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

Lu ZP, Xiao ZL, Yang Z, Li J, Feng GX, Chen FQ, Li YH, Feng JY, Gao YE, Ye LH, Zhang XD - Acta Pharmacol. Sin. (2015)

Bottom Line: As an oncogenic kinase, SPHK1 is associated with the development and progression of cancers.A positive correlation was found between the mRNA levels of SPHK1 and HBx in 38 clinical HCC samples (r=+0.727, P<0.01).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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT

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.

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The mRNA levels of HBx are positively associated with those of SPHK1 in clinical HCC tissues. (A) The correlation between HBx mRNA levels and SPHK1 mRNA levels was detected by qRT-PCR in clinical HCC tissues (P<0.01, r=0.727, Pearson's correlation). (B) The expression of SPHK1 at the levels of mRNA and protein was examined using RT-PCR and Western blot analysis in either the liver tissues or the HCC tissues of HBx-Tg mice.
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fig1: The mRNA levels of HBx are positively associated with those of SPHK1 in clinical HCC tissues. (A) The correlation between HBx mRNA levels and SPHK1 mRNA levels was detected by qRT-PCR in clinical HCC tissues (P<0.01, r=0.727, Pearson's correlation). (B) The expression of SPHK1 at the levels of mRNA and protein was examined using RT-PCR and Western blot analysis in either the liver tissues or the HCC tissues of HBx-Tg mice.

Mentions: It has been reported that the mRNA levels of SPHK1 are upregulated in liver cancer tissues16. We evaluated the relationship between HBx and SPHK1 in clinical HCC tissues. Our data showed that the mRNA levels of HBx were positively correlated with those of SPHK1 in 38 clinical HCC samples (P<0.01, r=0.727, Pearson's correlation) (Figure 1A). Further, we detected the expression of SPHK1 in the liver or HCC tissues from HBx-Tg mice. Interestingly, we found that SPHK1 was upregulated in 6-month-old HBx-Tg mice (Figure 1B). SPHK1 was especially highly expressed in liver cancer tissues from HBx-Tg mice aged 18 months, suggesting that HBx is able to upregulate SPHK1 in HBx-Tg mice. With these two findings, we conclude that the mRNA levels of HBx are positively associated with those of SPHK1 in clinical HCC tissues.


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

Lu ZP, Xiao ZL, Yang Z, Li J, Feng GX, Chen FQ, Li YH, Feng JY, Gao YE, Ye LH, Zhang XD - Acta Pharmacol. Sin. (2015)

The mRNA levels of HBx are positively associated with those of SPHK1 in clinical HCC tissues. (A) The correlation between HBx mRNA levels and SPHK1 mRNA levels was detected by qRT-PCR in clinical HCC tissues (P<0.01, r=0.727, Pearson's correlation). (B) The expression of SPHK1 at the levels of mRNA and protein was examined using RT-PCR and Western blot analysis in either the liver tissues or the HCC tissues of HBx-Tg mice.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814204&req=5

fig1: The mRNA levels of HBx are positively associated with those of SPHK1 in clinical HCC tissues. (A) The correlation between HBx mRNA levels and SPHK1 mRNA levels was detected by qRT-PCR in clinical HCC tissues (P<0.01, r=0.727, Pearson's correlation). (B) The expression of SPHK1 at the levels of mRNA and protein was examined using RT-PCR and Western blot analysis in either the liver tissues or the HCC tissues of HBx-Tg mice.
Mentions: It has been reported that the mRNA levels of SPHK1 are upregulated in liver cancer tissues16. We evaluated the relationship between HBx and SPHK1 in clinical HCC tissues. Our data showed that the mRNA levels of HBx were positively correlated with those of SPHK1 in 38 clinical HCC samples (P<0.01, r=0.727, Pearson's correlation) (Figure 1A). Further, we detected the expression of SPHK1 in the liver or HCC tissues from HBx-Tg mice. Interestingly, we found that SPHK1 was upregulated in 6-month-old HBx-Tg mice (Figure 1B). SPHK1 was especially highly expressed in liver cancer tissues from HBx-Tg mice aged 18 months, suggesting that HBx is able to upregulate SPHK1 in HBx-Tg mice. With these two findings, we conclude that the mRNA levels of HBx are positively associated with those of SPHK1 in clinical HCC tissues.

Bottom Line: As an oncogenic kinase, SPHK1 is associated with the development and progression of cancers.A positive correlation was found between the mRNA levels of SPHK1 and HBx in 38 clinical HCC samples (r=+0.727, P<0.01).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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus