In this study, we have provided the first evidence that genetic deletion and pharmacological inhibition of cerebral miR-15a/16-1 activity appears to reduce ischemic brain damage and improve neurological outcomes through anti-apoptotic and anti-inflammatory effects. pmol/g), were subjected to 1h of middle cerebral artery occlusion (MCAO) and 72h of reperfusion. The neurological scores, brain infarct volume, brain water content, and neurobehavioral tests were Isobavachalcone then evaluated and analyzed. To explore underlying signaling pathways associated with alteration of miR-15a/16-1 activity, major pro-inflammatory cytokines were measured by qPCR or ELISA and anti-apoptotic proteins were examined by western blotting. Results Genetic deletion of the miR-15a/16-1 cluster or intravenous delivery of miR-15a/16-1 antagomir significantly reduced cerebral infarct size, decreased brain water content and Rabbit Polyclonal to Uba2 improved neurological outcomes in stroke mice. Inhibition of miR-15a/16-1 significantly decreased the expression of the pro-inflammatory cytokines IL-6, MCP-1, VCAM-1 and TNF-, and increased Bcl-2 and Bcl-w levels in the ischemic brain regions. Conclusions Our data indicate that pharmacological inhibition of the miR-15a/16-1 cluster reduces ischemic brain injury via both upregulation of anti-apoptotic proteins and suppression of pro-inflammatory molecules. These results suggest that the miR-15a/16-1 cluster is a novel therapeutic target for ischemic stroke. manipulation of cerebral miR activity via intracerebroventricular or intravenous delivery of synthetic miR inhibitors and mimics has strengthened the rationale for the development of miR-based therapeutic drugs to reduce ischemic brain injury and promote post-stroke neurological recovery.9 MiR-15a and miR-16-1 are two highly conserved miRs that are located in a cluster 250bp apart on chromosome 13q14 in humans and only 54bp apart on chromosome 14 in mice. Generally, they act similarly by binding to their common mRNA targets, thus forming both a structural and functional cluster (the miR-15a/16-1 cluster).10, 11 The miR-15a/16-1 cluster was the first identified miR group associated Isobavachalcone with human carcinogenesis.10 Recently, dysregulation of plasma miR-15a/16-1 levels have been described in stroke patients,12 suggesting the potential for following this miR cluster as diagnostic and prognostic biomarkers. Beyond its use as biomarkers, inhibition of miR-15 has been shown to protect against myocardial infarction (MI),13 with strong findings that several pharmaceutical companies14 consider miR-15 one of the most important targets for miR-based drug development for improving post-MI recovery. However, the functional significance, molecular mechanisms, and future therapeutic potential of the miR-15a/16-1 cluster in stroke are poorly understood, and need further investigation. By using genetically-manipulated miR-15a/16-1 knockout mice and pharmacological intervention of cerebral miR-15a/16-1 levels, we have identified that cerebral miR-15a/16-1 functions as a critical negative regulator in ischemic stroke. Furthermore, we demonstrated that the miR-15a/16-1 cluster inhibits the anti-apoptotic genes bcl-2 and bcl-w, as well as promotes inflammatory responses to trigger ischemic brain damage. Materials and Methods All procedures using laboratory animals were approved by the University of Pittsburgh Institutional Animal Care and Use Committee, and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were randomly assigned to various experimental groups using a lottery box. All stroke outcome assessments were performed in blinded manner. Animal models Male homozygous miR-15a/16-1 knockout mice (kindly provided by Dr. Riccardo Dalla-Favera)11 and littermate wild-type controls, or male C57BL/6J mice (8-10w, 23-25g; Jackson Laboratory) were subjected to middle cerebral artery occlusion (MCAO) for 1h and followed by 1-7d reperfusion.8, 15 Regional cerebral blood flow was measured before, during and after MCAO. Animals that did not show a more than 75% CBF reduction or a less than 60% CBF reperfusion over baseline levels or died after ischemia induction (10% of stroke animals) were excluded from further experimentation. Intravenous administration of miR-15a/16-1 antagomir Immediately after onset of MCA occlusion or at 2h reperfusion after 1h MCAO, mice were injected with miR-15a/16-1 specific inhibitor/antagomir (a mixture of miR-15a and miR-16-1 antagomirs each at final concentration of 30 pmol/g) or scramble control (30 pmol/g) by tail vein.16 Sham-operated mice were also treated with either miR-15a/16-1 antagomir or scramble control. All mice were sacrificed 1-7 d after MCAO. Measurement of infarct volume, edema, neurological deficit and sensorimotor function After MCAO, brain slices were stained with 2% TTC to calculate the infarct volume. 8, 15 Brain water content was measured by using a dry-wet method.17 Neurological deficits were examined and scored on a 5-point scale.8, 15 Neurobehavioral outcomes were also determined by the rotarod test, foot fault test and adhesive tape removal test at Isobavachalcone 1, 3, 5 and 7 d after MCAO.15, 18 Real time PCR and TaqMan? miRNA assay IL-6, MCP-1, VCAM-1 and TNF- mRNAs were detected by a quantitative PCR.15 MiR-15a, miR-16-1 and miR-21 expressions were determined by TaqMan? MicroRNA Assay.8, 15 Western blot analysis and ELISA Total protein was isolated from mouse brains. The protein levels of Bcl-2, Bcl-w, Bcl-xl and -actin were determined by Western blot analysis. The concentrations of IL-6, MCP-1, VCAM-1 and TNF- were measured by ELISA kits. Statistical analysis Quantitative data were expressed as mean SEM..