Elsevier

Journal of Hepatology

Volume 49, Issue 6, December 2008, Pages 1029-1037
Journal of Hepatology

Identification of β-1,4-galactosyltransferase I as a target gene of HBx-induced cell cycle progression of hepatoma cell

https://doi.org/10.1016/j.jhep.2008.09.003Get rights and content

Background/Aims

The hepatitis B virus-encoded HBx protein contributes to hepatocarcinogenesis with largely unknown mechanisms. It is widely known that N-linked oligosaccharides on glycoproteins are structurally altered during malignant transformation and these alterations are often associated with malignant transformation of cells. β-1,4-galactosyltransferase I (GalT I) contributes to the biosynthesis of Galβ  4GlcNAc structure in the outer chain moieties of N-glycans.

Methods

The difference of GalT I expression between normal liver and hepatoma tissues were investigated; the effect of HBx on GalT I expression was investigated; the role of GalT I in hepatoma cell growth and HBx-induced hepatoma cell growth were investigated.

Results

GalT I was highly expressed in hepatocellular carcinoma and transcriptionally up-regulated by HBx, and functioned as a positive growth regulator in hepatoma cells. Furthermore, decreasing the expression of GalT I in hepatoma cells reduced the ability of tumor formation in vivo and inhibited HBx-induced cell cycle progression.

Conclusions

HBx-induced GalT I expression might contribute to HBx-mediated HCC development and progression.

Introduction

Hepatitis B virus is a major causative agent of acute and chronic hepatitis in humans and is closely associated with the incidence of human liver cancer [1], [2]. Among the four proteins that originate from the hepatitis B virus genome, HBx protein is a 17-kDa multifunctional regulatory protein and contributes to the development of hepatocellular carcinoma [2], [3], [4], [5]. However, the mechanisms of HBx-induced hepatocarcinogenesis remain largely unknown.

It is widely known that N-linked oligosaccharides on glycoproteins are structurally altered during malignant transformation and these alterations contribute to the biological functions of tumor cells [6], [7], [8]. Changes in the N-glycans structures of glycoproteins affect cell–cell and cell–extracellular matrix interactions, thereby affecting cell adhesion, cell migration and tumor malignancy [6], [9], [10], [11]. GalT I, a member of β-1,4-galactosyltransferase (GalT) family, is responsible for the biosynthesis of Galβ  4GlcNAc units in N-glycans and core 2 O-glycans of glycoproteins [12], including IgG, gp120 and serum α-fetoprotein [6], [13], [14], [15], [16]. GalT I-knockout mice exhibited growth retardation and inflammatory responses suppression, and showed significantly delayed wound healing with reduced re-epithelization, collagen synthesis and angiogenesis [12], [17], [18], [19], [20]. In addition, accumulated evidence indicated that GalT I was up-regulated in highly metastatic tumor and was involved in tumor cell migration and invasion [21], [22], [23], indicating the contribution of GalT I in tumor cell behavior.

Here, we show that GalT I was highly expressed in hepatoma and functioned as a positive role in hepatoma cell growth depending on its galactosylation activity. Furthermore, GalT I was transcriptionally up-regulated by HBx. Down-regulation of GalT I reduced the ability of hepatoma cell in tumor formation in vivo and inhibited HBx-induced cell cycle progression. These data indicate that GalT I might contribute to HBx-induced cell cycle progression of hepatoma cells.

Section snippets

Reagents

Restriction enzymes, Trizol reagent and the mammalian expression plasmid pcDNA3.0 were from Invitrogen. Anti-GAPDH antibody, anti-GalT I antibody, anti-mouse-HRP secondary antibody and anti-goat-HRP secondary antibody were from Santa Cruz Biotechnology. Anti-HBx antibody was purchased from Chemicon. Anti-β-Actin antibody was purchased from Sigma. We thank Prof. Lunxiu Qin (Zhongshan Hospital of Fudan University, China) and Prof. Aiguo Shen (Jiangsu Key Laboratory of Neuroregeneration, Nantong

The difference of GalT I expression between normal liver and hepatoma tissues

To investigate the contribution of GalT I in hepatoma development, we first detected the level of GalT I mRNA expression in normal liver and hepatoma tissues. Compared to that in normal liver tissues, GalT I mRNA expression was significantly increased in hepatoma tissues (Fig. 1A).

Accordingly, the level of GalT I protein was significantly increased in hepatoma tissues as compared to that in normal liver tissues (Fig. 1B). High expression of GalT I in hepatoma was confirmed in a series of

Discussion

It is widely known that N-linked oligosaccharides on glycoprotein are structurally altered during malignant transformation and that these alterations have a biological meaning [6], [32]. Here, we found that the Galβ1  4GlcNAc structure was significantly increased in hepatoma cell membrane compared to normal liver tissues. β-1,4-galactosyltransferase (GalT) family are the enzymes responsible for the biosynthesis of N-acetyllactosamine on N-glycans by transferring UDP-galactose to the terminal N

Acknowledgments

This work was supported by Shanghai Educational Development Foundation (2007CG02), Shanghai Rising-Star Program (08QA14013), the Opening Project of Institutes of Brain Science of Fudan University, National Natural Scientific Foundation of China (30870542 and 30700132), Shanghai Leading Academic Discipline Project (B110) and a Grant from the Development of Science and Technology of Shanghai (07ZR14011).

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    The authors who have taken part in the research of this paper declared that they do not have a relationship with the manufacturers of the materials involved either in the past or present and they did not receive funding from the manufacturers to carry out their research.

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