Introduction

Acute ischemic stroke (AIS), a critical and severe disorder, is the result of a sudden decrease in cerebral circulation.¹ Featured with high incidence, mutilation and death rate, AIS has threatened people’s health seriously.² Common pathological changes in the brain include the accumulation of toxins, global brain inflammation, cellular energy depletion and cell necrosis.^3,4 For AIS patients, intravenous infusion of alteplase remains the most recommended as well as the sole FDA-approved treatment.^5,6 However, the effectiveness of thrombolytic therapy is constrained by a short time frame, in addition to potential side effects.⁷ At the same time, few neurotherapeutic drugs are clinically available to treat more severe secondary brain injury, cerebral ischemia/reperfusion injury. This damage, in some cases, might be caused by the prompt blood-supply recovery as the main treatment for ischemic stroke.⁸ Therefore, there is an urgency for further exploration and new therapeutic approaches to AIS.

Iron is widely distributed and serves as a vital participant in numerous bioprocesses in the brain.⁹ Individuals exhibiting heightened serum ferritin levels typically face a more devastating prognosis within 24 h of cerebral ischemia.¹⁰ Ferroptosis is defined as a type of non-apoptotic cell death, usually distinguished by the iron-dependent buildup of lipid reactive oxygen species (ROS).^11–13 Both iron metabolism and lipid peroxidation signaling are now considered as primary factors of ferroptosis.¹² Long-chain acyl-CoA synthetase 4 (ACSL4), a member of the ACSL family, is capable of initiating lipid peroxidation and prone to inducing ferroptosis.¹⁴ ACSL4 and ferroptosis are closely associated with ischemia/reperfusion injury, and knockdown of ACSL4 or inhibition of ferroptosis have shown promising therapeutic potential.¹⁵ However, the specific regulatory mechanisms underlying this process remain unclear.

MicroRNAs (miRNAs) belong to a subgroup of small non-coding RNA, playing an essential role in regulating gene expression.¹⁶ They hold relevant competencies for modulating the level of specific gene through influencing stability of mRNA of interest or suppressing translational efficiency.¹⁷ These biomolecules can be detected in circulation and have been extensively explored as putative indicators for predicting, diagnosing and prognosing various physiological conditions.¹⁸ Previous studies have elucidated the biological functions of miR-342-5p in various conditions. For example, the overexpression of miR-342-5p in HER2+ breast cancer cells significantly impacted HER2 downstream signaling, cell motility, and mitochondrial stability.¹⁹ It was observed that miR-342-5p is upregulated in transgenic AD mice and speculated that its alteration may contribute to AD axonopathy by downregulating AnkG.²⁰ MiR-342-5p in exosomes was also reported to exert anti-apoptotic and cardioprotective effects by targeting caspase 9, an apoptosis-inducing factor.²¹ Nevertheless, there is limited research on the relationship between miR-342-5p and AIS.

Featured with a covalently closed loop structure, circular RNAs (circRNAs) are available to evade degradation caused by nuclease.²² Researchers have discovered plenty of endogenous circRNAs in mammalian cells, with some demonstrating high abundance and evolutionarily conservation.^23,24 Studies also have demonstrated that certain exonic circRNAs can function as miRNA sponges or modulate the content of linear protein-encoding RNA products by “mRNA trap” mechanisms.^23,25 These mechanisms allow circRNAs to exert control over gene expression and potentially affect various cellular processes.²³ For example, there were studies indicating that miR-197 could be sponged by circ_0001017 and regulate the cancer-related gene RHOB, which was helpful in ameliorating gastric cancer progression.²⁶ Another example is that CircEPS15 acts as a sponge of miR-24-3p to regulate mitophagy gene PINK1, enhancing neuronal protection in Parkinson’s disease.²⁷ Similarly, CircZXDC can absorb miR-125a-3p, leading to an upregulation of ABCC6 expression, which triggers endoplasmic reticulum stress and hence contributes to the thickening of the intima in Moyamoya disease vessels.²⁸ Previous study also shown the regulatory role of circRNA-miRNA-mRNA network on ferroptosis in stroke patients.²⁹ Even so, the specific contribution of circRNAs in AIS is still unclear.

In the current study, we identified miR-342-5p as downregulated in ischemic stroke patients by small RNA sequencing, and discovered the regulatory effect of circ_0008146/miR-342-5p/ACSL4 axis on ferroptosis by in vivo stroke models. Finally, we verified the expression relationship between miR-342-5p and ACSL4 in clinical samples, and provided a new potential treatment strategy for AIS.

