Original Article

Hepatitis B virus X protein promotes human hepatoma cell growth via upregulation of transcription factor AP2α and sphingosine kinase 1

Zhan-ping Lu, Ze-lin Xiao, Zhe Yang, Jiong Li, Guo-xing Feng, Fu-quan Chen, Ying-hui Li, Jin-yan Feng, Yu-en Gao, Li-hong Ye, Xiao-dong Zhang
DOI: 10.1038/aps.2015.38


Aim: Sphingosine kinase 1 (SPHK1) is involved in various cellular functions, including cell growth, migration, apoptosis, cytoskeleton architecture and calcium homoeostasis, etc. As an oncogenic kinase, SPHK1 is associated with the development and progression of cancers. The aim of this study was to investigate whether SPHK1 was involved in hepatocarcinogenesis induced by the hepatitis B virus X protein (HBx).
Methods: The expression of SPHK1 in hepatocellular carcinoma (HCC) tissue and hepatoma cells were measured using qRT-PCR and Western blot analysis. HBx expression levels in hepatoma cells were modulated by transiently transfected with HBx or psi-HBx plasmids. The SPHK1 promoter activity was measured using luciferase reporter gene assay, and the interaction of the transcription factor AP2α with the SPHK1 promoter was studied with chromatin immunoprecipitation assay. The growth of hepatoma cells was evaluated in vitro using MTT and colony formation assays, and in a tumor xenograft model.
Results: A positive correlation was found between the mRNA levels of SPHK1 and HBx in 38 clinical HCC samples (r=+0.727, P<0.01). Moreover, the expression of SPHK1 was markedly increased in the liver cancer tissue of HBx-transgenic mice. Overexpressing HBx in normal liver cells LO2 and hepatoma cells HepG2 dose-dependently increased the expression of SPHK1, whereas silencing HBx in HBx-expressing hepatoma cells HepG2-X and HepG2.2.15 suppressed SPHK1 expression. Furthermore, overexpressing HBx in HepG2 cells dose-dependently increased the SPHK1 promoter activity, whereas silencing HBx in HepG2-X cells suppressed this activity. In HepG2-X cells, AP2α was found to directly interact with the SPHK1 promoter, and silencing AP2α suppressed the SPHK1 promoter activity and SPHK1 expression. Silencing HBx in HepG2-X cells abolished the HBx-enhanced proliferation and colony formation in vitro, and tumor growth in vivo.
Conclusion: HBx upregulates SPHK1 through the transcription factor AP2α, which promotes the growth of human hepatoma cells.
Keywords: hepatitis B virus X protein; hepatocellular carcinoma; HepG2 cells; tumor xenograft model; sphingosine kinase 1; transcription factor AP2α; cell proliferation

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