Death Associated Protein Kinase 1 (DAPK) Suppresses Hepatocellular Carcinoma (HCC) Metastasis Via Phosphorylation Of DDX20 At Ser221.

DAPK is a calmodulin-regulated serine/threonine kinasewhich which is expressed as a loss of expression in hepatocellular carcinoma(HCC)patients,and DAPK is present in various studies.DAPK in malignant tumors(such as lung cancer,bladder cancer,etc.)are closely associated with metastasis.Futhermore,other studies in HCC samples showed that low expression of DAPK was associated with lymph node metastasis.However,the specific mode of action of DAPK on hepatoma cells and the mechanism of its regulation are still unclear.Objective:The purpose of this study is to investigate the mechanism of DAPK regulating DDX20 in hepatocellular carcinoma,and further study the molecular mechanism of DAPK regulating cellular function through DDX20,so as to clarify the clinical value of DAPK in the diagnosis and treatment of HCC.Methods:First of all,lentiviral transfection of Tet-pLKO-shRNA and liposome transfection of Tet-on system were used to construct stable Hep3 B hepatoma cell lines which could induce DAPK knockdown and overexpression,which could be used for cell proliferation and clone formation.Cell function tests,such as scratches and Transwell assay,cell function tests such as cell proliferation,colone formation,scratches,and Transwell assay.They were also used to detect the effects of decreased expression of DAPK gene on cell proliferation,cell clone formation,scratch migration and cell invasion.The stable Hep3 B hepatoma cell line,which could induce DAPK knockout,was used in proteomics experiment.The protein DDX20,which was regulated by DAPK,was screened by proteomics assay,The protein DDX20,whose expression and phosphorylation levels were regulated by DAPK,was screened by proteomics assay.and the expression of DDX20 was detected by Western Blot.The effect of DDX20 on the function of DAPK was detected by scratching migration and cell invasion after over-expression orover-expression of DDX20.By analyzing the stability of DAPK on DDX20 mRNA and DDX20 proteins,the regulation mode of DAPK on DDX20 and the effect of DAPK kinase activity on DDX20 stability were determined,which provided the basis for DDX20 as a downstream target of DAPK regulation.Experimental result:The results of the experiment show that the decrease of the expression of DAPK does not affect the proliferation and colony formation of Hep3 B cells,but the decrease of the expression of DAPK can promote the migration and invasion of the liver cancer cells,and the expression of the DDX20 protein is detected by Western blot with the change of the DAPK.It is consistent with the analysis of proteomics,and the DDX20 can reverse the function of DAPK on cell migration and invasion.DAPK does not have an effect on the mRNA of DDX20,but can regulate the stability of the DDX20 protein,and rely on the kinase activity of the DAPK,and the phosphorylation site of the DAPK on the DDX20 gene is determined by the phosphorylation site point mutation experiment.Based on the above experimental results,it is concluded that the DAPK can inhibit the invasion and migration of the liver cancer cells,and has no effect on the migration and the cloning of the cells,and the DAPK enhances the stability of its downstream protein DDX20 by its kinase activity and the DDX20 is involved in the regulation of the cellular function of the liver cancer cell by the DAPK.In addition,DAPK can inhibit the activity of Cdc42 in the liver cancer cells,and the DDX20 can replace the DAPK to inhibit the activity of the Cdc42,and the DAPK may also be involved in the regulation of the activation factor ikk-? through DDX20-NF-?B signal pathway.The DDX20,as a control factor downstream of DAPK,is involved in the control of the invasion and migration of the liver cancer cells by the DAPK,and provides a new molecular target for the treatment of the liver cancer.