Upregulation of miR-499a-5p Decreases Cerebral Ischemia/Reperfusion Injury by Targeting PDCD4

MiR-499a-5p was significantly downregulated in degenerative tissues and correlated with apoptosis. Nonetheless, the biological function of miR-499a-5p in acute ischemic stroke has been still unclear. In this study, we found that the plasma levels of miR-499a-5p were significantly downregulated in 64 ischemic stroke patients and negatively correlated with the National Institutes of Health Stroke Scale score. Then, we constructed cerebral ischemia/reperfusion (I/R) injury in rats after middle cerebral artery occlusion and subsequent reperfusion and oxygen–glucose deprivation and reoxygenation (OGD/R)-treated SH-SY5Y cell model. Transfection with miR-499a-5p mimic was accomplished by intracerebroventricular injection in the in vivo I/R injury model. We further found that miR-499a-5p overexpression decreased infarct volumes and cell apoptosis in the in vivo I/R stroke model using TTC and TUNEL staining. PDCD4 was a direct target of miR-499a-5p by luciferase report assay and Western blotting. Knockdown of PDCD4 reduced the infarct damage and cortical neuron apoptosis caused by I/R injury. MiR-499a-5p exerted neuroprotective roles mainly through inhibiting PDCD4-mediated apoptosis by CCK-8 assay, LDH release assay, and flow cytometry analysis. These findings suggest that miR-499a-5p might represent a novel target that regulates brain injury by inhibiting PDCD4-mediating apoptosis.


Introduction
Ischemic stroke is identified as a type of cerebrovascular disease accounting for nearly 90% of stroke cases with lower life quality of victims and huger public health burden (Benjamin et al. 2018;Luengo-Fernandez et al. 2013). Sudden insufficient blood flow to an area of the brain is the main characteristics of ischemic stroke, which could cause neuronal cell apoptosis, necrosis, and other metabolism-related disorders (Tobin et al. 2014). Despite the advances in rapid restoration of the blood supply for ischemic stroke, blood flow reperfusion, a process termed cerebral ischemia/reperfusion (I/R) injury seriously limits its development (Chomova and Zitnanova 2016). Thus, a better understanding of the molecular mechanisms underlying the pathological process of I/R injury may improve the ischemic stroke therapy.
Human programmed cell death 4 (PDCD4) was initially identified as a nuclear antigen gene and mapped at chromosome 10q24 (Soejima et al. 1999), which is further confirmed to be a novel tumor suppressor to show multifunctions inhibiting cell growth, invasion, and especially inducing apoptosis (Matsuhashi et al. 2019). Notably, PDCD4 was involved in several miRNAs-meditated I/R injury as follows: Knockdown of miR-21 or inhibition of hypoxia inducible factor 1 alpha (HIF-1α) abolished the Ischemic postconditioning-mediated attenuation of intestinal injury and apoptosis and the downregulation of PDCD4 (Jia et al. 2017). MiR-21 participates in H 2 O 2 -mediated gene regulation and functional modulation in cardiac myocytes via its target gene PDCD4 (Cheng et al. 2009). MiR-340-5p might exert neuroprotective effects during OGD/R injury by targeting PDCD4 (Zheng et al. 2020). In addition, PDCD4 silencing can activate the Akt signaling pathway and attenuate vascular endothelial cell injury caused by ischemia-reperfusion in the lower extremities in rats ). Our previous prediction analysis showed that PDCD4 was a target gene of miR-499a-5p. However, there were no data regarding the miR-499a-5p/PDCD4 axis in regulating cerebral I/R injury until now.
Therefore, the aims of the present study were to investigate the functional role of miR-499a-5p in neuronal injury induced by cerebral I/R using an in vivo middle cerebral artery occlusion (MCAO) rat model and in vitro OGD/R-induced SH-SY5Y cellular model. Moreover, we confirmed the direct targeting of miR-499a-5p on PDCD4 in neuronal injury.

