObjective:Previous studies on HCC methylation profiles have focused on liver tissue, and the heterogeneity of liver composition may impact the genuine methylation status of HCC. The aim of the present study was to establish the methylation profile of hepatocytes isolated from primary tissues, to elucidate the mechanism of hepatocarcinogenesis and to identify the potential biomarker for the diagnosis of HBHC.Methods and Findings:The global methylation profile of single hepatocytes isolated from liver tissue of HBHC was analyzed using Illumina Infinium Human Methylation27BeadChips. Combined bisulfite restriction analysis (COBRA) and bisulfite sequencing were used to validate the20significant hypermethylated genes identified.In this study, we found many noteworthy differences in the genome-wide methylation profiles of single hepatocytes of HBHC. Unsupervised hierarchical clustering analysis showed that hepatocyte methylation profiles could be classified according to three cell types:hepatocytes of HCC, adjacent hepatocytes and normal hepatocytes. Aberrant methylated genes were enriched in pathways including gap junction, calcium signaling pathway, nature killer cell mediated cytocoxicity, cell adhesion and cell communication. Among the20most hypermethylated genes in the hepatocytes of HBHC,7novel genes (WNK2, EMILIN2, TLX3, TM6SF1, TRIM58, HIST1H4F and GRASP) were found to be hypermethylated in HBHC and hypomethylated in paired adjacent liver tissues; and these findings have not been reported in previous studies on tissue samples.Conclusion:The methylation profile of purified hepatocytes of HBHC distinguishes significantly from that of adjacent non-cancerous hepatocytes and normal hepatocytes. Aberrant methylated genes in hepatocytes of HBHC were enriched in signal pathways associated with cell survival and resistant to apoptosis. The promoter hypermethylation of seven novel genes found in this study have the potential to be biomarkers for the diagnosis of HBHC. Part â…¡ The DNA methylation profile for prognosis of Hepatitis B Virus-Related Hepatocellular CarcinomaObjective:The prognosis of HCC patients is unsatisfactory even after liver transplantation (LT) due to the high rate of recurrence and metastasis. Even though the methylation profile of HCC with early recurrence was still of scarcity, it was also the promising biomarkers for HCC prediction of the risk of early recurrence. The aim of the present study was to establish the global methylation profile of HBHC with early recurrence; to find out aberrant methylated genes associated with poor prognosis; and finally to evaluate the effect of those new biomarkers in the prediction.Methods and Findings:In the present study, genome-wide DNA methylation profiles of HBHC subclasses with high-risk and low-risk were established, with adjacent liver tissues and normal liver tissues as control. Random variance model (RVM) f-test was used to filter1164differentially methylated genes from the four groups. Based on change tendency of methylation status under the four groups, we identify300genes associated with poor prognosis of HBHC. After combined the analysis of gene ontology analysis, KEGG pathway and the map of signal net,16genes were selected for future validation. With the verification of gene expression and promoter methylation in clinical samples, promoter hypermethylation of HIST1H2BH and SOCS2were found closely related with the prognosis of HBHC after LT, and SOCS2methylation status was an independent prognostic factor for disease-free survival of HBHC after LT.Conclusion:HBHC subclasses with early recurrence displayed distinct genome-wide DNA methylation profiles, with unique genes enriched in signal pathway and sig-net map associated with tumor invasion and metastasis. Promoter hypermethylation of HIST1H2BH and SOCS2were found closely related with the prognosis of HBHC after LT. And DNA methylation in the promoter region of SOCS2is an independent risk factor influencing HBHC recurrence after LT and thus may be used as a potential biomarker for tumor recurrence of HBHC after LT.
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