miR-26a prevents neural stem cells from apoptosis via β-catenin signaling pathway in cardiac arrest-induced brain damage

Neural stem cells (NSCs) transplantation is one of the most promising strategies for the treatment of CA-induced brain damage. The transplanted NSCs could differentiate into new neuron and replace the damaged one. However, the poor survival of NSCs in severe hypoxic condition is the limiting step to make the best use of this kind of therapy. In the present study, we investigated whether the overexpression of miR-26a improves the survival of NSCs in hypoxic environment in vitro and in vivo. In vitro hypoxia injury model is established in NSCs by CoCl2 treatment, and in vivo cardiac arrest (CA) model is established in Sprague-Dawley (SD) rats. Quantitative real-time polymerase chain reaction is used to detect the mRNA level and Western blot is used to examine the protein level of indicated genes. TUNEL staining and flow cytometry are applied to evaluate apoptosis. Dual-luciferase reporter assay is utilized to analyze the target gene of miR-26a. The expression of miR-26a is reduced in both in vitro and in vivo hypoxic model. MiR-26a directly targets 3′-UTR of glycogen synthase kinase 3β (GSK-3β), resulting in increased β-catenin expression and decreased apoptosis of NSCs. Overexpression of miR-26a in transplanted NSCs improves the survival of NSCs and neurological function in CA rats. MiR-26a prevents NSCs from apoptosis by activating β-catenin signaling pathway in CA-induced brain damage model. Modulating miR-26a expression could be a potential strategy to attenuate brain damage induced by CA.


Introduction
Cardiac arrest is an immense and sustained public health problem and more than 500,000 patients die from a cardiac arrest each year [1]. It induces the cessation of cerebral blood flow, which can result in brain damage. Patients who achieve return of spontaneous circulation (ROSC) after cardiac arrest (CA) have some degree of brain injury and impaired consciousness. Extensive investigations have been conducted to uncover the underlying pathophysiological mechanisms, and people found that the main threat of this disease can be attributed to massive neuron cell death after CA [2]. It is well established that neural stem cells (NSCs) are essential for regeneration of the nervous system, which secrete various neurotrophic factors and cytokines to improve microenvironment and maintain blood-brain barrier integrity, or differentiate into different cell types to compensate for hypoxia-induced cell death, strengthen the synapses connection, and establish new neural circuits to reduce hypoxic brain injury and finally ameliorate neurobehavioral recovery [3][4][5][6]. NSCs transplantation is one of the most promising strategies for the treatment of CA-induced brain damage [7]. However, NSCs are supersensitive to hypoxic condition induced by transient global cerebral ischemia, stroke and CA. NSCs that are exposed to such hypoxic environment easily undergo apoptosis, which is the limiting step to make the best use of NSCs transplantation [8,9]. Therefore, how apoptosis of transplanted NSCs could be inhibited in hypoxic environment is the most urgent issue to resolve when NSCs transplantation is applied to cure CA-inducing brain damage.
In recent years, extensive research about apoptosis was concentrated on microRNAs that function to modulate a wide variety of cellular processes by interfering with protein expression or mRNA degradation [10]. Among them, miR-26a is one of the important miRNAs that was reported to regulate apoptosis in various cell types [11,12]. It was shown that miR-26a helped to decrease pro-apoptotic signaling in hypoxic environment [13], inhibit hypoxia-induced tubular cell apoptosis [14] and attenuate cardiac ischemia reperfusion injury [15]. Interestingly, it was reported that miR-26a directly targeted the 3 -UTR of glycogen synthase kinase 3β (GSK-3β), which plays a crucial role in apoptosis by regulating β-catenin signaling pathway in various cell types [16][17][18], in human adipose-derived mesenchymal stem cells [19]. However, the exact role of miR-26a in regulating NSCs apoptosis in CA-induced brain damage model remains largely unexplored. The relationship between miR-26a and GSK-3β/β-catenin also remains elusive in CA-induced brain damage model.
In the present study, we investigated the anti-apoptosis effect of miR-26a on NSCs in hypoxic environment in vitro and in vivo, and revealed the underlying mechanism of the neuroprotective effects exerted by miR-26a. Our data showed miR-26a overexpression remarkably blocked NSCs apoptosis and ameliorated neurological function in our hypoxia model. The neuroprotective effects of miR-26a are mainly through activation of β-catenin signaling pathway via directly targeting 3 -UTR of GSK-3β, which result in increased β-catenin expression and decreased apoptosis of NSCs. Therefore, miR-26a/β-catenin axis may serve as a potential molecular target for the treatment of CA-induced brain damage.

