Protective mechanisms of microRNA-27a against oxygen-glucose deprivation-induced injuries in hippocampal neurons.

Hypoxic injuries during fetal distress have been shown to cause reduced expression of microRNA-27a (miR-27a), which regulates sensitivity of cortical neurons to apoptosis. We hypothesized that miR-27a overexpression attenuates hypoxia- and ischemia-induced neuronal apoptosis by regulating FOXO1, an important transcription factor for regulating the oxidative stress response. miR-27a mimic was transfected into hippocampal neurons to overexpress miR-27a. Results showed increased hippocampal neuronal viability and decreased caspase-3 expression. The luciferase reporter gene system demonstrated that miR-27a directly binded to FOXO1 3'UTR in hippocampal neurons and inhibited FOXO1 expression, suggesting that FOXO1 was the target gene for miR-27a. These findings confirm that miR-27a protects hippocampal neurons against oxygen-glucose deprivation-induced injuries. The mechanism might be mediated by modulation of FOXO1 and apoptosis-related gene caspase-3 expression.

Introduction

During the perinatal stage, ischemia and hypoxia are the main causes of infant hypoxic-ischemic brain damage (HIBD), which can lead to mortality and neurological disability in infants and young children and occurs in 1 to 6 of every 1,000 live-term births (Hristova et al., 2015; Yu et al., 2015; Lai et al., 2016). Fetal distress, hypoxia due to an umbilical cord around the neck, and intrauterine infection can lead to an insufficient oxygen and blood supply for fetal or neonatal brain tissue (Chen et al., 2013; Fitch et al., 2013). Approximately 40% of newborn infants with HIBD die during the neonatal period, and an additional 30% sustain lifelong neurological deficits, including cerebral palsy, epilepsy, and cognitive disabilities (Charon et al., 2015; Li et al., 2015a). Treatment of HIBD sequelae requires enormous efforts. However, even with the best management, there is often little improvement in the overall ability of these children (Liu et al., 2015a).

The discovery of microRNAs (miRNAs) brings a new understanding of HIBD, and provides novel strategies and targets for HIBD treatment. miRNAs are small, non-coding RNAs that are 17–25 nucleotides in length (Jiang et al., 2016). miRNAs serve as posttranscriptional regulators by binding to the targeting gene mRNA 3′untranslated region (3′UTR), thereby inducing degradation or translation inhibition (Li et al., 2015b; Zhou et al., 2016). An increased understanding of miRNAs brings novel insight into the regulation of gene expression, consequently playing regulatory roles in a wide variety of physiological and pathological cellular processes (Rajasekaran et al., 2015; Katsura et al., 2016).

During myocardial ischemia/reperfusion injury, miR-22 has been shown to play an important cardioprotective role, partly by regulating the CBP/AP-1 pathway to reduce cell apoptosis and inflammatory damage (Yang et al., 2016). Similarly, some miRNAs may be deregulated, thereby influencing pathological changes in the nervous system (Wu and Murashov, 2013; Lechpammer et al., 2015; Looney et al., 2015). Zhang et al. (2012) reported that hypoxia caused increased FasL expression, but decreased miR-21 expression in microglia, suggesting that miR-21 could play an important role in potential novel therapeutic interventions for cerebral hypoxic diseases associated with microglial activation.

Numerous studies have revealed that miR-27a plays an oncogenic role in the development and progression of human cancers (Park et al., 2015; Towers et al., 2015; Zhou et al., 2015b). Chen et al. (2014) reported that hypoxic injuries during fetal distress cause reduced miR-23b and miR-27b expression, which further inhibits Apaf-1 expression and regulates neuronal sensitivity to apoptosis. However, the biological roles of miR-27a in hypoxia and ischemia remain poorly understood.