Materials and Methods

Study Population and Samples

Blood samples were obtained from healthy controls and AIS patients who visited Nanjing First Hospital between January 2021 and September 2023. The AIS group were enrolled according to the following inclusion: (1) over 18 years of age; (2) admitted within 48 h after symptom onset. The symptom onset was defined as the time new neurological deficits appear. For wake-up stroke patients, it was defined as the last known normal time;^30,31 (3) confirmed by imaging tests utilizing MRI or CT. Subjects were excluded if: (1) severe comorbidities including the heart, liver, kidney and other vital organ failure or insufficiency; (2) a history of intracranial hemorrhage or tumor; (3) unsigned informed consent. The blood samples of AIS patients were collected within 24 h of admission to the hospital. After centrifugation at 500 g for a quarter hour, samples were obtained and then stored at −80 °C for future use. The control group consisted of individuals who visited our hospital seeking physical examinations. Blood samples were drawn from them in a similar manner to the AIS group. This study was in full compliance with the Declaration of Helsinki and was approved by the Ethics Committee of Nanjing First Hospital, Nanjing Medical University (No. KY20211011-05). All study participants provided written informed consent before enrollment in the study.

Small RNA Sequencing

Blood samples from two AIS patients and two normal individuals were collected for RNA sequencing. Firstly, total RNA was isolated from the aforementioned collected plasma with miRNeasy Mini Kit (Qiagen, Hilden, NRW, Germany). Small RNAs ranging from 18 to 30 nucleotides were obtained through gel electrophoresis from total RNA. Following that, 3’ and 5’ adapters were successively added. The RNAs with the adapters added were then reverse transcribed, and the cDNA products ranging from 140 to 160 base pairs were obtained to construct the cDNA library. Then RNAs were sequenced on the Illumina HiSeqTM 2500 by Gene Denovo Biotechnology Co. (Guangzhou, China). All the obtained data were processed normally, and the differentially expressed miRNAs were screened as per the criterion |log2 (FC)| > 1 and P<0.05. In addition, the sequencing results have been stored in the Gene Expression Omnibus (GSE254322).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis

Total RNA in the collected plasma from AIS patients and controls was extracted with miRNeasy Mini Kit (Qiagen, Hilden, NRW, Germany). Total RNA in mouse brain tissues was extracted with Trizol reagent (Invitrogen, CA, USA) at 24 h after MCAO/R modeling. Then, the content of extracted RNA was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Next, taking advantage of a reverse transcription kit supported by Takara Bio, the extracted RNA was reverse transcribed, resulting in the generation of ideal cDNA. The qRT-PCR reaction was conducted by the ABI7500 type sequence detection system (7500, ABI, USA) using the SYBR-Green method, and each well’s cycle threshold (CT) was recorded. β-actin (for circRNA) or U6 (for miRNA) served as the housekeeping gene, as appropriate. Lastly, the relative expressions of the genes were assessed by the 2^−ΔΔCt approach. The primers have been shown in Supplementary Table 1.

Functional Enrichment Analysis

Potential functions and signal paths of target genes for hsa-miR-342-5p were predicted by leveraging Gene Ontology (GO) enrichment analysis or Kyoto Encyclopedia of Genes and Genomes (KEGG) path analysis approaches using clusterProfiler (version 3.11) of R package. The biological processes, cellular components, and molecular functions of miR-342-5p target genes were identified in GO enrichment analysis. The significantly enriched Top 30 pathways based on FDR value were demonstrated in KEGG enrichment analysis.

Animals

C57BL/6J male mice (8–10 weeks, 20–25g) were provided by Beijing Zhishan Co., Ltd (Beijing, China). All of them were housed in a regulated environment with 60 ± 5% humidity and a temperature of 23 ± 1°C, under a daily 12-hour light and dark phase. Food and water were consumed ad libitum. Mice were randomly divided into each group. A minimum of six mice were examined at every time point, and the investigator was concealed from the group assignment. This study was approved by the Laboratory Animal Ethics Committee of Nanjing First Hospital, Nanjing Medical University (No. DWSY-23146450). The experimental protocol was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) Model

MCAO/R model, a well-established animal model for AIS, was constructed as described previously.^32,33 First of all, mouse was treated with 2% isoflurane in oxygen (RWD Life Science, Shenzhen, China) for anesthetization. After confirming anesthetic depth, a midline neck incision was made. Then, the internal, external and common carotid arteries were separated through blunt dissection. Following the ligation of the external carotid, a silicon-coated monofilament (Cinontech, Beijing, China) was carefully inserted through the internal carotid artery. When feeling slight resistance, stop the insertion of the monofilament. One hour later, monofilament was gently pulled down. The procedure was true to the Sham group except for suture insertion. At last, the post-operation mice were kept on a heating pad (37 ± 0.5 °C).

Reactive Oxygen Species (ROS), Malondialdehyde (MDA) and Lipid Peroxidation (LPO) Levels

At 24 h after MCAO/R modeling, cortical tissue samples were collected from the operation groups, along with the corresponding area from the Sham group. For measurement of ROS, the samples were washed with PBS and prepared into a single-cell suspension. For measurement of MDA and LPO, the samples were homogenized with PBS. The content of ROS (#E004), MDA (#A003) and LPO (#A106) was assessed using the corresponding assay kits, all of which were provided by Nanjing Jiancheng Bioengineering Institute. Special instructions were obtained in advance, and procedures were carried out following the manufacturers’ protocols.