Animals
Specific pathogen free (SPF) Sprague-Dawley (SD) rats were obtained from Medical School Animal Center of Sun Yat-sen University and raised in the SPF laboratory. All animal experiment protocols were approved by Animal Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University. The harm to animals was reduced to the minimal extent during the experimental process.

Isolation and culture of neural stem cells (NSCs)
The post-implantation (13.5 dpc) embryos were obtained from a pregnant female SD rats, then the hippocampus was dissected in phosphate buffer saline (PBS, pH 7.4, Gibco BRL) mechanically. The NSCs were suspended in DMEM/F12 complete medium (20 μg/ml EGF, 20 μg/ml bFGF, 1% B27, 2 mM/l glutamine, 5 IU penicillin and 5 μg/ml streptomycin), after centrifugation at 1000 rpm for 5 min. The collected NSCs were plated in 25 cm 2 flasks (2 × 10 8 /l), and renewed half of the liquid volume every 2-3 days. NSCs were passaged every 5-7 days and used to perform experiments after three passages. All these reagents were purchased from Gibco.

In vitro NSCs hypoxia injury model
NSCs were cultured in complete medium containing various concentration of CoCl 2 (0, 200, 400 and 600 μmol/l) for 24 h. Then, the concentrations for in vitro experiments were determined when obvious apoptosis was observed.

TUNEL assay
Apoptosis was measured by TUNEL assay according to manufacturer's instructions (Roche, Mannheim, Germany). NSCs were plated on polylysine coated slides and treated with CoCl 2 for 24 h. Then, the cells were rinsed with cold PBS, fixed with 4% paraformaldehyde for 20 min, washed once in 0.1% Triton X-100 for 5 min and incubated in 0.1% sodium citrate. After that, TUNEL was used to stain apoptotic cells and Hochest33342 was used to stain nucleus. Finally, the apoptotic cells were counted under the fluorescence microscope. Apoptosis index = TUNEL positive cells/Hochest33342 positive cells × 100%.

Flow cytometry assay
AnnexinV/7AAD Kit was purchased from BD and used to examine apoptosis of NSCs. NSCs were plated on six-well plate (1 × 10 5 /well) and incubated with CoCl 2 (400 μg/l) containing medium for 24 h. Then, NSCs were suspended with 1× suspension buffer, and incubated with Annexin V (5 μl) and 7AAD (5 μl) in dark for 15 min. After that, 1× suspension buffer was added and flow cytometry assay was performed to detect apoptosis within 1 h.

Transfection
Lentivirus was purchased from Genechem (Shanghai, China). In brief, a lentivirus vector system consisting of pGC-LV176 vector, pHelper 1.0 vector and pHelper 2.0 vector was used to knockdown miR-26a and a system including pGC-LV204 vector, pHelper 1.0 vector and pHelper 2.0 vector was used to knockdown miR-26a. For transfection, NSCs were plated on six-well plates. Six hours later, lentivirus (MOI = 10, 5E + 8 TU/ml) were used to infect cells to overexpress or knockdown miR-26a. Then, the cells were kept in culturing at 37 • C in 5% CO 2 . Twelve hours later, half of the liquid volumes were renewed.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Cells were seeded and then harvested for RNA isolation using RNAiso plus Kit (Takara) the next day. For miR-26a detection, primescript miRNA qPCR starter kit ver.2.0 (Takara, Tokyo, Japan) was used to synthesize cDNAs and perform quantitative real-time polymerase chain reaction (qRT-PCR) assays. Relative mRNA levels of miR-26a shown were normalized to the expression of U6B, and the miR-26a primer sequence was designed based on the miRNA sequence (Supplementary Table S1). For some other genes, synthesis of cDNAs using PrimeScriptTM RT Master Mix (Takara) and qRT-PCR assays using SYBR Premix EX Taq (Takara) were carried out according to manufacturer's protocols. Relative mRNA levels of each gene shown were calculated by 2 − Ct and normalized to the expression of the house keeping gene Actin. Primer pairs used for the amplification of β-actin, cyclin D1 and β-catenin in this study are provided in the Supplementary Table S2.

