HBx inhibits CYP2E1 gene expression via downregulating HNF4α in human hepatoma cells.

Hongming Liu,Guiyu Lou,Chongyi Li,Lixia Gan,Bin Xie,Arthur I. Cederbaum,Xiaodong Wang,Wang-Shick Ryu

doi:10.1371/journal.pone.0107913

Hongming Liu, Guiyu Lou + Show 6 more

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https://doi.org/10.1371/journal.pone.0107913

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Abstract

CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4α (HNF4α). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4α binding sequence located on −318 to −294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4α, we have further validated knockdown of HNF-4α significantly decreased CYP2E1expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4α expression, and HNF-4α levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4α protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4α which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4α-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis.

Highlights

Hepatocellular carcinoma (HCC) is the fifth most common cancer globally and the second leading cause of cancer death in China, a country with the largest HCC population in the world [1,2,3]
We found that ectopic overexpression of HBx inhibits Cytochrome P450 2E1 (CYP2E1) gene expression via downregulating hepatocyte nuclear factor 4a (HNF4a) in cultured HepG2 cells and promotes hepatoma cell growth
Since previous studies show that viral proteins play a critical role in HCC development, we analyzed the expression levels of Hepatitis B Virus (HBV) RNA and CYP2E1 in liver tissues from HCC patients. 5 pairs of HBV-positive primary HCC tissues, adjacent tissue of HCC and 5 cases of HBV-negative normal liver tissues were freshly resected from surgery

Summary

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer globally and the second leading cause of cancer death in China, a country with the largest HCC population in the world [1,2,3]. The risk factors for HCC have been well defined and included hepatitis virus infection, cirrhosis, and alcohol consumption. Gene array studies revealed that CYP2E1 was significantly down-expressed in HCC liver tissue [15]. In clinical specimens from 85 HCC patients, Ho et al found that 70% of the tumor tissues showed lower expression of CYP2E1, and decreased CYP2E1 is associated with poor prognosis of HCC [17]. These studies suggest that down-regulation of CYP2E1 may play an important role in HCC tumorigenesis. The mechanism of CYP2E1downregulation in HCC has so far not been elucidated

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