肝素酶介导的肝癌细胞与血管内皮细胞黏附及穿内皮迁移

Heparanase-mediated adhesion with vascular endothelial cell and trans-endothelial migration of hepatocellular carcinoma cells

目的:探讨肝素酶(heparanase,HPSE)对肝细胞癌(hepatocellular carcinoma,HCC)细胞与血管内皮细胞黏附及穿内皮迁移的影响。方法:选用人正常脐静脉内皮细胞株HUVEC-C,肝LO-2细胞,肝癌Hep G2、BEL-7402和HCCLM3细胞,用Real-time PCR筛选高表达HPSE的HCC细胞;设计4个HPSE基因RNA干扰(RNA interference,RNAi)序列、构建质粒,并筛选出最优RNAi质粒;用该RNAi质粒转染高表达HPSE的HCC细胞,同时设空白对照组、阴性对照组和未转染的HCC细胞组;黏附试验观察各组HCC细胞与HUVEC-C细胞的黏附情况;Transwell小室法观察HCC细胞穿HUVEC-C细胞迁移情况;癌细胞均以0.25%玫瑰红染色、光镜下观察;酶标仪法分别测定脱色后各组细胞D值并分析比较。结果:HCCLM3细胞HPSE mRNA表达水平显著高于肝LO-2、Hep G-2和Bel-7402细胞[(5.72±0.62)vs(1.05±0.09)、(2.65±0.31)和(3.43±0.58),均P<0.01)];设计的4个HPSE之RNAi载体,以si HPSE-3158对HPSE基因表达的抑制效果最佳(P<0.05)。RNAi组HCCLM3细胞与HUVEC-C细胞黏附率显著低于空白对照组、阴性对照组和未转染的HCCLM3细胞组[(0.31±0.04)vs(0.46±0.06)、(0.45±0.05)和(0.64±0.09),均P<0.01)]。RNAi组HCCLM3细胞穿HUVEC-C细胞迁移率也显著低于空白对照组、阴性对照组和未转染的HCCLM3细胞组[(0.28±0.03)vs(0.41±0.04)、(0.41±0.05)和(0.43±0.05),均P<0.05)]。结论:HPSE参与介导HCC细胞与血管内皮细胞的黏附及HCC细胞穿内皮迁移,可能是HCC微血管捕获、血行转移的重要因素。

Objective：To explore the effect of heparanase （HPSE） on adhesion with vascular endothelial cell and trans-endothelial migration of hepatocellular carcinoma （HCC） cells. Methods：Human normal umbilical vein endothelial cell HUVEC-C line , hepatic LO-2 cells, and hepatic carcinoma HepG2, BEL-7402 and HCCLM3 cells were selected to use. HCC cells with high expression of HPSE were screened using Real-time PCR. Four RNA interference （RNAi） sequences of HPSE gene were designed, and the corresponding plasmids were constructed respectively, and the RNAi plasmids with the highest efficiency were screened out, which were transfected into HCC cells with high expression of HPSE. At the same time, blank control （BC） group, negative control （NC） group and untransfected HCC cell group were set up. Adhesion test was used to detect adhesion situation of HCC cell and HUVEC-C cell in each group and Transwell assay was performed to determine the trans-HUVEC-C cell migration of HCC cells. The HCC cells were stained with 0.25% Rose Bengal and observed under inverted microscope. After decolorization, D values of the HCC cells in each group were determined and compared using an enzyme-labeled instrument. Results： Expression level of HPSE mRNA in the HCCLM3 cells was significantly higher than those in hepatic LO-2, HepG-2 and Bel-7402 cells （[5.72±0.62] vs [1.05±009], [2.65±0.31] and [3.43±0.58], all P〈0.01 ）. Of the designed 4 RNAi plasmids of HPSE, siHPSE-3158 plasmid showed the best inhibitory effect on expression of HPSE gene （P〈0.05）. The adhesion rate of HCCLM3 cells and HUVEC-C cells in RNAi group was significantly lower than those in BC, NC and untransfected HCCLM3 cell groups （[031±0.04] vs [0.46±0.06], [0.45±0.05] and [0.64±0.09], all P〈0.01）. The trans-HUVEC-C cell migration rate of HCCLM3 cell in RNAi group was also significantly lower than those in BC, NC and untransfected HCCLM3 cell group （[0.284±0.029] vs [0.41±0.04], [0.41±0.05] and [0.43±0.05], all P〈0.05）. Conclusion： HPSE might be involved in mediating the adhesion of HCC cells and vascular endothelial cells as well as trans-endothelial cells migration, which could be a key factor in the adhesion to vascular endothelial cells and hematogenous metastasis of HCC cells.