In recent years, research has explored the use of microRNA (miRNA) analysis in extracellular vesicles (EVs) as a minimally invasive strategy for the diagnosis and prediction of diseases. This is because miRNAs in EVs partly reflect the miRNA information and cellular status of the origin cells. However, not all intracellular miRNAs are internalized into EVs. Therefore, the miRNA information obtained from EVs is limited. To get more miRNA information, researchers at the National Institute of Advanced Industrial Science and Technology, Japan aimed to produce artificial EVs (aEVs) encapsulating Argonaute 2 (Ago2) miRNA-binding protein, which actively incorporate miRNAs within themselves. In this study, the researchers utilized the protein EPN-01, which is capable of releasing aEVs encapsulating it and associated proteins. This system enables them to obtain more miRNA species and increase each miRNA’s yield in the EV fraction. Furthermore, they examined whether miRNAs in the EV fraction using their system reflect the cellular condition. In cells treated with CoCl2, a reagent for inducing a hypoxia-mimic state, the researchers detected a change in the level of hypoxia marker miR-210 with aEVs. To the best of their knowledge, this is the first report on a method to increase the yield and variety of endogenous miRNAs in the EV fraction. This approach leads to improved accuracy of cell status assessment using miRNAs in EVs.
Schematic overview of the production of miRNA-containing extracellular vesicles
(A) EPN-01 nanocages containing miRNA-binding protein fused with Vpr and miRNAs budding at the plasma membrane. (B) Schematic of Ago2 truncation mutants fused with Flag-tag and Vpr. (C) Immunoblot analysis showing Ago2-FL and the truncated mutants fused with Vpr and Flag-tag and EPN-01 (Myc tag) harvested from transfected HEK293T cells (Cell) or the EV fraction of the cell culture supernatants (EVs). (D) HEK293T cells transfected with EGFP, Flag-Ago2-Vpr or Flag-MID-PIWI-Vpr plasmids were immunoprecipitated (IP) and analyzed by immunoblot analysis. (E) Relative level of let7a-5p co-immunoprecipitated with Ago2 or its truncated mutant MID-PIWI fused with Flag-tag and Vpr. Data shown were calculated using the comparative CT (ΔΔCT) method. After normalization to the input, the data were expressed relative to the mean of the control, which was then normalized to 1; results are representative of three independent experiments. Error bars represent the standard deviation. Statistical analysis was performed by one-way ANOVA and Tukey’s test. **p < 0.01.