Hepatitis B virus X gene induces human telomerase reverse transcriptase mRNA expression in cultured normal human cholangiocytes

Abstract

Aim: To study the transcriptional regulation of human telomerase reverse transcriptase (hTERT) mRNA in normal human cholangiocytes (HBECs) after hepatitis B virus X (HBx) gene transfection and to elucidate the possible mechanism of HBV infection underlying cholangiocarcinoma.

Methods: HBECs were cultured in vitro and co-transfected with a eukaryotic expression vector containing the HBx coding region and a cloning vector containing coding sequences of enhanced green fluorescent protein (EGFP) using lipid-mediated gene transfer. The transfection efficiency was determined by the expression of EGFP. The expressions of hTERT mRNA and HBx protein in HBECs were detected by RT-PCR and immunocytochemical stain, respectively.

Results: The transfection efficiencies were about 15% for both HBx gene expression plasmid and empty vector. No hTERT mRNA was expressed in HBECs when transfected with OPTI-MEM medium and empty vector, but a dramatic increase was observed for hTERT mRNA expression in HBECs when transfected with HBx expression vector. HBx protein was only expressed in HBECs when transfected with HBx expression vector.

Conclusion: HBx transfection can activate the transcriptional expression of hTERT mRNA. Cis-activation of hTERT mRNA by HBx gene is the primary mechanism underlying the proliferation, differentiation and tumorigenesis of biliary epithelia.

Figure 1

Transfection efficiency evaluated by EGFP-express-ing cells in the HBx gene transfected cell cultures and empty vector control. pEGFP 0.1 μg and pCMV-X (or pcDNA3) 2.9 μg were co-transfected into HBECs by Lipofectamine. Thirty-eight hours after transfection. EGFP-expressing cells were visible under fluorescence microscope in the HBx gene transfected cell cultures (A) and empty vector control (B), but not in the blank control (Under fluorescence microscope × 200).

Figure 2

Analysis of hTERT mRNA expression by RT-PCR. RT-PCR was performed on total RNA extracted from HBECs transfected with OPTI-MEN medium (1), pCMV-X (2) and pcDNA3 (3), respectively. M: DL2000 Marker.

Figure 3

Quantitative and relative changes of hTERT mRNA expression analyzed by NIH Image software. Dramatic expres-sion of hTERT mRNA was observed in HBECs when trans-ferred with pCMV- X vector (lane 2), but there was no hTERT mRNA expression in HBECs when transferred with OPTI-MEM medium (lane 1) and empty vector (lane 3).

Figure 4

HBx protein expression in transferred HBECs assayed by ultrasensitive immunocytochemistry. The blank vector and OPTI-MEN transfected HBECs showed no expression of HBx protein (A, C), but pale brown positive signals scattered in pCMV- X vector transferred HBECs (B). Immunocytochemis-try (S-P methods, × 200).