108 Preferential loading of thermal stress-associated microRNAs into extracellular vesicles: attempt to mitigate effects of heat stress in bovine granulosa cells

Increases in environmental temperatures, possibly associated with climate change, negatively affect physiological processes of reproductive function in dairy and beef cattle. Increasingly found in almost all biological fluids, extracellular vesicles (EVs) are broadly defined as conserved, bilipid, membrane-bound complexes that serve as a mass transit mechanism for cargos such as microRNA (miRNAs), mRNA, proteins, and lipids. More recently, EVs’ natural role in genetic information transfer has propagated their substantial interest as agents mediating multidirectional cellular crosstalk. Previously, it has been shown that granulosa cells (GC) subjected to heat stress (HS) are enriched with the miRNAs bta-miR-1246, bta-miR-374a, and bta-miR-2904. However, the specific role of those miRNAs in inducing a protective response has not yet been elucidated. Therefore, here we aimed to preferentially load these specific candidate miRNAs within GC-derived EVs, using electroporation, kit-based assays, and cellular overexpression. Electroporation of miRNA mimics was performed under a high-voltage method (500 V for 10 ms) using 1.5 µL of miRNA mimics, 200 µL of EVs and 800 µL of electroporation buffer. Similarly, the Exo-Fect™ siRNA/miRNA Transfection Kit was used according to the manufacturer’s protocol, for targeted loading of miRNA mimics in GC-derived EVs. Finally, in vitro-cultured bovine GCs were transfected using the candidate miRNA mimics as a means to imitate mature miRNA duplexes, needed for cellular overexpression, followed by isolation of the EVs from the conditioned medium post-overexpression. The loading efficiency of each method was then evaluated using real-time quantitative PCR. Accordingly, miR-1246 was abundantly detected in both electroporated EVs and their non-electroporated counterparts, leading to decreased efficiency due to a high preexisting copy number of miR-1246 within the EVs. In contrast, electroporation of miR-374a into EVs resulted in subsequent enrichment within electroporated EVs, compared with the non-electroporated counterparts. Alternatively, each specific miRNA was preferentially taken up and increased, compared to respective controls, when EVs were loaded through the Exo-Fect siRNA/miRNA Transfection kit: miR-1246 (2-fold increase), miR-374a (2-fold increase) and miR-2904 (>2-fold increase). Interestingly, GCs overexpressed with the three candidate miRNAs released EVs enriched with those miRNAs compared with EVs from GCs transfected with a respective mimic negative control. In conclusion, the loading efficiency of miRNAs into EVs is dependent on the amount of preexisting miRNA within the EVs, and the loading method of choice. The role of these specific miRNA-loaded EVs in enhancing stress tolerance in recipient GCs will be elucidated in the future.