The Effect Of PTTG1 On The Invasion Of Glioma Cells

Objective: Gliomas are the most common kind of primary malignant brain tumors in adults and present a spectrum of aberrantly aggressive phenotype. Numerous researches indicated that Pituitary tumor transforming gene1（PTTG1） was correlated with the severity and prognosis of tumors. However the specific mechanism of PTTG1 is not clear in glioma. In this study, we analyzed the protein expression of PTTG1 in several glioma cells by Western blot. After transfection, we detected the variation of invasiveness by Matrigel Invasion Assay and analyzed the phosphorylation of AKT and ARK5 by Western blot. Studying the mechanism provides the basis for clinical diagnosis and treatment.Methods: Human glioblastoma（U87, U251, LN-229, which belong to WHO grade IV） cell lines and normal human astrocytes（NHA） cell line were cultured in RPMI 1640 supplement with 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Lipofectamine was used to transfect PTTG1-siRNA: 5′-GACCCUGGAUGUUGAAUUG-3′ into glioma cell line U87, with U87 cell line transfected by scramble siRNA as a control group. Western blot was used to investigate the expressions of PTTG1 and phosphorylation of AKT and ARK5. Transwell invasion assay examined in vitro the variation of invasiveness of siPTTG1/U87 cells and Scr/U87 cells.Results:（1） Human glioblastoma cells and NHA cells were cultured in RPMI 1640 supplement with 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. We extracted the protein of logarithms plant cells. The results of Western blot showed that the expression of PTTG1 was higher in all glioma cell lines and none in NHA.（2） After transfection, we maintained the cells for 72 hours before we extracted the protein. The results of Western blot showed that the expression levels of PTTG1 of siPTTG1/U87 cells was obviously reduced compared to Scr/U87 cells.（3） After transfection, a Boyden chamber invasion assay was performed. After 24 h of incubation（37°C, 5% CO₂）, the non-invading cells were removed by wiping the upper side of the membrane, and the invading cells were fixed and stained. The number of invading cells was counted under a microscope in five predetermined fields. SPSS 16.0 statistical software package was used to perform all statistical analyses. Quantitative analysis of cell numbers revealed that the SiPTTG1/U87 cells had an invasion rate two times lower than that of Scr/U87 cells in response to 10ng/ml EGF.（4） After 5min with EGF stimulation, the phosphorylation of ARK5 and Akt was significantly enhanced in siPTTG1/U87 cells. However, whether or not the existence of EGF, the phosphorylation of ARK5 and Akt had no differences in SiPTTG1/U87.Conclusion: In glioma cells, PTTG1 is high expression and maybe have an important function in glioma cells invasion through Akt-ARK5 signaling pathway.