title: [Intervention of glypican-3 genetic transcription on anti-proliferative effect of hepatoma cells with high metastatic potentiality].
abstract: OBJECTIVE
  To explore the silencing glypican-3 (GPC-3) gene transcription by specific small hairpin RNA (shRNA) on the inhibition of hepatoma cells with high metastatic potentiality and hepatoma growth.
  
  
  METHODS
  After MHCC-97H cells were transfected with higher effective GPC-3-shRNA, GPC-3 mRNA was analyzed by multiple FG-RT-PCR or protein by Western blot. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine and sulforhodamine B assay, its migratory metastasis and invasiveness by wound healing or transwell chamber system and cell apoptosis was detected by Caspase-Glo(®) 3/7 Luminescence assay. Nude mice were subcutaneously injected with stable MHCC-97H cells for observing the forming time or volume of xenograft tumors. And the expressions of GPC-3, β-catenin, p-GSK3β and CyclinD1 were analyzed by immunohistochemistry.
  
  
  RESULTS
  After shRNA1 transfection with high efficiency (>80%), the expression of GPC-3 was down-regulated to 75.6% (t = 15.473, P < 0.001) at mRNA level in accordance with its protein, inhibiting cell proliferation (71.1%, t = 10.468, P < 0.001) notably, decreasing its migration (80.1%, t = 32.697, P < 0.001) and invasiveness (69.1%, t = 39.647, P < 0.001). β-catenin was down-regulated (67.7%, t = 18.4, P < 0.001) and Gli1 increased (53.5%, t = -4.824, P = 0.008) with its protein. The average forming time of subcutaneous tumors was 11.2 days (d) in the shRNA group and it was significantly longer (P < 0.01) than that in the control (5.3 d) or shRNA-neg (5.5 d) group. And the average volume (65.5 mm(3)) of tumors with decreased GPC-3, β-catenin, p-GSK3β, and cyclinD1 expressions in the shRNA group was significantly smaller (P < 0.01) than that in the shRNA-neg (365.7 mm(3)) or control (404.8 mm(3)) group, respectively.
  
  
  CONCLUSIONS
  Specific shRNA might intervene effectively the GPC-3 gene transcription and inhibit invasion and tumor growth. Thus GPC-3 may become a potential molecular target for hepatoma gene therapy.
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Qian
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Yu
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China.
authors.forename: Shaolin
authors.initials: S
authors.lastname: Lu
authors.affiliation: Research Center of Clinical Medicine, Nantong University, Nantong 226001, China. Email: yaodf@ahnmc.com.
authors.forename: Dengfu
authors.initials: D
authors.lastname: Yao
mesh_terms: D000818:Animals; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003857:Deoxyuridine / Q000031:analogs & derivatives; D015536:Down-Regulation; D020868:Gene Silencing; D053673:Glypicans; D006801:Humans; D008113:Liver Neoplasms; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014158:Transcription, Genetic; D014162:Transfection; D051176:beta Catenin
chemical_list: D053673:Glypicans; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051176:beta Catenin; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine