Effects Of X-ray Radiation And Combined With CCR7-siRNA On Proliferation, Invasion And Migration In Lung Carcinoma A549 Cells In Vitro

Objective:To study the effects of X-ray radiation on CC-Chemokine Receptor 7(CCR7) mRNA and protein expression in human lung adenocarcinoma A549 cells in vitro. Designing and screening the interference RNA against CCR7 mRNA (CCR7–siRNA) and transfection them into A549 cells to study the effects of X-ray radiation combined with CCR7-siRNA on proliferation, invasion and migration in A549 cells in vitro. The purposes are to research the mechanism of invasion and migration after radiation in A549 cells and to explore a novel treatment pattern of radiation therapy combined with RNA interference in lung cancer therapy.Methods:Cultured A549 cells were irradiated by x ray from Precise linear accelerator at dose rate of 442.89 cGy/min. The expression level of CCR7 mRNA and protein were tested separately by real-time PCR and Western blotting. The interference RNA targeted to CCR7 mRNA (CCR7-siRNA) were designed by Bioinformatics method and constructed CCR7- siRNA was transfected into A549 cells by lentiviral vectors in vitro. MTT method, Matrigel invasion experiment and Cell Scratche method were separately used to detect the ability of proliferation, invasion and migration of A549 cells in vitro.Results:1. The untreated A549 cells were used as control group. The proliferative capacity of A549 cells in 2 Gy group was higher than in the control group at 24 h and 96 h after radiation(0.087±0.002 VS 0.075±0.001, 0.449±0.009 VS 0.252±0.018, P<0.05), and lower than in the control group at 120 h after radiation (0.509±0.060 VS 0.738±0.087, P<0.05); After 24 h, in 4 Gy group, the level of proliferation was higher than in the control group(0.087±0.001 VS 0.075±0.001, P<0.05), and lower in the other groups exclude the 72 h group(0.088±0.008 VS 0.127±0.006, 0.182±0.012 VS 0.252±0.018, 0.571±0.064 VS 0.738±0.087, P<0.05); The level of proliferation in 6 Gy group at 48 h, 72 h and 120 h respectively were 0.072±0.002, 0.138±0.030, 0.365±0.024, and all of them were lower than the control group (P<0.05); The level of proliferation in 8 Gy group after 72 h respectively were 0.155±0.038, 0.139±0.015, 0.248±0.016, and lower than the control group (P<0.05).?2. Using Transwell model detect the invasion of A549 cells in vitro, there was no cells crossed the membrane in the control group. The cells numbers of crossed the membrane in 2, 4,6 and 8 Gy groups respectively were 22.67±3.32, 1.83±0.41, 3±1.10, 4.67±2.16. And the differences between treatment groups and the control group was significant(P<0.01), and the most numbers cells were observed in 2 Gy group (22.67±3.32, P <0.01); The migration capacity of A549 cells in 2, 4 and 6 Gy groups were higher than in the control group at 24 h after radiation (P<0.05), but lower in 8 Gy group (P<0.05); The level of migration was higher than the control group only in 2 Gy group at 48 h after radiation (P<0.05), and the differences between 2 Gy group and the other treatment groups were significantly (P<0.05).3. After be irradiated by x ray at absorbed doses of 4, 6 and 8 Gy, the expression levels of CCR7 mRNA and protein in A549 cells increased from 4 h after radiation, but decreased gradually after the peak; The expression of CCR7 mRNA in 4,6 and 8 Gy group, and of protein in 2 and 6 Gy group were still higher than in the control group at 72 h after radiation (P<0.05), then the expression of protein in 4 and 8 Gy group were no remarkably different compared to control group after radiation 48 h and 72 h (P>0.05).4. Using Bioinformatics method to design the interference RNA against CCR7 mRNA (CCR7-siRNA) and construct CCR7- siRNA lentiviral vectors, then transfected them into A549 cells; Compared with the control group, the CCR7-siRNA group could significantly reduce the expression level of CCR7 mRNA and protein in A549 cells (0.39±0.05 VS 0.87±0.04, 0.59±0.19 VS 1.00±0.00, P<0.05). Between designed couple of CCR7-siRNA sequences, the first sequence revealed better inhibitory effect than the other (0.39±0.05 VS 0.50±0.04, 0.59±0.19 VS 0.93±0.07, P<0.05). So the first CCR7-siRNA sequence was used for the follow-up experiment.5. Compare with the control group, the CCR7-siRNA combined radiation group could significantly reduce the expression levels of CCR7 mRNA and protein (0.56±0.05 VS 1.20±0.20, 0.32±0.06 VS 1.88±0.13, P<0.05) and could obviously inhibite the ability of proliferation, invasion and migration of A549 cells in vitro (P<0.05).Conclusions:In certain period, irradiation by x ray at some dose (2, 4, 6 and 8 Gy )could up-regulate the expression level of CCR7 mRNA and protein and promote the invasion and migration, but inhibit the proliferation of A549 cells in vitro. Designed CCR7-siRNA could significantly down-regulate the expression level of CCR7 mRNA and protein and inhibit the invasion and migration of A549 cells in vitro. The CCR7-siRNA combined radiation could significantly reduce the expression levels of CCR7 mRNA and protein and could obviously inhibit the ability of invasion and migration which reduced by radiation, and combine with irradiation inhibit the ability of proliferation of A549 cells in vitro (P<0.05). The results reveal a new treatment pattern for clinic lung cancer therapy.