Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans, France.
Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans, France.
J Control Release. 2020 Nov 10;327:429-443. doi: 10.1016/j.jconrel.2020.08.028. Epub 2020 Aug 24.
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.
微小 RNA (miRNA) 寡核苷酸治疗剂是治疗癌症的有效且有吸引力的药物，但它们在细胞内的转运、RISC 加工以及与靶 mRNA 的相互作用的动力学仍未得到很好的理解。此外，缺乏有效的载体阻碍了它们在临床上的转化。在这里，我们比较了用自制的脂多聚复合物（LPRi）或 RNAiMax 转染试剂转染 U87MG 神经胶质瘤细胞后，miRNA-133a 活性的动力学。为此，我们将共聚焦显微镜的 miRNA 细胞内转运研究与我们之前描述的在这里克隆到慢病毒表达载体中的 RILES miRNA-ON 报告系统（LentiRILES）相结合，用于转染细胞中 miRNA 活性的纵向分析。使用 LentiRILES 系统，我们报告了这两种转染试剂在 miRNA 传递动力学方面的显著差异。我们解析了 LPRi 传递 miRNA 的机制，并研究了 miRNA 内化和胞质加工的主要步骤。我们证明 LPRi 优先使用网格蛋白介导的内吞作用作为主要的内化途径，在孵育的前 3 小时后将 miRNA 释放到细胞质中，并将细胞质中的 miRNA 靶向 P 体，而一部分 miRNA 通过外泌体被运送到细胞外空间，外泌体被发现完全能够重新转染细胞。我们将 LentiRILES 细胞植入小鼠的大脑中，并通过对流增强递送方法将 LPRi.miRNA 递送到肿瘤中。活体小鼠的生物发光成像显示 miRNA 在神经胶质瘤肿瘤中的有效传递，证明了 miRNA 在体内的有效摄取、内化和 RISC 激活。总的来说，我们的研究提供了在神经胶质瘤背景下 miRNA 细胞内转运和加工的全面概述，并强调了 LPRi 在 miRNA 治疗中的潜在应用。
