短发夹状RNA抑制survivin基因在肝癌细胞中的表达  

Inhibition of survivin expression in liver cancer cells by shRNA

作　　者：闫歌[1] 黄爱龙[1] 唐霓[1] 张秉强[1] 蒲丹 向明确[1] 兰英华[1] 吴刚[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所,400010

出　　处：《中华肝脏病杂志》2003年第12期712-715,共4页Chinese Journal of Hepatology

基　　金：国家自然科学基金(30371599);国家杰出青年基金(B类)(30228026)

摘　　要：目的 构建抗凋亡因子survivin的短发夹状RNA(shRNA),观察其在肝癌细胞株HepG2、SMMC—7721中对survivin基因表达的抑制作用。方法 设计、合成两对survivin编码基因的反向重复序列,中间分别间隔4和8个nt,分别定向克隆至载体pTZU6+1的U6转录启动子下,构建pshRNA—survivin 1和pshRNA—survivin 2重组质粒,与表达survivin基因的质粒pEGFP—C1-survivin共转染肝癌细胞株HepG2和SMMC—7721,荧光显微镜下观察融合蛋白中GFP的表达以分析pshRNA—survivin对survivin基因表达的抑制作用。结果 酶切分析和测序证实pshRNA—survivin 1和pshRNA—survivin 2构建成功;两组shRNA对survivin的表达均有明显的抑制作用,抑制率达80％以上;两重组质粒在HepG2和SMMC—7721两种细胞中抑制目的基因的作用无明显差异;反向重复序列中间隔4或8个nt构建的shRNA,对抑制目的基因的表达无显著差异。结论 构建的pshRNA—survivin重组质粒能有效抑制survivin基因在肝癌细胞株HepG2、SMMC-7721中的表达。Objective To construct the plasmid containing short hairpin RNA (shRNA) of survivin in order to suppress the expression of survivin gene in HepG2 and SMMC-7721. Methods Two 20-21 bp reverse repeated motifs of survivin target sequence with 4 bp or 8 bp spacer were synthesized respectively and inserted into plasmid pTZU6+l to generate the plasmid pshRNA-survivinl and pshRNA-survivin2; plasmid pEGFP-C1-survivin and pshRNA-survivinl or pshRNA-survivin2 plasmid were cotransfected into liver cancer cell HepG2 and SMMC-7721 to detect effect of GFP expression respectively and analyze the inhibition of survivin gene. Results The recombinant plasmid pshRNA-survivinl and pshRNA-survivin2 were successfully constructed. The recombinant plasmids suppress the survivin expression by 80% in HepG2 and SMMC-7721. Conclusion The result showed that the short hairpin RNA of survivin can efficiently suppress it's expression in HepG2 and SMMC-7721.

关 键 词：短发夹状RNA SURVIVIN基因 肝癌 癌细胞 细胞凋亡 基因治疗 

分 类 号：R735.7[医药卫生—肿瘤]