Dual-luciferase reporter assay
The target genes of miR-26a were predicted using the database from targetscan (http://www.targetscan.org/). Wild-type and mutant GSK-3β 3 -UTR-mRNA sequences (Supplementary Table S3) were cloned into the pmiR-RB-REPORT™ luciferase reporter vectors, purchased from RiboBio (Guangzhou, China). 293T cells were seeded and cultured in the incubator for 24 h. Then, the luciferase vectors were mixed with miR-26a overexpression or knockdown plasmids and added into the medium. The cells were refreshed 6 h later, and luciferase substrates were added after another 48 h. About 30 μl stop reagent was added to stop the reaction after shaking for 10 min. Ten minutes later, the luminescence value was counted by a luminometer. All the experiments were repeated three times.

Experimental group design
SPF SD rats, male only, weighing 300 g were obtained from Medical Laboratory Animal Center of Sun Yat-sen University and raised in the SPF laboratory. The temperature of the SPF laboratory was kept at 25 • C. Animals were kept under a 12-h dark-light cycle and fed by standard laboratory pellet feed. All animals were divided into three groups: miR-26 overexpression group (CA+miR-26 overexpression NSCs), miR-26a knockdown group (CA+miR-26 knockdown NSCs) and control group (CA+negative control NSCs).

Cardiac arrest (CA) and cardiopulmonary resuscitation (CPR)
Rats were injected with 10% pentobarbital intraperitoneally (0.3 ml/100 g). Once the epiglottis was clearly identified, the angiocath tube was gently slipped into the trachea. The tube was then secured with a suture to the lip. The femoral artery was cannulated with PE-50 tubing. After that, vecuronium bromide (1 mg/kg) was administered from the femoral vein, and CA was defined as a mean arterial pressure (MAP) below 30 mm Hg. Six minutes after CA, chest compressions were started, which were delivered in a standardized manner by accustom-made thumper at a rate of 200/min, a depth of 1/3 of anterior-posterior chest diameter, and with an equal 1:1 compression/relaxation ratio. Animals were ventilated with 21% O 2 at a rate of 70/min (tidal volume: 0.65 ml/100 g) during resuscitation. O 2 concentration and ventilation were adjusted according to the obtained arterial blood samples. Adrenaline (2 μg/100 mg) was administered at the same time as chest compressions and repeated every 2 min, if necessary. After 2 min of cardiopulmonary resuscitation (CPR), electrocardiography was used to monitor the recovery, and defibrillator (5J) was applied if ventricular fibrillation was observed by electrocardiograph. CPR was repeated if no obvious effects were observed. Rats that restored supraventricular rhythm and showed MAP > 60 mm Hg (maintaining more than 10 min) were considered to have successful CPR. If ROSC (return of spontaneous circulation: defined as MAP > 40 mm Hg) was not achieved 15 min after resuscitation, the CPR was considered failing and rats were excluded.

Transplantation of NSCs
One hour after ROSC, rats were injected with 10% pentobarbital sodium intraperitoneally (0.3 ml/100 g). NSCs were injected into both right and left lateral ventricles by micro syringe at the rate of 1 μl/min (5 μl). The needle was kept in lateral ventricles for 5 min after injection. The room temperature was kept at 25 + − 0.5 • C during the experiment.

Neurologic deficit score (NDS) assay
Neurologic deficit score (NDS) assay was used to evaluate neurological function of rats among different groups and performed as previously described [20,21]. In brief, the neurologic deficits were scored from 0 (no observed neurologic deficit) to 500 (death or brain death) based on five aspects including consciousness, respiration, cranial nerve function, motor and sensory function and behaviors, and the specific scoring criteria were showed in the Supplementary Table S4. Ten rats were used per group in this assay. NDS was recorded before NSCs transplantation, and then NDS was further evaluated 24 h and 7 days after ROSC by three independent investigators who were blinded to the study respectively and the average was taken as the final score.

