RNA干扰Heparanase基因表达对胆囊癌侵袭性影响的体外研究  

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

作　　者：李三红[1] 刘会春[1] 周少波[2] 金浩[1] 

机构地区：[1]蚌埠医学院第一附属医院肝胆外科,安徽蚌埠233004 [2]蚌埠医学院第二附属医院普外科,安徽蚌埠233040

出　　处：《蚌埠医学院学报》2011年第12期1289-1292,1295,共5页Journal of Bengbu Medical College

基　　金：安徽省高等学校自然科学研究资助项目(KJ2009A163)

摘　　要：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HPA基因沉默效果最佳的质粒。通过划痕损伤试验和Transwell侵袭试验检测干扰沉默HPA表达后对人胆囊癌GBC-SD细胞侵袭性的影响。结果:质粒pcDNA6.2-GW/EmGFP-miR-HPA-2沉默人胆囊癌GBC-SD细胞HPA mRNA表达的效果最好。划痕损伤试验中胆囊癌GBC-SD细胞的迁移距离在12 h、24 h pcDNA6.2-GW/EmGFP-miR-HPA-2组均低于阴性对照组和空白组(P<0.05～P<0.01)。pcDNA6.2-GW/EmGFP-miR-HPA-2组中穿过Transwell小室基质的胆囊癌GBC-SD细胞数均明显低于阴性对照组和空白组(P<0.01)。结论:靶向人HPA基因miRNA重组质粒能有效抑制HPA基因的表达,抑制人胆囊癌GBC-SD细胞的侵袭能力。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.

关 键 词：胆囊肿瘤 RNA干扰 乙酰肝素酶 肿瘤侵袭 

分 类 号：R735.8[医药卫生—肿瘤]