The loss of hepatitis B virus receptor NTCP/SLC10A1 in human liver cancer cells is due to epigenetic silencing
Authors: Ibrahim Marwa K. , Liu Cheng-Der , Zhang Liyong , Yu Xiaoyang , Kim Elena S. , Liu Zhentao , Jo Sumin , Liu Yuanjie , Huang Yufei , Gao Shou-Jiang , Guo Haitao 
Published: September 2024

ABSTRACT

Human Na+-taurocholate cotransporting polypeptide (hNTCP) is predominantly expressed in hepatocytes, maintaining bile salt homeostasis and serving as a receptor for hepatitis B virus (HBV). hNTCP expression is downregulated during hepatocellular carcinoma (HCC) development. In this study, we investigated the molecular mechanisms underlying hNTCP dysregulation using HCC tissues and cell lines, and primary human hepatocytes (PHHs). Firstly, we observed a significant reduction of hNTCP in HCC tumors compared to adjacent and normal tissues. Additionally, h
NTCP
mRNA levels were markedly lower in HepG2 cells compared to PHHs, which was corroborated at the protein level by immunoblotting. Sanger sequencing confirmed identical sequences for h
NTCP
promoter, exons, and mRNA coding sequences between PHH and HepG2 cells, indicating no mutations or splicing alterations. We then assessed the epigenetic status of h
NTCP
. The h
NTCP
promoter, with low CG content, showed no significant methylation differences between PHH and HepG2 cells. Chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) revealed a loss of activating histone posttranslational modification (PTM) H3K27ac near the h
NTCP
transcription start site (TSS) in HepG2 cells. This loss was also confirmed in HCC tumor cells compared to adjacent and background cells. Treating HepG2 cells with histone deacetylase inhibitors enhanced H3K27ac accumulation and glucocorticoid receptor (GR) binding at the h
NTCP
TSS, significantly increasing h
NTCP
mRNA and protein levels, and rendering the cells susceptible to HBV infection. In summary, histone PTM-related epigenetic mechanisms play a critical role in hNTCP dysregulation in liver cancer cells, providing insights into hepatocarcinogenesis and its impact on chronic HBV infection.

IMPORTANCE

HBV is a hepatotropic virus that infects human hepatocytes expressing the viral receptor hNTCP. Without effective antiviral therapy, chronic HBV infection poses a high risk of liver cancer. However, most liver cancer cell lines, including HepG2 and Huh7, do not support HBV infection due to the absence of hNTCP expression, and the mechanism underlying this defect remains unclear. This study demonstrates a significant reduction of hNTCP in hepatocellular carcinoma samples and HepG2 cells compared to normal liver tissues and primary human hepatocytes. Despite identical h
NTCP
genetic sequences, epigenetic analyses revealed a loss of the activating histone modification H3K27ac near the h
NTCP
transcription start site in cancer cells. Treatment with histone deacetylase inhibitors restored H3K27ac levels, reactivated h
NTCP
expression, and rendered HepG2 cells susceptible to HBV infection. These findings highlight the role of epigenetic modulation in h
NTCP
dysregulation, offering insights into hepatocarcinogenesis and its implications for chronic HBV infection.