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

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-β23'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-β23'UTR共转染组荧光素酶活性较对照组低(P<0.05)。miR-212-3p-mimics+TGF-β23'UTR共转染组Has-miR-212 mRNA相对表达量最高,miR-212-3p-inhibitor+TGF-β23'UTR共转染组最低(P<0.05)。miR-212-3p-mimics+TGF-β23'UTR共转染组TGF-β2 mRNA相对表达量最低,miR-212-3p-inhibitor+TGF-β23'UTR共转染组最高(P<0.05)。miR-212-3p-inhibitor+TGF-β23'UTR共转染组TGF-β2蛋白相对表达量最高,miR-212-3p-mimics+TGF-β23'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。

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 RTPCR 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.