Effects Of Ebp1 Protein Silencing On Proliferation,Invasion And Migration Of Gastric Cancer HGC-27 Cells

Objective: Ebp1 is one of binding proteins for the Erb B3 receptor which the diverse cellular functions including apoptosis,cell proliferation,and differentiation.It has been reported that Ebp1 has been express in a different type of cancer and play an important role in the development of cancer.Previous research has been conducted showing that Ebp1 is also highly expressed in Gastric cancer cells but its exact role in Gastric cancer is still need to be study.Therefore,the main motive of this study was to investigate the Effects of Ebp1 Protein Silencing on Proliferation,Invasion,and Migration of Gastric Cancer HGC-27 Cell line.Furthermore,this study also provides experimental evidence for the diagnosis and prognosis of Gastric cancer.Methods: In this experiment,gastric cancer HGC-27 cell line was used to observe the silence of Ebp1 protein in the cells after 72 hours of infection with recombinant lentiviral vector.The experiment was divided into two groups: the Ebp1 silence group(KD group)and the virus-free control group(NC group).Western blotting was used to detect the expression of Ebpl protein in the two groups of cells.A significant decrease in the expression of Ebp1 protein indicated that the transfection was successful.After successful transfection,MTT method were perfomed to observe cell viability,plate cloning experiment were completed to observe cell proliferation and colony forming ability,flow cytometry were carry out to find out cell cycle and apoptosis,scratch test were carry out to observe cell migration ability,Transwell assay method was used to identify the invasion ability of the cells,and the western blotting method were carry out to detect the expression of the epithelial-mesenchymal transition related protein.Results:1.The western blot identification results showed that the expression of Ebp1 in the KD group was significantly decrease than that in the NC group,indicating that the Ebp1 silence in the gastric cancer HGC-27 cell was suscessfully constructed.2.The MTT test showed that the cell proliferation ability of the NC group is stronger than the KD group(*P<0.05).3.The plate cloning experiment showed that the cloning ability of the KD group was significantly reduced compared to the NC group(***P<0.001).4.The scratch test showed that the healing ability of the KD group was significantly reduced compare to the NC group,showing the migratory ability of HGC-27 cells was reduced after Ebp1 gene silencing.5.The transwell assay experiment showed that the invasion ability of the KD group was significantly decreased compared to the NC group,indicating that Ebp1 gene silencing reduced the invasion ability of HGC-27 cells.6.Flow cytometry test revealed that in the gastric cancer HGC-27 cell line,the ratio of cells in the G0/G1 phase of the KD group was significantly increased than that of the NC group,while the ratio of cells in the S phase and G2/M phase was significantly lower than that of the NC group.This indicate that cell was arrested in the G0/G1 phase.7.The Western blot method showed that: Compared with the NC group,N-cadherin,Vimentin,Snail and Slug proteins was found decreased in the KD group.But the expression of the E-cadherin proteins was found increased in KD group compared to the NC group.The increase of E-cadherin protein and decrease in the N-cadherin,Vimentin,Snail and Slug proteins means the decrease of epithelial-mesenchymal transition.Decrease in the epithelialmesenhymal transition state that reduction in the invasive and metastastic activities in KD group.Conclusion:1.Ebp1 gene silencing declines the proliferation,reduced the invasion and metastasis of Gastric cancer HGC-27 cell lines.2.Ebp1 induces epithelial-mesenchymal transition in gastric cancer cells.