Blood Samples of Patients with Acute Ischemic Stroke
Total 64 patients with acute ischemic stroke were enrolled from the 1st Affiliated Hospital, School of Medicine, Zhejiang University (Zhejiang Province, China), which were all clinically diagnosed by neurologists based on the medical imaging diagnostic methods (CT scanning and MRI), clinical symptoms, and neurological examination. The baseline characteristics of enrolled stoke patients are summarized at Table 1. Clinical severity was assessed at the third month after the stroke onset using the National Institutes of Health Stroke Scale (NIHSS) score (Special report from the National Institute of Neurological Disorders and Stroke. Classification of cerebrovascular diseases III 1990). The patients who received thrombolytic therapy or diagnosed as renal or liver failure, tumor, infectious, and hematologic disease were excluded. Equivalent number of age-and sex-matched patients without medical history of stroke were enrolled as controls. The peripheral blood sample was collected from patient/volunteer into tubes containing EDTA, which was immediately centrifuged at 1500×g for 10 min at 4 °C until further analysis. The use of the human blood samples for research purposes was approved by the Ethics Committee of the 1st

Construction of I/R Injury Rat Model
Healthy male Sprague-Dawley (SD) rats at the age of 25 weeks (220-250 g) were purchased from Vital River Laboratory Animal Technology Co. (Beijing, China) and housed in a standard environment with free access to food/ water. Rat model of cerebral I/R injury was established by 2 h of middle cerebral artery occlusion (MCAO) via intraluminal silicon-coated filament method following previously reported procedures (Begum et al. 2015). Briefly, rats were subcutaneously anesthetized with isoflurane (3% initially, 1 to 1.5% maintenance). Then, midline skin incision was made to carefully isolated the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Subsequently, a heparinized intraluminal silicon-coated filament (diameter 0.43 ± 0.02 mm) was inserted from ECA through the ICA until it reached the CCA, and finally blocked the origin of middle cerebral artery (MCA). After 2 h occlusion, the filament was slowly withdrawn, with subsequent 24 h of reperfusion. The rats in the sham group underwent the same operations with the exception of the filament insertion. Following depth of anesthesia, the brains of rats from different groups were quickly removed for further analysis.

Animal Groups
All rats were randomly assigned as the following three groups with six rats per group: (1) Sham group; (2) I/R group; (3) I/R + mimic group; (4) I/R + siNC group; and (5) I/R + siPDCD4 group, of which miR-499a-5p mimics, siNC, and siPDCD4 were synthesized by GenePharma Co., Ltd. (Shanghai, China), diluted with EntransterTM in vivo transfection reagent (Engreen, Beijing, China), and then intracerebroventricularly (i.c.v) injected into the cerebral cortex with a stereotaxic instrument while the rats were under anesthesia 3 days prior to MCAO.

2,3,5-Triphenyltetrazolium Chloride (TTC) Staining
TTC staining was conducted in accordance with a previous method to measure infarct volumes (Yang et al. 2018). In brief, the brains of rats from the above groups were quickly removed at 24 h after reperfusion, frozen at -20 °C for 30 min and then cut into 1.5-mm coronal sections with a sharp blade. The sections were incubated in 2% TTC solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C and then fixed in 10% formalin for 2 h. After photographed, the infarct volume calculation was performed blindly with Image-Pro Plus 6.0 analysis software according to the following formula: total infarct volume/total brain volume × 100%.

TdT-Mediated dUTP Nick-End Labeling (TUNEL) Staining
TUNEL staining was performed to examine the apoptosis of cortical neurons in rat brains by a commercially available kit (Roche Diagnostics Crop., Indianapolis, USA). Briefly, the cortex region brain tissues were fixed with 4% paraformaldehyde, embedded in paraffin and made into 5 μm thickness sections. Afterwards, the sections were digested with 2% proteinase K for 30 min, washed with PBS (3× for 5 min) and incubated with TUNEL reaction mixture at 37 °C for 30 min in darkness, followed by immerged into DAPI staining (Beyotime Biotechnology, China). Positive TUNEL staining (green) and DAPI staining (blue) were observed under a fluorescence microscope. The percentage of positive TUNEL staining was evaluated by determining the ratio of the number of TUNEL-positive cells to that of total cells in each field.

