Retrospective Cohort Study of Peripheral Blood miR-486-3p Levels in Patients with Acute Cerebral Infarction and Obstructive Sleep Apnea Syndrome

Summary

miR‑486‑3p is downregulated in acute cerebral infarction patients with obstructive sleep apnea syndrome, correlates negatively with disease severity and inflammatory markers, and predicts poor 1‑year prognosis, supporting its potential as a diagnostic and prognostic biomarker.

Abstract

Acute cerebral infarction (ACI) complicated with obstructive sleep apnea syndrome (OSAS) confers a high risk of stroke recurrence and death, yet effective biomarkers for this comorbid condition remain limited. This retrospective cohort study enrolled 270 patients with ACI between November 2020 and December 2023, who were classified into the ACI group (n = 100, apnea‑hypopnea index [AHI] < 5) and the ACI with OSAS group (n = 170, AHI ≥ 5), with the latter further stratified into mild (5 ≤ AHI < 15, n = 60), moderate (15 ≤ AHI < 30, n = 75), and severe (AHI ≥ 30, n = 35) subgroups. Serum miR‑486‑3p expression was measured by RT‑qPCR, and inflammatory markers, including tumor necrosis factor‑α (TNF‑α), C‑reactive protein (CRP), and hypoxia‑inducible factor‑1α (HIF‑1α), were quantified by ELISA. The diagnostic value of miR‑486‑3p was evaluated using receiver operating characteristic curve analysis, and its prognostic significance was assessed through Kaplan‑Meier survival analysis and Cox regression models. miR-486-3p was markedly lower in the comorbid group and demonstrated strong diagnostic ability (AUC = 0.927). Notably, a clear dose‑dependent trend was observed across OSAS severity subgroups, with miR‑486‑3p decreasing progressively and TNF‑α and CRP increasing stepwise from mild to severe OSAS. Spearman correlation analyses revealed that miR‑486‑3p was negatively correlated with AHI, NIHSS score, TNF‑α, and CRP. At 1‑year follow‑up, patients with low miR‑486‑3p expression had significantly worse functional outcomes compared with those with high expression. Multivariate Cox regression confirmed miR‑486‑3p as an independent protective factor for 1‑year prognosis. These findings suggest that miR‑486‑3p may serve as a novel diagnostic and prognostic biomarker for ACI patients with OSAS, potentially through its regulatory role in systemic inflammation, and warrant further validation in larger prospective cohorts.

Introduction

Obstructive sleep apnea syndrome (OSAS) is a common condition characterized by repeated interruptions in breathing during sleep¹. OSAS is often associated with comorbidities such as metabolic, cardiovascular, renal, pulmonary, and neuropsychiatric disorders². Beyond its general associations with cardiovascular and metabolic diseases, OSAS poses a specific and grave threat in acute ischemic stroke, complicating up to 70% of cases and increasing risks of neurological decline, mortality, and recurrent stroke^3,4,5. Acute cerebral infarction (ACI) constitutes the most prevalent form of stroke. Although significant progress has been made in ACI treatment in recent years, most patients still suffer from severe disability or even death⁶. The synergistic interplay between ACI combined OSAS exacerbates ischemic damage and systemic inflammation, substantially elevating risks of stroke recurrence, disability, and mortality. Consequently, identifying reliable biomarkers for the early diagnosis and management of this high-risk comorbid condition is of paramount importance.

MicroRNAs (miRNAs) have shown great promise as biomarkers in recent years. Among them, miR-486-3p is of great significance in neurovascular diseases. Previous research have shown that miR-486-3p expression is inhibited in obstructive sleep apnea (OSA)⁷. In addition, rabies virus glycoprotein-miR-486-3p-exosome treatment can alleviate neurobehavioral disorders, cerebral edema, blood-brain barrier damage, and neurodegeneration⁸, indicating that miR-486-3p plays a role in brain injury. After ACI, microglia in the damaged brain tissue are activated, promoting the secretion of inflammatory cytokines⁵. OSAS causes intermittent hypoxia symptoms, leading to activation of the sympathetic nervous system and oxidative stress, thereby triggering a systemic inflammatory cascade reaction⁹. miR-486-3p is closely related to inflammation¹⁰. These lines of evidence suggest that miR-486-3p may serve as a molecular link between OSAS-related hypoxia, systemic inflammation, and post-stroke recovery.

