Effect Of GRASP65 On JNK Signaling Pathway Regulating Proliferation,Apoptosis And Cell Cycle In Gastric Cancer Cells

Objective: To investigate the Effect of GRASP65 on JNK signaling pathway regulating proliferation,apoptosis and cell cycle in gastric cancer cells in vitro.Method: First,three GRASP65 siRNA(si-GRASP65)sequences or empty vector(si-NC)were transfected using lipofectamine 2000 to screen the optimal sequence.Then,western blot and RT-qPCR were carried out to detect the protein and mRNA expression of GRASP65,the experiment include 4 groups,which is NC group,NC+SP group,si-GRASP65 group,si-GRASP65+SP group.SP600125 was used when GRASP65 was successfully knockdown.Last,the proliferation rate of SGC7901 cells was assessed by CCK-8 assay.The cell apoptosis as well as cell cycles was detected by flow cytometry.As for the protein expression related to apoptosis,the western blotting assay was used.Results: Western blot and RT-qPCR shows the most pronounced gene silencing effect was observed following transfection with siRNA-GRASP65-homo-1230.SP600125 could inhibited the proliferation of the NC(negative control)group significantly(P <0.001),but had no significant effect on the si-GRASP65 group(P> 0.05).After 40μM treatment for 24 hours,the rate of apoptosis in NC group was significantly increased(P <0.01),but there was no significant change in si-GRASP65 group(P> 0.05).SP600125 induced a detectable increase in the proportion of the cell cycle in G2 phase in NC+SP group compared with NC group(P<0.001),the cells arrested in G2 phase of si-GRASP65+SP group were less than that of NC+SP group(P<0.05).The protein expression of cleaved caspase 3 level was significantly decreased in si-GRASP65 group after SP600125 treatment(P<0.01),while the protein expression of bcl-2 was increased significantly(P<0.01).The expression of GRASP65 was downregulated in NC+SP group compared with NC group,and there is no significantly difference between si-GRASP65 group and si-GRASP65+SP group.The protein expression of GM130,P-JNK was the same as GRASP65 in each group.The expression of c-jun in NC+SP group was lower than that in NC group,while its expression was higher in si-GRASP65+SP group than that in si-GRASP65 group.Conclusion: The present study shows that the effect of GRASP65 could alter the biomechanical properties of gastric cancer cells to inhibit proliferation and to induce apoptosis is mediated through JNK signaling pathway,and it is critical in JNK signaling pathway.The mechanism is regulating expression of bcl-2,cleaved caspase 3,c-jun protein by adjusting the phosphorylation status of JNK.