RNAi silencing of Villin2 suppresses invasion and migration of human gastric adenocarcinoma cell line AGS / 第三军医大学学报

ABSTRACT

Objective To investigate the effect of membrane-cytoskelet on linker Ezrin in the invasion and migration of human adenocarcinoma cells line AGS. Methods Two small hair RNA targeting Villin2 gene ( Ezrin coding gene) was designed,chemically synthesized and inserted into plasmid pGenesil-shRNA,then transformed into E. coli DH5 ?. The recombinant plasmid was extracted in middle quantity and transfected into AGS cells by LipofectamineTM 2000. Ezrin expression in AGS cells transfected by shRNA recombinant plasmid was measured by RT-PCR and Western blotting. AGS cells were transfected by effective shRNA plasmid and cultured in 1640 media containing G418 ( 800 ?g/ml) . RT-PCR and Western blotting were applied respectively to detect the Ezrin mRNA and protein expression to evaluate the blocking effect of shRNA-Ezrin. Cell migration and invasion was determined by scratch test and Transwell chamber assay. Results Our designed shRNA knocked down 93% Ezrin in AGS cells which had a stable expression of Ezrin small hairpin RNAs. The migrated cells number of Ezrin knocked-down group was 27. 67 ? 4. 50,which was significantly decreased compared with the control group ( 125. 50 ? 8. 33,P