Cell Culture, Transfection, and OGD/R Establishment
A neuron-like human-derived neuroblastoma cell line SH-SY5Y was provided by American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, USA) with 10% fetal bovine serum (FBS, Gibco) at 37 °C. The miR-499a-5p mimic, inhibitor, corresponding negative control miR-NC, small interference RNA against PDCD4 (siP-DCD4) and siNC were purchased from GenePharma Co., Ltd. The pcDNA3.1/PDCD4 vector was generated by inserting the open reading frame of PDCD4 without 3′UTR into the pcDNA3.1 vector (Sangon Biotech, Shanghai, China). Cell transfection or co-transfection was performed in SH-SY5Y cells, which at a density of 3 × 10 5 cells per well were seeded in six-well plates using Lipofectamine 2000 reagent (Invitrogen). After 48 h transfection, oxygen and glucose deprivation/reoxygenation (OGD/R) was performed as briefly described as follows: SH-SY5Y cells were subjected to 5 h of glucose-free DMEM in a pre-conditioned hypoxic condition (1% O 2 , 5% CO 2 , and 94% N 2 ), followed by 24 h of reoxygenation by replacing the hypoxic medium with standard medium under normoxic conditions.

Western Blot Analysis
Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology Co. Ltd., Shanghai, China) was employed to extract total protein from tissues or cells and protein concentration was examined using BCA Protein Assay reagent kit (Beyotime Biotechnology). A total of 30 μg of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene membrane (Millipore). The membranes were blocked with 5% non-fat milk for 2 h and incubated with primary antibodies against PDCD4, caspase-3, and GAPDH overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 2 h at room temperature. Protein bands were detected using an enhanced chemiluminescent reagent (Thermo Fisher Scientific).

Cell Viability Assay
SH-SY5Y cells at a density of 4000 cells per well were seeded into 96-well plates and incubated overnight at 37 °C. Next day, cells in each well were washed with PBS and incubated with 10 µl CCK-8 reagent (Dojindo Molecular Technologies, Dojindo, Japan) for 2 h at 37 °C. Afterwards, the absorbance was measured at 450 nm using a microplate reader and relative cell viability was expressed by setting the control group as "1". Each sample was analyzed in triplicate.

Cytotoxicity Assay
The release of lactate dehydrogenase (LDH) was analyzed by the LDH Cytotoxicity Assay Kit (Beyotime Biotechnology) for evaluation of cell cytotoxicity. In brief, SH-SY5Y cells were seeded into 96-well plates at a density of 4000 cells per well and incubated with LDH release regent (150 µL per well) at 37 °C for 1 h. After determining the absorbance at 490 nm, the LDH release rate was expressed as the percentage of the maximum enzymatic activity of the control sample. Each sample was analyzed in triplicate.

Flow cytometric analysis
Apoptotic cells were quantified by flow cytometry with Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA) in accordance with the manufacturer's instructions. Briefly, SH-SY5Y cells harvested by trypsin digestion and seeded into 6-well plates at a density of 2.0 × 10 5 cell per well. After washed with PBS, cells were re-suspended in 200 μL binding buffer containing 10 μL Annexin V-FITC and 5 μL PI for 30 min in the dark. The apoptotic cells (Annexin V positive) were distinguished by a flow cytometry. Each sample was analyzed in triplicate.

Caspase-3 Activity Assay
Colorimetric Assay Kits (R&D systems Co. Germany) were utilized to measure the caspase-3 activity in SH-SY5Y cells from different groups following the manufacturer's protocol.
In brief, cells were centrifuged at 10,000×g for 10 min for preparation of cell lysate supernatant. Then, the supernatant was incubated with 50 µL reaction buffer and 5 µL of caspase-3 substrate at 37 °C for 1.5 h. according to the absorbance at 405 nm, relative caspase-3 activity was expressed as fold change of caspase-3 activity in experimental group divided by that of control group. Each sample was analyzed in triplicate.

