RESUMO
OBJECTIVE: To investigate the effect of overexpression of glycosylphosphatidyl-inositol-specific phospholipase D (GPI-PLD) on the biological character of hepatocellular carcinoma cell line HepG2. METHODS: The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/ GPI-PLD was transiently transfected into HepG2 cell by lipid-media transfection. The untransfected HepG2 and HepG2 transfected with pcDNA3.1(+) were used as controls. After screening with G418, the single clone was obtained. The expression level of GPI-PLD mRNA in HepG2 was identified by reverse transcription polymerase chain reaction (RT-PCR). GPI-PLD activities were analyzed quantitatively by triton-X-114 partition with GPI anchored placental alkaline phosphatase (PLAP) as a substrate. Cell count was used to detect the proliferation of the 3 groups, and complement dependent cytotoxicity (CDC) effects were observed by the staining of trypan blue. Apoptosis cells were analyzed by flow cytometry. Carcinoembryonic antigen (CEA)was detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with HepG2 and pcDNA3.1(+)/HepG2 cell, the levels of GPI-PLD activities and its mRNA from pcDNA3.1(+)/GPI-PLD/HepG2 were increased with almost 2 to 5 times,respectively. The GPI anchored PLAP and CEA released into the medium by GPI-PLD, and the rate of CDC killing on the cells were significantly increased. However, the proliferative capacity was obviously decreased, and the typical apoptosis cells were presented in positive clones and its apoptosis rates were increased significantly. CONCLUSION: The stable cell line with overexpression of GPI-PLD has been constructed. The overexpression of GPI-PLD in these cells increases the sensitivity of these cells to CDC killing and impairs the proliferative capacity of cells, and promotes the apoptosis.
Assuntos
Apoptose/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fosfolipase D/biossíntese , Carcinoma Hepatocelular/patologia , Ativação do Complemento/genética , Citotoxicidade Imunológica/genética , Células Eucarióticas/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Fosfolipase D/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transfecção , Células Tumorais Cultivadas , Regulação para CimaRESUMO
OBJECTIVE: To investigate the roles of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in the pathogenesis of hepatocellular carcinoma (HCC). METHODS: The expression of the GPI-PLD in HCC was determined. The GPI-PLD gene was stably transfected in HepG2 cells and the proliferation of these cells was detected; CD55, CD59 and apoptotic cells were also analyzed. RESULTS: The serum GPI-PLD activities, the protein and mRNA levels of GPI-PLD in HCC patients were decreased by 40%, 60% and 56%, respectively. The killing rate of CDC against the positive clone cells was significantly increased, but the proliferative capacity was obviously decreased. The apoptotic rate in positive clones was increased. CONCLUSION: The expression of GPI-PLD decreases in HCC patients. The over-expression of GPI-PLD in HepG2 cells increases their sensitivity to CDC killing, impairs proliferative capacity and promotes apoptosis.