Creation of a six-fingered artificial transcription factor that represses the hepatitis B virus HBx gene integrated into a human hepatocellular carcinoma cell line

J Biomol Screen. 2013 Apr;18(4):378-87. doi: 10.1177/1087057112463066. Epub 2012 Oct 5.

Abstract

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection-related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection-related HCC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Carcinoma, Hepatocellular / metabolism*
  • Carcinoma, Hepatocellular / pathology
  • Cell Proliferation
  • Down-Regulation / genetics
  • Enhancer Elements, Genetic / genetics
  • Genes, Reporter
  • Genome, Human / genetics
  • Hep G2 Cells
  • Humans
  • Liver Neoplasms / metabolism*
  • Liver Neoplasms / pathology
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics
  • Protein Binding / genetics
  • Repressor Proteins
  • Trans-Activators / genetics*
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism*
  • Viral Regulatory and Accessory Proteins
  • Zinc Fingers*

Substances

  • Repressor Proteins
  • Trans-Activators
  • Transcription Factors
  • Viral Regulatory and Accessory Proteins
  • hepatitis B virus X protein