miR-1247-5p对Smad2基因的靶向调控作用

Targets regulation of Samd2 gene expression by miR-1247-5p

目的利用miR-1247-5p过表达模拟物mimics,研究miR-1247-5p对TGF-β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-β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提供了依据。

Objective By use of the miR-1247-5 p mimics which can over-express miR-1247-5 p,to explore its targeted effects on Smad2. Methods Use the commercially available miR-1247-5 p mimics and miR-1247-5 p inhibitors. Based on chemical technology to synthesize stem-loop structure RNA of Smad2 3 ＇-UTR which is complementary to miR-1247-5 p,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. Then we used the relative luciferase activity test,and Western blot test to validate the targeted regulatory relationship between miR-1247-5 p and Smad2 3 ＇-UTR. Results The recombinant plasmid of report-Smad2-3 ＇ UTR was constructed successfully,and Western blot test confirmed that the over-expression of miR-1247-5 p suppressed the expression level of Smad2 protein significantly（ P〈0. 05）; suppression of miR-1247-5 p expression increased the expressionlevel of Smad2 protein significantly（ P〈0. 05）. Conclusions miR-1247-5 p can directly target at the gene expression of Smad2. It regulates at the protein level,and lay the foundation of miRNA for further research on its signaling pathways.