However, its expression profile, diagnostic accuracy, and prognostic significance in ACI patients combined with OSAS have not been systematically investigated, and whether it correlates with OSAS severity or inflammatory mediators in a dose-dependent manner remains unknown. To address these gaps, this study evaluated the diagnostic value of miR-486-3p for OSAS in ACI patients, examined its dose-dependent relationship with OSAS severity and inflammatory markers (TNF-α, CRP, and HIF-1α), and assessed its prognostic utility for 1-year functional outcomes. To our knowledge, this is the first comprehensive study to demonstrate that miR-486-3p may serve as a novel diagnostic and prognostic biomarker for ACI-OSAS comorbidity, potentially through regulation of systemic inflammation.

Protocol

This study was approved by the Ethics Committee of Affiliated Hospital of Xuzhou Medical University (approval number: XYFY2024-KL238-01) and was exempt from the requirement to obtain additional informed consent from patients. The reagents and the equipment used are listed in the Table of Materials.

Patients and specimens
This study was a retrospective cohort study. From November 2020 to December 2023, a total of 270 patients undergoing ACI from Affiliated Hospital of Xuzhou Medical University were consecutively recruited. The patient selection process is summarized in Supplementary Figure 1. Eligible patients were identified through daily screening of all new admissions to the stroke unit. Symptom onset within 72 h prior to admission was verified by reviewing emergency medical records and confirmed by a certified neurologist through patient history and family interview. The sample size was calculated based on the ability to detect a significant difference in miR-486-3p levels between groups. Assuming an effect size of 0.5, a two-sided α of 0.05, and a power (1-β) of 0.80, a minimum of 64 patients per group was required. The final group sizes reflect the actual recruitment and the higher prevalence of OSAS in the stroke population, not a predetermined allocation. Patients with ACI combined OSAS were followed up on their prognosis for 1 year.

The inclusion criteria were: (1) Acute ischemic stroke with symptom onset within 72 h before admission, confirmed by magnetic resonance imaging. (2) The ability to comprehend and complete the study questionnaires and neurological assessments. (3) The severity of the ACI was assessed by the National Institutes of Health Stroke Scale (NIHSS) at admission. When the NIHSS score is <5, it is classified as mild ACI; when the NIHSS score is between 5 and 20, it is considered moderate ACI. Key exclusion criteria included a history of malignant tumors, pre-existing severe physical disability or psychiatric disorders, severe cardiac, pulmonary, or renal insufficiency, and major surgery or trauma within the preceding 3 months, active infections or autoimmune diseases; previous stroke within 6 months; immunosuppressive therapy; and prior diagnosis or treatment of OSAS (including continuous positive airway pressure therapy).

For MRI confirmation, all patients underwent standard brain MRI on a 3.0-T system, including the following sequences: T1-weighted, T2-weighted, fluid-attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), and apparent diffusion coefficient (ADC) mapping. Acute infarction was defined as the presence of a hyperintense signal on DWI with a corresponding hypointense signal on ADC. All MRI scans were interpreted by two experienced neuroradiologists who were blinded to the clinical data, with discrepancies resolved by consensus. NIHSS scoring was performed at admission by certified neurologists prior to any thrombolytic or endovascular treatment.

Fasting venous blood was collected between 24 h and 48 h after stroke onset, following an overnight fast of at least 8 h. Blood draws were performed between 6:00 AM and 8:00 AM to minimize circadian variation. A total of 5 mL of peripheral venous blood was collected into EDTA-coated vacuum tubes. Within 2 h of collection, blood samples were centrifuged at 1,500 × g for 15 min at 4 °C using a refrigerated centrifuge. The serum supernatant was carefully aliquoted into 1.5 mL RNase-free microcentrifuge tubes and immediately stored at −80 °C until RNA extraction. Repeated freeze-thaw cycles were strictly avoided; all aliquots were subjected to a maximum of one freeze-thaw cycle prior to analysis.

