Objective To evaluate the effect of microRNA (miRNA) on the activity of the 3' untranslated region (3'-UTR) of four and a half LIM domaim (FHL) 1 gene.Methods The 3'-UTR of FHL1 fragment amplified by PCR was cloned into luciferase-modified pcDNA 3.0 vector(pcDNA 3.0-luc).The miRNA targeting FHL1 3'-UTR was predicted by Target Scan5.1 and miRanda software.The reporter activity was quantitated after the luciferase promoter vector and miRNA eukaryotic expression vector were transferred into 293T cell line.Results DNA sequencing showed that the sequences of the cloned regions were correct.The luciferase activity of the reporter contruct treated with miR-200c,miR-146a and miR-146b-5p was decreased respectively by about 40％ compared with control,and miR-200c could reverse the luciferase activity after the absence mutants were cloned.Conclusion The FHL1 3'-UTR luciferase reporter vector has been successfully constructed.The luciferase activity of the reporter gene can be repressed by miR-200c、miR-146a and miR-146b-5p,and miR-200c may have direct effect on FHL1 3'-UTR.%目的 构建含有FHL1基因3'-UTR区的荧光素酶报告基因载体,用以检测与其相互作用的microRNA(miRNA).方法 PCR扩增出FHL1基因3'-UTR区片段,插入到经过改造的荧光素酶报告基因载体pcDNA 3.0中；利用Target Scan5.1软件预测可能与FHL1基因3'UTR区相互作用的miRNA,将miRNA与荧光素酶报告重组子共转染至293T细胞中,检测其荧光活性；构建针对荧光活性结果变化明显的miRNA的种子区缺失突变体,检测荧光活性.结果 测序结果表明,含有FHL1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,miR-200c、miR-146a、miR-146b-5p可使荧光素酶报告重组子的荧光素酶活性降低40％左右,并构建上述miRNA缺失突变体,miR-200c使荧光素酶活性回复.结论 成功构建了FHL1基因3'UTR区的荧光素酶报告基因载体,而miR-200c、miR-146a、miR-146b-5p可以抑制其荧光素酶活性,其中miR-200c可能直接作用于FHL1基因3'-UTR区.
展开▼
