Inhibition Effects Of RNA Interference On Expression Of Glucose-regulated Protein 78 In Human Colorectal Carcinoma Cell Line RKO

Objective:To construct the shRNA plasmid expression vector of GRP78 gene(PGPU6/GFP/Neo-GRP78, shRNA-GRP78) and to observe its interfering effect on the biological behaviors of human colorectal carcinoma RKO cells, including cell proliferation, migration, cell apoptosis.Methods:The shRNA plasmid expression vector (shRNA-GRP78) was transfected into human colorectal carcinoma RKO cells by Lipofectamine 2000, and the stable transfecant was selected by G418. The levels of GRP78 mRNA and protein were analyzed by fluorescent quantitative real-time PCR and Western blotting assays, respectively. The xCELLigence system was performed to detect the proliferation and migration of transfected cells in vitro, and FCM(Flow cytometry) was performed to detect the changes of cell apoptosis.Results:Quantitative real-time PCR and Western bloting assays showed that shRNA-GRP78 could significantly downregulate GRP78 expression in RKO cells(P<0.05). The shRNA-mediated downregulation of GRP78 expression could significantly reduce the activity of cell proliferation in vitro. Furthermore, analysis of apoptosis by FCM assay indicated that the apoptosis rate significantly increased after transfected shRNA-GRP78(P<0.05). But downregulation of GRP78 expression didn't affect the migration of RKO cells in our study.Conclusion:The shRNA plasmid expression vector specific for GRP78 (shRNA-GRP78) can evidently inhibit the expression of GRP78 both at mRNA and protein levels. The downregulation of GRP78 expression could significantly reduce the activity of cell proliferation in vitro and enhance apoptosis in RKO cells.