DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins

Abstract

Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2'-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.

Figure 1

(a) Immunofluorescence using anti-5mC antibody and integrated intensity fluorescence analysis revealed a decrease in global genome CpG methylation in HepG2 cells following treatment with 1 μM and 2.5 μM 5-aza-2dC when compared to the control group (p < 0.005). Error bars show standard deviation of fluorescence intensity in nuclei of each group. (b) Methylation levels at six CpG sites in the LINE-1 element were significantly decreased in treated groups as compared to control. Error bars show standard deviation between biological replicates. Asterisks in the stacked bars indicate statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001. (c) Cell cycle profiles for untreated cells and cells treated with 1 μM and 2.5 μM 5-aza-2dC 5-aza-2dC show statistically significant differences to the level of p < 0.01. Both concentrations of 5-aza-2dC induced a decrease in the proportion of cells in G1 and S-phase, and an increase in the proportion of cells in G2/M phase. The higher the concentration of 5-aza-2dC, the lower was the proportion of the cells in G1 phase and the higher was the proportion of the cells stalled in G2/M phase (p < 0.01). Error bars show standard deviation between biological replicates.

Figure 2. UPLC analysis of the N-glycome of HepG2 secretome.

The N-glycome from secretome of untreated HepG2 cells was separated into 20 chromatographic peaks (GP6-GP24) by hydrophilic interaction chromatography (HILIC). The structures of the N-glycans present in each peak were characterized by fractionation and subsequent MALDI-TOF-MS and LC-ESI-MS(/MS) analysis (see Suppl. Table 1). GP = glycan peak.

Figure 3. Contribution of individual glycan structures to the total N-glycome of the HepG2 secretome as measured after the treatment with the DNA methylation inhibitor 5-aza-2dC.

(a) The glycan structures under the peaks GP6, GP7, GP16, GP17, GP18 and GP20 were significantly changed following the treatment with 1 μM 5-aza-2dC. (b) The glycan structures under the peaks GP12, GP14, GP15 GP23 and GP24 were significantly changed following the treatment with 2.5 μM 5-aza-2dC. p < 0.05; GP = glycan peak.

Figure 4

Gene expression analysis performed using Glycosylation RT² Profiler PCR Array revealed glyco-genes that were down-regulated (a) or up-regulated (b) following the treatment with 1 μM and 2.5 μM 5-aza-2dC, respectively. Error bars indicate standard deviation between biological replicates.

Figure 5

(a) The consistent changes in the glycome of HepG2 secretome was a decrease in the quantity of the glycans with core fucose and tetra-antennary glycans, which could be explained by elevated MGAT3 gene expression. (b) Addition of a bisecting GlcNAc by the catalytic activity of GnT-III (the MGAT3 gene) inhibits the elongation of a glycan structure by inhibiting GnT-IV (the MGAT4 gene), GnT-V (the MGAT5 gene) and Fut8 (the FUT8 gene).

Figure 6. The expression level of the MGAT3 gene as measured following 5-aza-2dC treatment.

(a) Experiment 1 showed 3.4-fold and 5.4-fold up-regulation of the MGAT3 expression (p < 0.05) after the treatment with 1 μM and 2.5 μM 5-aza-2dC, respectively. (b) Experiment 2 showed 3.9-fold and 4.8-fold up-regulation of the MGAT3 expression (p < 0.01) after the treatment with 1 μM and 2.5 μM 5-aza-2dC, respectively. The experiments were done in five biological replicates. Error bars indicate standard deviation between biological replicates.

Figure 7. Methylation levels in the promoter/first intron of the MGAT3 gene as measured after 5-aza-2dC treatment.

(a) Schematic representation of the MGAT3 gene. The promoter (1,000 bp upstream from the transcription start site, indicated by an arrow) has one CpG island (CpG island 1), which is 734 bp long. Second CpG island (CpG island 2, 742 bp long) includes the first exon and intron. White boxes represent untranslated (UTR) exons, and the black box stands for coding exon. The location of the four regions, containing CpG sites analysed for methylation level, is represented as coloured boxes. (b) Decrease in methylation level at 10 CpG sites in the region 1 (p-values ranging from p < 0.001 to p < 0.005). (c) Decrease in methylation level at 5 CpG sites within the region 2 (p < 0.001). (d) Within the region 3, only the site CpG22 shows statistically significant decrease in methylation level following 1 μM 5-aza-2dC treatment (p < 0.05); (e) Decrease in methylation level in the region 4 (p-values ranging from p < 0.001 to p < 0.05). Asterisks in the stacked bar graphs indicate statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8. Methylation level of the MGAT3 promoter region in HepG2 cell line and in HCC.

Gene model and coordinates from the hg19 human genome assembly are shown, with location of CpG islands highlighted on a separate track. Methylation of the promoter region covered by the Illumina 450k platform is shown in colors corresponding to the methylation level. Differential methylation can be observed between probes located within CpG islands, with HepG2 cell line being hypomethylated in comparison to normal hepatocytes, as well as HCC when compared to adjacent tissue.