Superoxide Dismutase (SOD) Activity and Total Glutathione/Oxidized Glutathione (GSH/GSSG) Ratio

At 24 h after MCAO/R modeling, sample preparation was carried out as described above. Briefly, the samples were homogenized with PBS, then centrifuged to collect the supernatant. SOD activity (#A001), as well as GSH/GSSG ratio (#A061), were detected using the corresponding SOD Assay Kit and GSH/GSSG Assay Kit, all of which were provided by Nanjing Jiancheng Bioengineering Institute. Special instructions were obtained in advance, and procedures were carried out following the manufacturers’ protocols.

Luciferase Reporter Assays

TargetScan 7.2 online prediction website (https://www.targetscan.org/vert_72/) was used to predict consequential pairing of 3’-UTR sequence of ACSL4 and miR-342-5p. RNAhybird (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) was performed to predict the potential binding sites of miR-342-5p in circ_0008146. The WT or Mut of ACSL4 3’-UTR or circ_0008146 luciferase reporter constructs were co-transfected into HEK 293T cells along with miR-342-5p mimic or NC mimic using Lipofectamine 2000 as the transfection agent, respectively. Waiting for 2 d, harvested cells were examined using dual-luciferase reporter assay system supported by Promega.

Treatment with RAS-Selective Lethal 3 (RSL3)

RSL3 was administered as described previously.³⁴ RSL3 has been widely reported to be an inducer of ferroptosis.³⁵ Aim to assess the effect of miR-342-5p on ferroptosis under ischemic and secondary reperfusion conditions, the mice were randomly divided into 4 groups, consisting of the Sham group, MCAO/R group, MCAO/R + RSL3 + NC mimic group, and MCAO/R + RSL3 + miR-342-5p mimic group. RSL3 (S8155, Selleck, Houston, TX, USA), dissolved in the solvent dimethylsulfoxide (DMSO), was intraperitoneally injected into the mice in the later 2 groups at a dose of 30 mg/kg within 24 h before the MCAO/R operation.

Lateral Intracerebroventricular Injection

The LV-circ_0008146 and the negative control (NC) vector were supported by Genechem Company (Shanghai, China). The miR-342-5p mimics and NC mimics were obtained from GenePharma Company (Shanghai, China). At 3 days before MCAO/R operation, mice were anesthetized, and pedal reflexes were checked to confirm successful anesthesia. After shaving and disinfecting the surgical area, mice were fixed on a stereotaxic device (RWD Life Science, Shenzhen, China). Anterior fontanelle was considered as a reference point to pinpoint the right ventricle in mice (anteroposterior (AP), −0.5 mm; mediolateral (ML), −1.0 mm; dorsoventral (DV), −3.0 mm), where lentiviral vectors and miR-342-5p mimics or NC mimics were injected at a rate of 0.2 μL/min using a microsyringe (Hamilton, Nevada, USA). The microsyringe was left in place for a duration of 20 min before being withdrawn. Following the removal of the needle, bone wax was applied to seal the burr hole, and the incision was sutured closed, allowing the mice to recover.

Western Blot

At 24 h after MCAO/R modeling, the mouse brain tissues were collected. Firstly, the brain tissues were dissected using RIPA lysis buffer (Thermo Fisher Scientific, USA) on ice. Secondly, above samples were quantified using BCA protein assay kit (Thermo Fisher Scientific, USA). Thirdly, 30–40 µg protein was loaded per sample into wells of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, along with 5 µL PageRuler Prestained Protein Ladder (#26617, Thermo Fisher Scientific, USA). Later, the gels were run in 2 stages (stage 1: 80 V, 30 min; stage 2: 120 V, 60 min). Following electrophoresis, the proteins were transferred onto advanced activated polyvinylidene fluoride (PVDF) membranes (Merck Group, Darmstadt, HE, Germany). Subsequently, above membranes were blocked with 5% skim milk (BD, MD, USA) for 1 h at room temperature. Next, the membranes were placed on a rocker and incubated overnight at 4 °C with the diluted primary antibodies: anti-ACSL4 antibody (1:10,000, #ab155282, Abcam) and anti-β-actin antibody (1:1000, #4970, Cell Signaling Technology). Afterwards, the membranes were rinsed 3 times and then incubated on a rocker with diluted anti-rabbit secondary antibody (1:5000, #7074, Cell Signaling Technology) at room temperature for 2 h. Finally, the membranes underwent three additional washes before being imaged by Enhanced Chemiluminescence Plus kit (Thermos Fish Scientific, USA). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each experimental sample was normalized to β-actin.

Iron Assay

At 24 h after MCAO/R modeling, sample preparation was carried out as described above for ferrous iron assay using the iron assay kit (Abcam, Cambridge, UK). Samples preparation and assay procedures strictly followed the recommendations. In the end, the output was assessed on a colorimetric microplate reader (OD 593 nm).