Statistical Analysis
All quantitative data were presented as mean ± SD of three independent experiments. Data were evaluated using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Comparison on the expression of miR-499a-5p between ischemic stroke patients and matched healthy controls was performed using Wilcoxon signed-rank test. Correlation between miR-499a-5p and NIHSS scores was estimated by Pearson correlation test. Differential comparison was performed using the Student's t test for two group and one-way analysis of variance with Tukey's post hoc test for multiple groups. A value of p less than 0.05 was thought to be statistically significant.

Circulating miR-499a-5p Expression Level in Acute Ischemic Stroke Patients
To reveal the role of miR-499a-5p in acute ischemic stroke, we performed RT-qPCR analysis to analyze the expression level of miR-499a-5p in the blood samples derived from 64 cases of ischemic stroke patients and matched healthy controls. As shown in Fig. 1a, circulating miR-499a-5p expression level was significantly downregulated in stroke group compared with control group. We further analyzed the association between miR-499a-5p and NIHSS scores. The results of Pearson correlation test showed that miR-499a-5p expression level was negatively correlated with NIHSS scores (Fig. 1b, r = − 0.3802, p = 0.0046).

Ischemic Expression of miR-499a-5p and PDCD4 in Rat Brain Against Cerebral I/R Injury
To investigate the potential role of miR-499a-5p in brain I/R injury, the rats randomly received an intracerebroventricular injection of miR-499a-5p mimic prior to MCAO treatment.
RT-qPCR analysis first demonstrated that the expression of miR-499a-5p was significantly decreased in the I/R group compared with sham group, but notably increased after intracerebroventricular injection of miR-499a-5p mimic in I/R group (Fig. 2a). Meanwhile, we found miR-499a-5p overexpression remarkedly suppressed the increased expression of PDCD4 mRNA and protein in I/R group by RT-qPCR (Fig. 2b) and Western blot analysis (Fig. 2c, d), respectively.

Overexpression of miR-499a-5p Reduced the Infarct Damage and Cortical Neuron Apoptosis Caused by I/R Injury
Based on the constructed I/R injury rat model, we further investigate the role of miR-499a-5p cerebral infarction and the apoptosis of cortical neurons. According to the results from TTC staining (Fig. 3a, b), normal brain tissue was red, while the infarcted brain tissue was pale. The brain tissue of the Sham group was red with no signs of pale infarction identified. The infarct region was obviously observed in the brain of I/R groups in comparison with the Sham group, which was significantly reduced after intracerebroventricular injection of miR-499a-5p mimic in I/R group. TUNEL assay further showed more TUNEL-positive cells was observed in the brain cortical neurons sections from the I/R group, whereas miR-499a-5p mimic treatment induced significant decrease in the TUNEL-positive cells in I/R group (Fig. 3c). Furthermore, pro-apoptotic caspase-3 was found to be significantly upregulated in I/R group compared with Sham group, which was obviously attenuated after miR-499a-5p mimic injection in I/R group (Fig. 3d). These in vivo findings indicated that overexpression of miR-499a-5p could exert protective role against cerebral I/R injury by decreasing the infarct damage and cortical neuron apoptosis.

MiR-499a-5p Expression was Decreased in the In Vitro Model of OGD/R
To confirm the protective role of miR-499-5p in cellular OGD/R injury, we established OGD/R cell model in SH-SY5Y cells and performed a series of functional experiments to evaluate the constructed in vitro model of OGD/R. CCK-8 assay showed that cell viability was significantly reduced in OGD/R cells compared to those in normoxic control cells (Fig. 4a). Consistent with decreased cell viability, the levels of LDH release (Fig. 4b) and caspase-3 activity (Fig. 4c) were both significantly elevated when the SH-SY5Y cells under OGD/R conditions. What's more, flow cytometry further manifested the percentage of apoptotic cells was The protein level of PDCD4 was measured in SH-SY5Y cells transfected with miR-499a-5p mimics, inhibitor or miR-NC, followed by OGD/R treatment. Each value represented mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with miR-NC by Student's t-test remarkedly increased after OGD/R treatment (Fig. 4d).
Within the successfully constructed OGD/R cell model, we consistently observed that miR-499-5p expression was significantly downregulated in OGD/R group compared with control group (Fig. 4e).

