肝素酶调节对肝癌细胞粘附侵袭能力的影响

摘要

目的：探讨人肝素酶（HPSE）对肝癌细胞粘附、侵袭能力的影响。方法：RT-PCR、Western blot检测人肝癌细胞系BEL-7402、HepG2、HCCLM3中HPSE的表达。构建4个HPSE的miR载体pcDNATM6.2-GW/EmGFP-miR-HPSE（pcDNA-miR-HPSE）和表达载体pIRES2-EGFP-HPSE；分别转染HPSE高表达和低表达细胞。荧光显微镜观察转染效率，RT-PCR和Western blot检测转染细胞HPSE表达；酶标仪检测细胞粘附能力，Transwell小室检测侵袭转移能力。结果：HPSE mRNA和蛋白在在3种肝癌细胞中相对表达量均高于正常肝细胞（P<0.05），以HCCLM3表达最高，HepG2最低（P<0.05）；4种miR载体和表达载体鉴定符合要求；转染效率75%～85%；miR载体均能抑制HCCLM3细胞HPSE表达，以pcDNA-miR-HPSE-1抑制效果最佳（P<0.01）；表达载体可提高HepG2细胞HPSE表达（P<0.05）；HCCLM3细胞转染pcDNA-miR-HPSE-1后，粘附率、侵袭和迁移细胞数均下降约50%（P<0.01）；HepG2细胞转染pIRES2-EGFP-HPS后上述各指标分别升高近40%（P<0.05）。结论：不同性质的HPSE载体可对肝癌细胞粘附和侵袭转移能力进行双向调节；HPSE不但参与肝癌细胞侵袭转移，亦可能与肝癌细胞粘附有关。%Objective:To explore the effect of heparanase (HPSE) on the cell adhesion and invasion ability of hepatoma carcino-ma (HC) cell. Methods:HPSE expressions in human HC cell lines (BEL-7402, HepG2, and HCCLM3) were measured by real-time re-verse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Four recombinant miRNA vectors, pcD-NATM6.2-GW/EmGFP-miR-HPSE (pcDNA-miR-HPSE), were constructed and transfected into HCCLM3 cells. Full-length cDNA of HPSE gene was cloned into pIRES2-EGFP vector and transfected into HepG2 cells. Transfection efficiency was observed with fluores-cence microscope. HPSE expressions in transfected cells were measured by real-time RT-PCR and Western blot analysis. Adher-ence ability was determined with microplate reader, and invasion and migration abilities were detected with Transwell chambers. Re-sults:Both HPSE mRNA and protein relative expression levels were higher in the three types of HC cells than those in normal hepato-cyte (P<0.05). HPSE had the highest expression level in HCCLM3 cells and the lowest expression level in HepG2 cells (P<0.05). All five recombinant vectors met the experimental requirements. The transfection efficiencies were 75%-85%. The four miRNA vectors, pcDNA-miR-HPSE, significantly decreased HPSE expression in transfected HCCLM3 cells (P<0.05), and pcDNA-miR-HPSE-1 showed the best interference effect (P<0.05). Plasmid pIRES2-EGFP-HPSE increased HPSE expression in transfected HepG2 cells (P<0.05). After pcDNA-miR-HPSE-1 was transfected, the HCCLM3 cell adherence rate and the cell invasion and migration numbers dropped by almost 50%(P<0.01). After transfection of pIRES2-EGFP-HPSE, the HepG2 cell adherence rate and the cell invasion and migration numbers increased by nearly 40%(P<0.05). Conclusion:Different HPSE vectors could regulate bi-directionally the adher-ence, invasion, and migration abilities of transfected HC cells. HPSE may be related with adherence aside from invasion of HC cell.