title: 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.
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans and Inserm, Orléans, France, UMRE 4320, Faculté de Médecine, Université de Nice-Sophia-Antipolis, Nice, France, Inserm UMR 1087/CNRS UMR 6291, Université de Nantes, Faculté de médecine, L'institut du Thorax, Nantes F-44000 and In-Cell-Art, Nantes F44200, France.
authors.forename: Safia
authors.initials: S
authors.lastname: Ezzine
authors.forename: Georges
authors.initials: G
authors.lastname: Vassaux
authors.forename: Bruno
authors.initials: B
authors.lastname: Pitard
authors.forename: Benoit
authors.initials: B
authors.lastname: Barteau
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Malinge
authors.forename: Patrick
authors.initials: P
authors.lastname: Midoux
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
authors.forename: Patrick
authors.initials: P
authors.lastname: Baril
mesh_terms: D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007121:Immunocompetence; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D009133:Muscular Atrophy / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051598:Whole Body Imaging
chemical_list: D035683:MicroRNAs; D008156:Luciferases