Abstract 143: Exosomal miRNA Profiling Following Ischaemic Stroke

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Introduction: MicroRNA (miRNA) profiles are altered in patients with stroke. Recently, active inter-cellular transport of miRNAs in extracellular vesicles (EV), such as exosomes has been shown. We assessed whether miRNAs packaged in EVs differed in patients with stroke and patients without stroke and then performed in vitro and in vivo experiments to assess potential significance of dysregulated miRNAs.

Methods: We recruited patients with suspected stroke. Serum was obtained at 48h post-stroke. EVs were isolated from 200μL serum before RNA extraction. A miRNA microarray was performed (Openarray™ platform) (39 patients) to identify significantly dysregulated miRNAs in stroke vs non-stroke patients. Results were validated by Taqman™ real time quantitative polymerase chain reaction of specific miRNA (17 miRNAs, 169 patients). In vitro studies of selected miRNAs were performed on a rat neuronal cell line (B50) and a rat glial cell line (B92) subjected to 9h hypoxia (1% O2) and serum starvation with 24h reoxygenation in complete media (n=4-5/group). In vivo, total miRNA expression was quantified in serum (n=4) and in peri-infarct tissue of adult spontaneously hypertensive stroke-prone rats (SHRSP) following 45min transient middle cerebral artery occlusion (tMCAO) (n=6/group).

Results: The microarray identified 26 dysregulated miRNAs in stroke patients. Using Taqman™ levels of miRNAs 17, 20b and 93 were observed to be significantly (p<0.05) increased in stroke vs non-stroke patients. Patients with small vessel disease consistently had the highest levels. In vitro, a trend towards increased exosomal expression of miRNAs-17, 93 and 20b in B50 hypoxic cells was in contrast to small changes in total cell miRNA expression (vs. normoxic cells). In vivo, tMCAO did not induce significant modulation of miRNAs 17, 93 or 20b expression in SHRSP brain either 24h or 72h post-tMCAO. EVs isolated from the serum showed a significant increase in miRNA-93 expression at 72h post-tMCAO (p<0.05 vs. sham animals).

Conclusions: We have identified and validated changes in EV packaged miRNA expression in patients with stroke, particularly in small vessel disease. Our pre-clinical experiments corroborate the human data and support a functional role for these findings.

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