Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells - PubMed (original) (raw)
Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells
Zhen-Liang Qu et al. World J Gastroenterol. 2005.
Abstract
Aim: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.
Methods: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcription-polymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.
Results: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene.
Conclusion: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.
Figures
Figure 1
EGFP expressions in HepG2 (A and B) and QBC939 (C and D) cell lines under fluorescence microscope. After 36 h of transfection, EGFP-expressing cells were visible in group B (A and C) and group C (B and D), but not in group A.
Figure 2
Transfection efficiency evaluated by flow cytometry. The transfection efficiencies were about 46.4% for HepG2 (A) and 29.6% for QBC939 (B).
Figure 3
Analysis of hTERT mRNA expression by RT-PCR. RT-PCR was performed on total RNA extracted from both HepG2 (lanes 1-3) and QBC939 (lanes 4- 6) cell lines transfected with OPTI-MEM medium (lanes 1 and 4), pCMV-X (lanes 2 and 5) and pcDNA3 (lanes 3 and 6) respectively. M: DL 2000 marker.
Figure 4
Relative quantitative changes of hTERT mRNA in HepG2 (lanes 1-3) and QBC939 (lanes 4-6) cell lines. A significant higher expression of hTERT mRNA was observed in both HepG2 and QBC939 cells after being transfected with pCMV-X vector (lanes 2 and 5) compared to those transfected with OPTI-MEM medium (lanes 1 and 4) and pcDNA3 (lanes 3 and 6). a_P_<0.05 vs others.
Figure 5
Western blot analysis of HBx protein expression in transfected HepG2 (lanes 1-3) and QBC939 (lanes 4 -6) cell lines. A marked expression of HBx protein was observed in HepG2 and QBC939 cells when transfected with pCMV -X vector (lanes 2 and 5), but there were no such protein expression in those when transfected with OPTI-MEM medium (lanes 1 and 4) and blank vector (lanes 3 and 6). M: Protein marker.
Figure 6
HBx protein expression in transfected HepG2 cells as assayed by UltraSensitive™ immunocytochemistry (SP×200). The cells transfected with OPTI-MEM medium (A) and blank vector (C) showed no expression of HBx protein, while some cells transfected with pCMV-X vector (B) showed visible brown positive signals.
Figure 7
HBx protein expression in transfected QBC939 cells as detected by UltraSensitive™ immunocytochemistry (SP×200). The cells transfected with OPTI-MEM medium (A) and blank vector (C) showed no expression of HBx protein, but some cells transfected with pCMV-X vector (B) showed brown positive signals.
References
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