Elevated GPC3 level promotes cell proliferation in liver cancer

The aim of the present study was to investigate the biological role of glypican 3 (GPC3), and to identify its mechanism and clinical significance in the carcinogenesis of liver cancer. A total of 114 patients with liver cancer were involved. Their clinical data, hematoxylin and eosin-stained and Antigen Ki-67 protein (Ki-67) and GPC3 immunohistochemically-stained liver cancer tissue sections were analyzed to evaluate the correlation between the liver cancer proliferation, differentiation and GPC3 expression. Fluorescence microscopy, western blotting, MTT and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were performed in HepG2 and HLE cell lines to investigate the potential mechanisms of action. Among the 114 patients with liver cancer enrolled in the present study, 12 exhibited well-differentiated liver cancer, of which 6 (50%) were positive for GPC3. A total of 30 cases exhibited poorly differentiated liver cancer; 26 (87%) of these expressed GPC3 and 11 cases (37%) demonstrated strong positive expression levels. The other 72 liver cancer cases were moderately differentiated; 75% (54/72) of these expressed GPC3 and 12.5% (9/72) exhibited strong positive expression levels. There was a significant association between the levels of GPC3 expression and liver cancer differentiation (χ(2)=16.306, P=0.008). Ki-67 staining as the criteria of the liver cancer cell proliferation index also indicated a cross correlation between liver cancer proliferation and GPC3 levels. Among the 39 liver cancer samples with a cell proliferation index <5%, only 2.6% (1/39) exhibited strong positive GPC3 staining, but of the 16 cases with a high cell proliferation index >50%, 6 exhibited strong GPC3 staining (37.5%). The difference of cell proliferation indexes between cancer cells were well, moderate and poorly differentiated, and was markedly significant (χ(2)=26.334, P=0.002), and suggested that liver cancer cell proliferation was positively correlated with GPC3 expression (r=0.316, P=0.001). Consistently, in vitro analysis indicated that GPC3 promoted HepG2 and HLE cell growth, which was more apparent in HepG2 cells. The RT-qPCR results indicated that GPC3 promoted proliferation through the Hedgehog (Hh) pathway in HepG2 cells, but not in HLE cells. In the present study, it was demonstrated that patients with liver cancer with higher GPC3 levels exhibited poorer differentiation and higher proliferation levels. In vitro GPC3 may promote liver cancer cell lines proliferation through the Hh pathway.