Survivin小干涉RNA和GRIM-19共表达质粒的构建

Construction of co-expression plasmid of survivin-specific short haipin RNA and GRIM-19

目的构建survivin小干涉RNA和干扰素/维甲酸联合应用诱导细胞凋亡相关的基因(gene associated with retinoid interferon induced mortality-19,GRIM-19)共表达质粒为探讨联合基因对喉癌细胞凋亡和增殖的影响奠定基础。方法以survivin小干涉RNA重组质粒pGCsilencer-siRNA-survivin(简称psi-survivin)为模板设计PCR引物,PCR产物经测序后与线性化pCDNA3.1-GRIM-19(简称p-GRIM-19)连接,构建survivin小干涉RNA和GRIM-19共表达质粒pCDNA3.1-siRNA survivin/GRIM-19(简称p-siRNA survivin/GRIM-19)。将质粒用脂质体lipofetamine2000包裹转入人喉癌Hep-2细胞中,用半定量RT-PCR检测survivin和GRIM-19 mRNA的含量。结果构建的survivin小干涉RNA和GRIM-19共表达质粒p-siRNA survivin/GRIM-19经酶切电泳和测序证实碱基序列的正确性,p-siRNA survivin/GRIM-19转染人喉癌Hep-2细胞,从mRNA水平能降低survivin的表达,抑制率55.9%,同时增加GRIM-19表达,表明共表达质粒的构建是成功的。结论构建的siRNA survivin和GRIM-19共表达质粒从mRNA水平能降低survivin的表达,同时增加GRIM-19表达,为进一步实验奠定了基础。

OBJECTIVE To construct co-expression plasmid of survivin-specific short hairpin RNA and GRIM-19,and to explore the possibility of combined gene therapy in inducing apoptosis and proliferation.METHODS RT-PCR was used to obtain U6-siRNA-survivin fragment from pGCsilencer-siRNA-survivin（in short psi-survivin,sequencing）,then U6-siRNA-survivin was inserted into the expression plasmid pCDNA3.1-GRIM-19 multiclone sites to produce co-expression plasmid survivin-specific short haipin RNA and GRIM-19（in short p-si RNA Survivin/GRIM-19）.The electrophoresis and sequencing were carried out.Then it was transfected to human laryngeal carcinoma cell line Hep-2 by lipofectamine2000.The levels of mRNA before and after transfection were determined by RT-PCR.RESULTS RT-PCR showed that p-si RNA-Survivin/GRIM-19 could reduce survivin expression,the inhibition rates of survivin mRNA was 55.9%,meanwhile,increase GRIM-19 expression,which showed that the coexpression was successful.CONCLUSION The co-expression plasmid of survivin-specific short haipin RNA and GRIM-19 could partly inhibit the expression of survivin and increase the expression of GRIM-19.It could be used for related study of combined gene therapy.