›› 2012, Vol. 3 ›› Issue (3): 117-119.

• 论著 • 上一篇    下一篇

MT1-MMP miRNA 干扰载体的构建及沉默效果评价

杨聪翀1,2,朱丽芳1,2,宁天云1,2,刘来奎2   

  1. 1.
    2. 南京医科大学附属口腔医院基础教研室
  • 收稿日期:2012-07-03 修回日期:2012-09-03 出版日期:2012-09-25 发布日期:2012-10-08
  • 通讯作者: 刘来奎 E-mail:liulaikui@126.com
  • 基金资助:
    国家自然科学基金

Construction and evaluation of the effect of MT1-MMP miRNA interference vectors

  • Received:2012-07-03 Revised:2012-09-03 Online:2012-09-25 Published:2012-10-08
  • Contact: Lai-Kui LIU E-mail:liulaikui@126.com

摘要: 目的:构建膜型基质金属蛋白酶-1(membrane-type matrix metalloproteinase-1,MT1-MMP)基因miRNA干扰载体,检测其对靶基因的沉默效率。方法:设计4种MT1-MMP基因特异性miRNA寡聚单链DNA,利用载体构建试剂盒构建MT1-MMP基因特异性干扰质粒,挑选阳性克隆,测序无误后扩增,表明载体构建成功。抽提干扰质粒,用脂质体2000转染HEK293细胞,通过实时荧光定量PCR法检测干扰载体对靶基因的干扰效果。结果:本实验成功构建了MT1-MMP miRNA干扰载体,筛选出最有效的干扰载体X173-4,并检测出其对靶基因的沉默效率可达到75%。结论:本实验结果为进一步研究MT1-MMP在口腔癌侵袭转移机制中的作用提供实验基础。

Abstract: Objective:To construct specific miRNA expression vectors targeting membrane-type matrix metalloproteinase-1(MT1-MMP)gene and test the silent rates of four different vectors for MT1-MMP. Methods:Design four different specific oligo sequences of DNA targeting MT1-MMP, and then construct recombinant plasmids using BLOCK-iT? Pol II miR RNAi Expression Vector Kit with EmGFP. The positive ones were cloned. The transformed clones were sequenced and then amplified. The resultant correct plasmids were extracted and then transfected into the HEK293 cells by Lipofectamine 2000 to test the silent rates of MT1-MMP with real-time fluorogenic quantitative RT-PCR method.Results:The MT1-MMP miRNA interference vectors were constructed. The most effective interference vector was X173 - 4, and its target gene silencing efficiency can reach 75%. Conclusions:This study provides an experimental basis for further study on the role of MT1-MMP in invasion and metastasis of oral squamous cell carcinoma.

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