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. 2016 Sep 20;7(38):61199-61214.
doi: 10.18632/oncotarget.11284.

Comprehensive N-glycan profiles of hepatocellular carcinoma reveal association of fucosylation with tumor progression and regulation of FUT8 by microRNAs

Lei Cheng  1   2 , Shuhang Gao  3 , Xiaobo Song  4 , Weijie Dong  5 , Huimin Zhou  6 , Lifen Zhao  1 , Li Jia  1
Affiliations

Comprehensive N-glycan profiles of hepatocellular carcinoma reveal association of fucosylation with tumor progression and regulation of FUT8 by microRNAs

Lei Cheng et al. Oncotarget. .

Abstract

Glycosylation has significant effects on cancer progression. Fucosylation is one of the most important glycosylation events involved in hepatocellular carcinoma (HCC). Here, we compared N-glycan profiles of liver tumor tissues and adjacent tissues of 27 HCC patients to reveal the association between fucosylation and HCC progression, as well as verified the potential role of miRNA in regulating fucosylation. Mass spectrometry (MS) analysis showed pronounced differences of the N-glycosylation patterns and fucosylated N-glycans between the adjacent and tumor tissues. Different fucosyltransferase (FUT) genes were also identified in adjacent and tumor tissues, and two HCC cell lines with different metastatic potential. High-level expression of FUT8 was detected in tumor tissues and highly metastatic HCC cells. Altered levels of FUT8 in HCC cell lines significantly linked to the malignant behaviors of proliferation and invasion in vitro. Furthermore, using microRNA array, we identified FUT8 as one of the miR-26a, miR-34a and miR-146a-targeted genes. An inverse correlation was revealed between the expression levels of FUT8 and these miRNAs. Luciferase reporter assay demonstrated these miRNAs specifically interacted with the 3'UTR of FUT8 and subsequently down-regulated FUT8 expression-level. The forced expression of these miRNAs was able to induce a decrease in FUT8 levels and thereby to suppress HCC cells progression. Altogether, our results indicate that fucosylated N-glycan and FUT8 levels can be used as markers for evaluating HCC progression, as well as miRNAs may be involved in inhibition of fucosylation machinery through targeting FUT8.

