Heparanase Induced Necroptosis Of Liver Cancer Microvascular Endothelial Cells Under The Inhibition Of Caspase-8

Objective:To verify that heparanase(HPSE)of hepatocellular carcinoma can cause necroptosis of microvascular endothelial cells;and to explore the role and mechanism of Caspase-8 in HPSE induced necroptosis.Methods: HPSE RNAi lentivirus was transfected into HCCLM3 cell line to knock down HPSE.The qRT-PCR and Western-Blot analysis were used to verify the transfection efficiency.In order to verify the migration ability of liver cancer cells,transwell experiments were used.Human umbilical vein endothelial cells(HUVECs)were co-cultured with HCCLM3 liver cancer cells.The number of dead endothelial cells was observed by flow cytometry,and the mode of death was detected by an electron microscopy.The HUVECs were divided into four groups in total,including non-co-culture group,HPSE mock group,HPSE RNAi group and NEC-1 group.The contents of supernatant HPSE,TNF-? and caspase-8 were detected by ELISA assay,and the protein expressions of RIP1,RIP3,MLKL and caspase-8 in HUVECs were determined by Western-Blot method.Metastasis model of liver cancer in nude mice was established with intraperitoneal injection of HCCLM3 cells.HE staining and double-label immunofluorescence assays were performed to observe the microvascular invasion.Results:1.HPSE mRNA and protein expressions in RNAi lentivirus transfected HCCLM3 cells was significantly decreasd compared that with mock group(P<0.05).2.Transwell sassy showed that inhibition of HPSE reduced the migration ability ofhepatocellular carcinoma(P<0.05).3.HUVEC death increased after coculture with HCCLM3 cells for 48 hours(P<0.05),and electron microscopy showed that the dead cells had the characteristics of necroptosis.4.Supernatant TNF-? and HPSE levels in non-coculture group and HPSE RNAi group are less than those in coculture group(P<0.05 vs Blank,P<0.05 vs co-culture),and the RIP1,RIP3,MLKL and caspase-8 protein expressions of HUVECs in the non-coculture group,HPSE RNAi group and NEC-1 group are less than those in the coculture group(P<0.05 coculture vs Blank,P<0.05 HPSE-RNAi vs coculture,P<0.05 NEC-1 vs coculture).5.HE staining failed to detect obvious microvascular invasion in metastasis model of liver cancer,but double-label immunofluorescence assays showed that the expressions of p-MLKL in HPSE RNAi group and NEC-1 group were less than that in HCC group(P<0.05 HPSERNAi vs HCC group,P<0.05 NEC-1 vs HCC group).Conclusion: HPSE inhibition can reduce the migration ability of liver cancer cells.HUVECs cells co-cultured with HCCLM3 cells show necroptosis.HPSE can induce necroptosis of microvascular endothelial cells of HCC,and the inhibition of caspase-8may be necessary.Down-regulating HPSE can inhibit necroptosis by regulating TNF-?levels...