Effects Of Heparanase On Adhesion And Invasion Of Hepatocellular Carcinoma Cells And Location Of P Selectin In Endothelial Cells

Objective: Vitro experiments were performed to observe the adhesion and invision of hepatocellular carcinoma cell lines with different levels of heparanase expression,and to explore the effect of HPSE on the localization of P selectin cells in endothelial cells.Methods:1.Lentivirus cell transfection and identification:Hep G2 cells were used as experimental cell lines,HPSE overexpression and RNAi lentivirus were transfected into Hep G2 cells.The expression level of HPSE gene and protein in transfected cells was detected by qRT-PCR and Western-blot respectively;2.Cell function tests:the adhesion,proliferation,invasion and migration ability of transfected cells were detected respectively;3.Cell immunofluorescence assay:Human Umbilical Vein Endothelial Cells(HUVECs)were co-cultured with transfected cells,immunohistochemistry was used to observe the localization of P-selectin in HUVECs;4.ELISA experiment: the content of soluble CD62 P in the culture medium of non contact coculture was detected by ELISA.Results:1.qRT-PCR results: the expression of HPSE m RNA in Hep G2 cells transfected with Lv was significantly higher than that in NC in HPSE over expression group(P <0.05).In HPSE RNAi group,the expression of HPSE m RNA in Hep G2 cells transfected with Lv was significantly lower than that in NC(P <0.05)and the expression of HPSE m RNA in Hep G2 cells transfected with Lv-HPSE RNAi1 was lower than that in Hep G2 cells transfected with Lv-HPSE RNAi2?3;2.Western-blot results: in HPSE overexpression group,the expression of HPSE protein in Hep G2 cells transfected with Lv was significantly higher than that of NC;In HPSE RNAi group,the expression of HPSE protein in Hep G2 cells transfected with Lv was significantly lower than that in NC(P <0.05)and the expression of HPSE protein in Hep G2 cells transfected with Lv-HPSE RNAi1 was lower than that in Hep G2 cells transfected with Lv-HPSE RNAi2?3;3.Cell function tests: In HPSE overexpression group,the cell proliferation,adhesion,invasion and migration ability of Hep G2 transfected with Lv were significantly higher than those in NC(P <0.05);In HPSE RNAi group,the cell proliferation,adhesion and invasion ability of Hep G2 transfected with Lv were lower than NC(P <0.05);but the cell migration ability of Hep G2 transfected with Lv-HPSE RNAi1 was not statistically different from NC(P?0.05);4.Cell immunofluorescence assay: the expression of CD62 P in the cytoplasm of HUVECs was observed under different levels of HPSE expressed by transfection of Hep G2;5.ELISA experiment: In HPSE overexpression group and HPSE RNAi group,Hep G2 cells transfected with Lv the soluble CD62 P content in HUVECs culture supernatant affected by Hep G2 transfected with Lv were not statistically significant with NC(P?0.05).Conclusions: 1.Hep G2 cells transfected with HPSE overexpression and RNAi lentivirus can effectively up regulate and down regulate the expression of HPSE gene and protein respectively;2.Upregulation of HPSE gene expression in Hep G2 cells could enhance the proliferation,adhesion,invasion and migration ability of Hep G2 cells in vitro experiments;the downregulation of HPSE expression in Hep G2 cells could decreased the proliferation,adhesion and invasion ability of Hep G2 cells,but had no effect on the migration ability of Hep G2 cells;3.The expression of CD62 P in the cytoplasm of HUVECs was observed under different levels of HPSE expressed by transfection of Hep G2;4.ELISA experiment: In HPSE overexpression group and HPSE RNAi group,Hep G2 cells transfected with Lv were have no effect on the content of soluble CD62 P in the supernatant of HUVECs respectively.