MicroRNA-532-5p protects against cerebral ischemia-reperfusion injury by directly targeting CXCL1

We investigated the function of microRNA (miR)-532-5p in cerebral ischemia-reperfusion injury (CI/RI) and the underlying mechanisms using oxygen-glucose deprivation and reperfusion (OGD/R)-treated SH-SY5Y cells and middle cerebral artery occlusion (MCAO) model rats. MiR-532-5p levels were significantly downregulated in OGD/R-treated SH-SY5Y cells and the brain tissues of MCAO model rats. MiR-532-5p overexpression significantly reduced apoptosis, reactive oxygen species (ROS), and inflammation in the OGD/R-induced SH-SY5Y cells. Bioinformatics analysis using the targetscan and miRDB databases as well as dual luciferase reporter assays confirmed that miR-532-5p directly binds to the 3’UTR of C-X-C Motif Ligand 1 (CXCL1). Methylation-specific PCR (MSP) analysis showed that miR-532-5p expression was reduced in OGD/R-treated SH-SY5Y cells because of miR-532-5p promoter hypermethylation. Moreover, 5-azacytidine, a methylation inhibitor, restored miR-532-5p expression in OGD/R-treated SH-SY5Y cells. Brain tissues of MCAO model rats showed significantly increased cerebral infarction areas, cerebral water, neuronal apoptosis, and activated CXCL1/CXCR2/NF-κB signaling, but these effects were alleviated by intraventricular injection of miR-532-5p agomir. These findings demonstrate that miR-532-5p overexpression significantly reduces in vitro and in vivo CI/RI by targeting CXCL1. Thus, miR-532-5p is a potential therapeutic target for patients with CI/RI.


INTRODUCTION
Stroke is a form of brain injury due to cerebral vascular rupture or interruption in blood supply to the affected part of the brain, and it leads to long-term disability in nearly 40 million individuals world-wide every year [1,2]. In several cases, restoration of cerebral blood supply is associated with cerebral ischemia-reperfusion injury (CI/RI) [3,4]. The currently available treatments for CI/RI include surgery and drugs, but the efficacy of these treatments is limited [5,6]. Therefore, there is an urgent need for newer and more effective treatment strategies for CI/RI. MicroRNAs (miRNAs) are non-coding RNAs (22 nucleotides in length) which can modulate mRNA expression [7,8]. Several studies have shown that dysregulation of specific miRNAs influences CI/RI progression. For example, Liu et al. reported that miR-211 protected against CI/RI in the MCAO model rats by downregulating p53-upregulated modulator of apoptosis (PUMA) [9]. MiR-193b-3p protected against focal CI/RI injury by inhibiting 5-LOX expression [10]. Furthermore, miR-532-5p attenuates the progression of ischemic stroke through inactivating PTEN and upregulating PI3K/Akt signaling pathway [11,12]. However, the detailed mechanisms underlying the neuroprotective effects of miR-532-5p in CI/RI are not fully clear.
Therefore, this study aimed to investigate the mechanisms underlying the neuroprotective role of miR-532-5p during CI/RI using SH-SY5Y cells grown under oxygen-glucose deprivation and reperfusion (OGD/R) conditions and the cerebral artery occlusion (MCAO) rat model.

MiR-532-5p overexpression significantly reduced oxidative stress in OGD/R-treated SH-SY5Y cells
Flow cytometry analysis showed that ROS levels were significantly increased in SH-SY5Y cells upon OGD/R treatment, but were reduced by miR-532-5p overexpression (Figure 2A-2B). Furthermore, LDH and MDA levels were higher in the supernatants of OGD/R-treated SH-SY5Y cells than untreated controls, but were significantly reduced by miR-532-5p over-expression ( Figure 2C-2D). Moreover, SOD levels were decreased in the supernatant of OGD/R-treated SH-SY5Y cells than the corresponding controls, but were higher in miR-532-5p overexpressing SH-SY5Y cells treated with OGD/R ( Figure 2E). Taken together, these demonstrated that miR-532-5p upregulation inhibited OGD/R-induced oxidative stress.

MiR-532-5p overexpression decreased the levels of pro-inflammatory cytokines in the OGD/R-induced SH-SY5Y cells
We performed ELISA assays to estimate the secretion of pro-inflammatory cytokines in the supernatants of control and miR-532-5p overexpressing SH-SY5Y cells treated with or without OGD/R. The levels of IL-6, TNF-α, IL-1β and MPO were markedly higher in the supernatants of OGD/R-treated SH-SY5Y cells than the controls, but were significantly reduced in miR-532-5p overexpressing SH-SY5Y cells treated with OGD/R ( Figure 3A-3D). This suggested that miR-532-5p overexpression significantly inhibited the inflammatory response in SH-SY5Y cells.

