Effects Of PTTG1 On The Proliferation,invasion And Migration Of Lung Adenocarcinoma H1299 Cells After Irradiation

Objective:To detect the effect of pituitary tumor-transforming gene 1(PTTG1)on the proliferation,invasion and migration capacity of lung adenocarcinoma cells after radiation,and to explore the mechanism of its occurrence through changes in TGF-?1.Methods:? Construction of stable transfected cell lines:Transfection of three lentiviral vectors with different interfering sequences Lv-shPTTG1 into lung adenocarcinoma H1299 cells,silencing of PTTG1 gene,qRT-PCR and Western Blot detection of PTTG1 interference efficiency,screening out the best interference efficiency sequence,establish a stable transfection H1299P-cell line,and establish a non-specific sequence H1299 negative control cell line(H1299NC);? Using the CCK-8 method to detect PTTG1 knockdown H1299 cells at different X-ray doses(OGy,4Gy,8Gy)proliferation ability 48 hours after irradiation;?Transwell test was used to detect the cell invasion and migration ability of H1299 cells with silenced PTTG1 expression at 48 hours after irradiation with different X-ray doses(4Gy,8Gy);?Test by qRT-PCR and Western Blot experiment down-regulated PTTG1 expression of H1299 cells at different X-ray doses(OGy,4Gy,8Gy)after 48 hours of irradiation related gene and protein expression,including PTTG1,TGF-?1,and epithelial-mesenchymal transition marker N-Cadherin and MMP-9.Results:?The lentiviral vector Lv-shPTTG1 was transfected into lung adenocarcinoma H1299 cells,and the positive rate of PTTG1 was observed to be more than 90%by fluorescence microscopy.The interference efficiency of PTTG1 was detected by qRT-PCR and Western Blot and the sequence with the best interference efficiency was selected and successfully constructed.Stable transfected H1299P-cell line,and at the same time establish a non-specific sequence H1299 negative control cell line(H1299NC);?The proliferative ability of the cells was detected by the CCK-8 method:(1)Irradiation can reduce the proliferation ability of H1299 cells;(2)Down-regulation of PTTG1 expression has no significant effect on the proliferation ability of H1299 cells;(3)Inhibition of PTTG1 expression can reduce the proliferation ability of H1299 cells at 48 hours after X-ray(4Gy,8Gy)irradiation at different dose;?Detecting the invasion and migration ability of cells by Transwell experiment:Inhibiting the expression of PTTG1 can not only reduce the invasion and migration ability of H1299 cells,but also further reduce the invasion and migration ability after different doses of X-ray(0Gy,4Gy,8 Gy)irradiation;?Through qRT-PCR and Western Blot experiments to detect the expression of related genes and proteins in H1299 cells after different doses of X-rays(0Gy,4Gy,8Gy)for 48 hours.qRT-PCR showed that the expression of PTTG1,TGF-?1,N-Cadherin gene decreased after 4Gy irradiation;and the expression of PTTG1,TGF-?1,N-Cadherin and MMP-9 all decreased significantly after 8Gy irradiation;knocking down the expression of PTTG1 in H1299 cells can not only down-regulate the expression of TGF-?1,but also reduce the expression of N-Cadherin and MMP-9.After 4 Gy irradiation,the expression of PTTG1 and TGF-?1 genes decreased;and the expression of PTTG1,TGF-?1,N-Cadherin,and MMP-9 all showed a significant decrease after 8Gy irradiation.Western Blot showed that compared with OGy(unirradiated),the expression of TGF-(31,N-Cadherin and MMP-9 in H1299 cells decreased after 4Gy and 8Gy irradiation;knockdown the expression of PTTG1 in H1299 cells not only can down-regulation TGF-?1 expression but also reduce the expression of N-Cadherin and MMP-9.When the transfected group cells were irradiation after 4Gy,the expression of PTTG1,TGF-?1 and N-Cadherin were down-regulated.The expression of PTTG1,TGF-?1,N-Cadherin and MMP-9 all decreased after 8Gy irradiation.Conclusions:(1)Silencing the expression of PTTG1 in H1299 cells can not only reduce the invasion and migration ability of H1299 cells,but also reduce the proliferation,invasion and migration ability of H1299 cells at 48 hours after different doses of X-ray(4Gy,8Gy)irradiation;(2)The ability of PTTG1 to affect the proliferation,invasion and migration of lung adenocarcinoma H1299 cells after radiation may be related to the changes of TGF-?1.