In the present study, we investigated the expression of miR-27a in oxygen-glucose deprivation (OGD)/reoxygenation-exposed hippocampal neurons to better understand its role in HIBD. We overexpressed miR-27a to analyze the effects of miR-27a on viability and apoptosis of OGD-treated neuronal cells. Results from the present study could provide a better understanding of miR-27a as a potential therapeutic target in the treatment of hypoxic-ischemic encephalopathy.

Materials and Methods

Cell culture

The experimental procedures involving animals were conducted according to the institutional animal care guidelines of Nantong University, China, and were ethically approved by the Administration Committee for Experimental Animals, Jiangsu Province, China.

Isolation and culture of rat hippocampal neurons were accomplished according to previously described methods with minor modifications (Booth et al., 2016).

Rat primary hippocampal neuron cultures were prepared from embryonic day 18 (E18)–E19, specific-pathogen-free, Sprague-Dawley rat (Laboratory Animal Center of Nantong University, China) embryos (SYXK (Su) 2012-0031). In brief, after rats were sacrificed under anesthesia, their brains were quickly removed; the hippocampal tissues were harvested on a cold stage, and dissociated with trypsin-ethylenediamine tetraacetic acid (0.25%) into cell suspensions, which were plated onto poly-lysine-coated plates at a density of 1 × 106/mL. The cells were maintained in serum-free Neurobasal medium (Life Technologies, Carlsbad, CA, USA) with 2% B27 (Gibco, Carlsbad, CA, USA) for 7 days, after which half of the medium was replaced every 3 days. The cells were then subjected to immunohistochemistry for neurofilament protein and fibrillary acidic protein, revealing that cell cultures contained approximately 95% neurons.

Human embryonic kidney 293 (HEK293) cells were obtained from American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco's-modified Eagle's medium (DMEM, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco). When HEK293 cell growth reached 70% to 80% confluency, the cells were digested with 0.25% trypsin and passaged. The culture medium was replaced every other day, and the cells were passaged every 3 to 4 days. Cells in the logarithmic growth phase were collected for experiments. Cell growth was observed under an inverted microscope, and cells were cultured in an incubator with 5% CO2 and saturated humidity at 37°C.

OGD/reoxygenation modeling of hippocampal neurons

OGD/reoxygenation model of hippocampal neurons was established in accordance with previously described methods with minor modifications (Gu et al., 2016). In brief, the cells were transferred to glucose-free DMEM bubbled with 95% N2/5% CO2 in a sealed humidified modular incubator chamber (MIC-101, Billups-Rothenberg, Del Mar, CA) for 3 hours. For reperfusion, the exposure medium was replaced with neuronal culture medium, and the cells were incubated in a normoxic incubator for an additional 6–24 hours. At the end of cell treatment, the cell cultures were subjected to various assessments. Cells were divided into four groups. In three of the groups, hippocampal neurons were subjected to OGD/reoxygenation treatment for 6, 12, and 24 hours, respectively. In the fourth group, the cells were cultured in plain DMEM and neuronal culture medium with ambient oxygen for 6–24 hours, respectively, which served as the control (no exposure to OGD).

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

Hippocampal neurons were transfected with mi-27a mimics and mimic control (ctrl) (Ribobio, Guangzhou, Guangdong Province, China), respectively, using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to manufacture instructions.

Total RNA at 48 hours post-transfection, including miRNAs, was extracted using the TaqMan miRNA Isolation kit (Applied Biosystems, Foster, CA, USA). NanoDropND-1000 spectrophotometry (NanoDrop Tech, Wilmington, DE, USA) was used to measure RNA concentrations. PCR was then used to amplify miR-27a using SYBR Premix Ex TaqTM II (Perfect Real Time; TaKaRa, Tokyo, Japan) and miR-27a-specific primers. Primer sequences are shown in . All reactions were performed in triplicate. The relative expression of miR-27a was normalized to the internal reference U6. Data were analyzed using the 2−ΔΔCt method (Yu et al., 2012).

