Inhibition of miR-181a protects female mice from transient focal cerebral ischemia by targeting astrocyte estrogen receptor-α

Abstract

Whether the effect of miR-181a is sexually dimorphic in stroke is unknown. Prior work showed protection of male mice with miR-181a inhibition. Estrogen receptor-α (ERα) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17β-estradiol (E2) replacement after ovariectomy. Adult female mice were ovariectomized and implanted with an E2- or vehicle-containing capsule for 14d prior to 1h middle cerebral artery occlusion (MCAO). Each group received either miR-181a antagomir or mismatch control by intracerebroventricular injection 24h before MCAO. After MCAO neurologic deficit and infarct volume were assessed. Primary male and female astrocyte cultures were subjected to glucose deprivation with miR-181a inhibitor or transfection control, and E2 or vehicle control, with/without ESRα knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ERα target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ERα by miR-181a in astrocytes. Individually, miR-181a inhibition or E2 decreased infarct volume and improved neurologic score in female mice, and protected male and female astrocyte cultures. Combined miR-181a inhibition plus E2 afforded greater protection of female mice and female astrocyte cultures, but not in male astrocyte cultures. MiR-181a inhibition only increased ERα levels in vivo and in female cultures, while ERα knockdown with siRNA increased cell death in both sexes. Treatment with ERα TSB was strongly protective in both sexes. In conclusion, the results of the present study suggest miR-181a inhibition enhances E2-mediated stroke protection in females in part by augmenting ERα production, a mechanism detected in female mice and female astrocytes. Sex differences were observed with combined miR-181a inhibition/E2 treatment, and miR-181a targeting of ERα.

Keywords: Astrocyte; Estrogen; Ischemia-reperfusion injury; MCAO; MicroRNA; Stroke.

Figure 1

Experimental protocols for in vivo (A) and primary astrocyte (B) studies. Antag = antagomir; E2 = 17β-estradiol; GD = glucose deprivation; MCAO = 1 hr middle cerebral artery occlusion; MM-control = mismatch control; NS = neurological deficit score; OVX = ovariectomy; V = control vehicle.

Figure 2. Effect of miR-181a antagomir and E2 on injury from middle cerebral artery occlusion (MCAO)

(A) Serum E2 levels in female mice prior to and following ovariectomy (OVX), with and without 17β-estradiol (E2) replacement. (B) Coronal brain sections stained with Cresyl Violet to assess infarct volume in OVX mice treated with mismatch control (MM) or miR-181a antagomir (Antag), and E2 or vehicle (V), 24 hr following 1 hr MCAO; infarcted areas are lighter in color. Quantification of infarct volume (C) and neurological deficit (D) 24 hr following MCAO. All graphs show mean±SE, * = p<0.05 versus pre or control injury; # = p<0.05 versus Antag or E2 alone. N = 8–9 OVX animals per group. E2 = 17β-estradiol.

Figure 3. Effect of miR-181a inhibitor and E2 on in vitro glucose deprivation (GD) astrocyte injury

(A) Representative micrographs of cell death assessed by propidium iodide (PI, red) staining following GD injury in C6 cells treated with control scramble sequence (Con) or miR-181a inhibitor, and E2 or vehicle (V). All cell nuclei are stained blue with Hoechst. Quantification of cell death by LDH release from C6 cells (B), male primary astrocyte cultures (C), and female primary astrocyte cultures (D) treated with miR-181a inhibitor, E2, or both. Blocking ERα using small interfering RNA (siRNA) abolished the protection seen with miR-181a inhibitor and E2 in both male and female cultures (C, D). N = 8 cultures per experiment. All graphs = mean±SE, * =p<0.05 versus control; # =p<0.05 versus E2 or inhibitor alone; Ψ= p<0.05 versus same condition without siRNA. E2 = 17β-estradiol Inh = inhibitor. Bar =15 μm.

Figure 4. In vivo and in vitro ERα protein expression

(A) Quantification of miR-181a levels from brains of OVX mice. Quantification of ERα protein from brains of OVX mice (B), male (C) and female (D) primary astrocyte cultures. ERα levels were significantly (p<0.05) lower in cultures co-treated with ERα siRNA (C, D). N = 8 cultures per experiment. All graphs = mean±SE, * = p<0.05 versus control; # = p<0.05 versus all other conditions. Antag = miR-181a antagomir, Con = transfection control (scrambled sequence), E2 = 17β-estradiol, Inh = miR-181a inhibitor, V = vehicle.

Figure 5. Cell death following miR-181a/ERα binding inhibition in astrocytes

(A) Primary astrocytes stained with the nuclear dye DAPI (4′,6′-diamidino-2-phenylindole, blue, left panel) display 6-FAM^™ reporter fluorescence (green, middle panel) when transfected with miR-181a/ERα target site blocker (TSB). Transfection efficiency is quantified by blue/green co-localization (right panel). Bars = 15 μm. (B) Quantification of cell death from GD injury of primary male and female astrocytes treated with control transfection or miR-181a mimic and TSB scramble control sequence or miR-181a/ESRα TSB. N = 8 cultures per experiment, mean±SE, * = p<0.05 versus control.