Measurement of apoptotic NSCs in cerebral cortex by TUNEL staining
After 24 h of ROSC, animals were killed with an intravenous lethal dose of pentobarbital sodium. Five coronal sections were made from each injection site and then embedded into paraffin blocks. Apoptotic NSCs were stained by TUNEL (Roche) following the manufacturer's protocol and images were captured using a fluorescence microscope system with 400× magnification (Olympus BX51, Tokyo, Japan). Apoptotic cells were counted in three random fields from each picture.

Statistical analysis
All statistical procedures were performed with SPSS13.0 (SPSS, Chicago, IL, U.S.A.) and the values were presented as the mean + − SD. ANOVA with Bonferroni's correction was used for experiments involving more than two groups, and Student's t-test was used to analyze difference between two groups. A P-value of <0.05 was considered statistically significant.

Down-regulation of miR-26a in hypoxia injury model and CA model
In order to examine whether miR-26a is associated with the NSCs damage in hypoxic condition, we performed qRT-PCR to detect the relative mRNA levels of miR-26a in both hypoxia injury model and CA model. As shown Figure 1A, miR-26a declined remarkably in CoCl 2 -treated NSCs compared with control group (P<0.05). Consistently, in cerebral cortex of CA rats, a significant decrease of miR-26a was also observed 24 h after ROSC compared with control group ( Figure 1B). The reduced expression of miR-26a in hypoxic condition indicates miR-26a may play a pivotal role in CA-mediated brain damage and modulating apoptosis of NSCs in low oxygen environment.

MiR-26a inhibits hypoxia-induced apoptosis of NSCs in vitro
To further examine the role of miR-26a in hypoxia-induced apoptosis of NSCs, we infected NSCs with lentivirus to overexpress or down-regulate miR-26a and results of qRT-PCR indicated high efficacy of miR-26a overexpression and knockdown ( Supplementary Figures S1 and S2). As expected, TUNEL staining showed that CoCl 2 incubation induced apoptosis of NSCs when compared with control group (Figure 2A,B). Overexpression of miR-26a reduced approximately 15% of apoptotic NSCs, while knockdown of miR-26a increased approximately 30% of apoptotic NSCs compared with CoCl 2 -treated group. The negative control for lentivirus showed no difference of apoptotic NSCs, which indicates the lentivirus infection did not affect apoptosis of NSCs. The flow cytometry data presented similar results as TUNEL staining and further supported the inhibitory effect of miR-26a on hypoxia-induced apoptosis of NSCs ( Figure 2C,D). Additionally, the cell viability assay showed overexpression of miR-26a increased cell survival, while knockdown of miR-26a decreased cell survival comparing with CoCl 2 -treated group ( Figure 2E). Taken together, the data indicate miR-26a plays an important role in modulating hypoxia-induced apoptosis of NSCs.

MiR-26a inhibits hypoxia-induced apoptosis of NSCs in vivo
Then, we further examined whether miR-26a could block hypoxia-induced apoptosis of NSCs in vivo. We transplanted lentivirus-infected NSCs (miR-26a overexpression, miR-26a knockdown and negative control for lentivirus infection) into both right and left lateral ventricles of CA rats 1 h after ROSC. Twenty-four hours later, we detected the apoptotic NSCs in cerebral cortex. As shown in Figure 3A,B, more than 30% TUNEL positive NSCs were observed in the miR-26a knockdown group compared with control group. Overexpression of miR-26a attenuated more than 10% of TUNEL positive SNCs in lateral ventricles. The results suggest miR-26a could inhibit hypoxia that induced apoptosis of NSCs in vivo.

