RT Journal Article T1 Intracellular trafficking and functional monitoring of miRNA delivery in glioblastoma using lipopolyplexes and the miRNA-ON RILES reporter system. A1 Simion, Viorel A1 Henriet, Elodie A1 Juric, Viktorija A1 Aquino, Ruth A1 Loussouarn, Claire A1 Laurent, Yoan A1 Martin, Francisco A1 Midoux, Patrick A1 Garcion, Emmanuel A1 Pichon, Chantal A1 Baril, Patrick K1 Glioblastoma K1 Intracellular trafficking K1 Lipopolyplexes K1 P-bodies K1 miRNA monitoring system K1 microRNA AB MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy. YR 2020 FD 2020-08-24 LK http://hdl.handle.net/10668/16162 UL http://hdl.handle.net/10668/16162 LA en DS RISalud RD Feb 16, 2025