MicroRNAs (miRNA) are short (~22 nt) single stranded RNAs that downregulate gene expression. Although recent studies indicate extensive miRNA changes in response to ischemic brain injury, there is currently little information on the roles of specific miRNAs in this setting. Heat shock proteins (HSP) of the HSP70 family have been extensively studied for their multiple roles in cellular protection, but there is little information on their regulation by miRNAs. We used bioinformatics to identify miR-181 as a possible regulator of several HSP70 family members. We validated GRP78/BIP as a target by dual luciferase assay. In response to stroke in the mouse we find that miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive. Increased levels of miR-181a are associated with decreased GRP78 protein levels, but increased levels of mRNA, implicating translational arrest. We manipulated levels of miR-181a using plasmid overexpression of pri-miR-181ab or mimic to increase, and antagomir or inhibitor to reduce levels. Increased miR-181a exacerbated injury both in vitro and in the mouse stroke model. Conversely, reduced levels were associated with reduced injury and increased GRP78 protein levels. Studies in C6 cells show that if GRP78 levels are maintained miR-181a no longer exerts a toxic effect. These data demonstrate that miR-181 levels change in response to stroke and inversely correlate with levels of GRP78. Importantly, reducing or blocking miR-181a protects the brain from stroke.

Mitochondria are central to the execution of apoptosis, and the Bcl-2 protein family of pro- and anti-apoptotic proteins interacts with mitochondria to regulate apoptosis. Using bioinformatics we predicted that miR-181, a microRNA expressed in brain, could target the 3′UTRs of Bcl-2 family members Bcl-2-L11/Bim, Mcl-1, and Bcl-2. Using the luciferase reporter assay we confirmed these targets. We used mimic and inhibitor to alter miR-181a levels in primary astrocyte cultures and found miR-181a reduction was associated with increased Bcl-2 and Mcl-1 protein levels. Decreased miR-181a levels reduced glucose deprivation induced apoptosis, mitochondrial dysfunction, and loss of mitochondrial membrane potential in astrocytes.