Effect Of Protein Kinase WEE1 And Calmodulin Kinase CAMK1G On Proliferation And Migration Of Gastric Cancer

ObjectiveTo study the effect of protein kinase WEE1 and calmodulin kinase CAMK1 G on the proliferation and migration of gastric cancer cells and its mechanism,which lay the experimental foundation for exploring the mechanism of CAMK1G-WEE1 network on gastric cancer cells.Methods1.To observe the expression of WEE1 in cancer tissues and adjacent tissues using gastric cancer tissue microarray and immunohistochemical staining.2.The normal gastric epithelial cells GES-1 and gastric cancer cells SGC-7901 and BGC-823 were cultured in vitro.3.Western Blot was used to detect the protein expression of CAMK1 G and WEE1 in three kinds of cells.MK1775 was added to WEE1 high expressing cells for subsequent experiments.4.The WEE1 inhibitor MK1775 experiments were divided into blank group,DMSO group and MK1775 group.5.The appropriate concentration of MK1775 was screened out using MTT.BGC-823 cells were treated with 5 nmol/L,10 nmol/L,20 nmol/L and 40 nmol/L MK1775,respectively.6.Scratch test was used to detect the effect of MK1775 on the migration of gastric cancer cell line BGC-823.7.Cloning formation assay was used to detect the effect of MK1775 on the proliferation of gastric cancer cell line BGC-823.8.Western Blot was used to detect the expression of CAMK1 G,the phosphorylation status of CDK1 T-14,CDK1 Y-15 and the expression of EMT activators including SNAL1,SNAL2 and TWIST1 in BGC-823 cells treated with MK1775.9.Gene transfection: The pc DNA3.1-flag-camk1 g plasmid was transferred into BGC-823 cells using the transfection reagent Lip 2000.The experiment was divided into untransfected group,empty vector transfection group and the pc DNA3.1-flag-camk1 g transfection group.10.The growth curve of gastric cancer cell line BGC-823 cells after overexpression of CAMK1 G using MTT assay.11.The colony formation assay was used to detect the proliferation of BGC-823 cells after overexpression of CAMK1 G.12.The migration of gastric cancer cell BGC-823 was observed after overexpressed CAMK1 G using scratch test.13.Western Blot was used to detect the expression of SNAL1,SNAL2 and E-cadherin proteins.Results1.Immunohistochemistry results showed that WEE1 expression in cancer tissues was significantly higher than that in adjacent tissues(p<0.01),and the WEE1 expression increased with the increase of clinical stage.2.Western Blot results showed that WEE1 was expressed in both SGC-7901 and BGC-823 cells and was most prominent in BGC-823,but there is almost no expression in GES-1 cells.We selected WEE1 high expression BGC-823 cells for subsequent experiments.3.MTT assay was used to detect the growth curve of BGC-823 cells treated with different concentrations(5 nmol/L,10 nmol/L,20 nmol/L and 40 nmol/L)of MK1775.The results showed that MK1775 inhibited the proliferation of BGC-823 cells at time and concentration dependent manner.In the subsequent experiments,40 nmol/L MK1775 was selected as the optimal concentration to treat BGC-823 cells.4.The results of the scratch test showed that the migration ability of BGC-823 cells with MK1775 was lower than that in the blank group and the DMSO control group(p<0.01).5.The results of colony formation experiments showed that the proliferation of BGC-823 cells in MK1775 treatment group was significantly lower than that in the blank group and DMSO control group(p<0.01).6.Western Blot assay showed that the expression of CAMK1 G was decreased in MK1775 treated group,and the phosphorylation status of CDK1 T-14 and CDK1 Y-15 was decreased(p<0.01).EMT activators(SNAL1,SNAL2 and TWIST1)protein expression was reduced(p < 0.01).7.The expression of CAMK1 G in Western Blot showed that CAMK1 G expression was significantly higher in GSG-7901 cells and BGC-823 cells than in GES-1 cells,and highest in BGC-823 cells(p<0.01).8.The proliferative ability was increased in a time dependent manner in overexpression of CAMK1 G in BGC-823 cells(p<0.01).9.The results of colony formation experiments showed that the proliferation of BGC-823 cells after overexpression of CAMK1 G was significantly higher than that of the blank group and pc DNA3.1 empty vector transfection group(p<0.01).10.The results of the scratch test showed that the migration ability of BGC-823 cells after overexpression of CAMK1 G was significantly higher than that of the blank group and pc DNA3.1 empty vector transfection group(p<0.01).11.The expression of SNAL1 and SNAL2 protein in BGC-823 cells was increased by Western Blot(p<0.01),and the expression of E-cadherin protein was decreased(p<0.01).Conclusions1.The WEE1 inhibitor MK1775 inhibited the proliferation and migration of gastric cancer cell line BGC-823 by regulating EMT markers(SNAL1,SNAL2,TWIST1).2.Overexpression of CAMK1 G promoted the proliferation and migration of gastric cancer cell line BGC-823 by regulating EMT markers(SNAL1,SNAL2,E-cadherin).