Cell viability assay

To measure cell viability, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed according to the MTT kit (Sigma-Aldrich, St. Louis, MO, USA) instruction protocol. Briefly, hippocampal neurons were seeded in 96-well plates subjected to OGD/reoxygenation treatment for 6, 12, and 24 hours, respectively. The original media were discarded and fresh medium containing MTT (5 mg/mL) was added prior to incubation for an additional 4 hours. Optical density values were measured by spectrophotometry at 570 nm with an ELX-800 microplate reader (Bio-Tek Inc., Winooski, VT, USA). Three independent assays were conducted.

Cell apoptosis assay

Flow cytometry and Annexin V/propidium iodide (PI) staining were used to analyze cell apoptosis. After 12 hours of treatment (OGD/reoxygenation), cells were harvested and washed once or twice with PBS, followed by addition of Annexin V-fluorescein isothiocyanate (FITC) and PI staining reagents (BioVision, Cliniscience, Montrouge, France). The mixture was incubated in the dark for 15 minutes. After filtration with a screen cloth, the cells were analyzed using flow cytometry (BD Bioscience, San Jose, CA, USA). FITC+/PI– cells were considered early apoptotic cells. Flow cytometry CellQuest software (BD Bioscience) was used to quantify the cells, and Macquit software was used to analyze the data.

Luciferase reporter assay

The 3′-UTR sequence of FOXO1 (NM_001191846 in GenBank) was amplified from rat genomic DNA and subcloned into the luciferase gene in the luciferase reporter vector (Promega, Madison, CA, USA) at the restriction enzyme cleavage site. With appropriate primers, PCR amplification of the 3′-UTR sequence of FOXO1 generated different pGL3-Luciferase reporter vectors. The wildtype and mutant 3′-UTR sequences were confirmed by sequencing. HEK 293 cells were cultured under standard conditions and inoculated into a 96-well plate at 3 × 105 cells/mL (100 μL/well). The FOXO1 3′-UTR luciferase plasmid containing the FOXO1 3′-UTR, miR-27a mimic, or a negative control was transfected following the recommended protocol for the Lipofectamine 2000 transfection system (Invitrogen, Carlsbad, CA, USA), and a normal control was also included. After 48 hours of incubation, luciferase activities were measured using the luciferase reporter assay system (Promega, Madison, WI, USA) from cell lysates.

Western blot assay

Total proteins after 48 hours of treatment were extracted with a lysis buffer containing protease inhibitors (Promega) from primary neuronal cells and quantified using the Pierce™ Bicinchoninic Acid Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA). Protein (30 μg) was loaded into each lane and electrophoretically separated on a 10% sodium dodecyl sulphate-polyacrylamide gel, and transferred to polyvinylidene fluoride membrane (GE Healthcare, Little Chalfont, UK). The membrane was incubated with 5% (w/v) non-fat milk in Tris-buffered saline with Tween 20 (50 mM Tris-HCl, 100 mM NaCl, and 0.1% Tween-20, pH 7.4) at room temperature for 1 hour. The membranes were incubated with rabbit anti-rat pro-caspase-3 polyclonal antibody and rabbit anti-rat cleaved caspase-3 polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, USA), rabbit anti-rat FOXO1 polyclonal antibody (1:2,000; Abcam, Cambridge, MA, USA), and anti-rat β-actin monoclonal antibody (1:5,000; Sigma, St Louis, MO, USA), respectively, at 4°C overnight. The membrane was incubated with secondary donkey anti-rabbit horseradish peroxidase conjugated antibodies (1:2,000; Abcam) for 2 hours at room temperature. Immunoreactive proteins were visualized by an enhanced chemiluminescence-plus chemiluminescence reaction. The relative contents of pro-caspase-3, cleaved caspase-3, and FOXO1 were represented as the gray scale ratio of cleaved caspase-3/pro-caspase-3, and FOXO1/β-actin, and the gray scale was analyzed using QuantityOne software (Bio-Rad, Hercules, CA, USA).

