Glycan-related gene expression signatures in human metastatic hepatocellular carcinoma cells

Abstract

Human hepatocellular carcinoma (HCC) ranks second in cancer mortality in China; recurrence and metastasis have been the cause of the high mortality. Glycans on the cell surface play a pivotal role in tumor metastasis. The global alteration in the structure and composition of N-glycans during HCC metastasis remains unknown. To understand glycan alterations of glycoproteins by correlating the glycosyltransferase expression profile with glycan structure, we systematically used glycan profiling tools: glycogene microarray analyses of 115 genes, including glycotransferases, glycosidases and nuclear sugar transporters and lectin chips to investigate the glycan-related gene expression signatures in the high metastatic potential HCC cell line, HCCLM3, in comparison to the HCC cell line, Hep3B, with low metastatic potential. Of the 115 genes, 18 genes were up-regulated in high metastatic potential HCCLM3 cells in comparison to Hep3B cells, while 11 genes were down-regulated. The differentially expressed genes, such as ST3GalI, FUT8, β3GalT5, MGAT3 and MGAT5, were mainly involved in the synthesis of N-glycan and glycolipids, particularly the sialyl Lewis antigen. The results of the glycogene microarray analysis were further validated by quantitative real-time PCR analysis and lectin-based analysis. The differentially expressed glycogenes identified in this study may provide new insights and represent novel factors for investigating the functional role of cell surface carbohydrate-mediated HCC metastasis.

Figure 1.

Hierarchical clustering of glycan gene expression. Hep3B and HCCLM3 cells are well-separated, indicating that glycan genes are differentially expressed between the two cell lines.

Figure 2.

Relative mRNA levels of glycosyltransferase gene expression in HCCLM3 and Hep3B cells using real time-PCR.

Figure 3.

Differential membrane glycoprofiling of Hep3B and HCCLM3 cells. (A) Cy3-labeled membrane proteins of Hep3B and HCCLM3 cells were allowed to bind with lectin microarray. Bound membrane proteins were scanned with a fluorescent scanner. (B) Differential glycan profiling of Hep3B and HCCLM3 cells from lectin array. Predicted representative differential structures expressed in Hep3B and HCCLM3 were shown by monosaccharide symbols. ^*P<0.01. (C) Validation of microarray results of Hep3B and HCCLM3 cells by means of fluorescence labeling lectin staining.

Figure 4.

Functional subnetwork of differentially expressed glycogenes. The networks of genes with altered expression in HCCLM3 cells compared to Hep3B. Differentially expressed genes listed in Table II were analyzed by the IPA tool. The network shown was found to be significantly associated with cell adherence, apoptosis and angiogenesis. Gene products belonging to different functional classes are displayed with various shapes. Colored nodes are derived from the glycan gene array, and the white nodes are from the IPA database. The green represents a decreased expression in HCCLM3 whereas red indicates an increased expression. Solid lines represent direct interactions between 2 nodes and dashed lines represent indirect interactions. Lines beginning and ending on the same node mean self-regulation. Arrowheads mean node A acts on node B, while the edge without arrowheads means a binding relationship between two nodes.