应用表达载体介导siRNA抑制胰腺癌skp2基因表达的研究

首都医科大学学报 ›› 2008, Vol. 29 ›› Issue (5): 610-614.

应用表达载体介导siRNA抑制胰腺癌skp2基因表达的研究

任金帅¹, 李双², 宋茂民¹

1. 首都医科大学附属北京天坛医院普通外科;2. 中国人民解放军沈阳军区兴城疗养院疗养二科

收稿日期:2007-12-25 修回日期:1900-01-01 出版日期:2008-10-24 发布日期:2008-10-24
通讯作者: 宋茂民

Studies on the Inhibition of siRNA Expression Vector Mediated skp2 Expression in Human Pancreatic Cancer Cells

Ren Jinshuai¹, Li Shuang², Song Maomin¹

1. Department of General Surgery, Beijing Tiantan Hospital, Capital Medical University;2. Department of Convalescence, Xingcheng Convalescent Hospital, Shenyang Military District of PLA

Received:2007-12-25 Revised:1900-01-01 Online:2008-10-24 Published:2008-10-24

摘要/Abstract

摘要： 目的探讨表达载体介导小干扰RNA(siRNA)对人胰腺癌细胞系Capan-2skp2基因表达的抑制作用.方法构建靶向SKP2的siRNA质粒表达载体,采用Lipofectamine^TM 2000转染Capan-2细胞,实时定量PCR和Westernblot观察Capan-2细胞转染后SKP2mRNA和蛋白表达的改变.结果 PCR和DNA测序证实SKP2siRNA表达载体构建成功,分别转染Capan-2细胞后,skp2基因的mRNA和蛋白表达量与对照组相比均明显下降(P<0.05).结论所构建的靶向SKP2的siRNA质粒表达载体可以有效地阻断Capan-2细胞SKP2的mRNA和蛋白表达,为下一步以SKP2为靶点的胰腺癌的基因治疗实验研究奠定基础.

关键词: RNA干扰, SKP2, 胰腺癌

Abstract: Objective To construct a series of eukaryotic expression vector which can transcript shRNAagainst human skp2 gene and investigate the inhibitory effect of siRNAon skp2 expression in pancreatic caner cell line Capan-2.Methods Three pairs of hairpin-like oligonucleotides specific for human skp2 gene were designed and synthesized.The annealed oligonucleotides designed contain 5'-BamH Iand 3'-EcoR Ⅰ overhangs.Then they could be ligated into the eukaryotic expression RNAi-Ready pSIREN-RetroQvector.The recombined plasmids were verified by digestion with restriction endonuclease.Results The shRNAsequences were successfully inserted into the eukaryotic expression vector pSIREN-RetroQ.The sequence-specific siRNAefficiently down regulated the SKP2 expression at both mRNAand protein levels.The inhibition rate was 64.16% at mRNA level and 60.26% at protein level,respectively.Conclusion Three siRNA expression vectors were successfully constructed.The siRNAplasmid expression vectors against SKP2 can inhibit the SKP2 expression in Capan-2 cells efficiently.RNAi-mediated gene silencing of SKP2 can be a novel modality in gene therapy of pancreatic cancer.

Key words: RNA interference, SKP2, pancreatic cancer

中图分类号:

R735.9

任金帅;李双;宋茂民. 应用表达载体介导siRNA抑制胰腺癌skp2基因表达的研究[J]. 首都医科大学学报, 2008, 29(5): 610-614.

Ren Jinshuai;Li Shuang;Song Maomin. Studies on the Inhibition of siRNA Expression Vector Mediated skp2 Expression in Human Pancreatic Cancer Cells[J]. Journal of Capital Medical University, 2008, 29(5): 610-614.

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