中华皮肤科杂志

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乙酰肝素酶-siRNA表达载体的构建及其抑制黑素瘤细胞株体外侵袭能力的观察

刘晓艳1, 方红1, 羊正纲2, 阮黎明1, 方德仁1, 丁颖果1, 王懿娜1, 张好1, 蒋筱凌1   

  1. 1. 浙江大学医学院附属第一医院皮肤科, 杭州310003;
    2. 浙江大学医学院附属第一医院传染病研究所, 杭州310003
  • 收稿日期:2006-12-19 出版日期:2007-11-15 发布日期:2007-11-15
  • 通讯作者: 方红,email:Fanghongzy@sina.com E-mail:Fanghongzy@sina.com
  • 基金资助:
    浙江省教育厅资助项目(20051085)

Construction of small interfering RNA targeting heparanase gene and its inhibitory effect on the in-vasiveness of human malignant melanoma cell line A375 in vitro

LIU Xiao-yan1, FANG Hong1, YANG Zheng-gang2, RUAN Li-ming1, FANG De-ren1, DING Ying-guo1, WANG Yi-na1, ZHANG Yu1, JIANG Xiao-ling1   

  1. First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China
  • Received:2006-12-19 Online:2007-11-15 Published:2007-11-15

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摘要： 目的 构建针对人乙酰肝素酶(HPSE)基因的小干扰RNA(siRNA)及其表达载体,观察其对A375细胞HPSE基因的干扰作用及其对肿瘤细胞体外侵袭的抑制作用.方法 设计并构建了重组质粒pRNATU6.1/HPSE-siRNA,转染A375细胞,用实时荧光定量PCR法和蛋白质印迹法分别测定HPSE基因RNA和蛋白水平的表达变化,Matrigel侵袭实验观察A375细胞体外侵袭能力的改变.结果 将针对HPSE基因的siRNA的双链寡核苷酸片段正确克隆到pRNATU6.1载体;转染A375细胞,与对照组比较,HPSE-siRNA组HPSE基因和蛋白的表达均明显降低.转染后肿瘤细胞的体外侵袭能力与对照组相比受到明显抑制.结论 成功构建了针对HPSE基因的siRNA载体,HPSE-siRNA转染A375细胞可以显著降低细胞HPSE基因和蛋白的表达.

关键词: RNA干扰, 基因表达, 黑色素瘤, 乙酰肝素酶

Abstract: Objective To construct the small interfering RNA (siRNA) targeting heparanase gene and its expressing vector,and to observe its interference effect on the expression of heparanase gene and inhibitory effect on the invasive potential of human malignant melanoma A375 cells.Methods Three siRNAs were designed.The recombinant plasmid pRNATU6.1/heparanase-siRNA was designed and constructed. A375 cells were cultured,and transfected with pRNATU6.1/heparanase-siRNA.The cells treated with lipofectamine or Opti-MEM served as the controls.Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein in these treated A375 cells.The in vitro invasive potential of treated A375 cells was assessed by Matrigel gel assay.Results The siRNA targeting heparanase gene was successfully cloned to the eukaryotic expressing vector pRNATU6.1.The expression levels of both heparanase RNA and protein decreased significantly in siRNA-transfected A375 cells than those in the control cells.The in vitro invasive potential of siRNA-transfected cells was also significantly inhibited as compared with that of the control cells (P<0.01).Conclusions The siRNA targeting heparanase gene and its expressing vector are successfully constructed,and the designed siRNAs could significantly decrease the mRNA and protein expression of heparanase in A375 cells.

Key words: RNA interference, Gene express, Melanoma, Heparanase