Statistical analysis

All data were expressed as mean ± SEM from three independent experiments (each in duplicate). The Student's t-test and one-way analysis of variance followed by the post-hoc Scheffe's test were used for statistical analysis using the Stata 6.0 software package (Stata Corp., College Station, TX, USA). A value of P < 0.05 was considered statistically significant.

Results

Neuronal cell viability and miR-27a expression in OGD/reoxygenation-treated primary cultured hippocampal neurons

The MTT assay was used to analyze cell viability of hippocampal neurons after cell treatment. Results showed that during 6–24 hours of reoxygenation post-OGD, neuronal cell viability decreased compared with control (normoxia) (P < 0.05). Neuronal cell viability exposed to OGD/reoxygenation for 12 and 24 hours was significantly decreased compared with normoxia control (P < 0.01) (). Thus, we chose OGD/reoxygenation for 12 hours in the following experiments. After OGD treatment, primary hippocampal neurons exhibited morphological alterations, such as neurite disappearance and vacuolus emergence around the cell body (data not shown).

Neuronal cell viability and miR-27a expression in OGD-induced primary cultured hippocampal neurons.

(A) MTT assay was used to measure neuronal cell viability. (B) Expression of miR-27a was detected by qRT-PCR, and miR-27a expression was normalized to U6. Histogram shows cell viability or miR-27a expression in primary hippocampal neurons exposed to OGD/reoxygenation for 6, 12, and 24 hours, or normoxia, respectively. *P < 0.05, **P < 0.01, vs. normoxia control (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; qRT-PCR: quantitative real-time polymerase chain reaction; OGD: oxygen-glucose deprivation; h: hours.

To further confirm miR-27a expression in hippocampal neurons following OGD/reoxygenation, we detected miR-27a levels at various times by qRT-PCR. After neurons were induced by OGD/reoxygenation, the relative expression levels of miR-27a gradually decreased compared with control (normoxia) (P < 0.05; ).

Effects of miR-27a on viability and apoptosis of OGD/reoxygenation-treated hippocampal neurons

To investigate whether miR-27a dysregulation was sufficient to impact cell function against OGD-induced injury, hippocampal neurons were transfected with miR-27a mimic and non-targeting negative control (mimic ctrl) before OGD. Expression of miR-27a was detected by qRT-PCR in neurons transfected for 48 hours, showing significantly increased expression (5-fold) in neurons transfected with mimic compared with control (normoxia) (P < 0.01; ).

The effects of miR-27a on viability and apoptosis of OGD-exposed primary cultured hippocampal neurons.

(A) Expression of miR-27a was detected by qRT-PCR, and miR-27a expression was normalized to U6. (B) MTT assay was used to measure neuronal cell viability. (C) Flow cytometry was used to analyze neuronal cell apoptosis. Histogram shows miR-27a expression, neuronal cell viability, and cell apoptosis in primary hippocampal neurons transfected with miR-27a mimic, mimic control (ctrl), or untransfected (blank) exposed to OGD/reoxygenation for 12 hours or normoxia, respectively. *P < 0.05, **P < 0.01, vs. normoxia control, #P < 0.05, ##P < 0.01, vs. blank or mimic ctrl (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; qRT-PCR: quantitative real-time polymerase chain reaction; OGD: oxygen-glucose deprivation. Blank: Group with untransfected; mimic ctrl: group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.

The MTT assay was used to analyze miR-27a effects on OGD/reoxygenation-treated cell viability. Results showed that miR-27a overexpression promoted cell viability compared with untransfected control (blank) and mimic control (mimic ctrl) (P < 0.05; ).

Flow cytometry was used to test miR-27a effects on OGD/reoxygenation-treated cell apoptosis. As shown in , the percentage of apoptotic neurons transfected with miR-27a mimic was significantly less than in the untransfected control (blank) and mimic control (mimic ctrl) (P < 0.01).

