Investigation On The Expression And Function Of DAPK In Hepatocellular Carcinoma Cells

DAPK(Death associated protein kinase)is a Ca2+-calmodulin regulated Ser/Thr kinase.Abnormal expression of DAPK is closely associated with oncogenesis and metastasis of multiple malignant tumors(such as pulmonary carcinoma,breast carcinoma,cervical carcinoma etc.).But the function of DAPK in hepatocellular carcinoma(HCC)and the underlying mechanisms are still unclear.Objective:This study aims to find the clinical significance of DAPK in liver cancer and the regulatory role of DAPK in HCC cell lines.Methods:The protein expression of DAPK in HCC tissue and cells were detected using immunohistochemistry and western blot.The Tet-on inducible DAPK knockdown cell lines were created using lentivirus infection followed by puromycin selection.The effect of DAPK on proliferation,migration and invasion was examined using SRB assay,wound healing assay and transwell assay.Last,quantitative proteomics technology was employed to search proteins that may mediate DAPK function.Conclusion:Our work demonstrated that HCC patients with low DAPK expression are more likely to have a shorter 5-year survival rate,suggesting DAPK might act as a tumor suppressor in HCC.In p53 null Hep3B cells,DAPK cannot suppress cell proliferation,but was able to inhibit migration and invasion,indicating DAPK may repress metastasis.In order to further investigate the molecular mechanisms underlying DAPK mediated migration and invasion suppression,we have employed phospho-proteomics and normal proteomics to search for downstream effectors of DAPK.This project will not only help clarify the function and clinical application of DAPK in HCC,but also expand the understanding of the molecular mechanisms underlying DAPK function.