A mutant of HBx (HBxΔ127) promotes hepatoma cell growth via sterol regulatory element binding protein 1c involving 5-lipoxygenase

Abstract

Aim: Previously, we identified a natural mutant of hepatitis B virus X gene (HBx) with a deletion from 382 to 401 base pairs (termed HBxΔ127), which could potently enhance growth of hepatoma cells. In this study, we further investigated the mechanism of increased hepatoma cell growth that was mediated by HBxΔ127. Methods: We examined the effect of HBxΔ127 on the transcription factor sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS) in a model of HepG2-XΔ127 (or H7402-XΔ127) cells, which was stably transfected with HBxΔ127 gene in a human hepatoma HepG2 (or H7402) cell line. Results: Relative to wild type HBx, HBxΔ127 was able to potently activate SREBP-1c at the levels of promoter activity, mRNA and protein by a luciferase reporter gene assay, RT-PCR and Western blot analysis. Then, using the treatment with MK886, a specific 5-lipoxygenases (5-LOX) inhibitor, (or 5-LOX siRNA) we identified that 5-LOX was responsible for the upregulation of SREBP-1c by luciferase reporter gene assay, RT-PCR and Western blot analysis. Because FAS was a target gene of SREBP-1c, we further showed that HBxΔ127 was able to strongly activate the promoter activity of FAS and upregulated the mRNA expression level of FAS as well, by luciferase reporter gene assay and RT-PCR. In function, flow cytometry analysis revealed that FAS contributed to the growth of hepatoma cells that was mediated by HBxΔ127, using cerulenin (a FAS inhibitor). Conclusion: HBxΔ127 promotes hepatoma cell growth through activating SREBP-1c involving 5-LOX.

Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

“…Our laboratory has focused on the investigation of HBx-mediated hepatocarcinogenesis. Our findings show that arachidonic acid metabolism, extracellular signal-related kinases (ERK1/2), sterol regulatory element binding protein 1c, 5-lipoxygenase and fatty acid synthase are involved in HBx-induced cell proliferation [5,12,13] . However, the underlying mechanisms that mediate the effects of HBx on cell proliferation require further investigation.…”

“…HBx strongly enhances the growth of hepatoma cells in a system of stable transfection [5,12,13,32] . In the current study, we further investigated the mechanism that HBx employs to enhance the proliferation of hepatoma cells.…”

“…Previous studies have shown that HBx exhibits either apoptotic or proliferative activities in a context-dependent manner [33,34] . HBx promotes proliferation in stably transfected cells [5,12,13,32] . Therefore, we investigated the HBx-mediated proliferation in a model of stably HBx-transfected hepatoma HepG2-X cells.…”

“…The cells of HepG2, H7402, and HepG2.2.15, which constitutively replicated HBV [12] , were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (Life Technologies). Cultures were incubated at 37 °C in a humidified atmosphere with 5% CO 2 .…”

“…Cultures were incubated at 37 °C in a humidified atmosphere with 5% CO 2 . The engineered cells of HepG2-X/H7402-X (stably transfected with the pCMV-HBx plasmid), and HepG2-P/H7402-P (stably transfected with the empty pcDNA3.0 vector plasmid) were generated in HepG2 and H7402 cells, respectively, using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, as previously described [12] .…”