Effect Of Protein Kinase CAMK1G On Proliferation,Invasion And Migration Of Gastric Cancer Cells

ObjectiveThe experiment was conducted to verify whether Ca MK1G(Ca MK1G),one of the protein kinase family CAMK,has an effect on the proliferation,invasion and migration of gastric cancer cells,and to infer the mechanism of its occurrence.Methods1.Screening and culture of cell lines: Two kinds of gastric cancer cell lines BGC-823 and SGC-7901 were selected for culture.According to our previous research,compared with healthy gastric mucosal epithelial cell GES-1,CAMK1 G protein was up-regulated in SGC-7901 and BGC-823 cells,so the above two cell lines were selected for parallel experiment.2.Plasmid and siRNA transfection assay: the transfection reagent Lipo2000 was used to transfer the plasmid(pc DNA3.1-Myc-CAMK1 G recombinant plasmid)and CAMK1 G siRNA synthesized in the laboratory into the above two cell lines.Over-expression transfection was divided into three groups: blank group,empty vector group and pc DNA3.1-myc-CAMK1 G transfection group.SiRNA transfection was divided into blank group,transfection control group and CAMK1 G siRNA transfection group.3.MTT was used to verify the proliferation of gastric cancer cells after up-regulation and down-regulation of CAMK1 G.4.After transfection with CAMK1 G plasmid and si RNA,the cells were cloned and formed to observe the growth and proliferation of gastric cancer cells.5.By transfecting CAMK1 G plasmid and siRNA into two gastric cancer cell lines,the cell migration ability was observed by scratch test.6.Transwell experiment was used to observe the invasion and migration ability of CAMK1 G up-regulated and down-regulated in two gastric cancer cells.7.After CAMK1 G plasmid and siRNA were transfected into two gastric cancer cells by western blotting,the expression of EMT-related markers(Snailer,N-cadherin,E-cadherin)and cell cycle related markers(WEE1,CDK1-T14,CDK1-Y15,Cyclin B1)selected in this study were observed.Results1.Compared with blank group and empty vector group,the expression of CAMK1 G protein in overexpression group increased significantly(P < 0.01),while the expression of CAMK1 G protein in si RNA treatment group decreased significantly(P < 0.01).2.We can find that the overexpression of CAMK1 G promoted the proliferation of SGC-7901 and BGC-823 cells significantly(P < 0.01).The result was opposite after downregulating CAMK1G(P < 0.01)by MTT assay.3.In the result of clone formation,the number of clones of SGC-7901 and BGC-823 cells increased significantly after overexpression of CAMK1G(P < 0.01).However,the clone numbers of SGC-7901 and BGC-823 cells decreased significantly after CAMK1 G was down-regulated(P < 0.01).4.Scratch test results showed that the relative distance between scratches of SGC-7901 and BGC-823 cells was decreased and the cell mobility was increased(P< 0.01).However,down-regulating CAMK1 G increased the relative distance of scratches and decreased the mobility of SGC-7901 and BGC-823 cells(P < 0.01).5.Transwell pictures showed that the number of invasion and migration of gastric cancer cells increased significantly(P < 0.01).However,the number of invasion and migration of gastric cancer cells decreased significantly by downregulating CAMK1G(P < 0.01).6.Western blotting showed that compared with the control group,the expression of N-cadherin,Snail protein and MPF signaling pathway related proteins WEE1 and Cyclin B1 increased,while the expression of E-cadherin protein decreased,and the phosphorylation levels of CDK1-T14 and CDK1-Y15 increased(P < 0.01).Compared with the control group,the expression of N-cadherin,Snail,cyclin WEE1 and Cyclin B1 decreased,the expression of E-cadherin increased,and the phosphorylation levels of CDK1-T14 and CDK1-Y15 decreased(P < 0.01).ConclusionsOverexpression of CAMK1 G in the experiment can promote the proliferation,invasion and migration of gastric cancer cells.Downregulation of CAMK1 g resulted in the opposite result.It was speculated that it might be related to the effect of protein kinase CAMK1 G on epithelial mesenchymal transformation of gastric cancer cells by regulating WEE1.