Influences Of Autophagy Inhibition On Cell Migration And Invasion Ability And The Efficacy Of Gefitinib In Lung Adenocarcinoma Cells

Objective:To explore the influence of autophagy inhibition on migration and invasion of lung adenocarcinoma cells,autophagy related gene ATG5 was knockdown by small interference RNA in lung adenocarcinoma cells.The role of autophagy in the treatment of lung adenocarcinoma using gefitinib was also discussed.Methods:1.ATG5 siRNA-1,siRNA-2,siRNA-3 and NC-siRNA were transfected into lung adenocarcinoma cells A549,HCC827 and H1975.The knock-down effect of ATG5 expression at gene level and protein level were detected by Real Time-PCR and Western-Blot,and the autophagy inhibition effect of ATG5 siRNA-1,2,3 on three kinds of cells was detected by Western-Blot method.2.Cell migration test and Transwell chamber assay were applyed to detect the effect of autophagy inhibition on cell migration and invasion capacity in three kinds of lung cancer cells.3.The cell proliferation ability of lung adenocarcinoma cells transfected with ATG5 siRNA-2 and NC-siRNA were detected by CCK8 method.4.In lung adenocarcinoma cells A549,HCC827 and H1975,the cells were exposed to different concentrations of gefitinib for 48 hours after siRNA transfection.The OD value of each pore was determined by CCK8 assay.Results:1.Small molecule RNA interference technique was used to transfect ATG5 siRNA-1,siRNA-2,siRNA-3 and NC-siRNA into lung adenocarcinoma A549,H1975 and HCC827 cells.The results of Real-time PCR and Westren Blot showed that compared with negative control group,the expression of ATG5 in the experimental groups were significantly decreased at the mRNA level and protein level,and the highest rate of knock-down effection was ATG5 siRNA-2 groups.After ATG5 siRNA knocked down the expression of ATG5,the results of Western-Blot showed that the expression of p62/SQSTM1 increased and the ratio of LC3BII/I decreased.2.Autophagy inhibition down-regulated the ability of migration and invasion in lung adenocarcinoma cells.The results of cell scratch test showed that the migration distance of ATG5 gene knockdown group was significantly shorter than that of negative control group,and the results of Transwell cell migration test showed that the average number of migration cells in ATG5 knockdown group was significantly lower than that of negative control group.The results of Transwell cell invasion test showed that the number of cells passing through the Matrigel matrix glue in the ATG5 gene knockdown group was significantly lower than that in the negative control group.3.CCK8 assays results showed that in the three types of lung adenocarcinoma cells lines,there was no significant difference between control group cells and ATG5-siRNA-2 group cells.4.The results of CCK8 assays showed that the inhibitory rate of ATG5 knockout group was significantly higher than that of control group in the three kinds of lung adenocarcinoma cells after the treatment of Gefitinib for 48 h.Conclusion:Autophagy inhibition dereases the migration and invasion ability of lung adenocarcinoma cells,but has no obvious effect on the proliferation of lung adenocarcinoma cells.Autophagy inhibition can enhance the cytotoxic effect of gefitinib on lung adenocarcinoma cells.