MicroRNA-195与Smad7靶向关系的研究

Investigating the relationship between MicroRNA-195 and Smad7

目的:构建含有Smad7基因3'-UTR区的荧光素酶报告基因载体,利用双荧光素酶报告基因验证MicroRNA195与其潜在靶基因Smad7的靶向关系。方法:PCR扩增出Smad7基因3'-UTR区片段,以此构建含3'-UTR的荧光素酶报告基因载体(wild type,WT);将miRNA-195与荧光素酶报告重组子共转染至293T细胞中,双荧光素酶报告基因系统检测荧光素酶活性;构建含Smad7基因3'-UTR突变体(mutant,Mut)的荧光素酶报告基因质粒,检测荧光素酶活性变化。结果:测序结果表明,含有Smad7基因3'-UTR区的荧光素酶报告基因载体构建正确;荧光素酶活性实验表明,与对照组相比,miRNA-195可使含Smad7基因3'-UTR区的荧光素酶报告重组子的荧光素酶活性降低40%左右;而定点突变Smad7基因3'-UTR的荧光素酶报告重组子荧光活性未有明显变化。结论:成功构建了Smad7基因3'-UTR区的荧光素酶报告基因载体,而miRNA-195可以直接作用于Smad7基因3'-UTR区,抑制其荧光素酶活性。

Objective: The luciferase reporter vector containing 3’-UTR of Smad7 was constructed. Dual luciferase reporter gene system was applied to determine the association between miRNA-195 and its target gene Smad7. Methods: The 3’-UTR of Smad7 fragment amplified by PCR was cloned into luciferase vector. MiRNA-195 and the luciferase reporters containing 3’-UTR( Smad7-3’ UTR-WT) or mutant 3’-UTR( Smad7-3’UTR-Mut) of Smad7 were co-transfected into HEK293 T cells and dual luciferase reporter gene system was applied to test luciferase activity. Results: DNA sequencing showed that the sequences of the cloned regions were correct. The luciferase activity of Smad7-3’ UTR-WT plasmid treated with miRNA-195 was decreased to about 40% compared with control. The mutant 3’-UTR were no longer repressed by miRNA-195. Conclusion: The Smad7 3’-UTR luciferase reporter vector has been constructed successfully.MiRNA-195 can repress the luciferase activity of the reporter gene and had direct effect on Smad7 3’-UTR.