Neurobehavioral Tests

The neurobehavioral tests include the modified Neurologic Severity Score (mNSS), rotarod test, and corner test, all of which were performed before MCAO/R operation and on days 1, 3, and 7 after MCAO/R operation to evaluate neurobehavioral functions. Behavioral analyses were performed by personnel who were unaware of the study groups.

mNSS Assessment

All mice were rated by mNSS (scores 0–14) regarding the neurobehavior function and classified into slight injury (scores 1–4), moderate injury (5–9), and serious injury (10–14). This assessment encompassed motor, reflex and balance tests. The higher the score, the more severe the injury.

Corner Test

The corner test was used to evaluate sensorimotor asymmetry. Two 30cm × 20cm cardboards were positioned at a 30° angle with a narrow gap. The mice were positioned in the middle of the two cardboards, allowing their vibrissae on both sides to be stimulated when they entered the corner. The direction in which they turn back was recorded. Typically, the probability of turning in either direction was equal. However, following MCAO/R that resulted in contralateral limb deficits, mice tended to use their ipsilateral limbs (right side) to turn back. A stronger preference for one side indicates a more severe ischemic stroke outcome.

Rotarod Test

The rotarod test was utilized to test the overall motor performance. Mice were placed on a rotating rod and attempted to stay on it rather than falling onto a platform below. The speed progressively rose from 4 to 40 rpm over a 5-minute period. The shorter the duration, the poorer the motor function. The duration they stayed on the accelerating rotating rod was recorded.

2,3,5-Triphenyltetrazolium Chloride (TTC) Staining

TTC staining was adapted to measure cerebral infarction size. At 7 days after MCAO/R modeling, the mouse brains were gently sectioned into six pieces and immersed directly in 12-well plates containing the prepared TTC staining solution (Sigma-Aldrich, St. Louis, MO, USA). The plates were then incubated away from light in a 37 °C water bath for 0.5 h and subsequently fixed with 4% paraformaldehyde (Biosharp, Beijing, China). In the end, ImageJ (National Institutes of Health, Bethesda, MD, USA) was employed to process and analyze specific images, which contained normal tissue (dark red regions) and infarcted areas (white regions).

Nissl Staining

At 7 days after MCAO/R modeling, the mice were anesthetized and the specimen were obtained. 200 mL of normal saline and 4% paraformaldehyde (Biosharp, Beijing, China) was successively injected into the heart of the mice in each group. Afterward, the mouse brains were harvested, dehydrated, and embedded in paraffin. Subsequently, paraffin blocks were divided into 6–8 μm slices. Later, the Nissl staining of the mouse brain slices was performed following the manufacturer’s instructions using Nissl staining kit (Solarbio, Nanjing, China). Briefly, the slices were sequentially submerged in xylene, anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and double distilled water. Subsequently, the slices were stained with cresyl violet stain. Finally, the aforementioned dehydration steps were repeated in reverse order. The morphology of the ischemia-reperfusion area in the right cortex was examined under an Olympus microscope (Olympus, Tokyo, Japan).

Statistical Analysis

GraphPad prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) was utilized for all statistical analyses. Data are shown as mean ± standard deviation (SD). Statistical analysis included Student’s t-test (for two groups) or one-way ANOVA (for three or more groups) followed by Tukey’s post hoc test. Neurobehavioral tests were analyzed using two-way repeated-measures ANOVA followed by Tukey’s post hoc test. Receiver operating characteristic (ROC) curve was used to assess the diagnosis value of miR-342-5p and ACSL4 for AIS. The correlation between miR-342-5p and ACSL4 in AIS patients was analyzed by Pearson correlation coefficient. P < 0.05 was considered statistically significant.

Results

MiR-342-5p Was Markedly Reduced in the Plasma of AIS Participants

To investigate the involvement of miRNAs in AIS patients, we initially performed high-throughput small RNA sequencing in the plasma of 2 AIS patients and 2 healthy controls. Subsequently, 21 differentially expressed miRNAs (DEmiRNAs) were screened as per the criterion |log2 (FC)| > 1 and P<0.05 (Figure 1A), including 10 downregulated miRNAs and 11 upregulated miRNAs in AIS patients. Considering the potential heterogeneity of clinical samples, we focused on 6 human-mouse homologous miRNAs and further validated their expression in 10 healthy controls and 10 AIS patients. The results revealed a significant alteration in expression levels of 5 miRNAs in AIS patients. Notably, hsa-miR-342-5p exhibited pronounced downregulation (Figure 1B). The GO and KEGG functional enrichment analysis demonstrated that miR-342-5p were involved in multiple stroke-associated activities and signaling pathways, including the MAPK signaling pathway, mitochondrial autophagy, ferroptosis, apoptosis, etc. (Figure 1C and D) Hence, miR-342-5p was selected for further study.

