The Effect And Regulatory Mechanism Of DAPK1 On The Cancer Stem Cells And Epithelial-mesenchymal Transition Process In Colorectal Cancer

Part1 The effect of DAPK1 on the cancer stem cells and epithelial-mesenchymal transition process in colorectal cancerObjective: To determine the effect of DAPK1 on the cancer stem cell(CSCs)and epithelial-mesenchymal transition(EMT)process in colorectal cancer(CRC).Methods: Datasets from Oncomine cancer microarray database were utilized to identify the association of DAPK1 expression and CRC.Immunohistochemistry(IHC)was used to detect the expression of DAPK1 in 33 pairs of CRC tissue samples.Western blot and PCR were utilized to determine the association of DAPK1 expression and CD133 expression in CRC tissues.Also,the expression of DAPK1 in different CRC cell lines and normal colon mucosal epithelial cells was examined.Then the CRC cell lines SW480 and DLD1 was treated by TGF-β1 to induce EMT,and protein level of DAPK1 was determined by western blot.After the stable cell line with down-expression of DAPK1 was established with lentiviral vector,sphere culture was applied to examine the ability of sphere formation.CCK8 assay was used to examine the resistance of SW480 and DLD1 to chemotherapeutic agents including fluorouracil,cisplatin,and doxorubicin.Apoptosis rate of both cell lines was examined by flow cytometry.Expression of CD133,SOX-2 and CD44 were examined to determine the effect of DAPK1 down-expression on the stemness of CSCs.Then,transwell assay and wound-healing assay were utilized to determine the effect of DAPK1 down-expression on the migration of CRC cells.Immunofluorescence(IF)was applied to detect the expression of tight junction between cells.Western blot and PCR were applied to determine the expression of E-cadherin,Zo-1 and N-cadherin.IF was applied to confirm the expression of E-cadherin and N-cadherin on the membrane of cells.Tumor xenografts was used to determine the effect of DAPK1 down-expression on the tumorigenesis in vivo.Formed tumors were detected with CD133 by IHC and E-cadherin and Zo-1 by IF to determine the effect of DAPK1 down-regulation on the stemness of CSCs and EMT process in vivo.Results: Seven analyses from 4 independent studies accessed from the oncomine cancer microarray database were included to determine the association between DAPK1 expression and the risk of CRC.The expression of DAPK1 in CRC tissues was found to be significantly suppressed(median rank: 1840.0;p-value: 1.37E-4).On IHC analysis,we found a strong correlation of the expression rate of DAPK1 with the risk of CRC(66.7% vs.39.4%,P=0.48),as well as that of lymph node metastasis and cancer differentiation.Results of western blot and PCR indicated an inverse association between expression of CD133 and DAPK1.The expression of DAPK1 in normal colon epithelial cells NCM460 was higher than that in most of CRC cells,with the exception of DLD1.Moreover,the expression of DAPK1 in SW620(derived from the metastatic site)was significantly lower than that in SW480(derived from primary tumor of the same patient).Treatment of SW480 and DLD1 cells with TGF-β1 led to reduced expression of DAPK1.Down-regulation of DAPK1 expression could significantly improve the ability of sphere formation,improve the resistance with chemotherapeutic agents(fluorouracil,cisplatin,and doxorubicin)and decrease the apoptosis rate of SW480 and DLD1.Meanwhile,protein level of CD133,CD44 and SOX-2 were elevated in both SW480 and DLD1 cells with sh-DAPK1.In addition,silencing of DAPK1 significantly promoted the migration of SW480 and DLD1 cells in both transwell and wound-healing assays,decreased the intercellular tight junctions in immunofluorescence assay,suppressed the protein level of E-cadherin and Zo-1,and enhanced the expression of N-cadherin.In vivo,the volume of tumors was enhanced significantly,CD133 expression was increased,EMT markers E-cadherin and Zo-1 were decreased after knockdown of DAPK1 expression.Conclusion: Down-expression of DAPK1 could promote the metastasis and chemoresistance by promoting the stemness of CSCs and EMT process.Part 2 The mechanism of DAPK1 down-regulation promoting the stemness of CSCs and EMT process in colorectal cancerObjective: To explore the molecular mechanism of DAPK1 down-regulation promoting the stemness of CSCs and EMT process in colorectal cancer cells.Methods: After the expression of DAPK1 was knockdown,the activation of TGF-β pathway and Wnt pathway and the expression of Zinc finger E-box binding homeobox 1(ZEB1)were examined by western blot,PCR and IF.Then the expression of ZEB1 was knocked down with si RNA in sh-DAPK1 cell lines.Sphere culture was performed to examine the capability of sphere formation.CCK8 assay was used to examine the resistance to fluorouracil.Apoptosis rate of both cell lines was examined by flow cytometry.Wound-healing assay were utilized to determine the migration of cells.Immunofluorescence was applied to detect the expression of tight junction between cells.Western blot and PCR were applied to determine the expression of CD133,CD44,SOX-2,E-cadherin and Zo-1.TC-DAPK6,inhibitor of DAPK1,was used to treat CRC cells.Then,markers of EMT and CSCs were examined by western blot.Results: DAPK1 knockdown led to activation of TGF-β and Wnt pathways and increased expression of ZEB1.When ZEB1 was knocked down in sh-DAPK1 cell lines,migration distance was shorter in wound-healing assay,tight junctions between cells were restored,ability of sphere formation and resistance to fluorouracil restored,and apoptosis rate increased in ZEB1-silenced cells.Correspondingly,when the expression of ZEB1 was knocked down,the expression of CD133,CD44 and SOX-2 were decreased and the expression of E-cadherin and Zo-1 were increased.Treatment with inhibitor of DAPK1 could not affect the expression of markers of EMT and CSCs.Conclusion: Down-regulation of DAPK1 could promote stemness of CSCs and EMT process by activating ZEB1.DAPK1-ZEB1 may lie at the interface of TGF-β pathway and Wnt pathway.Targeted-therapies aimed at DAPK1-ZEB1 pathway may inhibit the chemoresistance and metastasis of CRC.