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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。
Objective To construct a luciferase reporter vector containing the 3’-UTR region of FHL1 gene to detect microRNAs (miRNAs) interacting with it. Methods The 3’-UTR region of FHL1 gene was amplified by PCR and inserted into pcDNA 3.0, a luciferase reporter gene vector. Target Scan5.1 software was used to predict miRNAs that might interact with the 3’UTR region of FHL1 gene. miRNA and luciferase reporter recombinants were co-transfected into 293T cells to detect the fluorescence activity of the recombinant plasmids. A mutation in the seed region of the miRNA with obvious change in fluorescence activity was constructed to detect the fluorescence activity. Results The sequencing results showed that the luciferase reporter gene vector containing 3’-UTR region of FHL1 gene was constructed correctly. The luciferase activity assay showed that compared with the control group, miR-200c, miR-146a and miR-146b-5p Luciferase reporter recombinants reduce the luciferase activity by about 40%, and construct miRNA deletion mutants, miR-200c luciferase activity. Conclusion The luciferase reporter gene vector of 3’UTR region of FHL1 gene was successfully constructed and miR-200c, miR-146a and miR-146b-5p could inhibit the luciferase activity. MiR-200c may directly affect the FHL1 gene 3’-UTR region.