title: Upregulation of DNMT1 mediated by HBx suppresses RASSF1A expression independent of DNA methylation.
abstract: The hepatitis B virus (HBV) X protein (HBx) plays a key role in the molecular pathogenesis of HBV-related hepatocellular carcinoma (HCC). However, its critical gene targets remain largely unknown. RASSF1A gene (Ras-association domain family 1A, RASSF1A), a tumor-suppressor gene, is frequently found to be hypermethylated and downregulated in HCC. In the present study, we investigated whether HBx is involved in the hypermethylation and downregulation of RASSF1A and we examined the potential regulation mechanisms. RT-PCR analysis was used to determine RASSF1A and HBx expression in 9 liver cell lines and the results showed that RASSF1A expression was relatively low in HBx-positive cells. Notably, RASSF1A was downregulated in HepG2.2.15 cells, as compared to HepG2 cells. Further analysis revealed that HBx transfection suppressed RASSF1A expression and HBx knockdown induced its expression. Enforced HBx suppressed RASSF1A and meanwhile induced DNMT1 and DNMT3B expression. In addition, RASSF1A is negatively regulated by DNMT1. ChIP analysis using an antibody against DNMT1 revealed that HBx enhanced the binding of DNMT1 to the RASSF1A promoter but the inhibition of RASSF1A by HBx is DNA methylation-independent as detected by methylation-specific PCR (MSP). Further studies using MSP and bisulfite genomic sequencing (BGS) revealed that no significant methylation changes were observed for regional methylation levels of RASSF1A in DNMT1 knockdown cells, although methylation levels of specific CpG sites at the predicted binding sites for the Sp1 and USF transcription factors were reduced. Additionally, RASSF1A was downregulated in HBV-associated HCC (HBV-HCC) as detected by RT-PCR and immunohistochemistry suggesting RASSF1A expression may be related to HBx in HCC and the clinical relevance of our observations. Collectively, our data showed that HBx suppressed RASSF1A expression via DNMT1 and offered a new mechanism of RASSF1A inactive in HCC in addition to the widely known DNA methylation, enriching the epigenetic mechanism by which HBx contributes to the pathogenesis of HBV-HCC.
authors.affiliation: Department of Medical Genetics and Developmental Biology, Medical School of Southeast University and the Key Laboratory of Developmental Genes and Human Diseases, Ministry of Education, Southeast University, Nanjing, Jiangsu, P.R. China.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Qiu
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Sen
authors.initials: S
authors.lastname: Lu
authors.forename: Yunwei
authors.initials: Y
authors.lastname: Song
authors.forename: Yingbin
authors.initials: Y
authors.lastname: Lao
authors.forename: Jiaojiao
authors.initials: J
authors.lastname: Hu
authors.forename: Hong
authors.initials: H
authors.lastname: Fan
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000821:virology*; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis* / Q000235:genetics; D019175:DNA Methylation; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D006515:Hepatitis B virus; D006801:Humans; D008099:Liver / Q000473:pathology; D008113:Liver Neoplasms / Q000235:genetics / Q000821:virology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D015854:Up-Regulation; D051720:Upstream Stimulatory Factors / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D051720:Upstream Stimulatory Factors; D054334:Viral Regulatory and Accessory Proteins; D000097584:DNA Methyltransferase 3B; C411762:RASSF1 protein, human; C497181:USF1 protein, human; C057628:hepatitis B virus X protein; C000619380:DNMT1 protein, human