Figure 1 MiR-342-5p was significantly reduced in the plasma of AIS patients. (A) Heatmap illustrating the differentially expressed miRNAs (DEmiRNAs) in AIS patients and control subjects by high-throughput sequencing. Red scale: higher expression. Blue scale: lower expression (n = 2 per group). (B) Validation of selected human-mouse homologous DEmiRNAs in the plasma from AIS patients and control subjects by qRT-PCR analysis (n = 10 per group). (C and D) GO and KEGG enrichment analysis of the target genes of hsa-miR-342-5p. Results were expressed as mean ± SD. NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 versus Control group.

MiR-342-5p Inhibited Oxidative Stress After Cerebral Ischemic/Reperfusion in Mice

It is reported that miR-342-5p reduces the volume of cerebral infarction and relieves the neurological function deficits in vivo.³⁶ Moreover, substantial evidence suggests that oxidative stress is detrimental in ischemic and reperfusion,³⁷ which may worsen neuronal damage and result in severe functional deficits.³⁸ It is known that inhibiting oxidative stress could be an effective approach to alleviate cerebral ischemic/reperfusion injury.^39,40 Hence, we speculated that miR-342-5p may have an effect on oxidative stress. We injected miR-342-5p mimics or NC mimics into the MCAO/R models and miR-342-5p was successfully overexpressed in the brain tissues (Figure 2A). Then, we measured multiple indicators including the levels of ROS, MDA, LPO, SOD and GSH/GSSG, aiming to determine the impact of miR-342-5p on oxidative stress. As expected, compared to the Sham group, the MCAO/R group presented a considerable increase in ROS, MDA and LPO. Besides, the administration of the miR-342-5p mimic effectively reduced aforementioned indicators, suggesting its potential in alleviating oxidative stress (Figure 2B-D). Similarly, the MCAO/R group showed a decrease in the SOD and GSH/GSSG levels in contrast to the Sham group, while the application of miR-342-5p mimic partly reversed such decrease (Figure 2E and F). These findings suggested that miR-342-5p mitigated oxidative stress of the brain tissue after cerebral ischemic/reperfusion in mice.

Figure 2 MiR-342-5p suppressed oxidative stress in MCAO/R mice. At 24 h post-modeling, the cortical tissues from each group were gathered to analyze the contents of miR-342-5p (A) using qRT-PCR (n = 6 per group). The ROS level (B), MDA level (C), LPO level (D), SOD activity (E) and GSH/GSSG ratio (F) were also measured using the corresponding detection kits (n = 6 per group). Results were expressed as mean ± SD of three independent experiments. ***P < 0.001 versus NC mimic group or Sham group; ##P < 0.01, ###P < 0.001 versus MCAO/R + NC mimic group.

MiR-342-5p Inhibited RSL3-Induced Ferroptosis in MCAO/R Mice

Ferroptosis engages multiple pathways related to oxidative stress and antioxidant defense.⁴¹ The functional enrichment analysis in Figure 1 further suggested the involvement of miR-342-5p in ferroptosis. Therefore, we proceeded to explore the in vivo correlation between miR-342-5p and ferroptosis. Targetscan online tool was utilized to predict the potential binding sites between miR-342-5p and ACSL4 (Figure 3A). The conducted dual-luciferase reporter assay demonstrated that the luciferase activity of the ACSL4 WT + miR-342-5p mimic group was observably impaired compared with the ACSL4 WT + NC mimic group, confirming the binding affinity between ACSL4 and miR-342-5p (Figure 3B). Injection of RSL3, the most potent inhibitor of GPX4, has been proven to effectively induce ferroptosis in previous studies.^42–44 Therefore, to investigate the effect of miR-342-5p on ferroptosis, the operated mice were injected with 30 mg/kg RSL3 based on the body weight. We examined both ACSL4 and Fe²⁺ levels and observed that treatment with RSL3 significantly elevated the contents of ACSL4 and Fe²⁺ in the MCAO/R mice, respectively. This funding indicates that RSL3 exacerbated ferroptosis of brain tissue induced by MCAO/R, while injection of miR-342-5p mimic partially reversed these changes (Figure 3C-E). In summary, the aforementioned results emphasized the capability of miR-342-5p in inhibiting ferroptosis following cerebral ischemic/reperfusion.

Figure 3 MiR-342-5p inhibited RSL3-induced ferroptosis in MCAO/R mice. (A) Putative biological targets between 3’ UTR of ACSL4 mRNA and miR-342-5p using Targetscan. (B) Luciferase reporter assay shows relative luciferase activity of HEK 293T cells co-transfected with ACSL4WT or ACSL4 Mut and miR-342-5p mimic or NC mimic, respectively (n = 3 per group). (C and D) Typical Western blot images and quantification of ACSL4 protein levels in each group. β-actin was used as an internal control (n = 6 per group). (E) Fe2+ contents in each group were measured using iron assay kit (n = 6 per group). Results were expressed as mean ± SD of three independent experiments. ***P < 0.001 versus NC mimic group or Sham group; ###P < 0.001 versus MCAO/R group; &&&P < 0.001 versus MCAO/R + RSL3 + NC mimic group.

