MicroRNA-200c contributes to injury from transient focal cerebral ischemia by targeting Reelin

Abstract

Background and purpose: MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin.

Methods: Brain infarct volume, neurological score and levels of miR-200c, reelin mRNA, and reelin protein were assessed in mice subjected to 1 hour of middle cerebral artery occlusion with or without intracerebroventricular infusion of miR-200c antagomir, mimic, or mismatch control. Direct targeting of reelin by miR-200c was assessed in vitro by dual luciferase assay and immunoblot.

Results: Pretreatment with miR-200c antagomir decreased post-middle cerebral artery occlusion brain levels of miR-200c, resulting in a significant reduction in infarct volume and neurological deficit. Changes in brain levels of miR-200c inversely correlated with reelin protein expression. Direct targeting of the Reln 3' untranslated region by miR-200c was verified with dual luciferase assay. Inhibition of miR-200c resulted in an increase in cell survival subsequent to in vitro oxidative injury. This effect was blocked by knockdown of reelin mRNA, whereas application of reelin protein afforded protection.

Conclusions: These findings suggest that the poststroke increase in miR-200c contributes to brain cell death by inhibiting reelin expression, and that reducing poststroke miR-200c is a potential target to mitigate stroke-induced brain injury.

Keywords: infarction, middle cerebral artery; microRNAs; reperfusion injury; stroke.

Figure 1

A, Brain miR-200c levels following intracerebroventricular (ICV) pre-treatment decrease with miR-200c antagomir and increase with mimic, relative to mismatch-control (Control). B, Representative Cresyl Violet stained brain sections from mice subjected to ICV pre-treatment, 1 hr MCAO, and 24 hrs of reperfusion. Regions of infarct are lighter in color. C, Effect of altering miR-200c levels on injury: quantification of infarct volume averaged over 4 levels demonstrated significant protection with miR-200c antagomir (Antag) pre-treatment compared to either Control or Mimic. D, Neurologic score in post-MCAO mice: miR-200c antagomir pre-treatment resulted in significantly improved neurologic outcome (lower score). * = p < 0.05 compared to control; n = 11–23 animals/group.

Figure 2

A, Time course of brain levels of miR-200c (), reelin mRNA () and uncleaved reelin protein () in mice subjected to 1 hr MCAO. B, Examples of immunoblots for reelin and actin following middle cerebral artery occlusion (MCAO) at the 3 hr reperfusion time point. While levels of miR-200c increased by 1 hr of reperfusion (A), levels of uncleaved reelin protein (A, B) decreased by 3 hrs, suggesting translational inhibition. * = p < 0.05 compared to sham control; n = 8 animals/group.

Figure 3

In mice pre-treated with ICV infusion of miR-200c antagomir, post-MCAO miR-200c expression in the brain significantly decreased relative to control (A). Although reelin mRNA remained unchanged (B), uncleaved reelin protein increased (C, D), suggesting silencing by miR-200c rather than degradation. * = p < 0.05 compared to pre-MCAO control; n = 8–11 animals/group.

Figure 4

Reln 3’UTR is a direct target of miR-200c. Predicted binding for miR-200c:Reln 3’UTR (A) was assessed by dual luciferase activity assay in N2a cells co-transfected with Renilla (Ren) Reln 3′UTR target reporter, Firefly (Ff) control reporter, plus either wild type miR-200c or seed mutant (SM). A reduction in luciferase activity with wild type but not SM control indicated that miR-200c recognizes the Reln 3′UTRs (B). Uncleaved reelin protein expression (C) decreases in N2a cells treated with mimic, and increases in cells treated with inhibitor, relative to mismatch control-treated cells. All cell culture experiments were performed in triplicate, * = p < 0.05 compared to control, n= 4–6 wells/condition.

Figure 5

A, Cell death following in vitro reperfusion injury (18 hrs serum deprivation plus 500 µM H₂O₂) increased in cells transfected with miR-200c mimic but decreased with miR-200c inhibitor. Co-transfection with Reln siRNA abolished the protective effect of miR-200c inhibition. B, Cell death following 24 hrs serum-deprivation plus 500 µM H₂O₂ was significantly attenuated by application of recombinant reelin protein. All cell culture experiments were performed in triplicate, * = p < 0.05 compared to control, n= 8 wells/condition.