Effects Of X-ray Combining With Plekmon-siRNA On Proliferation Of A549 Cells

1 Objective:The purpose of this paper was to study the dose and time effects of x-ray radiation on the expression of Pokemon gene and protein in human lung adenocarcinoma A549 cells. By constructing the Lentivirus expression vector of Pokemon-siRNA and stably transfecting A549 cells after sorting by flow cytometry, we investigated the effects of Pokemon-siRNA on Pokemon expression and proliferation of A549 cells after combining with x-ray radiation.So as to provide an experimental basis for the combination of radiotherapy and biology therapy in clinical application.2 Methods:Cultured human lung adenocarcinoma cancer cell line A549 in vitro. Constructed the Lentivirus expression vector of Pokemon-siRNA and transfected A549 cells, the stably transfected cells were sorted by flow cytometry. X-rays was used to irradiate A549 cells in various guoups. Fluorescent quantitative PCR was used to measure Pokemon mRNA expression and Western blotting was used to measure Pokemon protein level. The proliferation of cells was detected by methylthiazolyl tetrazolium (MTT).3 Results:1). Effects of X ray irradiation on the expression of Pokemon gene in human A549 cells of lung adenocarcinomaA549 cells were exposed to different doses of X-rays (2,4,6 and 8 Gy )and the relative level of Pokemon mRNA in the cells was detected by using Quantitative real-time PCR at 2, 4, 8, 12, 24, 48 and 72 h after irradiation. Compared with the control group, The expression of Pokemon mRNA trended to decrease after irradiated with 2,4,6 and 8 Gy in the earlier period(except 2 and 4 h after irradiated with 2 Gy) and increased on the advanced stage with the statistical difference at the most time points (3.4<t<154.76 P<0.05). The expression of Pokemon mRNA reached the peak at 48 h after irradiatied by 6 Gy (3.49±0.03, P<0.05).2). Effects of X ray irradiation on the expression of pokemon gene in human A549 cells of lung adenocarcinomaA549 cells were exposed to different doses of X-rays (2,4,6 and 8 Gy )and the relative level of Pokemon protein in the cells was detected by using Western-blotting at 2, 4, 8, 12, 24, 48 and 72 h after irradiation. Compared with the control group, The expression of Pokemon protein trended to increase and reached the peak at 8 h after irradiated by 2,4,6 and 8 Gy with the statistical difference at the most time points(4.18<t<89.64,P<0.05).The expression of Pokemon protein reached the peak at 8 h after irradiatied by 6 Gy (3.35±0.02,P<0.05).3).The depressive effects of X-ray combining with Pokemon-siRNA on proliferation of A549 cellsWe identified the Pokemon-siRNA1 and Pokemon-siRNA of the four designed Pokemon-siRNA had cloned exactly into PsicoR vector by gene sequencing, constructed Lentivirus expression vector, transfected A549 cells and sorted them by flow cytometry. The Pokemon-siRNA1 significantly decreased the expression of Pokemon mRNA (76%) and protein (66%), while The Pokemon-siRNA4was only 27%.Compared with the merely received radiation group, the Pokemon mRNA expression in the transfected Pokemon-siRNA combining with radiation group was significantly decreased (0.34±0.14 vs 3.51±1.42 , P<0.05). When Compared with the control group, the merely received radiation group(except the 3 day was not significant) and the merely transfected Pokemon-siRNA group, the proliferative capacity of A549 cells of the the transfected Pokemon-siRNA combining with radiation group was significantly suppressed in the 2~5 day (P<0.05) .4 Conclusions:1) Some doses of X-rays may increase the expression of Pokemon mRNA and protein in A549 cells.2) Pokemon-siRNA can supress the expression of Pokemon efficiently and inhibit the proliferation of A549 cells significantly.3) The Combination of Pokemon-siRNA and X-ray might inhibit the proliferation of A549 cells, which indicates the higher expression of Pokemon may repair the radiation damage and Pokemon-siRNA may enhance the radiation sensitivity of tumour cells.