基于双荧光素酶报告基因系统建立miR-140生物传感器-东南大学学报医学版

基于双荧光素酶报告基因系统建立miR-140生物传感器

单位：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

分类号：R789

出版年·卷·期（页码）：2014·33·第二期（133-137）

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-140 sensor。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 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.

参考文献：

[1] NAKASA T,NAGATA Y,YAMASAKI K,et al.A mini-review:microRNA in arthritis[J].Physiol Genomics,2011,43(10):566-570.
[2] CHI S W,HANNON G J,DARNELL R B.An alternative mode of microRNA target recognition[J].Nat Struct Mol Biol,2012,19(3):321-327.
[3] KROL J,LOEDIGE I,FILIPOWICZ W.The widespread regulation of microRNA biogenesis,function and decay[J].Nat Rev Genet,2010,11(9):597-610.
[4] IORIO M V,CROCE C M.MicroRNA dysregulation in cancer:diagnostics,monitoring and therapeutics.A comprehensive review[J].EMBO Mol Med,2012,4(3):143-159.
[5] 毛安琼,姚怡,段晓霞,等.MicroRNA与心血管疾病[J].医学综述,2012,18(6):801-804.
[6] PALL G S,HAMILTON A J.Improved northern blot method for enhanced detection of small RNA[J].Nat Protoc,2008,3(6):1077-1084.
[7] KIM S W,LI Z,MOORE P S,et al.A sensitive non-radioactive northern blot method to detect small RNAs[J].Nucleic Acids Res,2010,38(7):e98.
[8] ZHAO B,JIN L,WEI J,et al.A simple and fast method for profiling microRNA expression from low-input total RNA by microarray[J].IUBMB Life,2012,64(7):612-616.
[9] BENES V,CASTOLDI M.Expression profiling of microRNA using real-time quantitative PCR,how to use it and what is available[J].Methods,2010,50(4):244-249.
[10] 景花,宋沁馨,周国华.MicroRNA定量检测方法的研究进展[J].遗传,2010,32(1):31-40.
[11] CHENG A M,BYROM M W,SHELTON J,et al.Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis[J].Nucleic Acids Res,2005,33(4):1290-1297.
[12] HUANG J,ZHAO L,XING L,et al.MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation[J].Stem Cells,2010,28(2):357-364.
[13] GAROFALO M,CONDORELLI G L,CROCE C M,et al.MicroRNAs as regulators of death receptors signaling[J].Cell Death Differ,2009,17(2):200-208.
[14] 吴春彪,陆军,王宸.晚期糖基化终末产物在骨关节炎中的研究进展[J].现代医学,2012,40(3):356-360.
[15] MIYAKI S,NAKASA T,OTSUKI S,et al.MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses[J].Arthritis Rheum,2009,60(9):2723-2730.
[16] MIYAKI S,SATO T,INOUE A,et al.MicroRNA-140 plays dual roles in both cartilage development and homeostasis[J].Genes Dev,2010,24(11):1173-1185.

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