Glucose-regulated Protein78Contributes To The Proliferation Of Human Colorectal Carcinoma RKO Cells Via AKT And ERK Pathways

Objective:The shRNA plasmid expression vector with targeting GRP78gene (PGPU6/GFP/Neo-GRP78) was constructed to identify the influence of GRP78on proliferation and cell cycle distribution of human colorectal carcinoma RKO cells. To explore the relationship between GRP78and AKT-ERK signal pathway.Methods:Using lipofectamin2000, the shRNA plasmid expression vector was transfected into RKO cells, then the stably transfected cell line was established by G418selection. Western-blot was used to detect the expression of GRP78protein in RKO cells. After cultured in medium for24,48and72h, the cell viability rate of RKO cells was evaluated using cell counting Kit-8(CCK-8). Flow Cytometry (FCM) was performed to assess cell cycle distribution. Furthermore, Western-blot was used to detect the expression level of p-AKT, t-AKT, p-ERKl/2and t-ERKl/2protein.Results:The shRNA-GRP78can significantly decline the expression of GRP78protein in RKO cells. After cultured for24h, there was no statistical difference among three groups of RKO cells (P>0.05), after cultured for48and72h, silencing of GRP78can markedly inhibit the proliferation of RKO cells; Meanwhile, down-regulation of GRP78was associated with a decreased number of cells in phase S (P<0.05); After transfection with shRNA-GRP78, Western-blot showed that the phosphorylation of AKT and ERK was significantly attenuated (P<0.05).Conclusion:GRP78promoted the proliferation of colorectal carcinoma cells and influenced its cell cycle distribution; GRP78may contribute to the proliferation of RKO cells via AKT and ERK pathways.