Patient grouping
Participants were grouped based on their AHI scores from polysomnography. All recordings were performed in the hospital sleep laboratory between 10:00 PM and 6:00 AM for a minimum duration of 7 h. The monitored physiological channels included: electroencephalography (EEG; six channels), electrooculography (EOG; two channels), electromyography (EMG; chin and bilateral anterior tibialis), electrocardiography (ECG; single lead), nasal airflow (thermistor and pressure transducer), thoracic and abdominal respiratory effort (respiratory inductance plethysmography), and pulse oximetry (SpO2). The sleep study technician calculated AHI. Scoring of sleep stages and respiratory events was performed manually by a certified sleep technician according to the American Academy of Sleep Medicine (AASM) 2012 scoring manual, using the Somnolyzer 24×7 software. According to the widely accepted diagnostic criteria of the American Academy of Sleep Medicine, an AHI of 5 or more events per hour is the standard threshold for diagnosing OSA. Therefore, participants with an AHI < 5 were classified as the ACI group (n = 100), whereas those with an AHI ≥ 5 were classified as the ACI combined OSAS group (n = 170). Among those with AHI ≥ 5 (ACI combined OSAS group, n = 170), patients were further stratified into three severity subgroups based on established criteria: mild OSAS (5 ≤ AHI < 15, n = 60), moderate OSAS (15 ≤ AHI < 30, n = 75), and severe OSAS (AHI ≥ 30, n = 35). This stratification enabled evaluation of the dose‑dependent relationship among hypoxic burden, miR‑486‑3p expression, and inflammatory markers. Patients with prior OSAS treatment (e.g., CPAP) were not included in the study to avoid confounding of the baseline AHI measurement.

Prognosis assessment
The 1-year timepoint allows acute effects to resolve and the cumulative hypoxic burden of OSAS to manifest, providing a robust window to assess its prognostic impact. The clinical prognosis of patients with ACI combined with OSAS was evaluated by the modified Rankin Scale (mRS) after the onset of the disease. All mRS assessments were performed by certified neurologists who underwent standardized training prior to the study, using the structured mRS interview form. Inter-rater reliability was assessed with a kappa coefficient >0.85. The follow-up assessment was conducted through a scheduled outpatient clinic visit. For patients unable to attend in person, a structured telephone interview was performed by a trained research nurse using a standardized script. All assessors were blinded to the patients’ miR-486-3p levels and OSAS status. To maintain consistency between outpatient and telephone assessments, both formats used the same structured questionnaire, and all assessors completed a pre-study calibration exercise. Patients with scores of 0–2 were placed in the good prognosis group, and those with scores of 3–6 were placed in the poor prognosis group. No patients were lost to follow-up. The SAQLI, ESS, TNF-α, and HIF-1α were evaluated as prognostic variables rather than being used to define prognosis groups. The NIHSS, SAQLI, and ESS were administered by trained research staff. The SAQLI and ESS were administered as interviewer-administered questionnaires in a quiet room.

Scoring index
NIHSS is a quantitative scale for stroke¹¹, with higher scores indicating greater severity of ACI. SAQLI is a quality of life questionnaire for OSA on a scale of 1–7, with higher scores indicating worse quality of life, covering daily activities, social interactions, emotional states, symptoms, and treatment-related symptoms¹². ESS is used to examine patients' self-reported daytime sleepiness, with a score range from 0 to 24, and a score > 10 indicates daytime sleepiness¹³.

RNA extraction and reverse transcription
Total RNA was extracted from 200 µL of serum using TRIzol reagent. Briefly, serum samples were mixed with 800 µL of TRIzol, incubated at room temperature for 5 min, and centrifuged at 12,000 × g for 15 min at 4 °C. Chloroform (200 µL) was added, and the mixture was vigorously shaken for 15 s, incubated for 3 min, and centrifuged at 12,000 × g for 15 min at 4 °C. The aqueous phase was transferred to a fresh tube, and RNA was precipitated with 500 µL of isopropanol, incubated at −20 °C for 1 h, and centrifuged at 12,000 × g for 10 min at 4 °C. The RNA pellet was washed with 75% ethanol, air-dried, and dissolved in 20 µL of RNase-free water. RNA concentration and purity were assessed using a microvolume spectrophotometer, with an A260/A280 ratio between 1.8 and 2.0 accepted for downstream analysis. RNA integrity was verified by agarose gel electrophoresis.

Reverse transcription was performed using 500 ng of total RNA with the Reverse Transcription Kit in a total reaction volume of 20 µL, using random hexamer primers according to the manufacturer's protocol. The reverse transcription reaction was carried out at 25 °C for 10 min, 42 °C for 60 min, and 70 °C for 5 min. The resulting cDNA was stored at −20 °C until use.

