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首页> 外文期刊>World Journal of Gastroenterology >Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells.
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Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells.

机译：通过将乙型肝炎病毒X基因体外转染到人肝癌和胆管癌细胞中，端粒酶逆转录酶mRNA表达上调。

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摘要

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(R) 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 transactiv-ation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangioca-rcinomas after HBV infection.
机译：目的：研究HBx基因转染后人肝癌细胞系（HepG2）和胆管癌细胞系（QBC939）中人端粒酶逆转录酶（hTERT）mRNA表达的变化，并阐明HBx基因对hTERT基因转录调控的意义。致癌作用。方法：通过脂质介导的基因转导技术，将HepG2和QBC939细胞系与含有HBx编码区的真核表达载体和含有增强型绿色荧光蛋白（EGFP）编码序列的克隆载体共转染。转染后36小时，细胞中EGFP的表达被用作成功转染的指标。进行流式细胞术以确定转染效率。收获细胞，并使用TRIzol试剂提取总RNA。通过逆转录-聚合酶链反应检测hTERT mRNA在HepG2和QBC939细胞系中的表达。通过免疫细胞化学染色和Western印迹检测两种细胞系中HBx蛋白的表达。结果：流式细胞仪检测HBx基因表达载体和空白载体在HepG2细胞和QBC939细胞中的转染效率分别为46.4％和29.6％。与用OPTI-MEM培养基和空白载体转染的HBx基因表达载体相比，在用HBx基因表达载体转染的HepG2和QBC939细胞系中，hTERT mRNA的表达显着增加。免疫细胞化学染色和Western印迹仅在转染HBx基因后才在HepG2和QBC939细胞中表达HBx蛋白。结论：HBx基因转染可上调hTERT mRNA的转录表达。 HBx基因对hTERT基因的反式激活是HBV感染后肝癌和胆管癌的发病机制的新发现。

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