MiR-26a inhibits hypoxia-induced apoptosis by directly targeting GSK-3β
Previous studies showed that GSK-3β played a crucial role in regulating apoptosis of NSCs, and inhibition of GSK-3β conferred NSCs resistance to apoptotic stimulator [16][17][18]. Additionally, it was reported that miR-26a directly targeted the 3 -UTR of GSK-3β in human adipose-derived mesenchymal stem cells [19]. Therefore, we hypothesize that GSK-3β is involved in hypoxia that induced apoptosis of NSCs, and miR-26a can target GSK-3β in NSCs, resulting in inhibition of hypoxia-induced apoptosis. As expected, CoCl 2 treatment, which resulted in down-regulation of miR-26a ( Figure 1A,B), increased GSK-3β protein level ( Figure 4A,B). Moreover, knockdown of miR-26a in CoCl 2 -treated NSCs elevated GSK-3β expression further, while overexpression of miR-26a suppressed the expression of GSK-3β. Then, we performed dual-luciferase reporter assay to further evaluate the relationship between miR-26a and GSK-3β in NSCs. The construct of luciferase vector is shown in Figure 4C. The overexpression of miR-26a obviously suppressed luciferase activity in GSK-3β 3 -UTR (wild-type) transfected 293T cells, while knockdown of miR-26a increased the luciferase activity ( Figure 4D). Both overexpression and knockdown of miR-26a did not affect the luciferase activity in mutant GSK-3β 3 -UTR transfected 293T cells. The Western blot and luciferase data indicate that miR-26a regulates expression of GSK-3β by directly targeting the 3 -UTR. We then examined whether the anti-apoptosis effect of miR-26a is through regulation of GSK-3β. We used SB216763, a potent GSK-3β inhibitor, to block the function of GSK-3β. As shown in Figure 4E,F, inhibition of GSK-3β abolished the anti-apoptosis effect of miR-26a in CoCl 2 -treated NSCs. Taken together, the data conclude that GSK-3β is involved in hypoxia-induced apoptosis of NSCs and the anti-apoptosis effect of miR-26a is through suppression of GSK-3β.

MiR-26a regulates β-catenin and its target gene
It was reported that β-catenin is regulated by GSK-3β [22,23] and tightly connected with apoptosis [24]. Then, we detected whether the expression of β-catenin is regulated by miR-26a. Figure 5A shows that the overexpression of miR-26a increased the β-catenin protein level compared with only CoCl 2 -treated NSCs, while the knockdown of miR-26a decreased the β-catenin protein level. The mRNA level of β-catenin is consistent with the protein expression  ( Figure 5B). It is well established that cyclin D1 is the target gene of β-catenin. Then, we further examined both mRNA and protein level of cyclin D1. As expected, the expression of cyclin D1 correlates well with the expression of β-catenin ( Figure 5). The data suggested the anti-apoptosis effect of miR-26a might be through regulating β-catenin expression.

MiR-26a overexpressed NSCs improves the neurological function of CA rats
Even though stem cell therapy is currently one of the most promising strategies for the treatment of neuronal injury and degenerative neuronal disease [7], the hypoxic microenvironment in the brain is always the limiting step to make the best use of this kind of therapy. As it was shown that overexpression of miR-26a improved the survival of NSCs in our in vitro hypoxia injury model and in vivo CA model, we then tried to examine whether the miR-26a overexpressed NSCs could improve the neurological function of CA rats. As shown in Table 1, the transplantation of miR-26a overexpressed NSCs significantly improved the neurological function of CA rats, while no improvement was observed in CA rats that were transplanted with miR-26a down-regulated NSCs. Additionally, we also observed an improvement of neurological function in negative control group, but the improvement is not as significant as miR-26a overexpressed group. The reason is that miR-26a could help NSCs survive in the hypoxic condition, and the survived NSCs could differentiate into new neuron and replace the damaged one.

