153 Enhanced extracellular vesicle–mediated delivery of stress-associated miRNAs in bovine granulosa cells

Extracellular vesicles (EVs) hold an emerging and pertinent role as cargo carriers, mediating cell-cell communication via the transfer of bioactive molecules (e.g. miRNAs). The detailed cargo contents of EVs reflect the physiological status of cells under various conditions of stress. Previous attempts in our laboratory revealed the enrichment of three miRNAs (bta-miR-1246, bta-miR-2904, and bta-miR-374a) to be released from in vitro-cultured granulosa cells exposed to thermal stress. Until now, the regulatory role of these EV-coupled stress-associated miRNAs in modulating stress response is not known. Here, we comparatively assessed three approaches (cellular overexpression, electroporation, and Exo-Fect™) to load the aforementioned candidate miRNAs (bta-miR, 1246, bta-miR-2904, and bta-miR374a) into EVs derived from in vitro-cultured bovine granulosa cells in order to elucidate their packaging capacity, delivery, and functional significance in naïve recipient granulosa cells to regulate predicted pathways. For this, candidate miRNA mimics (5 nmol) were preferentially loaded into EVs in equal volumes (v/v) containing ~2.95E+09 particles/mL. Exogenous loading via electroporation was performed under a high-voltage method (500 V for 10 ms) using a Gene Pulser Xcell™ Electroporation System (Bio-Rad), while Exo-Fect™ was carried out according to the manufacturer’s protocol. Endogenous or passive loading was performed via transfection of parent cells with miRNA mimics, followed by the collection of modified EVs Experiments were carried out over 4 biological replicates with statistical differences between loading types analysed using one-way analysis of variance followed by Tukey’s multiple comparisons test. Loading efficiency results assessed using droplet digital PCR indicate the highest miRNA loading from Exo-Fect™; bta-miR-1246 (>1.29-fold increase), bta-miR-2904 (>1461-fold increase), and bta-miR-374a (330-fold increase), compared with electroporation and cellular overexpression; bta-miR-1246 (>0.04; 0.002-fold increase), bta-miR-2904 (>45; 0.08-fold increase), and bta-miR-374a (>9.72; 0.59-fold increase), respectively, when evaluated against nonmodified EVs In terms of target delivery, a concurring trend with Exo-Fect™ modified EVs transferring the highest miRNA; bta-miR-1246 (>7.24-fold increase), bta-miR-2904 (>1.51-fold increase), and bta-miR-374a (>478-fold increase) compared with EVs modified via electroporation and cellular overexpression; bta-miR-1246 (>1.06; 1.01-fold increase), bta-miR-2904 (>0.94; 1.42-fold increase), and bta-miR-374a (>1.35; 0.66-fold increase), respectively. In silico analysis revealed a single pathway (MAPK signaling pathway) to be regulated by genes dually targeted by all three candidate miRNAs. Importantly, the MAPK signaling pathway plays a crucial role in stress regulation and activation of events including the induction of apoptosis or cellular senescence, as well as control adaptive responses to intracellular and extracellular stress. Collectively, the results obtained indicate that the Exo-Fect™ approach is superior in both loading efficiency and target delivery compared with both electroporation and cellular overexpression of stress-associated miRNAs. Further studies will aim to investigate the intracellular trafficking and bioavailability of target miRNAs using the aforementioned approaches, in the functional modulation of cellular response to stress.