MiR-532-5p
reduces MCAO-induced brain pathology in the in vivo rat CI/RI model Finally, we established the in vivo middle cerebral artery occlusion (MCAO) rat model to determine the function of miR-532-5p in CI/RI. MiR-532-5p level was notably higher in miR-532-5p agomir group ( Figure 7A). MiR-532-5p levels were reduced in the brain tissues of MCAO group rats (P < 0.01), but were higher in the miR-532-5p agomir plus MCAO group ( Figure 7A). Furthermore, MCAO treatment significantly increased cerebral infarction area and cerebral water, but these effects were significantly reduced by miR-532-5p overexpression AGING ( Figure 7B-7D). Moreover, TUNEL assay showed that MCAO treatment significantly increased apoptosis in the rat brain tissues, but these effects were markedly decreased by miR-532-5p overexpression ( Figure 7E-7F). The levels of CXCL1, CXCR2 and p-p65 proteins were significantly upregulated in the brain tissues of the MCAO group rats, but these effects were reversed by miR-532-5p overexpression ( Figure 8A-8D). Taken together, these data demonstrated that miR-532-5p significantly reduced brain pathology in the rat model of CI/R.

DISCUSSION
MicroRNAs are involved in multiple human diseases, and their dysregulation plays a critical role in CI/RI progression [9,16]. This research demonstrated that miR-532-5p significantly reduced apoptosis in OGD/Rinduced SH-SY5Y cells and MCAO-induced brain pathology in the CI/RI model rats. Previous reports revealed that miR-532-5p played a neuroprotective role during ischemic stroke [11,12]. Our data were in line with these recent findings and further confirmed that miR-532-5p plays a protective role in CI/R. In contrast, a previous study suggested that miR-532-5p upregulation promoted proliferation of breast cancer cells [17]. This suggests contrasting roles for miR-532-5p in different cell types. Furthermore, OGD/R treatment increased miR-532-5p promoter methylation, thereby reducing miR-532-5p levels. Promoter methylation-mediated downregulation of miRNAs is  commonly observed in tumorigenesis. For example, Ni et al. reported promoter methylation-associated downregulation of miR-638 in endometrial carcinoma [18]. Our findings are consistent with these results. Furthermore, our results confirm that OGD/R downregulated miR-532-5p level in SH-SY5Y cells by increasing miR-532-5p promoter methylation.
MicroRNAs regulate biological functions by decreasing expression of their target genes [19,20]. This research confirmed that CXCL1 mRNA was the target of miR-532-5p in CI/RI-induced SH-SY5Y cells. CXCL1 is over-expressed in several human diseases and is a member of the chemotactic superfamily [21,22]. CXCL1 promotes inflammation and progression of several cancers [23]. Our study demonstrated that CXCL1 promotes CI/RI progression. Furthermore, our findings show that miR-532-5p reduces CI/RI progression by targeting CXCL1. Wang et al. indicated that miR-532-5p inhibited glioma cell proliferation by targeting CSF1 [24]. CXCL1/CXCR2 (C-X-C motif chemokine receptor 2) mediated signaling promotes progression of inflammatory responses [25]. Manjavachi et al. reported that CXCL1 and CXCR2 suppressed the progression of peripheral neuropathy in rats [26]. Our study demonstrated that upregulation of miR-532-5p suppressed CI/RI through the CXCL1/CXCR2 pathway. CXCL1 is known to be a crucial mediator in show the methylation status of the CpG islands in the miR-532-5p promoter sequence in SH-SY5Y cells treated with OGD/R, 5-Aza or OGD/R + 5-Aza and untreated controls. 'U' refers to PCR with primers specific to unmethylated sequence and 'M' refers to PCR with primers specific to methylated sequence. (C) RT-qPCR analysis shows the relative expression levels of miR-532-5p in SH-SY5Y cells treated with OGD/R, 5-Aza or OGD/R + 5-Aza and untreated controls. (D) RT-qPCR analysis shows the relative expression levels of CXCL1 mRNA in SH-SY5Y cells treated with OGD/R, 5-Aza or OGD/R + 5-Aza compared to untreated controls. ** P < 0.01 compared to control; # P < 0.05, ## P < 0.01 compared to OGD/R. All experiments were performed in triplicate.
AGING inflammation and organ injury [27], and CXCR2 is the downstream protein of CXCL1 [28,29]. In a previous study, CXCL1/CXCR2 pathway could lead to the injury of brain tissues [15]. Our research was consistent to the previous reports, suggesting that miR-532-5p could reverse OGD/R-induced cell growth inhibition via mediation of CXCL1/CXCR2 pathway. On the other hand. We also found that miR-532-5p inactivated NF-κB, which is a key mediator of tumorigenesis and inflammatory responses [28,30]. Moreover, p65, a key protein in NF-κB signaling, and it is a crucial player in nervous and immune systems [31]. Ni et al. reported that the crosstalk between NFkB-dependent astrocytic CXCL1 and neuronal CXCR2 played a key role in chronic pain [32]. Our research confirmed that miR-532-5p overexpression alleviated CI/RI pathology in rat brains by inhibiting CXCL1/CXCR2/NF-κB signaling pathway.
Our study has few limitations. Firstly, our study focused only on the CXCL1/CXCR2/NF-κB signaling pathway. Secondly, we identified CXCL1 mRNA as the only

