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

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-? (ER?) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17?-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 ESR? knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ER? target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ER? 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 ER? levels in vivo and in female cultures, while ER? knockdown with siRNA increased cell death in both sexes. Treatment with ER? 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 ER? 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 ER?.

SUBMITTER: Stary CM

PROVIDER: S-EPMC5561665 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Stary Creed M CM Xu Lijun L Li Le L Sun Xiaoyun X Ouyang Yi-Bing YB Xiong Xiaoxing X Zhao Jing J Giffard Rona G RG

Molecular and cellular neurosciences 20170515

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 ...[more]

PMID: 28522364

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Project description:Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases. Experiment Overall Design: affy platform; replicate samples; two treated samples, two placebo. Experiment Overall Design: MIAME compliance: All samples were run in commercial arrays from Affymetrix, using Rat Genome U34A GeneChip arrays as described in the Affymetrix web site. The sample information is described below. Likewise, the Affymetrix protocol is detailed and referenced above. These descriptions include all information currently considered under the MIAME supportive guidelines, with which the JHMI Microarray Core Facility abides in all its procedures. Experiment Overall Design: Methodology. Expression profiling using DNA microarrays (One round amplification): Experiment Overall Design: Experimental Design and Sample Information. Transient focal cerebral ischemia was induced in ovariectomized female rats with and without replacement with 17beta-estradiol by intraluminal middle cerebral artery occlusion (MCAO), as described in the manuscript. At 6 and 24 hours after 2-hour MCAO, total RNA was prepared from the ischemic and contralateral cerebral cortex and striatum between coronal levels 2 and 3 mm relative to Bregma, which represent the core of MCA territory. RNA samples from 3 animals were combined into one pooled sample and hybridized to Affymetrix RG-U34A GeneChip, as described below. Each microarray experiment was performed in duplicate using two independent pooled samples per group (6 animals per group at each time point, 2 pooled samples for each brain region per time point). Experiment Overall Design: Microarray Hybridization Protocol. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA from control and experimental tissue are processed using the RNA amplification protocol described by Affymetrix (Affymetrix Expression Manual). Briefly, 5 micrograms of total RNA were used to synthesize first strand cDNA using oligonucleotide probes with 24 oligo-dT plus T7 promoter as primer (Proligo LLC, Boulder, Colorado) and the SuperScript Choice System (Invitrogen, Carlsbad, California). Following the double stranded cDNA synthesis, the product was purified by phenol-chloroform extraction, and biotinilated anti-sense cRNA was generated through in vitro transcription using the BioArray RNA High Yield Transcript Labeling kit (ENZO Life Sciences Inc., Farmingdale, New York). 15 ug of the biotinilated cRNA were fragmented at 94 C for 35 min (100mM Tris-acetate, pH 8.2, 500mM KOAc, 150mM MgOAC), and 10ug of total fragmented cRNA were hybridized to the Affymetrix U34A GeneChip arrays for 16hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was then used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinilated cRNA. The staining was then amplified using goat IgG as blocking reagent and biotinilated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate. Experiment Overall Design: Quality Control. To ascertain the quality control of the total RNA from the samples, we used the Agilent Bioanalyzer, Lab on a Chip technology, and confirmed the rRNA ratios and clean run patterns of the samples. Likewise, this technology is used to confirm the quality of the RNA in the form of cRNA and fragmented cRNA. To assess the QC of the hybridization, GeneChip image, and comparison between chips, we confirmed the following parameters: scaling factor values within comparable range; low background values (50-95); high percentage of present calls (between 30 and 55%); consistent 3â/5â ratios of GAPDH as representation of housekeeping genes, and presence or absence of Bio B and C as internal spike controls.

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

Publications

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

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