The Effect And Mechanism Of GRASP65 Expression On Proliferation,Apoptosis,Migration And Invasion Of Gastric Cancer

Objective: To investigate the effect and mechanism of GRASP65 expression on proliferation,apoptosis,migration and invasion of gastric cancer cells from human in vitro.Method: First,Western blot and RT-q PCR were carried out in three different differentiated gastric cancer cells(MKN-45,SGC-7901,MKN-28)and normal gastric mucosa cells(GES-1)to test the expression of GRASP65 protein and mRNA.Then,specific siRNA fragments and plasmid vectors were used through Lipofectamine 2000 to transient transfect MKN-45 cells and MKN-28 cells to knockdown and knockup GRAPS65 expression,respectively.Western blot and RT-q PCR were used to test GRAPS65 expression and confirm a success transfection.The experiment was divided into down-regulated part and up-regulated part,which includes 3 groups,experimental group(GRASP65 siRNA group/GRASP65 plasmid group),negative control group(Control siRNA group/Empty vector group)and black control group(MKN-45/MKN-28),respectively.Finally,CCK-8,flow cytometry,Transwell migration and invasion experiments were used to test the ability of cell proliferation,apoptosis,migration and invasion after transfection,and Western blot was used to test GM130,MMP-9 and MMP-2 protein expressions in each groups.Results: Western blot and RT-qPCR results showed that the expression of GRASP65 protein and mRNA in MKN-45 and SGC-7901 were much more than in MKN-28 and GES-1.MKN-45 and MKN-28 were chosen as experimental cells.Western blot and RT-qPCR results demonstrated that GRASP65 had been efficiently regulated through corresponding treatments.In addition,CCK-8,flow cytometry,Transwell chamber assays results showed that GRASP65 knockdown can suppress growth,promote apoptosis,inhibit migration and invasion of gastric cancer cells in vitro,while these results of GRASP65 knockup go opposite.In addition,the expression of GM130,MMP-9 and MMP-2were changed with the change of GRASP65 expression after transfection.Conclusion : GRASP65 knockdown could significantly suppress growth,promote apoptosis,inhibit migration and invasion of gastric cancer cells from human in vitro,and the mechanism may be associated with the decrease of GM130 expression.