Short hairpin RNA mediated glypican-3 silencing inhibits hepatoma cell invasiveness and disrupts molecular pathways of angiogenesis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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, b-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.</p><p><b>RESULTS</b>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 less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 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 b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 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 less than 0.001).</p><p><b>CONCLUSION</b>GPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.</p>

Effects of inhibited IGF-IR expression on proliferation and apoptosis of human hepatocellular carcinoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic value of inhibiting the expression of insulin-like growth factor-I receptor (IGF-IR) using picropodophyllin (PPP) by studying the effects on proliferative and metastatic potentials of human hepatocellular carcinoma (HCC) using an in vitro cultured cell system.</p><p><b>METHODS</b>IGF-IR expression in human HCC cell lines (Bel-7404, Bel-7402, HepG2, and Huh-7) and human hepatocytes (L02) was assessed at baseline (pre-treatment) and after PPP treatment by western blotting. Changes in cell cycle were analyzed by flow cytometry and in cell viability by sulforhodamine B staining. Early apoptosis was detected by annexin-V/FITC and propidium iodide double-staining assay. Caspase-3/7 activity was suppressed by z-VAD-FMK and analyzed by homogeneous luminescence assay. Effects on cell motility were tested by wound-scratch test. Between-group differences were assessed by t-test or one-way analysis of variance.</p><p><b>RESULTS</b>IGF-IR was markedly up-regulated in all HCC cell lines (vs. non-hepatoma hepatocytes). HCC cells with PPP-inhibited IGF-IR showed time-dependent decreases in cell motility and viability. After treatment with PPP for 24 hours, the proportion of HCC cells in G1 phase was 2.1% +/- 0.4%, in S phase was 11.0% +/- 0.7%, and in G2/M phase was 87.1% +/- 0.6%, and no healing was observed in the wound-scratch assay. The PPP treatment induced cell apoptosis, as evidenced by enhanced caspase-3/7 activity; the proportion of annexin-V+/PI- cells was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (16.4% +/- 0.4% vs. 5.8% +/- 0.2%, t = 14.05, P less than 0.01). After z-VAD-FMK treatment, the apoptosis rate was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (11.3% +/- 0.7% vs. 5.8% +/- 0.2%, t = 11.83, P less than 0.01).</p><p><b>CONCLUSION</b>IGF-IR is associated with proliferation, cell motility, and apoptosis of HCC cells, and may be a promising molecular target for HCC.</p>