Role Of The Heparinase /SDC1/TGF-β Axis In Promoting HCC Metastasis Of Vascular Endothelial Cells

Objective: To explore the definitive evidence of heparinase(HPSE)-mediated endothelialto-mesenchymal transition(Endo MT)in promoting hepatocellular carcinoma metastasis;and to explore the presence of HPSE-mediated Endo MT in the HPSE/SDC1/TGF-β axis.Methods: 1.cellular lentivirus transfection(Lv): According to the results of previous experiments,Liver cancer cell line HCCLM3 with the highest HPSE expression and umbilical vein endothelial cell HUVECs were selected as the experimental cell lines,Lentiviruses with HPSE and SDC 1 knockdown,respectively,The expression levels of HPSE and SDC 1 gene and its protein in the transfected cells were determined by RT-q PCR and Western-blot technology,respectively;2.Non-contact co-culture model: Through the establishment of the non-contact co-culture model,The effect of different expression of HPSE on the morphology and metabolism of endothelial cells was examined.3.Cell function experiment: the invasion and migration ability of transfected cells were detected by transendothelial cell migration experiments;4.Cell immunofluorescence: human umbilical vein vascular endothelial cells(HUVECs)and transfected cells were cocultured by noncontact,and the relevant genes in HUVECs were observed by cellular immunofluorescence;5.ELISA experiment: the content of soluble TGF-β in the chamber medium under noncontact co-culture was detected by ELISA.Results: 1.RT-q PCR results: in the HPSE reduction group(SH),The expression of HPSE m RNA in Lv HCCLM3 cells transfected with HPSE was significantly lower than that of the negative control(NC)(P ﹤ 0.05);In the SDC1 knockout group(SH),The SDC1 m RNA expression of LV in HUVECs cells transfected with SDC1 was significantly lower than that of the negative control(NC)group(P﹤0.05);Western-Blot result: In the HPSE knockdown(SH)group,The protein expression of HPSE of HCCLM3 cells with HPSE was significantly lower than that of the negative control(NC)group;In the SDC 1 knockout group(SH),The protein expression of SDC 1 in Lv HUVECs cells transfected with SDC1 was significantly lower than that in the negative control(NC)group.2,Results of non-contact co-culture model: SDC1 expression of endothelial cells co-cultured with high HPSE expression was significantly higher than the HPSE knockdown(SH)group(P﹤0.05);And simultaneously accompanied by increased SCD1 expression,The reduction in the endothelial cell marker CD31 content,The rise in the content of the interstitial cell marker VIMENT;By comparing the phenotype of endothelial cells after coculture with high HPSE expressing HCCLM3cells(NC)group with the HPSE knockdown(SH)group,There are significant spindle shape changes,Cells were wrinkled in a fibroblast-like pattern.3,cell function experiments: in the HPSE knockdown group,Lv HCCLM3 cells transfected with HPSE had less proliferation,invasion and migration capacity than the NC group,The difference was statistically significant(P﹤0.01);And the migration capacity of HCCLM3 cells in the group containing Lv with HUVECs transfected with SDC1 was less than that in the NC group,Statistically significant difference(P ﹤ 0.01);4.cellular immunofluorescence experiment: under the action of transfected HCCLM3 with different HPSE expression levels,That SDC1 was expressed on the HUVECs cellular membrane to varying degrees,Meanwhile,it shows that TGF-β is expressed in the HUVECs cell nucleus to varying degrees,The fluorescence intensity changes of the three factors are positively correlated;5,ELISA experiment: the content of soluble SDC1 in the HUVECs culture supernatant of HCCLM3 cells in the HPSE knockdown group,compared with the NC group,There was no significant difference(P﹥0.05);What cells are transfected The amount of soluble TGF-β in the clear solution was significantly insignificant when compared with the NC group(P﹥0.05).Conclusion: Reducing the content of HPSE in HCCLM3 cells could mediate the decrease of SDC1,thus further reducing the expression of TGF-β;HPSE / SDC1 axis plays an important role in TGF-β regulation and enhances the invasion and metastasis ability of HCC cells by promoting interstiization of vascular endothelial cells.