Effects Of Transcription Factor DEC1 On The Angiogenesis Of Endothelial Cells And Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is a serious threat to human life and is one of the most common malignant cancers in the word.However, due to the high frequency of metastasis or recurrence after surgery makes traditional treatments and prognosis increasingly limited. It is remain a major problem of HCC for mortality. Angiogenesis is one of the most important factors that affect tumor metastasis. Neovascularization transport nutrients and excrete metabolites for tumor tissue, and also provides a good channel to tumor invasion and metastasis. Many reports indicate that a variety of signaling molecules involved in the angiogenesis, VEGFA and MMP9 are the most important pro-angiogenic factors.DEC1 (Differentiated embryonic chondrocyte gene), belongs to the member of basic helix-loop-helix transcription factor, has been reported to be involved in cartilage formation, neurogenesisIn, cell differentiation and tumorigenesis. DEC1 is widely expressed in some tumor derived cell lines, as well as some tumor tissues, and is also related to cell proliferation, apoptosis, and differentiation of tumor cells. Since angiogenesis is an important factor of tumor recurrence and metastasis. Study of angiogenesis is beneficial to looking for a new therapeutic target, and the significance of DEC 1 in angiogenesis still remains unknown.Part I The roles of DEC 1 on the proliferation,migration and angiogenesis of endothelial cell.Purpose:To investigate the roles of DEC1 on the proliferation, migration and angiogenesis of endothelial cell.Methods:We established DEC1 overexpression cell lines by using Flag-DEC1 plasmid, and we established DEC1 knockdown cell lines by using shDEC1 lentivirus. The transfect efficiency was detected by western blot. Endothelial cell proliferation was measured by MTT assay. The migration was detected by wound scratch and transwell assays. And the angiogenesis was assessed by matrigel tube formation assay. The target genes were measured by quantitative real-time PCR at mRNA levels and by Western blot at protein levels.Results:The proliferation of endothelial cell transfected with Flag-DEC1 was slower compared with those transfected with Flag-CMV2. In contrast, the proliferation of cells transfected with shDEC1 was significantly stimulated. Migration assay showed that wound scratch in Flag-DEC1 group was wider compared to the control group and the numbers of endothelial cells in Flag-DEC1 group passed the membrane was decreased. In contrast, wound scratch in shDEC1 group had much narrower space compared to the control groups and the numbers of endothelial cells in shDEC1 group passed the membrane was increased. In tube formation assay, the relative tube length of endothelial cells transfected with Flag-DEC1 decreased by 48.28% compared with control group. However, compared with shRNA group, tube length of endothelial cells transfected with shDECl increased by 84.19%. Angiogenic factors VEGFA, MMP9 were significantly decreased in endothelial cells transfected with Flag-DEC1 both in mRNA and protein levels. In contrast, angiogenic factors were obviously increased in HUVECs transfected with shDECl.Conclusion:Overexpression of DEC1 inhibites the proliferation, migration and angiogenesis of human umbilical vein endothelial cell and knockdown of DEC1 promotes the proliferation, migration and angiogenesis of human umbilical vein endothelial cell. DEC1 regulates angiogenesis probably by affecting the expression of angiogenic factors (VEGF, MMP9). What’s more, overexpression of DEC1 inhibites the proliferation, migration and angiogenesis of EAhy926 endothelial cells.Part Ⅱ The roles of DEC 1 in angiogenesis of hepatocellular carcinoma cellsPurpose:To investigate the roles of DEC1 in angiogenesis of hepatocellular carcinoma cellsMethods:We established DEC1 overexpression and DEC1 knockdown hepatocellular carcinoma cells by using Flag-DEC1 plasmid and shDEC1 lentivirus respectively. We next cultured HUVECs with conditional medium containing supernatants from DEC1 overexpression and DEC1 knockdown hepatocellular carcinoma cells. The migration was detected by transwell assay. And the angiogenesis was assessed by matrigel tube formation assay. The target genes were measured by quantitative real-time PCR at mRNA levels and by Western blot at protein levels.Results:Transwell assay showed that the migration of HUVEC cells cultured by 97-L shDEC1 supernatant was increased. Tube formation assay indicated that the angiogenesis of HUVEC cells cultured by 97-L shDEC1 supernatant increased significantly compared with control group. However, when the HUVECs were cultured by 97-H Flag-DEC1 supernatant, the numbers of HUVECs passed the membrane was decreased and the relative tube length of HUVECs decreased obviously. Angiogenic factors VEGFA, MMP9 were significantly increased in 97-L cells transfected with shDECl both in mRNA and protein levels compared with 97-L cells transfected with shRNA. In contrast, angiogenic factors were obviously decreased in 97-H cells transfected with Flag-DEC1 compared with the control group.Conclusion:The results of our in vitro studies suggest that downregulation of DEC1 in HCC cell promotes cell migration and tube formation of HUVECs, and upregulation of DEC1 inhibits cell migration and tube formation of HUVECs. Overexpression of DEC1 decreases the expression of VEGFA and MMP9, which makes sense for the inhibition of angiogenesis of hepatocellular carcinoma cells.