Fucoidan Elevates MicroRNA-29b to Regulate DNMT3B-MTSS1 Axis and Inhibit EMT in Human Hepatocellular Carcinoma Cells

Abstract

Accumulating evidence has revealed that fucoidan exhibits anti-tumor activities by arresting cell cycle and inducing apoptosis in many types of cancer cells including hepatocellular carcinoma (HCC). Exploring its effect on microRNA expression, we found that fucoidan markedly upregulated miR-29b of human HCC cells. The induction of miR-29b was accompanied with suppression of its downstream target DNMT3B in a dose-dependent manner. The reduction of luciferase activity of DNMT3B 3'-UTR reporter by fucoidan was as markedly as that by miR-29b mimic, indicating that fucoidan induced miR-29b to suppress DNMT3B. Accordingly, the mRNA and protein levels of MTSS1 (metastasis suppressor 1), a target silenced by DNMT3B, were increased after fucoidan treatment. Furthermore, fucoidan also down-regulated TGF-β receptor and Smad signaling of HCC cells. All these effects leaded to the inhibition of EMT (increased E-cadherin and decreased N-cadherin) and prevention of extracellular matrix degradation (increased TIMP-1 and decreased MMP2, 9), by which the invasion activity of HCC cells was diminished. Our results demonstrate the profound effect of fucoidan not only on the regulation of miR-29b-DNMT3B-MTSS1 axis but also on the inhibition of TGF-β signaling in HCC cells, suggesting the potential of using fucoidan as integrative therapeutics against invasion and metastasis of HCC.

Keywords: DNMT3B; EMT; MTSS1; fucoidan; hepatocellular carcinoma; miR-29b.

Figure 1

Inhibitory effects of fucoidan on the cell viability and clonogenicity of human HCC cells. (A) Four HCC cells and (B) two immortalized normal hepatocytes were incubated for 48 h with various concentrations of fucoidan (0, 100 and 200 μg/mL), and the proliferation of cell was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Values are expressed as the mean ± standard error of three independent experiments. (C) SK-Hep1 and HepG2 cells were treated with fucoidan at 200 μg/mL and the clonogenicity was measured. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (* p < 0.05 and ** p < 0.01, compared with the control group).

Figure 2

Inhibitory effect of fucoidan on the invasion of human HCC cells. SK-Hep1 and HepG2 cells were treated with fucoidan at a dose of 200 μg/mL for 24 h and the invasiveness was measured by two-chamber transwell assay. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (* p < 0.05 and ** p < 0.01, compared with the control group).

Figure 3

Fucoidan increases the expression of miR-29b. L02, SK-Hep1 and HepG2 cells were treated with fucoidan (0, 100 and 200 μg/mL) for 48 h and the relative miR-29b expression levels were measured by quantitative RT-PCR. Data were normalized to the U6 signal. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (* p < 0.05 and ** p < 0.01, compared with respective control group; ^## p < 0.01, compared with the basal level of L02).

Figure 4

Fucoidan increases miR-29b to target the 3′-UTR region of DNMT3B and repress its expression in human HCC cells. (A) Schematic representation of 3′-UTRs of DNMT3B showing putative miR-29b target site; (B) After transfection, the DNMT3B-dependent luciferase activity of HCC cells was measured by Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA) 48 h after the treatment as indicated. The experiments were performed in triplicate. The values were normalized to the Renilla luciferase activity (transfection efficiency control) and plotted as relative luciferase activity. SCR: scrambled control. Each group of fucoidan-treated samples was normalized against each untreated control. The data are representative of three separate experiments and are presented as the mean ± SD; error bars indicate SD. Significant differences are shown (* p < 0.05 and ** p < 0.01, compared with the control group).

Figure 5

Fucoidan regulates the DNMT3B-MTSS1 axis to inhibit EMT in HCC cells. After treatment with fucoidan for 48 h, the mRNA (A) and protein (B) levels of DNMT3B, MTSS1, E-cadherin, and N-cadherin were analyzed by RT-PCR and Western blot, respectively. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used a loading control.

Figure 6

Fucoidan suppresses TGF-β signaling pathway of HCC cells and prevents degradation of extracellular matrix. (A) After 48 h of treatment, fucoidan dose-dependently down-regulated the TGF-β receptor 1, 2 (TGF-βR1, 2), phospho-Smad2/3 (p-Smad2/3) and Smad4 protein levels but increased the inhibitory Smad protein, Smad7. (B) Fucoidan dose-dependently decreased the protein levels of MMP2 and MMP9 while increased that of tissue inhibitor of metalloproteinase-1 (TIMP-1). The HCC cells were treated with fucoidan (0, 100, 200 μg/mL) for 48 h, and the cell lysates were then collected for Western blotting. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control.

Scheme 1

The proposed molecular mechanism of action related to the inhibition of invasion and metastasis of HCC cells by fucoidan. EMT: epithelial to mesenchymal transition; ECM: extracellular matrix.