miRNA Profiling of the Ischaemic Penumbra

MicroRNAs (miRNAs) are small non-coding RNA molecules (20 - 24 nucleotides) that inhibit mRNA translation. Demonstrated to have key roles in normal CNS development & function, they have also emerged to have effecter roles in the pathogenesis & endogenous repair mechanisms following stroke. To select 2 miRNAs to modulate therapeutically we profiled miRNA expression of the evolving (24h) & final (72h) peri-infarct tissue of adult spontaneously hypertensive stroke-prone rats (SHRSP) following 45 min transient middle cerebral artery occlusion (tMCAO). T2-weighted magnetic resonance imaging (MRI) was used for accurate dissection of the peri-infarct tissue, with equivalent brain regions taken from time-matched shams (n=6/group). Of the 754 miRNAs evaluated (TaqMan® human miRNA microarray card v3.0 Applied Biosystems) 89 were determined as differently regulated following tMCAO. 22 of these miRNAs were relevant in stroke & were thus validated by Taqman® qRT-PCR using specific probes (n=9 /group). 5 miRNAs were successfully validated; miR-34b & miR-520b were selected as miRNAs of interest due to their novelty, time of endogenous regulation & targets. An in vitro study to determine whether upregulation/knock-down of these miRNAs would demonstrate functional effects in classical hypoxic pathways was performed. A rat neuronal cell line (B50) & glial cell line (B92) were subjected to 9hr hypoxia (1% O2 -serum) & 24h reoxygenation (+serum) +/- miR-34b or miR-520b regulation. Upregulation of either miRNA in B50 cells demonstrated a reduction in apoptosis, assessed qualitatively by Caspase-3 immunocytochemistry & quantitatively by cell death detection ELISA (p<0.01 vs hypoxic non-treated cells (NTC)). Upregulation of either miRNA in B92 cells significantly reduced superoxide production, assessed by electron paramagnetic resonance (p<0.001 vs hypoxic NTC). MiR-520b significantly lowered levels of lipid peroxidation in B92 cells, assessed by malondialdehyde assay, & both were significantly effective in B50 cells (p<0.01 vs hypoxic NTC). These data suggest miR-34b & -520b upregulation ameliorates damage following hypoxia/reperfusion in cerebral cell lines. Future studies will assess the effect of modulating these miRNAs in vivo.