Figure 6. MiR-532-5p overexpression suppresses OGD/R-induced inflammation in SH-SY5Y cells via CXCL1/CXCR2/NF-κB axis.
(A) Representative western blot shows the expression of CXCL1, CXCR2, p65 and p-p65 proteins in control or miR-532-5p agomirtransfected SH-SY5Y cells, treated with or without OGD/R. (B-C) The histogram plots show the expression levels of CXCL1 and CXCR2 proteins relative to β-actin protein levels in control or miR-532-5p agomir-transfected SH-SY5Y cells treated with or without OGD/R. (D) The histogram plot shows the levels of p-p65 relative to p65 levels in control or miR-532-5p agomir-transfected SH-SY5Y cells treated with or without OGD/R. (E) SH-SY5Y cells were transfected with NC or pcDNA3.1-CXCL1. The efficiency of cell transfection was detected by RT-qPCR. (F) The viability of SH-SY5Y cells was tested by CCK-8 assay. ** P < 0.01 compared to control; ## P < 0.01 compared to OGD/R. ^^P < 0.01 compared to OGD/R plus miR-532-5p agomir group. All experiments were performed in triplicate.
AGING target of miR-532-5p in CI/RI. Therefore, future investigations are needed to identify other critical signaling pathways and target mRNAs of miR-532-5p in CI/RI.
In conclusion, miR-532-5p suppressed CI/RI progression by targeting CXCL1. Moreover, our results suggest that miR-532-5p overexpression may be therapeutically beneficial for ischemic stroke patients.

Cell culture
Human neuroblastoma cell (SH-SY5Y) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA), and and rat adrenal medulla pheochromocytoma cell lines (PC-12) were obtained from Chinese Academy of Sciences (Shanghai, China).

OGD/R treatment
SH-SY5Y cells were first cultured in deoxygenated glucose-free DMEM medium using a hypoxic vessel maintained at 37°C for 4 h (95% N2 and 5% CO2). Then, the cells were transferred onto high glucose DMEM medium containing 10% FBS and cultured under normoxic conditions (5% CO2) at 37°C for 24 h as previously described [33].

CCK-8 assay
The control (NC) or miR-532-5p agomir-transfected SH-SY5Y cells (5 × 10 3 per well) were seeded overnight and then treated with normoxia or OGD/R for 48 h. Then, we added 10 μl CCK-8 reagent (Beyotime, Shanghai, China) into each well and incubated the cells for another 2 h at 37°C. Finally, we measured the absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific).

Cellular apoptosis
The control or miR-532-5p-transfected SH-SY5Y cells were treated with normoxia or OGD/R. Then, they were trypsinized, washed with PBS, resuspended and stained with 5 μl Annexin V and propidium (PI) in the dark for 15 min. The stained cells were analyzed in a BD flow cytometer (BD, Franklin Lake, NJ, USA) and the proportion of apoptotic cells (Annexin V + PI + and Annexin-V + PI − ) were analyzed using a FACS software.

