title: [Short hairpin RNA mediated glypican-3 silencing inhibits hepatoma cell invasiveness and disrupts molecular pathways of angiogenesis].
abstract: 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, 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.
  
  
  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 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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Dan-dan
authors.initials: DD
authors.lastname: Yu
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Mei-juan
authors.initials: MJ
authors.lastname: Yan
authors.forename: Xing
authors.initials: X
authors.lastname: Gu
authors.forename: Li-wei
authors.initials: LW
authors.lastname: Qiu
authors.forename: Zhi-zhen
authors.initials: ZZ
authors.lastname: Dong
authors.forename: Deng-fu
authors.initials: DF
authors.lastname: Yao
authors.forename: Shao-lin
authors.initials: SL
authors.lastname: Lu
mesh_terms: D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D020868:Gene Silencing; D053673:Glypicans / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D009361:Neoplasm Invasiveness; D009389:Neovascularization, Pathologic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: C495270:CTNNB1 protein, human; C105937:GPC3 protein, human; D053673:Glypicans; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D051176:beta Catenin; C505407:CASP3 protein, human; D053148:Caspase 3