| Objective To investigate whether anchoring effect can reduce the the ability of cell invasion and vasculogenic mimicry and offer a new potential anti-tumor metastasis and angiogenesis therapy program.Methods The GPI-PLD gene eukaryon expression vector pcDNA3.1(+) /GPI-PLD produced by our laboratory was transfected into HepG2 cells by cationic polymer transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3.1(+) were used as control. After screening with G418, we got GPI-PLD positive clones. Expression of GPI-PLD mRNA in HepG2 was detected by RT-PCR. The GPI-PLD activities were estimated by partitioning in triton-X-114 with placental alkaline phosphatase (PLAP) as the substrate. The CD87 released from cells membrane before and after transfection GPI-PLD were detected by ELISA. GPI-anchored CD87 on cells before and after transfection GPI-PLD were analyzed by flow cytometer. Combination of immunocytochemistry with Western-blot affirmed the membrane surface expression of Ephrin-Al. Transwell assay were used to evaluate the vitro invasion ability of HepG2 cell line which were effected by CD87,Ephrin-Al. In order to observe whether HepG2 cell for their ability to form tube-like networks which were stained positively with periodic acid-Schiff (PAS), HepG2 cell line were cultured in three-dimensional culture containing matrigel.Results RT-PCR analysis showed that the HepG2 cell line with over-expression of GPI-PLD gene were established. The stably over-expression of GPI-PLD gene was compared with HepG2 cells and pcDNA3.1(+)/HepG2 cells, expression of GPI-PLD mRNA in pcDNA3.1(+)/GPI-PLD HepG2 cells was significantly up-regulated (P<0.05). GPI-PLD activities in pcDNA3.1(+)/GPI-PLD/HepG2 cells were obviously increased, compared with HepG2 or pcDNA3.1(+)/HepG2 cells, GPI-PLD activities in pcDNA3.1(+) /GPI-PLD/HepG2 cells were 14.24±6.27% while the other two groups were 6.92±4.34%,8.36±5.63%, separately (P<0.05). The CD87 released from pcDNA3.1(+)/GPI-PLD/HepG2 cells on cell membrane was significantly increased and GPI-anchored CD87 on cell membrane was decreased. ELISA assay detected on the group culture supernatants CD87 level in pcDNA3.1(+)/GPI-PLD/HepG2 cells was 4.16±2.37 pg/ml,and the other two groups were 1.73±1.89 pg/ml,2.08±1.57 pg/ml separately (P<0.05). Each group was analyzed by flow cytometry showed the cell membrane GPI anchored CD87 was significantly positive, however, the average fluorescence intensity values of pcDNA3.1(+)/GPI-PLD/HepG2 was smaller than the other two groups. The average fluorescence intensity values of pcDNA3.1(+)/GPI-PLD/ HepG2 was 0.62±0.110, and the other two groups were 1.91±0.130 and 1.86±0.091, separately (P<0.05). Immunocytochemistry and Western blotting showed that Ephrin-Al expression in pcDNA3.1(+)/GPI-PLD/HepG2 cells was down-regulated compared with other two groups. Transwell assay found that the solution role of GPI-PLD can reduce vitro invasive ability of HepG2 cells. The invasion rate of pcDNA3.1(+)/GPI-PLD/HepG2 cells was significant decreased by 30.6%,27.3% comparing with HepG2 or pcDNA3.1(+)/HepG2 cells (P<0.01). HepG2 cell lines were cultured for their ability to form tube-like networks in three-dimensional culture containing matrigel three days after, observing the untransfected HepG2 cells and transfected HepG2 cells with pcDNA3.1(+) were shown mosaic-like growth and formed tube-like network structures while the GPI-PLD transfected cells evenly were shown dispersed growth; after 7 days based on three-dimensional culture, vascular-like structures of HepG2 cells were stained positively with periodic acid-Schiff (PAS) in untransfected HepG2 group and transfected HepG2 group with pcDNA3.1(+), and transfected HepG2 group with GPI-PLD positive pattern was not obvious.Conclusion The stable HepG2 cell line with over-expression of GPI-PLD gene can increase the activity of GPI-PLD and the GPI-anchored CD87,ephrin-Al in the surface reduced. GPI-PLD gene effectively decreased HepG2 vitro invasion and influenced the ability of vasculogenic mimicry (VM). |
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