MiR-499a-5p Exerted Protective Effects Against OGD/R-Induced Injury in SH-SY5Y Cells
Since the expression of miR-499a-5p was downregulated in OGD/R cell model, we then performed gain-of-function assays in SH-SY5Y cells by transfecting with miR-499a-5p mimic, followed by OGD/R treatment. RT-qPCR analysis Relative luciferase activities were analyzed in SH-SY5Y cells 48 h after co-transfection of miR-499a-5p mimic and either wild-type or mutant PDCD4 3′UTR. **p < 0.01, compared with miR-NC by Student's t-test; (C) RT-qPCR and (D) Western blot analysis was used to determine the expression of PDCD4 in SH-SY5Y cells trans-fected with miR-499a-5p mimic or miR-NC, and then was exposed to OGD/R conditions. (E) RT-qPCR and (F) Western blot analysis was used to determine the expression of PDCD4 in SH-SY5Y cells transfected with miR-499a-5p inhibitor or miR-NC, and then was exposed to OGD/R conditions. Each value represented mean ± SD of three independent experiments. **p < 0.01, compared with Control; #p < 0.05, ##p < 0.01, compared with OGD/R by one-way analysis of variance with Tukey's post hoc test confirmed the successfully transfection of miR-499a-5p mimic in SH-SY5Y cells, as demonstrated by remarkedly increased miR-499a-5p expression (Fig. 5a). Then the effects of miR-499a-5p on cell viability, LDH release and apoptosis were evaluated in OGD/R cell model. The results from CCK-8 assay indicated that miR-499a-5p mimic transfection obviously increased the cell viability in OGD/R cells (Fig. 5b). The LDH release was significantly reduced after miR-499a-5p overexpression in OGD/R cell model (Fig. 5c).
In addition, miR-499a-5p overexpression notably decreased the cell apoptosis in SH-SY5Y cells after OGD/R treatment, as reflected by caspase-3 activity assay (Fig. 5d) and flow cytometry (Fig. 5e). Moreover, we performed loss-of-function assay to confirm the protective effects of miR-499a-5p against OGD/R-induced injury. Our data demonstrated that knockdown of miR-499a-5p (Fig. 5f) significantly decreased cell viability (Fig. 5g) and enhanced the caspase-3 activity (Fig. 5H) in OGD/R cell model. To further confirm apoptosis, we measured the protein level of caspase-3. As shown in Fig. 5i, miR-499a-5p mimic transfection obviously downregulated, while inhibitor transfection upregulated the protein level of caspase-3, compared with miR-NC transfection in OGD/R cell model.

