Association of glypican-3 expression with growth signaling molecules in hepatocellular carcinoma

Abstract

Aim: To clarify the association of glypican-3 (GPC3) expression with Wnt and other growth signaling molecules in hepatocellular carcinoma (HCC).

Methods: Expression of GPC3, Wnt, matrix metalloproteinases (MMPs), sulfatase (SULF)1, SULF2, and other growth signaling molecules was analyzed in HCC cell lines and tissue samples by real-time reverse transcription-polymerase chain reaction, immunoblotting, and/or immunostaining. Expression of various genes in GPC3 siRNA-transfected HCC cells was analyzed.

Results: GPC3 was overexpressed in most HCCs at mRNA and protein levels and its serum levels were significantly higher in patients with HCC than in non-HCC subjects (P < 0.05). Altered expressions of various MMPs and growth signaling molecules, some of which were correlated with GPC3 expression, were observed in HCCs. Down-regulation of GPC3 expression by siRNA in GPC3-overexpressing HCC cell lines resulted in a significant decrease in expressions of MMP2, MMP14, fibroblast growth factor receptor 1, insulin-like growth factor 1 receptor. GPC3 expression was significantly correlated with nuclear/cytoplasmic localization of beta-catenin.

Conclusion: These results suggest that GPC3, in conjunction with MMPs and growth signaling molecules, might play an important role in the progression of HCC.

Figure 1

Semiquantitative reverse transcription-polymerase chain reaction analysis of glypican-3 in hepatocellular carcinoma cell lines. Expression of glypican-3 (GPC3) mRNA was analyzed in hepatocellular carcinoma cell lines by semiquantitative reverse transcription-polymerase chain reaction. GAPDH served as a control.

Figure 2

Real time reverse transcription-polymerase chain reaction analysis of glypican-3 in hepatocellular carcinoma cell lines. cDNA was used for quantitative polymerase chain reaction (PCR). The amount of PCR products was determined by reading the midpoint of the linear portion of the S-shaped real-time curves, called the Ct point or threshold cycle. The Ct refers to the number of cycles it takes a sample to reach a specific fluorescence threshold.

Figure 3

Down-expression of various molecules in glypican-3 siRNA-transfected hepatocellular carcinoma cell line. A: mRNA levels analyzed by real-time reverse transcription-polymerase chain reaction. The mRNA expressions of matrix metalloproteinase (MMP)2, MMP14, fibroblast growth factor receptor 1 (FGFR1), and insulin-like growth factor 1 receptor (IGF1R) were significantly down-regulated in HepG2 cells treated with glypican-3 (GPC3) specific siRNA (P < 0.05). Bar: SE; B: Protein levels analyzed by immunoblotting. The protein expressions of MMP2, MMP14, FGFR1, and IGF1R were significantly down-regulated in HepG2 cells treated with GPC3 specific siRNA; C: MMP2 activity analyzed by gelatin zymography. Conditioned media of siRNA-transfected HepG2 were used for gelatin zymography.

Figure 4

Immunohistochemistry for glypican-3 in liver tissue. A: Normal liver negative for glypican-3 (GPC3); B: Hepatocellular carcinoma positive for GPC3. Original magnification, × 200. Expression of GPC3 was immunohistochemically analyzed with an anti-human GPC3 mouse monoclonal antibody. GPC3 protein was expressed in the cytoplasm and/or membrane of carcinoma cells.

Figure 5

Immunohistochemistry for β-catenin in hepatocellular carcinoma tissue. A: Membrane staining, cytoplasmic staining, nuclear staining of β-catenin. Expression of β-catenin was immunohistochemically analyzed with an anti-human β-catenin monoclonal antibody. Original magnification, × 200; B: Association of nuclear/cytoplasmic localization of β-catenin with glypican-3 (GPC3) expression.

Figure 6

Serum glypican-3 levels. Serum glypican-3 (GPC3) levels were measured using a commercially available sandwich ELISA kit. Bar: Standard error. Serum GPC3 levels were significantly higher in patients with hepatocellular carcinoma (HCC) than in non-HCC subjects (P < 0.05). HBV: Hepatitis B virus.

Figure 7

Associations of glypican-3 expression with matrix metalloproteinases and growth signaling molecules in hepatocellular carcinoma. A summary diagram is shown. MMPs: Matrix metalloproteinases; SULF2: Sulfatase 2; GPC3: Glypican-3; FGF: Fibroblast growth factor; IGF: Insulin-like growth factor.