Transformation of human liver L-O2 cells mediated by stable HBx transfection

Abstract

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-O2 cells stably transfected with HBx as a model. Methods: Plasmids encoding HBx were stably transfected into immortalized human liver L-O2 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 γ-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-κB, 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.

Hepatitis B virus X protein accelerates hepatocarcinogenesis with partner survivin through modulating miR-520b and HBXIP

“…To demonstrate the significance of survivin up-regulation in HBx-induced hepatocarcinogenesis, we successfully generated an engineered cell line model (termed LO2-X-S) that LO2 cells stable transfected with both HBx and survivin. We previously reported that 3 of 8 mice injected with LO2-X cells stably transfected HBx grew tumors [29]. Strikingly, in this study we observed that all 8 mice injected with LO2-X-S cells formed tumors.…”

Tumor microenviroment and hepatocellular carcinoma metastasis

“…Despite the absence of a dominant oncogene encoded by HBV genome, HBV X protein (HBx), a key regulatory multifunctional protein of the virus, has been reported to exert a direct hepatocarcinogenic effect in the development of HCC . HBx deregulates a wide variety of host genes related to cell proliferation, cell cycle progress, apoptosis, metastasis, protein degradation pathways, and genetic stability . Previous studies have demonstrated that HBx is able to promote migration and invasion of hepatoma cells by upregulation of many proteins, such as forkhead box protein M1 (FoxM1), OPN, forkhead box protein M1 (FoxM1), MMPs, chemokine [C‐X‐C motif] ligand (MIG), and deregulation of intercellular adhesion …”

Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-ERK1/2

“…We have found that HBx was able to regulate many genes associated with signal transduction pathways, cell cycle, metabolism and proliferation, and this regulation may be involved in the carcinogenesis associated with HBV [21]. Furthermore, in certain contexts, stable HBx transfection resulted in a malignant phenotype in human liver L-O2 cells in vivo and in vitro [22].…”