Effects Of VEGF On Vasculogenic Mimicry In Human Hepatocellular Carcinoma Cells In Vitro

ObjectiveTo study the effect of vascular endothelial growth factor (VEGF) on migration, invasion ability and vasculogenic mimicry (VM) of human hepatocellular carcinoma PLC cells in vitro by adding various concentrations of VEGF-A and trans feet VEGF plasmid.Methods1. Choose the human hepatocellular carcinoma cell lines PLC, cultered in37℃, a humidified5%CO2incubator. We devided the cells into five groups:control group and adding various concentrations (10μg/L、30μg/L、60μg/L、100μg/L) of VEGF-A group.2. We detected the migration ability of the human hepatocellular carcinoma PLC cells by wound-healing assay when treated with various concentrations of VEGF-A.3. We detected the invasion ability of the human hepatocellular carcinoma PLC cells by Transwell assay when treated with various concentrations of VEGF-A.4. We detected the activity of MMP-2and MMP-9of the human hepatocellular carcinoma PLC cells when treated with various concentrations of VEGF-A by Gelatin zymography assay.5. We detected the ability of VM formation of the hepatocellular carcinoma PLC cells in three-dimensional cultures when treated with various concentrations of VEGF-A.6. We detected the expression of VM markers VE-cadherin of the human hepatocellular carcinoma PLC cells after treated with various concentrations of VEGF-A by Western blot.7. Reverse transcription polymerase chain reaction(RT-PCR) was performed to determine the mRNA expression level of VE-cadherin in the human heaptocellular carcinoma PLC cells after treated with various concentrations of VEGF-A.8. Transient transfect VEGF plasmid into the human hepatocellular carcinoma PLC cells, The effect of transfection was observed by Western blot assay.9. Wound-healing assay was performed to determine the migration ability of the human hepatocellular carcinoma PLC cell after transfected VEGF plasmid.10. Transwell assay was performed to determine the invasion ability of the human hepatocellular carcinoma PLC cells after transfected VEGF plasmid.11. Gelatin zymography assay was performed to detected the activity of MMP-2and MMP-9of the human hepatocellular carcinoma cell PLC after transfected VEGF plasmid.12. We detected the ability of VM formation of the human hepatocellular carcinoma PLC cells in three-dimensional cultures when transfected VEGF plasmid.13. Western blot was performed to detected the expression of VE-cadherin in the hepatocellular carcinoma PLC cells when transfected VEGF plasmid.14. RT-PCR was performed to determine the mRNA expression level of VE-cadherin in the hepatocellular carcinoma PLC cells when transfected VEGF plasmid.Results1. The migration distance of the human hepatocellular carcinoma PLC cells was significantly longer than that of control group by wound-healing assay(P<0.05), when treated with various concentrations of VEGF-A.2. The PLC cells treated with various concentrations of VEGF-A were found through the membrane more than the control group in a Transwell assay(P<0.05).3. The activity of MMP-2and MMP-9of the human hepatocellular carcinoma PLC cells was increased in does-dependent manner after treated with various concentrations of VEGF-A by Gelatin zymography assay(P<0.05).4. The ability of VM formation of the human hepatocellular carcinoma PLC cells was increased significantly in three-dimensional cultures when treated with various concentrations of VEGF-A,the correlation between the level of VEGF-A and the quantities of the pipelines were positive significantly(r=0.929).5. The expression of VE-cadherin of the human hepatocellular carcinoma PLC cells were increased significantly by Western blot and RT-PCR when treated with various concentrations of VEGF-A(P<0.05). 6. The expression of VEGF is upregulated after transfected with VEGF plasmid into the human hepatocellular carcinoma PLC cells When observed by Western blot assay(.P<0.05).7. The migration distance of the hepatocellular carcinoma PLC cells was significantly longer than that of control group by Wound-healing assay after transfected VEGF plasmid (P<0.05)8. The PLC cells transfected with VEGF plasmid were found through the membrane more than the control group in a Transwell assay(P<0.05).9. The activity of MMP-2and MMP-9of the hepatocellular carcinoma PLC cells was increased after transfected VEGF plasmid(P<0.05).10. Compared with the control group, the hepatocellular carcinoma PLC cells can form pipeline structure after transfected VEGF plasmid.11. The expression of VE-cadherin of PLC were increased significantly by Western blot and RT-PCR after transfected VEGF plasmid(P<0.05).ConclusionVEGF can promote the migration, invasion and the ability of VM formati-on in human hepatocellular carcinoma PLC cells, VEGF is the important regu-lator in the process of VM formation in hepatocellular carcinoma, it probably promote VM formation in human hepatocellular carcinoma by upregulating M-MPs and VE-cadherin, and then make the tumor tissues develope diversified microcirculation, and promote the invasion and metastasis of tumor. This research may have significant value in laying the foundation for a more explicit anti-tumor angiogenesis therapy.