RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression

Abstract

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.

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy

“…Such a saturation of the read-out signals in the pRILES transfected cells was not observed in our previous study [18]. To investigate this point, we made a dual comparison between the RILES system that employed the luciferase gene as reporter gene and the RINES system that employed hNIS as reporter gene.…”

MicroRNA-Based Drugs for Brain Tumors

“…This system uses a bacterial transcriptional repressor, Cysteine metabolism repressor (CymR), to suppress a gene-of-interest under the control of an operator sequence. By introducing a miRNA target sequence in the CymR-gene, CymR expression can be reduces in a specific miRNA rich environment, which in-turn leads to the expression of the gene-ofinterest [79]. Zika virus has shown glioblastoma stem-like cells (GSC)-specific replication and oncolytic activity [80].…”

Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

“…Recently, we [ 85 ] and other teams [ 86 , 87 ] developed an alternative approach to monitor positively the endogenous expression pattern of miRNAs in cells in vitro and in vivo . The systems are based on engineered regulatable expression systems, also known as genetic switches, such as the Tet-Krab, Tet R [ 88 ] and the Cumate [ 89 ] gene-switch systems.…”

“…Depending on the nature of the regulatable expression system used, the transgene expression can therefore be turned “ON” or “OFF” by adding the inducer in the culture media or in the drinking water of mice. We [ 85 ] reasoned that placing expression of the transcriptional regulator directly under the control of the endogenous RNAi machinery rather than an exogenous molecule would be an alternative way to turn-ON expression of the transgene. Consequently, if a reporter gene was used as a transgene, the system would generate optical signals reflecting the expression pattern of miRNAs.…”