Effects of GPI-PLD Gene on Invasioness and Vasculogenic Mimicry of the Hepatoma Carcinoma Cell Line HepG2
|School||Central South University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Glycosylated phosphatidylinositol - specific phospholipase D Vasculogenic mimicry Cell invasion Urokinase-type plasminogen activator receptor ( CD87 ) Eph family of receptor interacting protein A1 (Ephrin-A1)|
Purpose by improve the GPI-PLD activity, increase the GPI anchor given protein in the cell membrane, such as CD87, ephrin-Al molecules of the release of the number, to observe the solution anchor given effect whether able to reduce HepG2 cells invasion and tumor vascular mimicry generation's ability, for tumor treatment proposed new anti-metastatic and anti-angiogenic program. The room with a cationic polymer have constructed the eukaryotic expression vector pcDNA3.1 () / GPI-PLD transfected into HepG2 cells, the cell group to non-transfected HepG2 cells group transfected with empty vector pcDNA3.1 () control, positive cell clones were screened by G418. GPI-PLD gene by RT-PCR identified mRNA expression levels using of homemade with complete GPI structure placental alkaline phosphatase (PLAP) substrate, quantitative detection of GPI-PLD activity. ELISA to detect the expression of CD87 release, flow cytometry cell surface GPI-anchored CD87 molecule changes; immunocytochemistry combined with western blot identification of the membrane surface expression of the ephrin-A 1. Transwell method to detect CD87, ephrin-Al HepG2 cell invasion ability; build a three-dimensional culture models of HepG2 cells using Matrigel the PAS staining whether the formation of vessel-like structures. The results of RT-PCR assay showed that stable transfection of GPI-PLD, non-transfected empty plasmid group of GPI-PLD gene transfected and transfected mRNA expression levels of GPI-PLD expression of the transfected group was significantly lower than the level (P lt; 0.05) .GPI-PLD GPI-PLD activity of the transfected cells was significantly higher than that of non-transfected group and the activity of cells transfected with empty plasmid group. GPI-PLD activity of GPI-PLD transfection group reached 14.24 ± 6.27%, while the other group of cells GPI-PLD (?) Tongue is very low, 6.92 ± 4.34% and 8.36 ± 5.63%, respectively, with a statistically significant (P lt; 0.05). CD87 concentration in the medium supernatant measured by ELISA GPI-PLD transfection group 4.18 ± 0.95pg/ml, and the other two groups were 1.48 ± 0.76 pg / ml and 1.84 ± 0.63 pg / ml, the difference was statistically significant (P <0.05). Flow cytometric analysis of each group on the cell membrane the GPI anchor of CD87 molecules were positive, but the values ??of the average fluorescence intensity GPI-PLD transfection group to be smaller than the other two groups. GPI-PLD transfection group average fluorescence intensity of 0.62 ± 0.110, while the other two groups were 1.91 ± 0.130 and 1.86 ± 0.091, the difference was statistically significant (P <0.01). Immunocytochemistry, western-blot results show the expression levels of ephrin-Al GPI-PLD transfection group and decreased significantly compared to the other two groups (P lt; 0.05) in Transwell GPI-PLD solutions anchored role weakened HepG2 cells in vitro invasion ability, GPI-PLD invasion force transfected group compared with untransfected and transfected with empty plasmid group were reduced by 30.6%, 27.3%, statistical significant difference (P lt; 0.01). The HepG2 cells Devascularization state three-dimensional culture results observed, three days after inoculation, no transfection group and transfected with empty plasmid group presents a mosaic-like growth, the network-like structure, GPI-PLD transfected cells evenly dispersed growth; 7 days transfection group and transfected with empty plasmid group by PAS staining, no visible ring PAS positive pattern, GPI-PLD transfection group positive pattern is not obvious. HepG2 cells stably transfected conclusion GPI-PLD enables GPI-PLD overexpression and its GPI-PLD activity was significantly increased. Meanwhile, the anchor of cell surface CD87, ephrin-Al number of molecules are significantly reduced. GPI-PLD can reduce the ability to generate human hepatoma HepG2 cell lines in vitro invasion capacity and vascular mimicry mechanism with CD87, Ephrin-Al GPI-PLD solutions anchored.