Targeting Association Between Circ_0008146 and miR-342-5p

Next, we tried to investigate the upstream mechanism regulating miR-342-5p expression in ischemic stroke. It is widely recognized that circRNAs may function as miRNA sponges, influencing miRNA levels according to the ceRNA hypothesis,⁴⁵ so circRNA and target miRNA usually demonstrate an opposite expression trend and biological function in certain diseases. A previous report showed that circ_0008146 was upregulated in MCAO/R mice and promoted apoptosis and neuroinflammation.⁴⁶ Considering the downregulation and beneficial effects of miR-342-5p in stroke conditions, circ_0008146 was selected and tested as a potential upstream circRNA in our successive studies. To assess the regulatory relationship between circ_0008146 and miR-342-5p, we employed RNAhybrid to speculate the potential binding sites, and the minimum free energy (mfe) is below −25 kcal/mol (Figure 4A). Subsequently, we conducted a luciferase reporter gene assay to verify the binding affinity of circ_0008146 to miR-342-5p. The luciferase activity of HEK 293T cells co-transfected with circ_0008146 WT and miR-342-5p mimic turned out to be lower than in cells co-transfected with NC mimic (Figure 4B). To confirm the in vivo effect of circ_0008146 on miR-342-5p, we overexpressed circ_0008146 in established MCAO/R models using lentiviral vectors. Subsequent qRT-PCR results verified the overexpression efficiency of circ_0008146 in MCAO/R mice (Figure 4C). Then, we found that the increased expression of circ_0008146 could lead to a reduction in miR-342-5p levels in the mouse brain tissues (Figure 4D). In view of these results, circ_0008146 may serve as a sponge of miR-342-5p and suppress the expression of miR-342-5p in MCAO/R mice.

Figure 4 Targeting association between circ_0008146 and miR-342-5p. (A) Speculated targets between circ_0008146 and miR-342-5p using RNAhybrid (mfe: −25.5 kcal/mol). (B) Luciferase reporter assay shows relative luciferase activity of HEK 293T cells co-transfected with circ_0008146 WT or circ_0008146 Mut and miR-342-5p mimic or NC mimic (n = 3 per group). (C and D) Circ_0008146 and miR-342-5p content in each group was measured using qRT-PCR (n = 6 per group). Results were expressed as mean ± SD of three independent experiments. **P < 0.01 versus NC mimic group; ***P < 0.001 versus Sham group; ###P < 0.001 versus MCAO/R + LV-NC group.

Overexpression of Circ_0008146 Exacerbated Brain Infarction in MCAO/R Mice

The assessment of neurobehavioral function affected by the injection of circ_0008146 was conducted using the mNSS score, corner test, and rotarod test as previously described. Furthermore, circ_0008146 has been repeatedly suggested to be related to a bad outcome. Generally, the group treated with LV-circ_0008146 exhibited significantly higher neurological deficit scores, a higher number of right turns in the corner test and shorter latency time to fall in the rotarod test by contrast with LV-NC group (Figure 5A-C). The cerebral infarct volume was visualized using TTC staining. The stained brain tissues from the LV-circ_0008146 treated group exhibited a significantly larger infarct size ratio compared to those from the LV-NC treated group (Figure 5D and E). Nissl staining was employed to detect histological changes and neuronal damage. The MCAO/R+LV-circ_0008146 group demonstrated more injured cells and neuronal loss compared to the MCAO/R+LV-NC group (Figure 5F). All these findings suggested that overexpression of circ_0008146 promoted brain injury in MCAO/R mice.

Figure 5 Overexpression of circ_0008146 exacerbated neurological deficit in MCAO/R mice. The mNSS score (A), corner test (B), and rotarod test (C) were performed at day 0, 1, 3, and 7 after MCAO/R operation to evaluate the neurobehavioral function (n = 12 per group). (D and E) The effects of circ_0008146 on infarct volume were evaluated by TTC staining at 7 days after reperfusion (n = 6 per group). (F) Typical images of Nissl staining for the ischemia-reperfusion area in cortex (n = 6 per group). Results were expressed as mean ± SD of three independent experiments. ***P < 0.001 MCAO/R group versus Sham group; ##P < 0.01, ###P < 0.001 MCAO/R + LV- circ_0008146 group versus MCAO/R + LV-NC group.

