基于双荧光素酶报告基因系统建立miR-140生物传感器

目的：利用双荧光素酶报告基因系统构建可检测miR-140活性的生物传感器。方法：首先，在psiCHECK-2双荧光报告基因质粒的多克隆位点插入miR-140成熟体的4个拷贝反义序列，构建miR-140 sensor。其次，将miR-140 sensor和miR-140 mimics共转染HEK-293 T细胞，利用双荧光素酶报告基因系统检验miR-140 sensor的功能。最后，转染miR-140 sensor至大鼠骨髓间充质干细胞（ rat MSCs ），分析在成软骨诱导中miR-140的活性变化，并与miR-140的表达水平相比较。结果：在HEK-293T细胞中，相对于阴性对照组，miR-140 sensor与miR-140 mimics共转能明显降低49％（20 nmol· L-1）和65％（50 nmol· L-1）的荧光活性。将转染miR-140 sensor的rat MSCs成软骨诱导7 d后，荧光活性降低43％，提示miR-140活性升高，与RT-qPCR法检测的miR-140表达水平相一致。结论：构建的miR-140 sensor是一种简单方便有效的miRNA传感器，可用于检测miR-140活性。

Dual luciferase reporter system-based miR-140 sensor

Objective：To construct a dual luciferase reporter system based sensor for detecting miR-140 activity. Methods：Firstly, we inserted four copies of miR-140-5p complementary sequences into the multiple cloning site （MCS） of psiCHECK-2 luciferase reporter vector to construct miR-140 sensor.Subsequently, miR-140 sensor and miR-140 mimics were co-transfected into HEK-293T cells and its function was validated by luciferase reporter assay . Finally, miR-140 sensor was also transfected into rat bone marrow stromal cells （ rat MSCs ） and then miR-140＆amp;nbsp;activity was analyzed comparatively with the expression of miR-140 in chondrogenesis .Results：49% （20 nmol · L-1 ） and 65%（50 nmol· L-1 ） reporter activity was decreased in HEK-293T cells relative to NC, when miR-140 sensor was co-transfected with miR-140 mimics.An apparent decrease in reporter activity （43%） was observed at 7 days after chondrogenic induction of rat MSCs compared with control , which reflected an increased miR-140 activity and was consistent with the expression of miR-140 by RT-qPCR method .Conclusion：miR-140 sensor is proved to be an easy , convenient and effective miRNA sensor for detecting miR-140 activity.