miR-27a-3p protects against blood-brain barrier disruption and brain injury after intracerebral hemorrhage by targeting endothelial aquaporin-11

Abstract

Previous studies have reported that miR-27a-3p is down-regulated in the serum of patients with intracerebral hemorrhage (ICH), but the implication of miR-27a-3p down-regulation in post-ICH complications remains elusive. Here we verified miR-27a-3p levels in the serum of ICH patients by real-time PCR and observed that miR-27a-3p is also significantly reduced in the serum of these patients. We then further investigated the effect of miR-27a-3p on post-ICH complications by intraventricular administration of a miR-27a-3p mimic in rats with collagenase-induced ICH. We found that the hemorrhage markedly reduced miR-27a-3p levels in the hematoma, perihematomal tissue, and serum and that intracerebroventricular administration of the miR-27a-3p mimic alleviated behavioral deficits 24 h after ICH. Moreover, ICH-induced brain edema, vascular leakage, and leukocyte infiltration were also attenuated by this mimic. Of note, miR-27a-3p mimic treatment also inhibited neuronal apoptosis and microglia activation in the perihematomal zone. We further observed that the miR-27a-3p mimic suppressed the up-regulation of aquaporin-11 (AQP11) in the perihematomal area and in rat brain microvascular endothelial cells (BMECs). Moreover, miR-27a-3p down-regulation increased BMEC monolayer permeability and impaired BMEC proliferation and migration. In conclusion, miR-27a-3p down-regulation contributes to brain edema, blood-brain barrier disruption, neuron loss, and neurological deficits following ICH. We conclude that application of exogenous miR-27a-3p may protect against post-ICH complications by targeting AQP11 in the capillary endothelial cells of the brain.

Keywords: aquaporin; blood–brain barrier; brain; brain injury; endothelium; intracerebral hemorrhage; miR-27a-3p; microRNA (miRNA); neurological deficit; post-transcriptional regulation.

Figure 1.

MiR-27a-3p is down-regulated in the serum of patients with ICH. The levels of miR-27a-3p in the serum of ICH patients (n = 26) and healthy subjects (n = 22) were determined by real-time PCR.

Figure 2.

Intracerebroventricular administration of the miR-27a-3p mimic improves behavioral performance in rats with ICH. A and B, corner turn test (A) and limb placement test (B) were performed 24 h after ICH and miRNA treatment (n = 12/group). **p < 0.01.

Figure 3.

Intracerebroventricular administration of the miR-27a-3p mimic reduces brain edema and maintains BBB permeability following ICH. A, the levels of miR-27a-3p in rat serum, perihematomal tissue, and the hematomal region were measured by real-time PCR 24 h after ICH (n = 6/group). B, brain water content was measured 24 h after ICH induction and miRNA treatment (n = 6/group). C, Evans blue extravasation assay was performed to assess BBB permeability following ICH (n = 6/group). *p < 0.05; **p < 0.01.

Figure 4.

The miR-27a-3p mimic inhibits leukocyte infiltration into the perihematomal area. A and B, immunofluorescent staining of MPO was performed to detect leukocyte infiltration (n = 5/group; scale bars = 33.3 μm). **p < 0.01.

Figure 5.

The miR-27a-3p mimic inhibits neuronal apoptosis and microglia activation in the perihematomal area. A, TUNEL staining was performed to detect apoptotic cells in the perihematomal zone (scale bars = 33.3 μm). B, dying neurons in the perihematomal area were labeled by Fluoro-Jade B stain (scale bars = 33.3 μm). C, immunohistochemical staining of OX-42, a marker of activated microglia, in the perihematomal zone (scale bars = 33.3 μm). D–F, statistical analyses of the TUNEL apoptotic ratio (D), Fluoro-Jade B–positive cells (E), and OX-42–positive cells (F) in the perihematomal area were performed based on the above staining images (n = 5/group). *p < 0.05.

Figure 6.

miR-27a-3p directly targets the 3′ UTR of the AQP11 transcript. A and B, 24 h after ICH, the levels of AQP11 mRNA and AQP11 protein in the perihematomal tissues were determined by real-time PCR and Western blotting, respectively (n = 6/group). β-Actin was used as an internal control. C and D, rat BMECs were isolated and transfected with miR-NC, the miR-27a-3p mimic, or the miR-27a-3p inhibitor, and the levels of AQP11 mRNA and AQP11 protein were measured 24 h after transfection. β-Actin was used as an internal control. E, sequence of AQP11_3′ UTR. F and G, alignment of miR-27a-3p with the 3′ UTR of AQP11. The miR-27a-3p binding site in the 3′ UTR of AQP11 was mutated (ACUGUGA to CUGUCCA). H, a Dual-Luciferase reporter assay was performed in BMECs to verify direct targeting of miR-27a-3p on the 3′ UTR of AQP11. The in vitro experiments were repeated three times. *, p < 0.05; **, p < 0.01.

Figure 7.

Antagonizing miR-27a-3p increases BMEC monolayer permeability and blocks BMEC proliferation and migration. A, the fluorescence intensity of FITC–dextran 20 that passed through a BMEC monolayer over 120 min was measured. B, an MTT assay was performed to assess the proliferation of BMECs that were transfected with miR-NC or the miR-27a-3p inhibitor. C, a scratch wound assay was conducted to evaluate the motility of BMECs. The experiments were repeated three times. *, p < 0.05; **, p < 0.01 versus miR-NC-transfected cells.