RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression.
RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression.
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DOI：
10.1093/nar/gkt797
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发表时间：
2013-11
影响因子：
14.9
通讯作者：
Baril P
中科院分区：
文献类型：
--
作者：
Ezzine S;Vassaux G;Pitard B;Barteau B;Malinge JM;Midoux P;Pichon C;Baril P
需要新的方法来研究microRNA（miRNA）生物学的复杂性，特别是它们在生理病理条件下的动态调节。本文中，一种新的基于质粒的RNAi诱导荧光素酶表达系统（RILES）被工程化以监测内源性RNAi机制的活性。当RILES在靶细胞中转染时，感兴趣的miRNA抑制转录阻遏物的表达，并因此开启荧光素酶报告基因的表达。因此，细胞中的miRNA表达通过生物发光信号的发射来标记，所述生物发光信号可以使用标准生物发光设备来监测。我们通过监测小鼠骨骼肌中myomiRs-133，-206和-1以及肝脏中miRNA-122的表达来验证这种方法。生物发光实验证明了与通过定量RT-PCR（qPCR）检测的miRNA表达模式相关的稳健的定性和定量数据。我们进一步证明了在肌萎缩的发展过程中miRNA-206表达的调节是个体依赖性的，时间调节的，并且比qPCR产生的信息更复杂。由于RILES是简单和通用的，我们相信，这种方法将有助于更好地了解miRNA生物学，并可以作为一个合理的开发新一代的可调控的基因表达系统与潜在的治疗应用。
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, −206 and −1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.
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影响因子：
14.9
作者：
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通讯作者：
Searles CD
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