短发夹状RNA抑制survivin基因在肝癌细胞中的表达

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—survivin1和pshRNA—survivin2重组质粒，与表达survivin基因的质粒pEGFP—Cl—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—survivin1和pshRNA—survivin2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。

Inhibition of survivin expression in liver cancer cells by shRNA

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.