PDCD4 was a Potential Target of miR-499a-5p
The online TargetScan program was first employed to predict the targets of miR-499a-5p. Among these predicted targets, we selected five targets of miR-499a-5p. As shown in Fig. 1S-A, the binding sites between miR-499a-5p and four top targets (MRPS35, ARGLU1, PFN2, and HNRNPC) were displayed. Western blot analysis showed that the protein levels of MRPS35, ARGLU1, PFN2, and HNRNPC were downregulated in different degrees after miR-499a-5p mimic transfection compared with siNC transfection in OGD/R cell model (Fig. 1S-B). next, we focused on PDCD4, closely associated with the occurrence of apoptosis, has been involved in several miRNAs-meditated I/R injury (Cheng et al. 2009;Zheng et al. 2020), which thus was selected as a potential target of miR-499a-5p. As shown in Fig. 6a, the binding sites between miR-499a-5p and PDCD4 were displayed. Then, Fig. 7 Knockdown of PDCD4 reduced the infarct damage and cortical neuron apoptosis caused by I/R injury. The rats randomly received an intracerebroventricular injection of siPDCD4 or siNC prior to MCAO treatment, with Sham as control (n = 6 per group). (A) Analysis of the volumes of infarction in rat brain tissues in the above two groups. (B) The apoptosis of cortical neurons was evaluated by TUNEL staining. (C) Measurement of caspase-3 protein levels in the above two groups using Western blotting. ***p < 0.001, compared with siNC by Student's t-test dual luciferase reporter assay was performed to confirm PDCD4 as a target of miR-499a-5p. As depicted in Fig. 6b, the luciferase activity of WT PDCD4 in the miR-499a-5p mimic group was significantly reduced in comparison with the miR-NC group. However, there was no remarkable difference in relation to the luciferase activity of MUT PDCD4 between the miR-NC and miR-499a-5p groups. Next, we analyzed the expression of PDCD4 in OGD/R cell model. Both the RT-qPCR (Fig. 6c) and Western blot analysis (Fig. 6d) consistently demonstrated that OGD/ R-induced upregulation of PDCD4 mRNA and protein expression was significantly reversed by miR-499a-5p mimic transfection in SH-SY5Y cells. MiR-499a-5p inhibitor transfection obtained the opposite results in SH-SY5Y cells (Fig. 6e, f). These results suggested that miR-499a-5p negatively regulated PDCD4 expression via binding its 3′-UTR.

Knockdown of PDCD4 Reduced the Infarct Damage and Cortical Neuron Apoptosis Caused by I/R Injury
We next investigate the role of PDCD4 in I/R injury in vivo. According to the results from TTC staining (Fig. 7a), intracerebroventricular injection of siPDCD4 dramatically decreased the infarct area in MCAO group compared with siNC group. TUNEL assay indicated that knockdown of PDCD4 induced significant decrease in the TUNEL-positive cells in I/R group (Fig. 7b). Consistently, the protein level of pro-apoptotic caspase-3 was obviously attenuated after siPDCD4 injection in I/R group compared with siNC injection (Fig. 7c).

