Background and Goal of Study:
MicroRNAs (miRNAs) provide posttranscriptional control of gene expression. These short, noncoding RNAs bind the 3’ untranslated region (3’UTR) of target messenger RNAs (mRNAs) causing mRNA degradation or translational inhibition. Recent studies demonstrate extensive miRNA changes following cerebral ischemia in animals and humans. To advance understanding individual miRNAs must be studied, and their targets identified. Since HSP70 chaperone family members and pro-survival members of the BCL2 apoptosis regulatory family are major protectors against stress induced cell death, we identified a miRNA that could influence these key proteins. The goal of this study was to validate potential targets of miR181 and assess the possible role of miR181 in outcome from ischemic brain injury.
Materials and Methods:
Using computational miRNA target prediction algorithms we identified potential miR-181 targets in 3’UTRs of HSP70 and BCL2 family members. Luciferase assays were used to confirm these targets. miRNA levels were assessed by quantitative RT-PCR, mRNA levels by RT-PCR, protein levels by western. Focal ischemia was transient suture occlusion of the middle cerebral artery (MCAO) in mice, infarct assessed by cresyl violet staining. miR181 levels were altered by intracerebroventricular injection of plasmid or antagomir. Primary cultures of astrocytes were used to assess the effect of miR-181 against in vitro ischemic injury.
Results and Discussion:
Luciferase assays confirmed that GRP78, the endoplasmic reticulum member of the HSP70 family, and antiapoptotic protein Bcl-2 are both targeted by miR-181, luciferase activity was reduced with wild type but not with mutated miRNA. In response to stroke miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive, while protein levels of GRP78 show inverse changes. Increasing miR-181 levels using plasmid overexpression increased infarct volume significantly in mouse stroke. Conversely, reducing levels using antagomir was associated with reduced injury. We previously found protection by overexpression of GRP78 or Bcl-2 was associated with reduced oxidative stress and preserved mitochondrial function. Protection due to reduction of miR-181 in vitro was accompanied by increased GRP78 and Bcl-2 protein levels, reduced oxidative stress assessed with the fluorescent dye hydroethidine, and better preserved mitochondrial membrane potential using the fluorescent dye tetramethylrhodamine methyl ester.
Levels of miR-181 change in response to stroke, and manipulating levels of miR-181 changes infarct volume. Reduced miR-181 levels are associated with increased GRP78 and Bcl2, and protection from ischemic injury in vivo and in vitro. miRNAs may provide a novel therapeutic strategy for stroke protection.
Supported by NIH grants NS 053898 and GM 49831.
© 2012 European Society of Anaesthesiology