Effect Of The Overexpression Of Glycosylphosphatidylinositol-specific Phospholipase D Gene On Hepatocellular Carcinoma Cell Line HepG2

Objective: To investigate effect of the overexpression of glycosyl-phosphatidylinositol-specific phospholipase D (GPI-PLD) gene on thehepatocellular carcinoma cell line HepG2. These effects include cellproliferation, apoptosis, complement dependent cytotoxicity and releaseof glycosylphosphatidylinositol(GPI) anchored proteins on the plasmamembrance of cell by GPI-PLD. To provid theoretical foundation ofGPI-PLD gene therapy for hepatocellular carcinoma.Method: The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/GPI-PLD produced by our laboratory was transfected into HepG2cell by lipid-media transfection. Untrasfected HepG2 and HepG2transfected with pcDNA3.1(+) were used as control. After screeningG418, the single clone was got. Expression level of mRNA of GPI-PLDin HepG2 cell was identified by reverse transcription polymerase chainreaction (RT-PCR), MTT was employed to detect proliferation of threegroups, morphological assessment of apoptosis was examined by lightand fluorescence microscope, GPI-PLD activity levels of cells, aredetemined through Triton X-114 phase partitioning by using GPIanchored placental the alkaline phosphatase(PLAP), produced by ourlaboratory, as substrate, the GPI anchored PLAP detected by spectro-photometric method, and complement dependent cytotoxicity (CDC)effects were observed by staining of trypan blue, The GPI anchored CEAwas detected by ELISA.Rusults:1. RT-PCR results and GPI-PLD activities showed that the HepG2cell line with overexpression of GPI-PLD gene was constructed.①Afterthe G418 screening, expression of GPI-PLDmRNA in pcDNA3.1(+)/GPI-PLD/ HepG2 cell compare with pcDNA3.1(+)/HepG2 cell andHepG2 cell obviously increased, the difference has the significance (P＜0.05), the GPI-PLD mRNA levels showed by IA ratio in three groupswere 0.926±0.124, 0.164±0.057 and 0.205±0.045 separately.②AfterGPI-PLD gene transfected into HepG2 cell, GPI-PLD activities in pcDNA3.1(+)/GPI-PLD/HepG2 cell compare with pcDNA3.1(+)/HepG2cell and HepG2 cell obviously increased, the difference has thesignificance significance (P＜0.01), each group of cell GPI-PLDactivities(%) respectively be 13.4±4.44, 4.82±2.99 and 6.52±2.34.③After GPI-PLD gene transfected into HepG2 cell, the GPI anchoredPLAP, CEA were released by GPI-PLD, on the group culture superna-tants CEA level in pcDNA3.1(+)/GPI-PLD/HepG2 cell compare withpcDNA3.1(+)/HepG2 cell and HepG2 cell obvious increased, thedifference has the significance significance (P＜0.01), PLAP detectedfrom sonicated cells and culture supernatants, maximal PLAP activities incell lysates on HepG2 and pcDNA3.1(+)/HepG2 cell, and maximal PLAPactivities in cell supernatants on pcDNA3.1(+)/GPI-PLD/HepG2.2. The effects of overexpression of GPI-PLD gene on hepatocellularcarcinoma cell line HepG2.①Proliferative capacity in pcDNA3.1(+)/GPI-PLD/HepG2 cell compare with pcDNA3.1(+)/HepG2 cell andHepG2 cell obviously decreased.②The cell rate of CDC killing frompcDNA3.1(+)/GPI-PLD/HepG2 cell compare with pcDNA3.1(+)/HepG2cell and HepG2 cell obviously increased, the difference has thesignificance significance (P＜0.01), the rates of CDC killing(%) in threegroups were 16.4±2.10,5.7±1.15 and 5.9±1.39 separately.③pcDNA3.1(+)/GPI-PLD/HepG2 cell compare with pcDNA3.1(+)/HepG2cell and HepG2 cell, pcDNA3.1(+)/HepG2 cell, pcDNA3.1(+)/GPI-PLD/HepG2 cell present typical apoptosis.Conclusion:1. The stable HepG2 cell line with overexpression of GPI-PLD genewas constructed.2. This study provid GPI-PLD gene anti-tumor effect, GPI-PLDgene up-regulated GPI-PLD activities, then hydrolyze the GPI anchoredprotein from HepG2 tumor cells, increased sensitivity of these cells toCDC killing, impaired proliferative capacity of cell and promoteapoptosis. This study provid theoretical and experimental foundation fortumorous gene therapy.