Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells.
OBJECTIVE
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.


CONCLUSIONS
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.
Qu
Zhen-Liang
ZL
Department of Surgery, Tianjin Nankai Hospital, Tianjin 300100, China. zlqu2002@hotmail.com
Zou
Sheng-Quan
SQ
Cui
Nai-Qiang
NQ
Wu
Xian-Zhong
XZ
Qin
Ming-Fang
MF
Kong
Di
D
Zhou
Zhen-Li
ZL
D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D004268:DNA-Binding Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006509:Hepatitis B / Q000235:genetics / Q000473:pathology / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D019098:Telomerase / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D019098:Telomerase