RT-qPCR
miR-486-3p expression was detected using a SYBR Green–based real-time PCR kit for microRNA quantification on a real-time quantitative PCR (qPCR) system. The reaction mixture contained 10 µL of 2× SYBR Green Master Mix, 2 µL of cDNA template (diluted 1:5), 1 µL of each primer (10 µM), and nuclease-free water to a final volume of 20 µL. The primer sequences are as follows (from 5'-3'): miR-486-3p-forward: TCGGCAGCGGGGCAGCUCAGU, reverse: CTCAACTGGTGTCGTGGA; and U6-forward: CTCGCTTCGGCAGCACA, reverse: AACGCTTCACGAATTTGCGT. The qPCR cycling conditions were: 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. Each sample was run in triplicate, and a no-template control was included in each run. Specificity of amplification was confirmed by melting curve analysis from 60 °C to 95 °C. The cycle threshold (Ct) was automatically determined by the QuantStudio 6 Pro software (v1.5) using the default threshold setting. miR-486-3p level was calculated with the 2^-ΔΔCT method and normalized to U6. U6 was validated as a stable reference gene in our serum samples using the NormFinder algorithm to ensure consistency across groups. Inter-assay and intra-assay coefficients of variation were <5%.

ELISA
The serum concentrations of C-reactive protein (CRP), TNF-α, and HIF-1α were detected using the ELISA kit. The experimental procedures were carried out in accordance with the manufacturer's instructions. Briefly, serum samples were thawed on ice and diluted 1:2 with the provided dilution buffer. Samples and standards were added to pre-coated 96-well plates (100 µL/well) and incubated for 2 h at 37°C. After washing four times with 300 µL of wash buffer, 100 µL of biotin-conjugated detection antibody was added and incubated for 1 h at 37°C. Following another wash step, 100 µL of horseradish peroxidase-conjugated streptavidin was added and incubated for 30 min at 37°C in the dark. The chromogenic substrate (tetramethylbenzidine, 100 µL) was added and incubated for 15 min at 37°C protected from light. The reaction was terminated with 50 µL of stop solution, and absorbance was measured at 450 nm using a microplate reader. The concentrations were calculated from standard curves generated with known concentrations of recombinant proteins. Each sample was measured in duplicate, and the mean value was used for analysis. Quality control samples provided by the manufacturer were included in each plate; results were accepted only when QC values fell within the manufacturer's specified range.

Quality control and reproducibility
All laboratory assays were performed in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines for RT-qPCR and the appropriate reporting standards for ELISA. Major instruments were calibrated according to the manufacturers' schedules. Negative and positive control samples were included in each batch of RT-qPCR and ELISA runs to monitor plate-to-plate variability. All data were recorded and managed using Microsoft Excel, and the corresponding author will provide detailed experimental protocols and raw data upon reasonable request.

Statistical analysis
Data were analyzed using SPSS 23.0 and a statistical and graphing software. Normal distribution was assessed by the Shapiro–Wilk test. Continuous variables are presented as mean ± SD or median (IQR), and were compared by t‑test or Mann–Whitney U test, as appropriate. Categorical variables were compared by the χ² test. Correlations were examined using Pearson or Spearman analyses based on the data distribution. The predictive value was determined using the ROC curve and verified with 5-fold cross-validation. The optimal cutoff value for miR-486-3p was determined by maximizing the Youden index. Pearson and Spearman correlation analyses were used to assess correlations. For prognostic analysis, patients were stratified by the median miR‑486‑3p expression; Kaplan–Meier curves with the log‑rank test were used to compare survival outcomes between groups. Variables with P < 0.1 in univariate Cox regression were entered into the multivariate Cox model. P < 0.05 was considered statistically significant.

Results

Baseline characteristics in the ACI group and the ACI combined OSAS group
As shown in Table 1, the ACI group consisted of 68 males and 32 females, with an average age of 62.84 ± 12.65 years, and the ACI combined OSAS group included 116 males and 54 females, with an average age of 61.31 ± 11.25 years. The ACI combined OSAS group had significantly higher AHI, NIHSS scores, and CRP levels. However, the two groups showed comparable distributions of age, gender, BMI, hypertension, DM, car...