Estimation of intracellular reactive oxygen species
After treatments, SH-SY5Y cells were incubated in buffer containing ROS-sensitive dye, DCFDA (Beyotime, Shanghai, China) as previously described [34] for 20 min. Then, the cells were centrifuged at 300 Representative western blot shows the expression levels of CXCL1, CXCR2, p65 and p-p65 proteins in brain tissues of sham, miR-532-5p agomir, I/R, and I/R plus miR-532-5p agomir group rats. (B-C) The histogram plots show the levels of CXCL1 and CXCR2 proteins relative to β-actin levels in brain tissues of sham, miR-532-5p agomir, I/R, and I/R plus miR-532-5p agomir group rats. (D) The histogram plot shows the levels of p-p65 relative to p65 in brain tissues of sham, miR-532-5p agomir, I/R, and I/R plus miR-532-5p agomir group rats. ** P < 0.01 compared to Sham; ## P < 0.01 compared to I/R. All experiments were performed in triplicates. AGING xg, washed and resuspended in 1X PBS and analyzed by FACS using a BD flow cytometer (BD, Franklin Lake, NJ, USA). The relative levels of ROS were measured based on DCFDA intensity with appropriate controls.

In vivo MCAO model rats
Forty eight Wistar rats (aged 6 weeks, Chinese Academy of Sciences, Shanghai, China) were purchased from Vital River (Beijing, China) and housed in a dedicated SPF facility. The rats were randomly divided into four groups (n = 12 per group): (1) sham-operated (Sham); (2) miR-532-5p agomir; (3) I/R (MCAO), and (4) I/R plus miR-532-5p agomir. The rats in groups 3 and 4 were anesthetized with 45 mg/kg pelltobarbitalum natricum. Then, focal cerebral ischemia was induced by MCAO treatment as previously described [35]. The filament was removed after 1 h and reperfusion was performed. The rats in groups 1 and 2 suffered the same protocol, while it needs no filament insertion. We then injected saline by intra-ventricular injections into rats belonging to groups 1 and 3 , whereas, rats belonging to groups 2 and 4 received intra-ventricular injection containing miR-532-5p agomir as previously described [36]. The rats were sacrificed after 3 days. Then, we harvested the whole brain tissues and determined the cerebral infraction area, water content and other parameters. The animal experiments were performed in line with the NIH guidelines for the care and use of laboratory animals and the protocol approved by the Ethics Committees of Southeast University (No. SU20190507).

TUNEL staining
The paraffin sections were permeabilized and treated with 50 μl TUNEL reaction mixture (Sigma Aldrich, St. Louis, MO, USA) for 60 min at 37°C with no light. Then, the slides were treated with 50 μl peroxidase (POD, Beyotime) for 30 min, followed by incubation with diaminobenzidine substrate solution (50 μl, DAB, Beyotime) for 10 min at 25°C. The TUNEL-positive apoptotic cells in the brain tissues were analyzed using an optical microscope (Tokyo, Japan).

Brain water content detection
Briefly, the cerebral hemispheres were separated quickly after harvesting the rat brains and their wet weights were assessed. Then, the cerebral hemispheres were dried at 120°C for 48 h and the dry weight was estimated. The water content of the brain was calculated as follows: [(wet weight-dry weight)/wet weight] × 100%.

2, 3, 5-Triphenyltetrazolium chloride (TTC) staining
In brief, brain tissues from the four different rat groups were frozen for 30 min. Then, the brain tissues were cut and treated with 2% TTC solution (Sigma) for 20 min. Subsequently, the sections were fixed and photographed under a light microscope. The cerebral infarction area was expressed as follows: total infarct volume/total brain volume × 100%.

Statistical analysis
The data are expressed as means ± standard deviation (SD) of at least three independent experiments (12 rats per group in animal study). The data between two groups was analyzed using Student's t-test. The differences between multiple groups (≥ 3) were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test. P < 0.05 means statistically significant. Graphpad prism version 8.0 was used for the statistical analysis.

AUTHOR CONTRIBUTIONS
Yuanyuan Shi, Yun Xu and Pinglei Pan conceived and supervised the study; Zhongquan Yi and Panwen Zhao performed the experiments and analyzed the data. All authors approved the final version of the manuscript. were treated with miR-532-5p agomir, OGD/R or OGD/R plus miR-532-5p. Then, the viability of PC-12 cells was tested by CCK-8 assay.
(B) The apoptosis of PC-12 cells was tested by flow cytometry. ** P < 0.01 compared to Control; ## P < 0.01 compared to OGD/R. All experiments were performed in triplicates.