Circ_0008146 Contributed to Ferroptosis in Cerebral Infarction via Sponging miR-342-5p to Regulate ACSL4

Based on the above results, we attempted to further elucidate the role of the circ_0008146/miR-342-5p/ACSL4 axis on ischemic stroke. In the MCAO/R mice, the overexpression of circ_0008146 with lentiviral vectors resulted in increased levels of ROS, MDA and LPO, along with a decrease in SOD activity or GSH/GSSG ratio. However, the administration of miR-342-5p partially reversed these effects (Figure 6A-E). Similarly, circ_0008146 overexpressing promoted the iron accumulation in the mouse brain due to cerebral ischemic/reperfusion, whereas treatment with miR-342-5p partly attenuated the iron accumulation due to stroke (Figure 6F). As a promoter and marker of ferroptosis, ACSL4 was also evaluated through Western blot. As anticipated, the obtained outcomes unveiled an enhanced expression of the ACSL4 protein in the group administered with circ_0008146. However, treatment with miR-342-5p resulted in the down-regulation of ACSL4 protein levels compared to the group treated with circ_0008146 alone in MCAO/R mice (Figure 6G and H). Taken together, these results revealed the possible regulation of circ_0008146/miR-342-5p/ACSL4 axis on ferroptosis in MCAO/R mice.

Figure 6 The circ_0008146/miR-342-5p/ACSL4 axis regulated ferroptosis in AIS induced by MCAO/R. ROS level (A), MDA level (B), LPO level (C), SOD activity (D), GSH/GSSG ratio (E) and Fe2+ level (F) of mouse brain tissues were measured using the corresponding detection kits (n = 6 per group). (G and H) Typical Western blot images and quantification of ACSL4 protein levels in each group. β-actin was used as an internal control (n = 6 per group). Results were expressed as mean ± SD of three independent experiments. ***P < 0.001 versus Sham group; ##P < 0.01, ###P < 0.001 versus MCAO/R + LV-NC + NC mimic group. &P < 0.05, &&P < 0.01, &&&P < 0.001 versus MCAO/R + LV-circ_0008146 + NC mimic group.

Hsa-miR-342-5p and ACSL4 Could Be Clinical Biomarker for AIS Patients

To investigate the clinical value of miR-342-5p/ACSL4 axis in diagnosing AIS, we recruited another 37 healthy controls and 32 AIS patients and measured the hsa-miR-342-5p levels and ACSL4 mRNA levels in their plasma by qRT-PCR. Consistent with Figure 1, plasma miR-342-5p levels in AIS participants were significant lower compared with the control group (Figure 7A). Also, ACSL4 mRNA levels were higher in the plasma of AIS patients than the healthy controls (Figure 7B). ROC curves showed that both miR-342-5p (area under the curve, 0.786) and ACSL4 mRNA (area under the curve, 0.813) levels have a good ability to discriminate the AIS patients and healthy individuals (Figure 7C), demonstrating the potential of miR-342-5p and ACSL4 in diagnosing AIS. Interestingly, we found that in AIS patients, the plasma miR-342-5p levels were negatively correlated with the ACSL4 mRNA levels (Figure 7D), which revealed that the targeting relationship between miR-342-5p and ACSL4 may apply to AIS patients. Finally, we divided AIS patients into two groups according to stroke severity evaluated by National Institutes of Health Stroke Scale (NIHSS) score: mild group (NIHSS < 5) and moderate to severe group (NIHSS ≥ 5), and compared the levels of miR-342-5p and ACSL4 mRNA in these two groups. As shown in Figure 7E and F, miR-342-5p in plasma was relatively lower in moderate to severe group than mild group, whereas the moderate to severe group has higher ACSL4 mRNA plasma levels. In general, these clinical results indicated the potential of miR-342-5p and ACSL4 as diagnostic markers and therapeutic targets for AIS patients.

Figure 7 Hsa-miR-342-5p and ACSL4 in the plasma could be clinical biomarker for AIS. (A and B) MiR-342-5p and ACSL4 mRNA levels were measured in the plasma of 37 healthy controls and 32 AIS patients using qRT-PCR. (C) ROC curves demonstrated the diagnostic value of miR-342-5p and ACSL4 for AIS. (D) The negative correlation between miR-342-5p and ACSL4 mRNA in the plasma of the 32 AIS patients detected by qRT-PCR. (E and F) The 32 AIS patients were divided into mild group (NIHSS < 5) and moderate to severe group (NIHSS ≥ 5), and miR-342-5p or ACSL4 mRNA levels were measured in the plasma using qRT-PCR. Results were expressed as mean ± SD.

Discussion

AIS is characterized by a high rate of disability and mortality, while there is still a significant unmet need for treatment of the large AIS population.^47,48 MiRNAs belong to small, non-coding RNA molecules and are found to be important in the regulation of gene expression after transcription.¹⁶ Most of miRNAs are involved in regulating mRNA through its degradation, in addition to controlling protein expression.¹⁸ In very recent times, a large number of miRNAs were detected and speculated to serve as potential therapeutic targets for AIS.¹⁷ For example, it was found that activating miR-337-3p/Nampt exerted neuroprotective effects.³² Exogenous miRNA-181b could activate the PTEN/Akt signal pathway and promoted angiogenesis, alleviating neuronal damage in ischemic stroke.⁴⁹ Moreover, blood miRNAs are good predictors of stroke risk.⁵⁰ Herein, we conducted small RNA sequencing and qRT-PCR analysis and detected the pronounced downregulated miRNA, hsa-miR-342-5p. This finding was consistent with previous studies, which demonstrated that miR-342-5p was significantly downregulated in both MCAO/R mice and OGD/R cells. Also, miR-342-5p has shown a protective effect against neuronal apoptosis.^36,51 Nevertheless, the functions and mechanisms of miR-342-5p in AIS have been relatively underexplored.