Discussion
In the present study, we reported that circulating miR-499a-5p expression level was significantly downregulated in stroke patients and negatively associated with NIHSS scores. By constructing the in vivo MCAO rat model, we further confirmed that the expression of miR-499a-5p was rapidly increased in the rat brain tissues after MCAO. Subsequently, rats were given intracerebroventricular injection of miR-499a-5p mimic and the data showed that overexpression of miR-499a-5p significantly effectively reduced brain infarct size and the apoptosis of cortical neurons. Consistently, miR-499a-5p expression has been shown to be remarkedly decreased after myocardial I/R injury . Similarly, miR-499a-5p may attenuate TNF-αinduced human nucleus pulposus cell apoptosis (Sun et al. 2019). These data indicated that miR-499a-5p suppressed the cerebral I/R injury might by decreasing MCAO-induced apoptosis.
As previously demonstrated, the neuron-like humanderived neuroblastoma cell line SH-SY5Y is a recognized as the most commonly used tool in five models of ischemiarelated injury, including OGD, H 2 O 2 -induced oxidative stress, oxygen deprivation, glucose deprivation, and glutamate excitotoxicity because of its human origin, catecholaminergic neuronal properties, and ease of maintenance (Liu et al. 2018b). Here, we further established an in vitro model of I/R injury with SH-SY5Y and determined the effect of miR-499a-5p on cell viability and apoptosis. Our data showed that overexpression of miR-499a-5p protected SH-SY5Y cells against OGD/R-induced injury, as reflected by elevated cell viability and decreased LDH release, apoptosis rate accompanied decrease in caspase-3 expression and caspase-3 activity. Knockdown of miR-499a-5p obtained Fig. 9 Overexpression of PDCD4 reversed the protective role of miR-499a-5p against OGD/R-induced injury. SH-SY5Y cells were co-transfected with miR-499a-5p mimic and PDCD4 overexpression plasmid or empty vector. (A) The relative expression of PDCD4 was determined in the above treated SH-SY5Y cells. (B) CCK-8 assay was utilized to analyze cell viability. (C) The release of lac-tate dehydrogenase (LDH) was analyzed in the above treated SH-SY5Y cells. (D) Flow cytometry was used to analyze cell apoptosis in the above treated SH-SY5Y cells. (E) Colorimetric assay of caspase-3 activity in the above treated SH-SY5Y cells. Each value represented mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001, compared with mimic + vector by Student's t-test the opposite results. As our best knowledge, I/R injury can induce apoptosis of tissues and organs, which is an important pathological manifestation of I/R injury affecting tissues and organs (Lopez-Neblina et al. 2005). Apoptosis usually occupies a crucial role in the pathogenesis of ischemic brain injury and the subsequent reperfusion damage (He et al. 2016;Machado et al. 2009). Similar to miR-499a-5pinduced suppression of apoptosis, miR-499a-5p could suppress the apoptosis of human nucleus pulposus cells and degradation of their extracellular matrix by targeting SOX4 (Sun et al. 2019). Downregulation of CD38 by miR-499a-5p significantly decreased the apoptosis and the LDH activity of H9C2 cells under hypoxia-reoxygenation conditions ). In addition, miR-499a-5p participated in circ_0079593 ) and lncRNA secretory carrier membrane protein 1 (SCAMP1) (Zong et al. 2019) regulating glioma cell proliferation, motility and apoptosis.
Since PDCD4 is closely associated with the occurrence of apoptosis (Matsuhashi et al. 2019). In our constructed in vivo and in vivo models, we found that PDCD4 expression was significantly upregulated compared with the corresponding control groups. As expected, we confirmed that the 3′UTR region of PDCD4 contains a binding site for miR-499a-5p and miR-400a-5p negatively regulated the PDCD4 expression. Furthermore, knockdown of PDCD4 reduced the infarct damage and cortical neuron apoptosis caused by I/R injury. PDCD4 knockdown imitated, while overexpression reversed the effects of miR-499a-5p on cell viability, LDH release, cell apoptosis, and caspase-3 activity in SH-SY5Y cells after OGD/R treatment. Similarly, PDCD4 silencing can attenuate vascular endothelial cell injury caused by ischemia-reperfusion in the lower extremities in rats ). In line with our data regarding on the role miR-499a-5p/PDCD4 axis in I/R injury, PDCD4 was demonstrated to be a downstream regulator of miR-21 (Jia et al. 2017;Cheng et al. 2009) and miR-340-5p regulating ischemic postconditioning-mediated attenuation of intestinal injury and apoptosis (Zheng et al. 2020). According to these facts, we concluded that miR-499a-5p exerted the protective role against OGD/R-induced injury might by targeting PDCD4 in SH-SY5Y cells.
In summary, the present study confirmed that the expression of miR-499a-5p was decreased during cerebral I/R injury in vivo and in vitro. Our in vitro transfection experiments show that elevated miR-499a-5p expression can rescue the apoptosis of SH-SY5Y cells under OGD/R condition. We further demonstrated that miR-499a-5p exerted the protective role against OGD/R-induced injury by targeting PDCD4 in SH-SY5Y cells. The data of this study suggest that targeting miR-499a-5p/PDCD4 axis may provide a potential therapeutic option for clinical treatment of ischemic stroke.
Funding Not applicable.

Data Availability
The data in this study are available in this published article.

Conflict of interest
The authors declare that they have no competing interests.
Ethical Approval The use of the human blood samples for research purposes was approved by the Ethics Committee of the 1st Affiliated Hospital, School of Medicine, Zhejiang University (Approval number: ZU349-2; 2017.8.16).

Consent to Participate
All participants signed the written informed consent, which is in accordance with the Declaration of Helsinki.