FOXO1 is a direct target of miR-27a

miRNAs exert their biological functions through suppression of target genes (Yu et al., 2012). Thus, it is necessary to identify the miRNA and gene target pairs. Online software analysis showed that FOXO1 might serve as the target gene (http://www.targetscan.org/). To verify whether the predicted miR-27a target was located in the FOXO1 3′UTR, we constructed a luciferase plasmid containing FOXO1 3′UTR and mutated FOXO1 3′-UTR, and the plasmid, together with miR-27a mimic and mimic control, was co-transfected into HEK293 cells. Relative luciferase activity was significantly decreased by miR-27a when wildtype FOXO1 3′UTR was present ().

FOXO1 is a direct target of miR-27a.

(A) Schematic representation of a section of FOXO1 3′-UTR showing the putative miR-27a target site. (B) The resulting histogram shows relative luciferase activities in each group. **P < 0.01, vs. mimic ctrl (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). 3′-UTR: 3′ Untranslated region. Mimic ctrl: Group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.

Effect of miR-27a on caspase-3 activation in OGD/reoxygenation-treated primary cultured hippocampal neurons

Western blot assay was applied to analyze pro-caspase-3 and cleaved caspase-3 expression in neurons after OGD/reoxygenation. Cleaved caspase-3 is the active subunit of pro-caspase-3 (Cui et al., 2016). Expression of cleaved caspase-3 significantly increased in neurons treated with OGD/reoxygenation compared with normoxia (P < 0.01), while pro-caspase-3 expression slightly decreased. Additionally, miR-27a inhibited cleaved caspase-3 expression after OGD/reoxygenation compared with untransfected control (blank) and mimic control (ctrl) (P < 0.05; ).

Effect of miR-27a on caspase-3 activation in OGD-exposed primary cultured hippocampal neurons.

Representative western blot image (the upper) and resulting histogram (the lower) show cleaved caspase-3 expression in primary hippocampal neurons transfected with miR-27a mimic, mimic ctrl, or untransfected (blank) after exposure to OGD/reoxygenation for 12 hours or normoxia, respectively. *P < 0.05, P < 0.01, vs. normoxia control; NRR-11-1285-g001 P < 0.01, vs. blank control or mimic ctrl (mean ± SEM; experiments were performed in triplicate; one-way analysis of variance followed by post-hoc Scheffe's test). OGD: Oxygen-glucose deprivation; Blank: group with untransfected; Mimic ctrl: group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.

Effect of miR-27a on FOXO1 expression in neuronal cells

Western blot assay was used to measure FOXO1 expression in neuronal cells. Results showed significantly reduced FOXO1 expression in neurons transfected with miR-27a mimic compared with untransfected control (ctrl) and mimic control (ctrl) (P < 0.01; ).

Effect of miR-27a on FOXO1 protein expression in rat primary hippocampal neurons.

Representative western blot image (the upper) and resulting histogram (the lower) show FOXO1 protein expressions in neurons transfected with miR-27a mimic, mimic ctrl, or untransfected, respectively. **P < 0.01, vs. untransfected ctrl and mimic ctrl (mean ± SEM; experiments were performed in triplicate; Student's t-test). Untransfected ctrl: Normal group; Mimic ctrl: group transfected with miR-27a mimic control; miR-27a mimic: group transfected with miR-27a mimic; ctrl: control.

Discussion

HIBD is a clinical condition in the neonate, resulting from oxygen deprivation around the time of birth, and a major public health issue globally with long-term effects on the family, health care system, and society (Orrock et al., 2015; Luckman et al., 2016; Xie et al., 2016). The involvement of miRNAs in regulating various cellular processes, including cell proliferation, apoptosis, and differentiation, provides novel insights in disease research, diagnosis, prognosis, and treatment (Yang et al., 2014; Chandrasekaran and Bonchev, 2016; Jeon and Osborne, 2016). Brain protection in neonatal infants remains a challenging priority and represents a medical requirement that has yet to be met (Berger et al., 2015; Lai et al., 2016). In the present study, miR-27a levels measured by qRT-PCR decreased in primary rat hippocampal neurons after OGD/reoxygenation, indicating that miR-27a might be involved in HIBD.

