ShRNA抑制gankyrin基因在肝癌细胞中表达的研究

Suppression the Expression of Gankyrin in Human Hepatocellular Carcinoma Cells by shRNA

目的构建靶向癌基因gankyrin的短发夹状RNA(shRNA),观察其在肝癌细胞株HepG2中对gankyrin基因表达的抑制作用。方法设计、合成三对针对gankyrin的shRNA序列,连接到带有人U6启动子的载体质粒pGenesil-1中,构建重组质粒pGenesil-1-Gankyrin1、pGenesil-1-Gankyrin2、pGenesil-1-Gankyrin3,稳定转染HepG2细胞,采用RT-PCR及W estern b lot检测对gankyrin表达的抑制效果。结果酶切分析和测序证实pGenesil-1-Gankyrin1、pGenesil-1-Gankyrin2、pGenesil-1-Gankyrin3构建成功;其中pGenesil-1-Gankyrin2、pGenesil-1-Gankyrin3对gankyrin的表达有明显的抑制作用。结论构建的pGenesil-1-Gankyrin重组质粒能有效抑制gankyrin基因在肝癌细胞株HepG2中的表达。

Objective To construct the plasmid containing short hairpin RNA（shRNA） targeting at gankyrin in order to inhibit the expression of gankyrin in HepG2 cell line. Methods Three couples of shRNA sequences targeting at gankyrin were synthesized and inserted into plasmid pGenesil-1 under U6 promoter. The recombinant plasmids pGenesil-1-Gandyrin 1, pGenesil-1-Gankyrin 2 and pGenesil-1-Gankyrin 3 were constructded and the plasmids were stably transfected into HepaG 2 cell line. The gene expression inhibition was assessed by TR-PCR and Western blot analysis. Results The recombinant plasmids pGenesil-1-Gankyrin 1, pGenesil-1-Gankyrin 2 and pGenesil-1-Gankyrin 3 were constructed successfully and identified by enzymatic digestion and sequencing. The expression of gankyrin was suppressed significantly by pGenesil-1-Gankyrin 2 and pGenesil-1-Gankyrin 3 in HepG2. Conclusion The expression of gankyrin in HepG2 cells can be suppressed effciently by the recombinant plasmid pGenesil-1-Gankyrin.