High-throughput Sequencing Revealing Differential Expression of Exosomal miRNAs Derived from Rat Cortical Neurons Under Ischemia/reperfusion Injury
Sung-Chun. Tang, Chien-Sung. Chiang, Jiann-Shing. Jeng.
National Taiwan University Hospital, Department of Neurology, Taipei, Taiwan 

Abstract Text
Background- Stroke involves in multiple cellular signaling pathways that leads to neuronal death. Exosomes are implicated in mediating interactions with cellular environment via their cargos of proteins, lipids, mRNAs and miRNAs. Our study investigates changes of exosomal miRNAs from primary cortical neurons with oxygen and glucose deprivation (OGD) injury.

Method- Exosomes were isolated from the medium of rat primary cortical neurons. The expression alterations of exosomal miRNAs before and after OGD injury were analyzed via next generation sequencing.We defined cut-off > 100 normalized reads and |log2 (mean fold change)| >1 as significant changes associated with OGD. 

Result-The purified exosomes were examined by transmission electron microscopy, which showed typical round particles ranging from 50 to 200 nm in diameter. Immunoblot analysis of the nanovesicle precipitates detected CD63 and Hsp70, selective exosome markers, validating the successful recovery of exosomes. miRNA-enrichment analysis identified a total of 31 dysregulated miRNAs between normoxia and post OGD injury, of which 29 and 2 miRNAs were found to be significantly upregulated and downregulated, respectively. Furthermore, MiR-26b-5p stood out as one of the most significantly upregulated miRNAs and the finding has been validated via repeated miRNA-seq, real-time PCR.

Conclusion- There were significant changes of specific miRNAs in neuronal exosomes in response to OGD/R and their functional roles in ischemic brain injury warrant further investigation.

Keywords stroke, exosome, neuron