MicroRNA-212-3p与靶基因转化生长因子-β2基因3'UTR的关系研究*

Relationship between miR-212-3p and the 3’UTR of its potential target gene TGF-β2*

摘要:

目的?构建转化生长因子-β2（TGF-β2）基因3'UTR双荧光素酶报告质粒，分析microRNA-212-3p（miR-212-3p）与其潜在靶基因TGF-β2的靶向关系。方法?将TGF-β2 3'UTR片段构建至双荧光素酶报告载体pYr-MirTarget上，随后将重组质粒与miR-212-3p核苷类、无核苷类及其抑制剂共同转染Saos-2细胞（人成骨肉瘤细胞），通过双荧光素酶报告系统检测荧光素酶活性，RT-PCR检测Hsa-miR-212及TGF-β2基因的mRNA相对表达量，Western blotting检测TGF-β2蛋白相对表达量。结果?miR-212-3p-mimics+TGF-β2 3'UTR共转染组荧光素酶活性较对照组低（P?<0.05）。miR-212-3p-mimics+TGF-β2 3'UTR共转染组Has-miR-212 mRNA相对表达量最高，miR-212-3p-inhibitor+TGF-β2 3'UTR共转染组最低（P?<0.05）。miR-212-3p-mimics+TGF-β2 3'UTR共转染组TGF-β2 mRNA相对表达量最低，miR-212-3p-inhibitor+TGF-β2 3'UTR共转染组最高（P?<0.05）。miR-212-3p-inhibitor+TGF-β2 3'UTR共转染组TGF-β2蛋白相对表达量最高，miR-212-3p-mimics+TGF-β2 3'UTR共转染组最低（P?<0.05）。结论?该实验成功构建了TGF-β2基因3'UTR荧光素酶重组质粒，而且miR-212-3p可以与TGF-β2基因的3'UTR结合，抑制荧光素酶活性，由此推断TGF-β2是miR-212-3p的靶基因。

Abstract:

Objective To construct the dual-luciferase reporter plasmids of the 3'-untranslated region (3'UTR) of transforming growth factor-β2 (TGF-β2) gene, and to verify the correlation between miR-212-3p and its potential target gene TGF-β2.?Methods?The 3'UTR of TGF-β2 was cloned into luciferase reporter vector pYr-MirTarget, and the recombinant plasmids were transfected with miR-212-3p mimics, NC mimics or inhibitor into Saos-2 cells (human osteosarcoma cells). The luciferase activity was detected by dual-luciferase reporter assay, and mRNA expression levels of Hsa-miR-212 and TGF-β2 and the protein expression level of TGF-β2 were detected via RT-PCR and Western blotting, respectively.?Results?The luciferase activity was decreased in Saos-2 cells into which TGF-β2 recombinant plasmids and miR-212-3p mimics were transfected compared to that in the control group (P < 0.05). The expression level of Hsa-miR-212 mRNA was the highest whereas the expression level of TGF-β2 mRNA was the lowest in Saos-2 cells transfected with miR-212-3p mimics and TGF-β2 recombinant plasmids among all the groups. However, the expression level of Hsa-miR-212 mRNA was lower and that of TGF-β2 mRNA was higher in Saos-2 cells transfected with miR-212-3p inhibitor and TGF-β2 recombinant plasmids than those in the other groups (P < 0.05). Besides, TGF-β2 protein level was the highest in Saos-2 cells transfected with miR-212-3p inhibitor and TGF-β2 recombinant plasmids while the lowest in those with miR-212-3p mimics and TGF-β2 recombinant plasmids (P < 0.05).?Conclusions?The luciferase reporter plasmids of the 3'UTR of TGF-β2 gene could be successfully constructed, and miR-212-3p can bind to the 3'UTR of TGF-β2 gene to inhibit its luciferase activity, which suggests that TGF-β2 is a target gene of miR-212-3p.