| Hepatocellular carcinoma (HCC), the major manifestation of primary liver cancer, is one of the most frequent and malignant diseases worldwide. HCC is frequently diagnosed at advanced stages with intrahepatic metastasis or vascular invasion and has a poor prognosis with a high mortality rate. Chronic infection of hepatitis B virus (HBV) is the leading cause of HCC in China, and the encoded protein of the HBV X gene (HBx) plays a critical role in the tumorigenesis of HBV-related HCC. However, the molecular mechanisms involved in HBx-mediated hepatocarcinogenesis remain unclear. E-cadherin is often observed to be aberrantly expressed in many cancers such as live cancer, prostate cancer and gastric cancer. The aberrant expression is considered to be regulated through epigenetic silencing mechanisms and to play a critical role in the development of metastasis in cancer. Enhancer of zeste homolog 2 (EZH2), component of the polycomb-repressive complex 2 (PRC2), plays a crucial role in tumorigenesis through trimethylation of lysine 27 of histone H3 (H3K27mer3) caused gene silencing. Several studies have shown that elevated EZH2 protein expression can repress E-cadherin expression by catalyzing H3K27mer3 in prostate cancer, breast cancer and gastric cancer. It has been reported that HBx can significantly repress E-cadherin expression by inducing the aberrant DNA methylation of CpG island in the e-cadherin promoter region, thereby promoting cell migration and invasion. Our question is if HBx represses E-cadheirn through EZH2-H3K27mer3 pathway as well in HCC.In this study, the mechanism of HBx promoting cell migration and repressing E-cadherin expression was studied based on the AML12-EGFP-HBx cell, which could steadily express HBx in vivo. Additionally, our results showed that EZH2 has potential side effect when it is used as a therapy target of HBV-related HCC.Firstly, the AML12-EGFP-HBx cell line with stable expression of EGFP-HBx protein was established, and its biological property was examined. It was found that the HBx fused with EGFP, like native HBx, did not affect the HBx-mediated activation of CMV promoter and the PI3K/Akt pathway, and could induce perinuclear mitochondrial clustering, suggesting that EGFP-HBx expressed in our experiment is functionally active. MTT assay, scratch assay and the nude mouse transplantation tumor experiment demonstrated that stable expression of HBx could significantly inhibit the proliferation of AML12 cell and had no effect on the cell malignant transformation. However, HBx could significantly promote the migration and motility of AML12 cell by altering the cellular morphology and inducing pseudopod formation.Secondly, the E-cadherin expression levels in AML12-EGFP-HBx cell line and the liver tissue from hbx-transgenic mice were examined and found severely repressed by stable but not transient expression of HBx in AML12 cell. Then, MSP assay showed that the promoter region of e-cadherin exhibited hypermethylation in AML12-EGFP-HBx cell. Moreover, the E-cadherin expression in AML12-EGFP-HBx cell line could be restored by treatment of DNA methyltransferase inhibitor 5'-Aza-2'dC. These results suggested that HBx repressed the E-cadherin expression via inducing hypermethylation of e-cadherin promoter.EZH2 plays an important role in E-cadherin regulation in prostate cancer, breast cancer and gastric cancer by catalyzing H3K27mer3 modification. In order to evaluate the regulatory effect of EZH2 on E-cadherin in liver cancer, EZH2 was knocked-down or over-expressed in HepG2 cell line, and then the E-cadherin expression level was analyzed by real-time PCR and Western Blotting. Results of both experiments showed that the E-cadherin expression increased with stable knockdown of EZH2 and reduced with the overexpression of EZH2 in HepG2 cell, indicating that EZH2 has the regulatory effect on E-cadherin expression in liver cancer. To investigate whether the EZH2 participates in the process of HBx regulated E-cadherin expression, the scratch assay was performed and results showed that the promoting effect of HBx on cell migration was attenuated by knocking down EZH2 in AML12-EGFP-HBx cells. Moreover, the inhibitory effect of HBx on E-cadherin expression was attenuated without impacting the hypermethylation of e-cadherin promoter after knocking down EZH2. Treatment with 5'-Aza-2'dC in AML12-EGFP-HBx cells led to rescue of E-cadheirn expression and knockdown of EZH2 could significantly inhibit the re-silencing of E-cadherin expression and de novo methylation of e-cadherin promoter after the removal of demethylating agents in AML12-EGFP-HBx cell. These results indicated that EZH2 had an important role in HBx regulated E-cadherin expression through coordinating the EZH2-H3K27mer3 pathway and DNA methylation pathway. To evaluate the molecular mechanism of HBx regulated EZH2 expression, we first detected the EZH2 expression level in HepG2.2.15 cell and found that HBV replication in HepG2.2.15 cell could dramatically boost the EZH2 expression and H3K27mer3 modification, and knockdown of the HBx expression could reduce this effect, suggesting that HBx had an important role in elevated EZH2 expression. Next, we used HeLa, HepG2 and AML12 cells to investigate the role of HBx in regulating the expression of EZH2, and confirmed that HBx could directly upregulate the expression of EZH2 and H3K27mer3 modification in these cells with dose-dependent manner by transient transfection experiment. To further investigate the mechanism of HBx regulated EZH2 overexpression, the 2500bp regulatory sequences upstream the first exon of the mezh2 and hezh2 gene were amplified from AML12 and HepG2 genomic DNA and constructed into the luciferase reporter plasmid respectively. The luciferase activity of the pmezh2 and phezh2 promoter significantly increased in AML12 cell or HepG2 cells co-transfected with HBx expression plasmid and reporter plasmids, and deleting the -486/-214 promoter region of pmezh2 decreased HBx-induced promoter activation by nearly 50%. The -486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease of HBx-induced activation of the pmezh2 promoter and EZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates EZH2 expression by trans-activating the ezh2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx. EZH2 had been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. Our data also suggested that EZH2 had an important role in HBx repressed E-cadheirn expression, indicating EZH2 could be a potential target of HCC therapy. However, the effect ofEZH2 on HBV replication should be firstly determined because of the high prevalence of HBV infection of HCC carrier in China. In this study, we analyzed the effect of EZH2 on HBV replication using the linear monomeric HBV genome replication system, which allows the production of circular HBV DNA. We found that HBV cccDNA-bound H3 histone is trimethylated on lysine 27 and organized into minichromosomes. The production of HBV serum markers and HBV transcripts were significantly inhibited by EZH2 expression in HepG2 cell, indicating that the HBV replication could be regulated by EZH2 through H3K27mer3 modification. These results suggested that we should consider the risk of HBV reactivation when using EZH2 as HCC therapy target.In conclusion, our studies revealed that HBx could upregulate EZH2 expression by trans-activating the E2F1 transcription factor and promote the cell migration by the EZH2-H3K27mer3 pathway and DNA methylation pathway. In addition, we found the HBV replication could be regulated by EZH2 expression, thereby indicating the risk of HBV reactivation when using EZH2 as HCC therapy target. Our data provide new epigenetic evidences for the carcinogenic effect of HBx and HCC therapy.
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