Effect Of GRASP65 On Grastric Cell MKN-45 Through JNK Signaling Pathway And Its Mechanism

Objective:To investigate the effects of Golgi reassembly and stacking protein 65 on the proliferation,apoptosis,cycle and invasion of gastric cancer cell MKN-45 through the JNK signaling pathway.Method:WB and IF detection of GRASP65 expression in three types of cells,and a cell line with high expression of GRASP65 was selected.siRNA was transfected into GRASP65 overexpressing gastric cancer cell lines to reduce the expression of GRASP65 protein.Western blot and RT-qPCR were used to screen out the best knockdown siRNA fragments for subsequent experiments.After knocking down the expression of GRASP65 in gastric cancer cells,JNK signaling inhibitor SP600125 was added to each group of cells.It has four groups: knockdown plus inhibitor group(SI-1565 + SP6 group),knockdown group(SI-1565 group),untransfected plus inhibitor group(SI-NC + SP6 group),and untransfected(SI-NC)group.Apply cell counting kit8 to detect the proliferation of four groups,and FCM to detect the cell apoptosis and the changs of cells.Usetranswell method to detect the invasion of four groups.Use westernblotting to detect GRASP65,P21,caspase3 and other proteins in each group of cells.Results:Western blot and immunofluorescence results showed that GRASP65 was highly expressed in MKN-45 cell.Westernblotting and RT-qPCR results showed that SI-1565 had the best inhibitory effect on GRASP65 in MKN-45 cell.From the results of cck8,compared with SI-NC group,the cell proliferation ability in the control plus inhibitor group(SI-NC + SP6)was significantly reduced(P <0.01).However,SI-1565 +SP6 and SI-1565 group showed no significant difference(P> 0.05).Flow cytometry results showed that after the addition of JNK signaling inhibitors,the control plus inhibitor group(SI-NC + SP6)had significantly higher apoptotic capacity than the control group(SI-NC)(P <0.001).However,the SI-1565 + SP6 group has no statistical significance with the SI-1565group(P> 0.05).Compare SI-NC SP6 group with SI-NC group,and SI-1565+ SP6 compared with SI-1565 group,cells were significantly blocked in G2 period.From the results of Transwell,it can be seen that the cell invasion ability of the SI-NC + SP6 group is significantly weaker than that of the SI-NC group(P <0.05),while the SI-1565 + SP6 group has no statistical significance with the SI-1565 group(P> 0.05)..Analyze the westernblotting results,the cyclin A,and bcl-2 proteins in the cells of each group were consistent with the changes in GRASP65 protein expression.The expression of P21 and cleaved caspase3 was opposite to that of GRASP65 protein.Conclusion:GRASP65 may affect the biological behavior of MKN-45 through JNK signaling pathway,such as proliferation,invasion and so on,cycle and invasion.It may be related to the change of GRASP65 in the phosphorylation status of JNK signaling pathway,which in turn affects the expression of proteins such as proliferation,apoptosis,and cycle,thereby affecting the biological behavior of MKN-45.