| Objective:To explore the effect of heparanase(HPSE)on apoptosis and necroptosis of microvascular endothelial cells of hepatocellular carcinoma(HCC).Methods:HCC cell line with high expression of HPSE was selected using real-time quantitative RT-PCR by RT-PCR and Western blot analysis.A lentiviral vector containing the RNA interference sequence of target gene was constructed and transfected into HCC cells.Its transfection efficiency was detected.Human umbilical vein endothelial cells(HUVECs)were obtained from ATCC,and used as microvascular endothelial cells(MVECs)of HCC.The HUVEC was cultured in Transwell till compact monolayer is formed.Trans-endothelial migration test was performed using transfected HCC cells with high expression of HPSE,and the migration rate was detected.HCC cells and HCC cells were used for non-contact co-culture experiments.Twenty-four hours later,the survival rate of endothelial cells was determined by CCK8,endothelial cell genomic DNA was extracted for gel electrophoresis,the apoptosis was detected via flow cytometryvia flow cytometry using Annexin V-APC single staining and cell morphology was observed by fluorescence microscope.Results: 1.The HPSE mRNA expression abundance of HPSE mRNA in SMMC7721,Huh7 and LCCLM3 cells were(9.53±0.08),(12.78±0.07)and(12.20±0.15),respectively,with the highest level in Huh7 cells;the HPSE protein expression is also highest in the cells.Huh7 cells was selected for following experiment.2.The lentiviral vector of HPSE-RNAi(LV-HPSE-RNAi(1249-2))was successfully constructed to transformed into Huh7 cells(shHPSE group).The fluorescence microscope showed the infection rate of Huh7 cells was over 70% with normal cell morpholgy;The HPSE mRNA expression levels in control group(shCtrl group)and shHPSE group were 1 and 0.31,respectively(P<0.05).The HPSE protein expression abundance was lower in hHPSE group than in shCtrl group.These findings suggested the LV-HPSE-RNAi(1249-2)can significantly decrease the expression of HPSE.3.The fluorescence microscope showed CD34 stained HUVECs formed compact monolayer in Transwell.The migration rates in two groups 0.62 and 0.40,respectively,and migration rate in shHPSE group was significantly lower than that in shCtrl group(P<0.05).The finding suggested RNAi can significantly decrease the migration rate of Huh7 cells.4.After co-cultue,the the survival rates of endothelial cell in two groups were 0.768 and 1.240,respectively.The the survival rate in shHPSE group was significantly higher than that in shCtrl group(P<0.05).It suggested HPSE can reduce the survival rate of HUVECs and increase cell death rate.5.Gel electrophoresis showed the DNA fragments of endothelial cells in the two groups was similar,and the main fragment length was greater than 10 kb.6.The apoptotic indices of endothelial cells in the two groups were(14.47±0.50)and(6.61±1.51),respectively.The indices in shHPSE group was significantly lower than that in shCtrl group(P<0.05).It indicated HPSE can promote the apoptosis of endothelial cells,and RNAi can inhibit apoptosis.7.Fluorescence microscopy showed that the endothelial cells of sh HPSE group had good extension,uniform size and tight arrangement,and no cell necrosis and debris were observed.In shCtrl group,the volumes of partial cell reduced,and the cell gap was enlarged;partial cell was swollen,and membrane rupture and spilling out of cell contents were observed.It was suggested that HPSE may induce apoptosis and necroptosis of vascular endothelial cells,and RNAi can inhibit this the death of endothelial cell.Conclusion:HCC cells overexpress HPSE,and HPSE expression level of Huh7 cells is higher than those of other 2 kinds of HCC cells.RNAi lentiviral vector can significantly inhibit HPSE expression and trans-endothelial migration of Huh7 cells.HPSE can significantly reduce the survival rate of non-contact co-cultured vascular endothelial cells,and promote apoptosis and necroptosis,which can be inhibitted by RNAi vectors. |
PDF Full Text Request