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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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HBx promotes proliferation of hepatoma cells through SPHK1 in vitro. (A) The effect of SPHK1 on HBx-enhanced proliferation of HepG2 cells was measured with MTT assays. (B) The effect of SPHK1 on HBx-enhanced clonogenicity of HepG2 (or LO2) cells was detected by clone formation assays. bP<0.05, cP<0.01.
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fig4: HBx promotes proliferation of hepatoma cells through SPHK1 in vitro. (A) The effect of SPHK1 on HBx-enhanced proliferation of HepG2 cells was measured with MTT assays. (B) The effect of SPHK1 on HBx-enhanced clonogenicity of HepG2 (or LO2) cells was detected by clone formation assays. bP<0.05, cP<0.01.

Mentions: Next, we evaluated the role of SPHK1 in promoting proliferation of hepatoma cells mediated by HBx. MTT assays showed that HBx was able to accelerate the proliferation of hepatoma cells. However, SPHK1 siRNA could block the promotion of cell proliferation mediated by HBx (Figure 4A). A colony formation assay showed that HBx increased the size and number of the colonies of LO2 and HepG2 cells. In contrast, SPHK1 siRNA blocked this event (Figure 4B), suggesting that SPHK1 is involved in the HBx-enhanced proliferation of hepatoma cells. We further verified these data in vivo using a tumor xenograft model. We observed that HBx accelerated the growth of tumors transplanted into nude mice, but the treatment with si-SPHK1 could markedly block the tumor growth (Figure 5A–5C). The expression levels of HBx and SPHK1 were confirmed by Western blot analysis in the tumor tissues of mice (Figure 5D). In addition, immunohistochemistry staining of Ki-67, a marker of proliferation, further attested that interference by SPHK1 blocked the HBx-enhanced tumor growth (Figure 5E), suggesting that SPHK1 plays a crucial role in the tumor growth mediated by HBx. With these two findings, we conclude that HBx promotes hepatoma growth through SPHK1 in vitro and in vivo.


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)

HBx promotes proliferation of hepatoma cells through SPHK1 in vitro. (A) The effect of SPHK1 on HBx-enhanced proliferation of HepG2 cells was measured with MTT assays. (B) The effect of SPHK1 on HBx-enhanced clonogenicity of HepG2 (or LO2) cells was detected by clone formation assays. bP<0.05, cP<0.01.
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Related In: Results  -  Collection

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

fig4: HBx promotes proliferation of hepatoma cells through SPHK1 in vitro. (A) The effect of SPHK1 on HBx-enhanced proliferation of HepG2 cells was measured with MTT assays. (B) The effect of SPHK1 on HBx-enhanced clonogenicity of HepG2 (or LO2) cells was detected by clone formation assays. bP<0.05, cP<0.01.
Mentions: Next, we evaluated the role of SPHK1 in promoting proliferation of hepatoma cells mediated by HBx. MTT assays showed that HBx was able to accelerate the proliferation of hepatoma cells. However, SPHK1 siRNA could block the promotion of cell proliferation mediated by HBx (Figure 4A). A colony formation assay showed that HBx increased the size and number of the colonies of LO2 and HepG2 cells. In contrast, SPHK1 siRNA blocked this event (Figure 4B), suggesting that SPHK1 is involved in the HBx-enhanced proliferation of hepatoma cells. We further verified these data in vivo using a tumor xenograft model. We observed that HBx accelerated the growth of tumors transplanted into nude mice, but the treatment with si-SPHK1 could markedly block the tumor growth (Figure 5A–5C). The expression levels of HBx and SPHK1 were confirmed by Western blot analysis in the tumor tissues of mice (Figure 5D). In addition, immunohistochemistry staining of Ki-67, a marker of proliferation, further attested that interference by SPHK1 blocked the HBx-enhanced tumor growth (Figure 5E), suggesting that SPHK1 plays a crucial role in the tumor growth mediated by HBx. With these two findings, we conclude that HBx promotes hepatoma growth through SPHK1 in vitro and in vivo.

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