Discussion

Patients with OSAS experience repeated airway collapse and obstruction, leading to intermittent hypoxia (IH) and excessive daytime sleepiness (EDS), which can cause neuronal damage¹⁴. OSA is highly prevalent after ACI, affecting approximately 72% of patients, and significantly worsens cardiovascular risk¹⁵. ACI constitutes a spectrum of neurological deficits arising from localized cerebral ischemia and hypoxia secondary to inadequate perfusion‌¹⁶

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3.0T MRI Scanner	Siemens Healthineers, Germany	MAGNETOM Skyra	Brain imaging for acute cerebral infarction confirmation
96-well ELISA Microplates	Shenzhen Xinbosheng Biological Co., Ltd., China	XBS-PLATE	Antigen-antibody reaction for ELISA detection
AASM Clinical Practice Guidelines for Sleep-Disordered Breathing	American Academy of Sleep Medicine	2020 full guideline	Standardized AHI diagnostic and OSAS severity stratification criteria
Calgary Sleep Apnea Quality of Life Index (SAQLI) Chinese validated version	University of Calgary	2021 translated edition	Quality-of-life assessment for OSAS complicated ACI patients
EDTA-K2 5 mL Vacuum Blood Collection Tubes	BD Biosciences, USA	367863	Peripheral venous blood collection for serum separation
Epworth Sleepiness Scale (ESS) Chinese validated version	Johns Hopkins University	2018 revised edition	Evaluation of daytime sleepiness in stroke patients
GraphPad Prism 9	GraphPad Software, USA	Version 9.4.1	Scatter plots, boxplots, ROC curve visualization
Human CRP ELISA Kit	Shenzhen Xinbosheng Biological Co., Ltd., Shenzhen, China	XBS-H001	Quantitative detection of serum C-reactive protein
Human HIF-1α ELISA Kit	Shenzhen Xinbosheng Biological Co., Ltd., Shenzhen, China	XBS-H007	Quantitative detection of serum hypoxia-inducible factor-1α
Human TNF-α ELISA Kit	Shenzhen Xinbosheng Biological Co., Ltd., Shenzhen, China	XBS-H003	Quantitative detection of serum TNF-α inflammatory factor
IBM SPSS Statistics 23.0	IBM, Armonk, NY, USA	Version 23.0	All statistical analyses (t-test, correlation, ROC, Kaplan–Meier, Cox regression)
Microplate Spectrophotometer	Thermo Fisher Scientific (Waltham, MA, USA)	Multiskan FC	Absorbance reading for ELISA plates at 450 nm
miR-486-3p & U6 qPCR Primers	Sangon Biotech, Shanghai, China	Custom synthesis	Target and reference gene amplification for qPCR
miScript SYBR Green PCR Kit	Qiagen GmbH, Hilden, Germany	218073	Real-time quantitative PCR amplification of miR-486-3p and U6
NIH Stroke Scale (NIHSS) Chinese Validated Version	National Institute of Neurological Disorders and Stroke (NINDS), USA	Standard clinical edition	Baseline neurological severity assessment for ACI patients
PSG Sleep Scoring Software	BMC Medical Co., Ltd., China	v3.2	Automated preliminary scoring of sleep respiratory events
QuantStudio 6 Pro Real-Time PCR System	Applied Biosystems, Thermo Fisher Scientific (Waltham, MA, USA)	QuantStudio 6 Pro	RT-qPCR amplification, melting curve acquisition, Ct value recording
QuantStudio Real-Time PCR Software	Applied Biosystems, USA	v1.7	qPCR raw data export, Ct value analysis
Refrigerated Bench Centrifuge	Xiangyi Centrifuge Instrument Co., Ltd., Hunan, China	TDZ5-WS	Serum separation from whole blood (2700 × g, 4 °C)
RevertAid RT Reverse Transcription Kit	Thermo Fisher Scientific, Waltham, MA, USA	K1691	Reverse transcription of serum miRNA into cDNA
RNase-free 1.5 mL Microcentrifuge Tubes	Axygen, USA	MCT-150-C	Aliquoting and storage of separated serum
TRIzol RNA Isolation Reagent	Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA	15596026	Total RNA extraction from human serum samples
Ultra-low Temperature Freezer	Haier Biomedical, Shandong, China	DW-86L388	Long-term serum sample storage at −80 °C
YH-600B Polysomnography Monitor	BMC Medical Co., Ltd., Beijing, China	YH-600B	Overnight sleep monitoring and AHI calculation for OSAS diagnosis