The Role Of Heparanase In The Process Of Capillary Capture In Hepatocellular Carcinoma

Objective: To explore the molecular evidence of heparanase(HPSE)-induced cell adhesion between hepatocellular carcinoma(HCC) cells and capillary endothelial cells, and search for the cellular evidence of HPSE-caused capillary capture in HCC.Method: 1) The recombinant vector including the interference sequences of HPSE was designed then fulfilling the cell transient transfection with liposomal transfection method. Through Real-time quantitative PCR and Western Blot, the RNAi sequences with highest efficiency was chosen; The HPSE over-expression lentiviral vector was constructed and detecting the infection efficiency through Real-tine quantitative PCR and Western Blot.2) Transfecting the Hep G-2 cells in 96-well plate with the optimal RNAi recombinant vector to inhibit HPSE gene expression. Meanwhile, non-transfected group, negative control group and idling group were set for comparison. Observing the adhesion levels between Hep G-2 cells and endothelial cells HUVEC-C in different groups, then staining with 0.25% Rose Bengal, measuring the OD levels of each well for analysis after decolorization.3) MHCC97-H cells were infected with HPSE over-expression lentiviral vector. Observing the adhesion levels between Hep G-2 cells and endothelial cells HUVEC-C in 96-well plate as well as the trans-endothelial migration condition in the Transwell chamber in transfected groups, non-transfected group and negative control group. Cells were stained with 0.25% Rose Bengal, after decolorization, the OD levels of each well were obtained for analysis.Result: 1) Successfully screened the optimal HPSE RNAi sequences, si HPSE-3158, the over-expression of HPSE gene in MHCC97-H caused by lentiviral vector infection was also demonstrated.2) Ruling out the influence of the endothelial cells, the adhesion rate between Hep G-2 cells and endothelial cells was higher in the non-transfected group(0.351±0.030)when compare with the transfected group(0.239 ± 0.019,P<0.01). In negative control group(0.291 ± 0.027) and idling group(0.297 ± 0.032) the adhesion rates were also lower than the non-transfected group(p<0.05),but higher than the transfected group(p<0.05). There was no significant difference between negative control group and idling group(p>0.05).3) Ruling out the influence of the endothelial cells, the adhesion rate between MHCC97-H cells and endothelial cells was higher in the over-expression group(0.512±0.049) comparing with the other two groups(p<0.01), and the adhesion rate showed no difference between negative control group(0.370 ± 0.027) and non-transfected group(0.377±0.050,p>0.05).4) Ruling out the influence of the endothelial cells,MHCC97-H cells showed the highest trans-endothelial migration ratio in the HPSE over-expression group(0.431 ± 0.043) rather than the other two groups(p<0.01), while there was no significant difference between negative control group(0.306 ± 0.026) and non-transfected group(0.295 ±0.044,p>0.05).Conclusion: Through RNAi and gene over-expression methods, our findings suggested that Heparanase(HPSE) might play an important role in the adhesion between HCC cells and capillary endothelial as well as the trans-endothelial migration and was involved in the process of capillary capture during the formation of HCC.