Transformation of human liver L-02 cells mediated by stable HBx transfection  

作　　者：Wei-ying ZHANG Na CAI Li-hong YE Xiao-dong ZHANG 

机构地区：[1]Department of Cancer Research, Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China [2]Department of Biochemistry, The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China

出　　处：《Acta Pharmacologica Sinica》2009年第8期1153-1161,共9页中国药理学报（英文版）

基　　金：Acknowledgements This project was supported by grants from the National Basic Research Program of China （973 Program, No 2007CB914802, No 2007CB914804, No 2009CB521702） and the National Natural Science Foundation （No 30670959）.

摘　　要：Aim： To explore the mechanism of hepatocarcinogenesis associated with the hepatitis B virus X protein （HBx）, we investigated the role of HBx in transformation using human liver L-02 cells stably transfected with HBx as a model. Methods： Plasmids encoding HBx were stably transfected into immortalized human liver L-02 cells and rodent fibroblast NIH/3T3 cells. The expression of alfa-fetoprotein （AFP）, c-Myc, HBx, and survivin in the engineered cells was examined by Western blotting. The malignant phenotype of the cells was demonstrated by anchorage-independent colony formation and tumor formation in nude mice. RNA interference assays, Western blotting, luciferase reporter gene assays and flow cytometry analysis were performed. The number of centrosomes in the L-O2-X cells was determined by y-tubulin immunostaining. The effect of HBx on the transcriptional activity of human telomerase reverse transcriptase （hTERT） and hTERT activity in L-O2-X cells and/or 3T3-X cells was detected by the luciferase reporter gene assay and telomerase repeat amplification protocol （TRAP）. Results： Stable HBx transfection resulted in a malignant phenotype in the engineered cells in vivo and in vitro. Meanwhile, HBx was able to increase the transcription of the NF-KB, AP-1, and survivin genes and to upregulate the expression levels of c-Myc and survivin. Abnormal centrosome duplication and activated hTERT were responsible for the transformation. Conclusion： Stable HBx transfection leads to genomic instability of host cells, which is responsible for hepatocarcinogenesis; meanwhile, transactivation by the HBx protein contributes to the development of hepatocellular carcinoma （HCC）. The L-O2-X cell line is an ideal model for investigating the mechanism of HBx-mediated transformation.Aim： To explore the mechanism of hepatocarcinogenesis associated with the hepatitis B virus X protein （HBx）, we investigated the role of HBx in transformation using human liver L-02 cells stably transfected with HBx as a model. Methods： Plasmids encoding HBx were stably transfected into immortalized human liver L-02 cells and rodent fibroblast NIH/3T3 cells. The expression of alfa-fetoprotein （AFP）, c-Myc, HBx, and survivin in the engineered cells was examined by Western blotting. The malignant phenotype of the cells was demonstrated by anchorage-independent colony formation and tumor formation in nude mice. RNA interference assays, Western blotting, luciferase reporter gene assays and flow cytometry analysis were performed. The number of centrosomes in the L-O2-X cells was determined by y-tubulin immunostaining. The effect of HBx on the transcriptional activity of human telomerase reverse transcriptase （hTERT） and hTERT activity in L-O2-X cells and/or 3T3-X cells was detected by the luciferase reporter gene assay and telomerase repeat amplification protocol （TRAP）. Results： Stable HBx transfection resulted in a malignant phenotype in the engineered cells in vivo and in vitro. Meanwhile, HBx was able to increase the transcription of the NF-KB, AP-1, and survivin genes and to upregulate the expression levels of c-Myc and survivin. Abnormal centrosome duplication and activated hTERT were responsible for the transformation. Conclusion： Stable HBx transfection leads to genomic instability of host cells, which is responsible for hepatocarcinogenesis; meanwhile, transactivation by the HBx protein contributes to the development of hepatocellular carcinoma （HCC）. The L-O2-X cell line is an ideal model for investigating the mechanism of HBx-mediated transformation.

关 键 词：HBX TRANSFORMATION HEPATOMA genomic instability stable transfection 

分 类 号：Q78[生物学—分子生物学] S511.03[农业科学—作物学]