Discussion
NSCs transplantation is one of the most promising strategies to treat CA-induced brain damage [7]. However, the poor survival of NSCs in severe hypoxic condition limits efficacy of this treatment. Therefore, how apoptosis could be inhibited to improve survival of transplanted NSCs in hypoxic environment is the most urgent issue to resolve. MiR-26a is one of the most important miRNAs that was extensively investigated and tightly connected to apoptosis. It was reported that miR-26a could prevent endothelial cell apoptosis by directly targeting TRPC6 in the setting of atherosclerosis [25]. Lee et al. [26] also showed that miR-26a could inhibit apoptosis of human liposarcoma through inhibiting RCBTB1. On the contrary, it is also reported that miR-26a could promote apoptosis in renal cancer and tongue squamous cell carcinoma [27,28]. In hypoxic condition, miR-26a also plays both positive and negative role in regulating apoptosis. Some researchers have revealed that miR-26a helped to decrease pro-apoptotic signaling [13], modulate innate immune response [29], promote proliferation of vascular endothelial cells and angiogenesis [30,31], inhibit hypoxia-induced tubular cell apoptosis [14] and attenuate cardiac ischemia reperfusion injury [15]. Some other researchers have reported that miR-26a could promote apoptosis of hypoxic rat neonatal cardiomyocytes [31]. The information suggests that miR-26a plays a complicated and important role in regulating apoptosis and manipulating CA-induced brain damage. In the present study, we used qRT-PCR to detect the mRNA level of miR-26a in hypoxic condition in vitro and in vivo. In CoCl 2 -treated NSCs, miR-26a declined remarkably compared with control group (P<0.05). Consistently, a significant decrease of miR-26a was also observed in CA rats 24 h after ROSC. The down-regulation of miR-26a in hypoxic condition indicates this gene may play a pivotal role in CA induced NSCs apoptosis.
Then we examined the anti-apoptosis effect of miR-26a in our model. We found that overexpression of miR-26a significantly reduced apoptosis of CoCl 2 -treated NSCs, while knockdown of miR-26a further increased apoptosis of CoCl 2 -treated NSCs. In the CA rat model, the miR-26a overexpressed NSCs are resistant to CA-induced apoptosis, while miR-26a down-regulated NSCs are more sensitive to CA-induced apoptosis. These results imply that miR-26 could block apoptosis induced by hypoxia and overexpression of miR-26a in NSCs helps NSCs to better survival in such severe hypoxic environment.
It is well known that GSK-3β is a pivotal regulator of apoptosis [17]. Inhibition of GSK-3β makes NSCs more resistant to apoptotic stimulators [16][17][18]. Interestingly, it is reported that miR-26a could directly target the 3 -UTR of GSK-3β in human adipose-derived mesenchymal stem cells [19]. Therefore, we tested whether miR-26a regulates the expression of GSK-3β in hypoxic condition. The data show overexpression of miR-26a decreased GSK-3β protein level, while the knockdown of miR-26a elevated GSK-3β expression. This suggests miR-26a could also target GSK-3β in NSCs in hypoxic condition. The dual-luciferase reporter assay further confirms that GSK-3β is the target of miR-26a, as the overexpression of miR-26a obviously suppressed luciferase activity in GSK-3β 3 -UTR (wild-type) transfected 293T cells, while knockdown of miR-26a increased the luciferase activity. Additionally, our data also show that the anti-apoptosis effect of miR-26a is dependent on GSK-3β. GSK-3β inhibitor, SB216763, could abolish the anti-apoptosis effect of miR-26a in CoCl 2 -treated NSCs.
β-Catenin is another important regulator of apoptosis. The inhibition of β-catenin leads to increased apoptosis in various types of cancer [32]. In NSCs, β-catenin plays an important role in regulating differentiation and apoptosis of NSCs. It is reported that β-catenin could be regulated by GSK-3β [22,23]. As miR-26a directly targets GSK-3β decreasing GSK-3β expression, miR-26a can also manipulate the expression of β-catenin. As expected, we observed the overexpression of miR-26a increased the β-catenin protein level, while the knockdown of miR-26a decreased the β-catenin protein level. Moreover, the expression of cyclin D1, a β-catenin target gene, correlates well with the expression of β-catenin. The data allow us to conclude that miR-26a directly targets 3 -UTR of GSK-3β, resulting in increased β-catenin expression in NSCs.
Even though miR-26a overexpression obviously decreased hypoxia-induced NSCs apoptosis, the protective effects on neurological function still need to be evaluated. We transplanted miR-26a overexpressed or down-regulated NSCs into CA rats, and examined the neurological function of these rats. As expected, miR-26a overexpression significantly improved the neurological function of CA rats, while no improvement was observed in CA rats that were transplanted with miR-26a down-regulated NSCs. MiR-26a suppresses apoptosis and improves survival of NSCs in the hypoxic condition. The increased NSCs could differentiate into new neuron and replace the damaged one in the injured brain region.
In sum, miR-26a could directly targets 3 -UTR of GSK-3β leading to activation of β-catenin signaling pathway, which inhibits NSCs apoptosis and improves NSCs survival in hypoxic condition. The increased NSCs help repair or replace the injured neuron in the damaged brain region. Therefore, manipulating miR-26a expression could provide a new target for the treatment of CA-induced brain damage, which is one of the major threats to human life and kill too many people each year [33,34].