基础医学与临床 ›› 2016, Vol. 36 ›› Issue (4): 445-450.

• 研究论文 • 上一篇    下一篇

miR-1247-5p对Smad2基因的靶向调控作用

张议心1,王利新2,徐广贤1,杨丽3,赵瑾3,郭文伟3,刘新兰2   

  1. 1. 宁夏医科大学检验学院
    2. 宁夏医科大学总医院
    3. 宁夏医科大学
  • 收稿日期:2015-09-17 修回日期:2015-11-12 出版日期:2016-04-05 发布日期:2016-03-29
  • 通讯作者: 徐广贤 E-mail:599040064@qq.com
  • 基金资助:
    国家自然科学基金地区基金项目;宁夏科技支撑项目;宁夏自然科学基金

miR-1247-5p targets regulation of Samd2 gene expression

  • Received:2015-09-17 Revised:2015-11-12 Online:2016-04-05 Published:2016-03-29

摘要: 目的 利用miR-1247-5p过表达模拟物mimics,研究miR-1247-5p对TGF-?信号通路中关键蛋白Smad2的靶向调控作用。方法 合成模拟miR-1247-5p过表达的模拟物mimics,及抑制miR-1247-5p表达的抑制物inhibitors;同时构建与miR-1247-5p互补靶基因Smad2的3'非翻译区,将其克隆到线性化的Report荧光报告载体中。将测序鉴定阳性的Report-Smad2 3'-UTR重组质粒,与miR-1247-5p的mimics/inhibitors共转染至HEK-293T细胞,通过relative luciferase activity实验验证靶向关系,并通过Western blot实验进一步检测Smad2蛋白表达水平。结果 成功构建Report-Smad2 3'-UTR重组质粒,Western blot实验表明转染miR-1247-5p mimics组中Smad2蛋白表达下调(P<0.05);而转染miR-1247-5p inhibitors组中,Smad2蛋白表达上调(p<0.05)。结论 证实miR-1247-5p直接靶向TGF-?信号通路中关键蛋白Smad2的表达,在蛋白水平对其进行调控,为进一步研究miRNA在细胞通路中的调控作用奠定了基础。

关键词: miR-1247-5p, TGF-?信号通路, Smad2基因, 肿瘤

Abstract: Objective:Use the miR-1247-5p mimics which can over-expression miR-1247-5p and explore its targeted effects on Smad2 which is a key protein in TGF-? signaling pathway.Methods: Use the miR-1247-5p mimics and miR-1247-5p inhibitors Synthesized by the company.Based on chemical technology to synthetic stem-loop structure RNA of Smad2 3'-UTR which is complementary to miR-1247-5p,and cloned it into the linearized Report plasmid which was digested by restriction enzyme,then sequenced,the positive recombinants were transfected into HEK-293 T cells;use the relative luciferase activity test,and Western blot test to validate the targeted regulatory relationship between miR-1247-5p and Smad2 3'-UTR. Results: The recombinant plasmid of Report-Smad2-3'UTR were constructed successfully,and Western blot test confirmed that the over-expression of miR-1247-5p suppressed the expression level of Smad2 protein significantly(P<0.05);suppression of miR-1247-5p expression increased the expression level of Smad2 protein significantly(P<0.05).Conclusion:miR-1247-5p can directly target on the gene expression of Smad2.It is regulated at the protein level,construct the foundation of miRNA for regulation of the further research on signaling pathways.

Key words: miR-1247-5p, TGF-? signaling pathway, Smad2 gene, tumour