The Effect Of EGFR Silencing By RNA Interference On The Behaviors And Chemosensitivity Of AGS Gastric Cell Line

ObjectiveThe purpose of this study is to investigate the effect of EGFR silencing by RNA interference on the behaviors such as proliferation,migration,invasion apoptosis and cell cycle in AGS gastric cell line, in addition,we also study how the chemosensitivity changes when EGFR is knocked down and how does the mechanism work. Methods1. Three small interference RNA were designed and then transfected into the AGS gastric cancer line, the transfection efficiency was calculated by fluorescence counting, the expression levels of EGFR mRNA and protein were examined by RT-PCR and western blotting2. Cck-8 colorimetry was applied to analysis the effect of EGFR silencing on the proliferation and chemosensitivities of chemotherapeutic drugs in gastric cancer cells3. Transwell model was constructed to investigate the effect of EGFR silencing on the migration and invasion in AGS gastric cancer line.4. Flow cytometry was employed to observe the apoptosis rate and cell cycle after EGFR was silenced.5. Western blotting was used to detect the changes of several proteins on EGFR pathway after EGFR was silenced. Results1. The transfection efficiency is 87%, three siRNA sequences we used can interference EGFR mRNA and protein in AGS gastric cancer line compared with control group.2. Compared with control group, cell proliferation was significantly inhibited when EGFR was silenced. Inhibition ratio was higher at 48 hours than that at 24 hours. EGFR silenced gastric cells show stronger chemotherapy sensitivity to DDP and PTX.3. EGFR silenced gastric cells show weaker migration and invasion ability than control group.4. EGFR silenced gastric cancer cells show a higher proportion of apoptosis and necrosis than control group, G0/ G1 phase ratio of cancer cells increased significantly.5. Decreased expression of AKT and phosphorylated AKT was detected in EGFR silenced cells, but no difference was detected in the expression of Erk and phosphorylated Erk Conclusions1. Silencing EGFR can inhibit gastric cancer cells proliferation, migration and invasion, it can also disturb cell cycle and induce cells apoptosis and necrosis.2. The effect of inhibiting EGFR in gastric cells may be through the PI3K- AKT pathway rather than Erk pathway.