title: Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7.
abstract: OBJECTIVE
  To build GPC3 gene short hairpin interference RNA (shRNA) slow virus vector, observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer.
  
  
  METHODS
  Hepatocellular carcinoma cell line Huh-7 was transfected by a RNA interference technique. GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR. Targeted GPC3 gene sequences of small interfering RNA (siRNA) PGC-shRNA-GPC3 were restructured. Stable expression cell lines of siRNA were screened and established with the help of liposomes (lipofectamine(TM2000)) as carrier transfection of human liver cell lines. In order to validate siRNA interference efficiency, GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot. The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects of GPC3 gene on Huh-7 cell proliferation and apoptosis were observed.
  
  
  RESULTS
  In the liver cancer cell lines Huh-7, GPC3 gene showed high expression. PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation. Stable recombinant plasmid transfected into liver cancer cell lines Huh-7 can obviously inhibit GPC3 mRNA expression level.
  
  
  CONCLUSIONS
  The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.
authors.affiliation: Department of General Surgery, the Second Affiliated Hospital of Jianghan University (Wuhan City Fifth Hospital), Wuhan, Hubei, China.
authors.forename: Chang-Jiang
authors.initials: CJ
authors.lastname: Lei
authors.affiliation: Wuhan Institute of Hematology, the Second Affiliated Hospital of Jianghan University (Wuhan City Fifth Hospital), Wuhan, Hubei, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Yao
authors.affiliation: Integrated Department, Branch of Jianqiao, Hanyang Affiliated Hospital of Wuhan University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Qing-Yun
authors.initials: QY
authors.lastname: Pan
authors.affiliation: Department of General Surgery, the Second Affiliated Hospital of Jianghan University (Wuhan City Fifth Hospital), Wuhan, Hubei, China.
authors.forename: Hao-Cheng
authors.initials: HC
authors.lastname: Long
authors.affiliation: Department of General Surgery, the Second Affiliated Hospital of Jianghan University (Wuhan City Fifth Hospital), Wuhan, Hubei, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Pediatrics, the Second Affiliated Hospital of Jianghan University (Wuhan City Fifth Hospital), Wuhan, Hubei, China. Electronic address: asdasq555@126.com.
authors.forename: Shu-Ping
authors.initials: SP
authors.lastname: Zheng
authors.affiliation: Department of General Surgery, the Second Affiliated Hospital of Jianghan University (Wuhan City Fifth Hospital), Wuhan, Hubei, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zeng
authors.affiliation: Department of General Surgery, the Second Affiliated Hospital of Jianghan University (Wuhan City Fifth Hospital), Wuhan, Hubei, China.
authors.forename: Jian-Bin
authors.initials: JB
authors.lastname: Huang
keywords: GPC3 gene; Liver cancer cell lines; RNA interference; Slow virus carrier