TGFβR3基因3’UTR双荧光素酶报告载体的构建及其与miR-let-7a靶向关系的验证

Construction of TGFβR3 gene 3'UTR dual luciferase reporter vector and targeting verification between TGFβR3 and miR-let-7a

目的:构建Ⅲ型转化生长因子β受体(TGFβR3)基因3’非编码区(3’UTR)双荧光素酶报告载体,并分析其与miR-let-7a的靶向关系。方法:根据micro RNA靶基因预测软件targetscan获取miR-let-7a与TGFβR3基因3’UTR潜在的互补结合位点;PCR扩增出TGFβR3基因3’UTR序列并克隆至pGEM-T载体,定点突变潜在互补结合位点后双酶切pGEM-T载体获取目的片段并插入至双荧光素酶报告载体获得野生型和突变型重组双荧光素酶报告载体,测序鉴定;将2种载体分别与miR-let-7a mimics或miR-let-7a NC共同转染HEK293T细胞,收集细胞后通过双荧光素酶报告系统检测各组荧光素酶活性。结果:经测序鉴定成功构建野生型和突变型重组荧光素酶报告载体。双荧光素酶报告系统检测显示,共转染miR-let-7a mimics和野生型双荧光素酶报告载体的荧光素酶活性比miR-let-7a NC组明显降低(P<0.05),而共转染突变型双荧光素酶报告载体后其荧光素酶活性差异无统计学意义(P>0.05)。结论:成功构建TGFβR3基因3’UTR双荧光素酶报告载体,并证实TGFβR3基因是新发现的miR-let-7a直接靶向调控基因。

Objective： To construct a luciferase reporter vector containing the 3’untranslated region （3’UTR） of TGFβR3 gene and evaluate the targeting correlation between TGFβR3 and miR-let-7a. Methods： The potential complementary binding sites of miR-let-7a and TGFβR3 were predicted by targetscan. The 3’UTR fragment of TGFβR3 amplifed by PCR was frstly cloned into pGEM-T vector and the potential complementary binding sites was mutated. Then, the amplifed fragment and the mutated fragment of TGFβR3 3’UTR were sub-cloned into pmirGLO dual-luciferase miRNA target expression vector within the PmeI/SalI restriction sites. The luciferase reporter vector and miR-let-7a mimics/miR-let-7a NC were co-transfected into HEK293T cells respec-tively. The relative luciferase activity was detected by luciferase reporter assay. Results： Results of double en-zyme digestion and DNA sequencing confrmed that the luciferase reporter vector was successfully constructed. The relative luciferase activity of TGFβR3 3’UTR reporter vector and miR-let-7a mimics co-transfected group were decreased signifcantly comparing with miR-let-7a NC group （P〈0.05）, but the relative luciferase activity was no signifcance in TGFβR3 3’UTR mutated reporter vector. Conclusion： The luciferase reporter vector con-taining the 3’UTR-WT or 3’UTR-MUT of TGFβR3 is constructed successfully. And TGFβR3 is a direct novel target gene of miR-let-7a.