Previous studies have shown that the combined effects of hemodynamic changes, as well as the excitotoxicity impact of excitatory amino acids, nitric oxide, intracellular calcium overloads, and free radical injury, result in neuronal structural damage and apoptosis, eventually leading to neurological functional defects and loss (Chang et al., 2013; Beppu et al., 2014; Du et al., 2014; Ginet et al., 2014; Li et al., 2015c). Although apoptosis occurs after hypoxia and ischemia in both the embryonic and adult stages, a large number of studies have suggested that apoptosis after brain injuries caused by hypoxia and ischemia plays a more prominent role in the neofetus than in the adult (Gao et al., 2015; Jantzie et al., 2015; Xie et al., 2016). Alleviating or reducing neuronal apoptosis in the neofetal brain is key for treating HIBD. In the present study, transfection of miR-27a mimic into hippocampal neurons resulted in miR-27a overexpression, and the effects of miR-27a on cell survival/viability and apoptosis were measured by MTT assay and flow cytometry. Interestingly, we found that upregulated miR-27a promoted cell survival and reduced OGD/reoxygenation-induced neuronal apoptosis, suggesting that miR-27a might play an important role in neuroprotection against OGD by reducing neuronal apoptosis.

Apoptosis involves a series of gene activation, expression, and regulation (Teo et al., 2015; Zhang et al., 2015b). The essential step in the mitochondria-dependent apoptosis pathway is the formation of apoptosomes, consisting of cytochrome c, apoptotic protease activating factor-1, pro-caspase-9, and adenosine triphosphate (Monian and Jiang, 2015; Zhang et al., 2015a; Zhou et al., 2015a). miR-27a/b are endogenous inhibitor factors of apoptotic protease activating factor-1 expression and have been shown to regulate sensitivity of neurons to apoptosis (Chen et al., 2014). Hypoxic-ischemic injury also triggers proteolytic processing of pro-caspase-3, resulting in the formation of a 17-kDa subunit of cleaved caspase-3 (active caspase-3) (Hattori et al., 2015; Liu et al., 2015b). In the present study, western blot assay was applied to analyze pro-caspase-3 and cleaved caspase-3 expression in neurons after OGD/reoxygenation. Additionally, miR-27a inhibited cleaved caspase-3 expression in neurons after OGD/reoxygenation, while pro-caspase-3 expression slightly decreased.

FOXO1 is an important transcription factor for regulating the oxidative stress response, cell proliferation, and apoptosis, and is widely expressed in multiple organs, such as the liver, fat, and skeletal muscle (Kinoshita et al., 2016; Luo et al., 2016). Zhao et al. (2015) indicated that propofol might exert its antioxidative effect through FOXO1 in H9c2 cells. Our online software results showed that FOXO1 might be a target gene for miR-27a. A fluorescence reporter gene system was used to verify the location of the predicted miR-27a target in the FOXO1 3′UTR. Relative luciferase activity was significantly decreased by miR-27a when wildtype FOXO1 3′UTR was present. This reduction was sequence-specific, because the relative luciferase activity did not decline as sharply in UTRs that contained a mutant binding site compared with a wildtype binding site. Western blot assay revealed that miR-27a significantly reduced FOXO1 expression.

In conclusion, miR-27a might play an important role in neuroprotection against OGD/reoxygenation by modulation of apoptosis-related gene caspase-3 expression and FOXO1. The neuroprotective effect of miR-27a suggests a possible use in the treatment of HIBD in the future.

Table 1

Primer sequences for quantitative real-time polymerase chain reaction