[Short hairpin RNA mediated glypican-3 silencing inhibits hepatoma cell invasiveness and disrupts molecular pathways of angiogenesis].

Objective To construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms.Methods GPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2,MHCC-97H,and Huh7.shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting,respectively.The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays,on migration by wound healing (scratch) assay,on invasion by transwell chamber assay,and on apoptosis by luminescence assay of caspase-3/7 activity.The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor,GLI1,and the beta-catenin Wnt signaling factor,β-catenin),immunofluorescent staining (for the insulin-like growth factor-Ⅱ,IGF-Ⅱ),and ELISA (for the vascular endothelial growth factor,VEGF).Pairwise comparisons were made by the independent sample t-test,and multiple comparisons were made by one-way ANOVA.Results In all cell lines,transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (％ reduction as compared to the non-target control shRNA:HepG2,89.2 + 6.0％,t =-25.753,P ＜ 0.001; MHCC-97H,75.3 + 4.9％,t =-26.487,P ＜ 0.001; Huh7,73.6 ± 4.6％,t =-27.607,P ＜ 0.001); the GPC-3 protein levels were similarly reduced.The GPC-3 shRNA-silenced cells showed significantly reduced proliferative,migratory and invasive capacities,as well as significantly increased apoptosis.The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of β-catenin mRNA (HepG2,46.9 + 0.6％; MHCC-97H,67.5 + 2.7％; Huh7,56.3 + 8.4％) and significant up-regulation ofGLI1 mRNA (HepG2,49.2 ± 28.6％; MHCC-97H,54.6 ± 24.4％; Huh7,31.6 ±15.7％).At 72 h after transfection,the HepG2 cells showed significant down-regulation of VEGF protein (54.3 + 1.5％,t =46.746,P ＜ 0.001).Conclusion GPC-3 contributes to migration,invasion,angiogenesis,and apoptosis of hepatoma cells,possibly through its interactions with the Wnt/β-catenin and Hedgehog signaling pathways.GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma. Key words: Carcinoma, hepatocellular;  Signal transduction;  Glypican-3;  Short hairpin RNA; Angiogenesis