RNA干扰Heparanase基因表达对胆囊癌侵袭性影响的体外研究

目的:利用RNA干扰技术沉默人胆囊癌GBC-SD细胞中乙酰肝素酶(heparanase,HPA)基因的表达,探讨抑制HPA基因后对GBC-SD细胞侵袭性的影响.方法:构建靶向人HPA基因miRNA重组质粒,采用脂质体转染法将质粒转染入人胆囊癌GBC-SD细胞,RT-PCR检测转染前后HPA mRNA表达情况,选出对HP...

Effects of heparanase gene silencing by RNA interference on invasion of gallbladder carcinoma in vitro

Objective：To investigate the effects of heparanase（HPA） gene silencing by miRNA interference on invasion and migration of GBC-SD cells of gallbladder carcinoma.Methods：Four pairs of single-stranded recombinant plasmid miRNA oligo-DNA targeting human HPA gene sequence were synthesized and transfected into GBC-SD cells by lipofectamineTM 2000.HPA mRNA expression was detectede by RT-PCR before and after transfection,the recombinant plasmid which showed the best silencing effect for further experiments was selected.Transwell invasion assays and wound scarification assays were performed to examine the migration and invasive ability of GBC-SD cells.Results：Recombinant plasmid pcDNA6.2-GW/EmGFP-miR-HPA-2 showed the best silencing effects.The migration distance in wound scarification assays of GBC-SD cells transfected with recombinant plasmid pcDNA6.2-GW/EmGFP-miR-HPA-2 at 12h,24h was significant shorter than negative control and blank groups（P0.05-P0.01）.The invasive ability of GBC-SD cells transfected with pcDNA6.2-GW/EmGFP-miR-HPA-2 was decreased significantly when compared with negative control and blank groups（P0.01）.Conclusions：The GBC-SD cells transfected with recombinant plasmid pcDNA6.2-GW/EmGFP-miR showed decreased HPA mRNA expression,as well as inhibited invasion and migration in vitro.