Keywords: fucosyltransferase (FUT) gene; human hepatocellular carcinoma cell lines; microRNAs; tumor progression.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. The profiles of N-Glycan composition in HCC tumor and adjacent tissues
(A) MALDI-TOF MS spectra of N-glycans from HCC tumor and adjacent tissues were shown respectively. N-glycans were released by PNGase F and permethylated. (B) Histograms of relative intensities of the differential N-glycan signals were observed (the histograms represents only signal intensities but not the quantities). The signals indicated with Arabic numerals are summarized in Table 1.
Figure 2
Figure 2. Differential expression of FUT family in HCC tissues and HCC cell lines
The mRNA levels of FUT family were analyzed by qRT-PCR. (A) Tumor tissues expressed higher levels of FUT4, FUT5, FUT8, FUT11 and POFUT2 mRNA than adjacent tissues. (B) MHCC97H cells with high metastatic potential expressed higher levels of FUT4, FUT5, FUT6 and FUT8 mRNA than MHCC97L cells with low metastatic potential. Data are the means ± SD of triplicate determinants.
Figure 3
Figure 3. FUT8 correlates with HCC cells progression in vitro
(A) FUT8 transcript was decreased apparently in MHCC97H cells by shRNA treatment. The distinct reduction of FUT8 was observed at mRNA and protein levels by qRT-PCR and western blot analysis (*P < 0.05). (B) Differential FITC-LCA binding profiles of MHCC97H control and MHCC97H shFUT8 cell lines using flow cytometry. Histograms of fluorescence intensities of cells with specific carbohydrate expression as determined. (C) After full-length sequences transfection, FUT8 mRNA and protein levels were increased notably in MHCC97L cells by qRT-PCR and western blot analysis (*P < 0.05). (D) Flow cytometry analysis showed α-1, 6 fucosylation level detected by FITC-LCA on the cell surface, was also increased in MHCC97L FUT8 cells. (E) Growth curves of MHCC97H shFUT8 cells were compared to control cells with the CCK-8 assay. (F) Transwell cell migration and invasion assays were performed to compare cell migration and invasion between MHCC97H shFUT8 cells and MHCC97H control. (G) Growth curves of MHCC97L FUT8 cells were compared to control cells with the CCK-8 assay. (H) In vitro Transwell cell migration and invasion analysis was performed. MHCC97L FUT8 cells were significantly more migratory and invasive (*P < 0.05) than MHCC97L mock cells. Data are the means ± SD of triplicate determinants (*P < 0.05).
Figure 4
Figure 4. MiR-26a, miR-34a and miR-455-3p as negative regulators of FUT8
(A) miR-26a, miR-34a and miR-455-3p expression were significantly decreased in 27 HCC compared with the corresponding adjacent tissues using qRT-PCR analysis. (B) The expression of miR-26a, miR-34a and miR-455-3p was examined by qRT-PCR in the MHCC97H, MHCC97L and L02 cell lines. (C) Relationship between miR-26a, miR-34a and miR-455-3p levels and FUT8 mRNA expression in 27 HCC tumor and 27 adjacent tissues. (D) FUT8 expression was significantly increased in 27 HCC compared with the corresponding adjacent tissues using qRT-PCR analysis. (E–G). The nucleotides sequence of the target site of miRNAs in FUT8 3′-UTR was shown; Luciferase assay for the direct targeting of 3′-UTR of FUT8 by miR-26a, miR-34a and miR-455-3p. The wide-type and mutant miRNA target sequences of FUT8 were fused with luciferase reporter and transfected into 293T cells, transfected with miRNA mimic and NC mimic. The mean of the results from the cells transfected with the NC mimic was set at 100. Each bar represents the relative luciferase activity (*P < 0.05). (H) FUT8 was analyzed by qRT-PCR and western blot in MHCC97H cells treated with miR-26a, miR-34a and miR-455-3p mimic (*P < 0.05). (I) FUT8 was analyzed by qRT-PCR and western blot in MHCC97L cells treated with miR-26a, miR-34a and miR-455-3p inhibitor (*P < 0.05). Data are the means ± SD of triplicate determinants.
Figure 5
Figure 5. Effect of miR-26a, miR-34a and miR-455-3p mimic on cell progression in MHCC97H cells
(A) The expression of miR-26a, miR-34a and miR-455-3p was studied by qRT–PCR in MHCC97H cells transfected with the mimics (*P < 0.05). (B) Transfection of miR-26a, miR-34a and miR-455-3p mimic in MHCC97H cells inhibited cellular viability as revealed by CCK-8 assay (*P < 0.05). (C) Overexpression of miR-26a, miR-34a and miR-455-3p inhibited the growth of MHCC97H cells with in focus formation assay. (D) Ki67 expression was detected by immunofluorescence staining in MHCC97H cells treated with miRNA mimic transfection. Red fluorescence: Ki67; DAPI staining for nuclear DNA. (E) Transwell cell migration and invasion assays were used to compare cell migration and invasion between miRNA mimic- and NC mimic-transfected cells. The data were mean ± S.D. of three separate transfections (*P < 0.05).
Figure 6
Figure 6. Effect of miR-26a, miR-34a and miR-455-3p inhibitor on cell progression in MHCC97L cells
(A) The expression of miR-26a, miR-34a and miR-455-3p was studied by qRT–PCR in MHCC97L cells transfected with the inhibitor (*P < 0.05). (B) MHCC97L cells were transfected with miR-26a, miR-34a and miR-455-3p respectively. Expression of miRNA was confirmed by qRT–PCR. B, Growth curves of miRNA inhibitor-transfected cells were compared to NC inhibitor cells with the CCK-8 assay (*P < 0.05). (C) Morphology foci were observed at the microscope and photographed. (D) Ki67 expression was detected by immunofluorescence staining in MHCC97L cells treated with miRNA inhibitor transfection. Red fluorescence: Ki67; DAPI staining for nuclear DNA. (E) Knockdown of miR-26a, miR-34a and miR-455-3p significantly increased the migration and invasion of MHCC97L cells. The data were mean ± S.D. of three separate transfections (*P < 0.05).
Figure 7
Figure 7. MiR-26a, miR-34a and miR-455-3p mediates MHCC97H cells tumorigenesis by targeting FUT8
(A) MHCC97H cells were transfected with miRNA mimics or NC mimic and implanted subcutaneously into the right flank of each nude mouse respectively (n = 6). One week later, the mouse in groups were injected intratumorally with miRNA mimics or NC mimic three times per week for 3 weeks respectively. miR-26a, miR-34a and miR-455-3p inhibited the tumorigenesis of MHCC97H cells in vivo. The tumor volumes and weight were reduced in miRNA mimic groups to compare with NC mimic group (*P < 0.05). (B) The expression levels of Ki67 and FUT8 were analyzed by IHC in tumors.
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