Featured with increased metabolic activity and susceptibility to ischemic stroke, brain is prone to suffer from oxidative damage.⁵² Substantial evidence suggests that oxidative stress exerts a negative influence on AIS and subsequent reperfusion injury.^37,51,53 In a study on lung injury, miR-342 inhibited oxidative stress in human bronchial epithelial cells by targeting KLF5, suggesting inhibition of oxidative stress may also be the mechanism by which miR-342-5p exerts its neuroprotective effect in AIS.⁵⁴ In our study, we discovered that attenuated oxidative stress and ameliorated neurological deficits were a reflection of increased miR-342-5p. This positive effect of miR-342-5p on AIS is consistent with the earlier findings. Oxidative stress has been reported to correlate with ferroptotic responses, and furthermore, the accumulation of lipid peroxides serves as a biochemical signature of ferroptosis.⁴¹ Considering the crosstalk between oxidative stress and ferroptosis, we focused on the effect of miR-342-5p on ferroptosis using ferroptosis inducer, and discovered that miR-342-5p inhibited RSL3-induced ferroptosis in MCAO/R mice. To the best of our knowledge, this is the first study connecting miR-342-5p and ferroptosis with cerebral ischemia. As a direct target of miR-342-5p, ACSL4 is involved in intracellular synthesis of lipid peroxide substrates and is considered to be a classic marker of ferroptosis.⁵⁵ We verified the negative correlation between miR-342-5p and ACSL4 in AIS patients, and found that miR-342-5p and ACSL4 have good diagnostic value for AIS. The different expression of miR-342-5p and ACSL4 in mild and moderate to severe groups also suggests that regulation of miR-342-5p/ACSL4 may be a potential therapeutic approach for AIS.

Considering the effect of miRNA dysregulation on ischemia-reperfusion injury, it is necessary to explore the upstream mechanism of miR-342-5p expression. In recent years, the circRNA/miRNA sponging mechanism theory proposes that circRNAs could competitively bind to miRNAs, thereby preventing miRNA interaction with mRNA targets. There is mounting evidence indicating that circRNAs are engaged in regulating pathophysiological processes of ischemic stroke by acting as miRNA sponges.^56–58 For example, circZfp609 promotes OGD/R-induced astrocyte injury by influencing BACH1 expression through sponging miR-145a-5p.⁵⁹ Also, circDLGAP4 was reported to sponge miR-143 in endothelial cells, and reduce the blood-brain barrier damage caused by ischemic stroke.⁶⁰ In the previous study by Li et al, circ_0008146 was identified as upregulated in the cerebral cortex of MCAO/R mice, and its expression significantly decreased after treatment. Initial cell experiments suggest its involvement in inflammation and apoptosis promotion. Therefore, we suspected that circ_0008146 is upstream of miR-342-5p regulation, which was validated in our study.⁴⁶ In fact, circRNAs are key factors in influencing the risk of oxidative-stress-related diseases.^61–63 In our study, we identified that the overexpression of circ_0008146 exacerbated cerebral infarction and impaired functional behavior, which is the first time to validate the detrimental effect of circ_0008146 on stroke in an in vivo model. Moreover, ferroptosis may be a key pathological process through which circ_0008146 exacerbates cerebral ischemia-reperfusion injury. Despite the limited research on circ_0008146, our study provided a potential direction for more detailed mechanistic studies in the future.

Some limitations must be acknowledged in our study. Firstly, the sample size of both normal controls and AIS patients is relatively small. Hence, our results need to be confirmed with a larger cohort in future studies. Secondly, our experiments were mainly conducted on animal models, which can more accurately simulate the complex environment in vivo, but we did not perform cellular experiments to further explore and validate the mechanisms involved. We plan to design and conduct such experiments to address this aspect in subsequent research. Thirdly, in our study, ACSL4 is considered to be the key protein in which miR-342-5p exerts ferroptosis inhibition, but considering the complex regulatory mechanism of ferroptosis related genes, whether miR-342-5p exerts its role through other proteins related to ferroptosis remains to be further investigated.

Conclusion

Our study provided evidence that the administration of miR-342-5p can effectively mitigate brain damage resulting from ferroptosis. Furthermore, our study elucidated the underlying mechanism of circ_0008146 on exacerbating neuronal damage after AIS via the circ_0008146/miR-342-5p/ACSL4 axis. These findings suggest that such an axis holds promise as an available and practical therapeutic focus in AIS.