基于双荧光素酶报告基因系统建立miR-140生物传感器  

Dual luciferase reporter system-based miR-140 sensor

作　　者：高原[1] 邹阳[2] 于影[2] 林金德[1] 张春丽[1] 温传俊[2] 沈干[3] 

机构地区：[1]南京医科大学附属友谊整形外科医院科教办,江苏南京210029 [2]南京师范大学生命科学学院分子细胞生物学研究所,江苏南京210023 [3]南京医科大学第二附属医院整形外科,江苏南京210003

出　　处：《东南大学学报（医学版）》2014年第2期133-137,共5页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金面上项目(81071480)

摘　　要：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-140sensor。其次,将miR-140 sensor和miR-140 mimics共转染HEK-293T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。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.

关 键 词：miR-140生物传感器 荧光素酶 实时荧光定量聚合酶链反应 成软骨诱导 

分 类 号：R789[医药卫生—口腔医学]