Background: Micro RNAs (miRNAs) could target multiple mRNAs, repressing the protein translation. We report acute changes in humoral miRNAome in a murine thromboembolic stroke model (eMCAo), and demonstrate the benefits of miRNA therapy in improving cerebral blood flow (CBF).
Methods: Non-biased micro RNA (miRNA) array and bioinformatics analysis was performed in plasma collected at 4h post-eMCAo from male mice (C57/B6, 16-weeks). Individual PCR for miRNAs was also performed in brain tissues at 24h post-eMCAo. Moreover, frozen human plasma samples collected at ~4.5h post-stroke were also used for miRNA analysis. Finally, the miRNA mimic that was predicted to target genes of our interest was also tested in vivo and in vitro.
Results: Principal component analysis (PCA) of the miRNA-array showed ~68% variance in the humoral miRNAome 4h after eMCAo in mice, and a significant change in Stroke vs. Sham groups (Cut off value >2 fold; p<0.05). Of interest, the hairpin precursor of miR-449b was downregulated (~2.35 fold, p<0.05) at 4h post-eMCAo, while the mature miR-449b was also significantly reduced at 24h post-eMCAo. Mature miR-449b was significantly reduced in human stroke plasma, too. In human brain endothelial cells, miR-449b mimic downregulated gene expressions of both plasminogen activator inhibitor (PAI-1) and alpha 2- antiplasmin (α-AP) only in hypoxia but not during normoxia. Therefore, we finally tested the cholesterol-conjugated miR-449b mimic in the murine eMCAo model. Pre-treatment with miR-449b mimic (8 mg/kg bwt) increased the absolute CBF and reduced edema (as determined by MRI), and also improved the neurological outcomes and reduced % infarct volume (p<0.05).
Results: The miR-449b mimic could be a possible therapy to suppress aberrant gene expressions of PAI-1 and α-AP, which will allow more spontaneous reperfusion and benefits from low dose tPA.