Abstract SNACC-80

Return to Poster Listing

Inhibition of miR-200c Decreases Injury Severity and Improves Neurological Outcome Following Transient Focal Cerebral Ischemia

Stary C, Xu L, Sun X, Leong J, White R, Ouyang Y, Giffard R
Stanford University, Stanford, CA, USA

Introduction: Stroke remains a leading cause of death and disability. MicroRNAs (miRs) are endogenous, non-coding RNAs that regulate gene expression by inhibiting translation of target mRNAs. miR-200c increases in the brain following transient cerebral ischemia, however its role in post-stroke brain injury is unclear. In other organ systems, miR-200c contributes to cell death by silencing pro-survival genes. We hypothesized that inhibiting the post-stroke increase in miR-200c would minimize infarct volume and improve neurological outcome following transient focal cerebral ischemia.

Methods: The contribution of miR-200c to injury following transient cerebral ischemia was examined in vivo by subjecting male CB57/B6 mice (n = 11-23) to 1 hr of middle cerebral artery occlusion (MCAO) following pre-treatment with intracerebroventricular (ICV) infusion of miR-200c antagomir, mimic or control. Infarct volume and neurological score were assessed following 24 hrs of reperfusion. Next, we assessed the role of miR-200c in neuronal cell death in an in vitro model of reperfusion injury. LDH release was assessed in N2a cells transfected with miR-200c mimic/inhibitor and subjected to 24 hrs serum deprivation + 500 uM H202. Finally we validated a functional role in neuronal cell death for a predicted target of miR-200c, the neurotrophic protein reelin, by: 1) dual luciferase assay; 2) assessing the post-MCAO time-course of reelin protein expression in ICV pre-treated mice; and, 3) examining the effect of reelin mRNA knockdown with siRNA on neuronal cell survival following in vitro reperfusion injury.

Results: Relative to sham-surgery treated animals, in brains of mice subjected to MCAO miR-200c significantly increased (18.8±6.9 fold) by 1 hr of reperfusion, which was blocked by antagomir pre-treatment (0.18±0.5 fold). miR-200c antagomir resulted in a significant decrease in infarct volume by 34±12%, and improved 24 hr neurological score. Pre-treatment with mimic had no significant effect on either outcome measure. MCAO alone caused a significant decrease in brain levels of reelin protein expression (0.81±0.06 fold), which was reversed by antagomir pre-treatment (1.38±0.05 fold). Targeting of reelin by miR-200c was verified with dual luciferase assay whereby reelin-reporter activity decreased following co-transfection with wild-type miR-200c (0.40±0.04 fold) but not seed mutant control (1.09±0.18 fold). Treatment of N2a cells with miR-200c mimic increased cell death relative to control (130±3%), while inhibitor was protective (78±2%). Knockdown of reelin mRNA abolished the protective effect of miR-200c inhibitor and approximated cell death with mimic (132±9.5%).

Conclusions: These findings suggest that reducing the post-stroke increase in miR-200c is protective, due at least in part to decreasing targeted inhibition of reelin by miR-200c. Future directions to determine the therapeutic potential of post-stroke miR-200c inhibition will include exploring less invasive methods to deliver miR-based treatments and examining the effect of miR-200c inhibition on long-term neurobehavioral outcome.

Supported by the Stanford Department of Anesthesia’s T32 Training Grant NIH GM089626 to CMS and by NIH Grants NS080177 and NS084396 to RGG.

Back to Top