靶向FHL1的microRNA初步筛选研究

发布时间：2018-01-19 07:20

  本文关键词： FHL基因 荧光素酶报告基因 转录活性　出处：《军事医学》2013年06期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to construct a luciferase reporter gene vector containing the FHL1 gene 3r-UTR region. MicroRNAs and miRNAs interacting with them were detected. Methods the FHL1 gene 3- UTR region was amplified by PCR. Inserted into the modified luciferase reporter gene vector pcDNA 3.0; The Target Scan5.1 software was used to predict the miRNA that might interact with the 3UTR region of the FHL1 gene. MiRNA and luciferase reporter recombinant were co-transfected into 293T cells to detect their fluorescence activity. In order to detect the fluorescence activity of miRNA, we constructed the mutant of miRNA with obvious changes in fluorescence activity. Results the sequencing results showed that. The luciferase reporter gene vector containing the FHL1 gene 3 + -UTR region was constructed correctly. The results of luciferase activity test showed that miR-200cn miR-146a was higher than that of control group. MiR-146b-5p could reduce the luciferase activity of luciferase report recombinant by about 40%, and construct the miRNA deletion mutants mentioned above. Conclusion the luciferase reporter gene vector of FHL1 gene 3UTR was constructed successfully, and miR-200cnmmiR-146a was successfully constructed. MiR-146b-5p can inhibit the luciferase activity of FHL1 gene, and miR-200c may directly act on the 3- UTR region of FHL1 gene.
【作者单位】： 广西医科大学;军事医学科学院放射与辐射医学研究所;军事医学科学院生物工程研究所;
【基金】：中国博士后科学基金面上资助项目(201150M1523)
【分类号】：R346
【正文快照】： FHL1属于FHL(41/2骜骀LIM结构域,four and ahalf LIM domain)家族,该家族共有5个成员,即FHL1、FHL2、FHL3、FHL4、FHL5/ACT(activator ofCREM intestis),其蛋白结构中含有4骀骜LIM结构域,属于LIM超家族。LlM是3种转录因子Lin-11、Isl-1和Mec-3的缩写,由约55个氨基酸残基组成,，

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