Expression of microRNA-21 in acute ischemic stroke: relationship with inflammatory cytokines, clinical severity, and clinical outcome

Ashari Bahar; Muhammad Akbar; Andi Kurnia Bintang; Muhammad Nasrum Massi; Rusdina Bte Ladju; Agussalim Bukhari; Jumraini Tammasse; Wijoyo Halim; Gita Vita Soraya; Irawan Satriotomo

doi:10.12688/f1000research.156542.1

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Research Article

Expression of microRNA-21 in acute ischemic stroke: relationship with inflammatory cytokines, clinical severity, and clinical outcome

[version 1; peer review: 2 approved with reservations]

Ashari Bahar ^1-4, Muhammad Akbar^2,4, Andi Kurnia Bintang^2,4, [...] Muhammad Nasrum Massi⁵, Rusdina Bte Ladju^2,6,7, Agussalim Bukhari⁸, Jumraini Tammasse^2,4, Wijoyo Halim^1,9, Gita Vita Soraya^4,10, Irawan Satriotomo ^11,12

Ashari Bahar ^1-4, Muhammad Akbar^2,4, [...] Andi Kurnia Bintang^2,4, Muhammad Nasrum Massi⁵, Rusdina Bte Ladju^2,6,7, Agussalim Bukhari⁸, Jumraini Tammasse^2,4, Wijoyo Halim^1,9, Gita Vita Soraya^4,10, Irawan Satriotomo ^11,12

PUBLISHED 07 Oct 2024

Author details Author details

¹ Division of Neurointervention and and Endovascular Therapy, Brain Center, Dr Wahidin Sudirohusodo Hospital, Makassar, South Sulawesi, Indonesia
² Hasanuddin University Hospital, Makassar, South Sulawesi, Indonesia
³ Faculty of Medicine, Hasanuddin University Graduate School, Doctoral Program, Makassar, South Sulawesi, Indonesia
⁴ Department of Neurology, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, Indonesia
⁵ Department of Microbiology, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, Indonesia
⁶ Department of Pharmacology, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, Indonesia
⁷ Faculty of Medicine, Hasanuddin University Medical Research Center, Makassar, South Sulawesi, Indonesia
⁸ Department of Clinical Nutrition, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, Indonesia
⁹ Department of Neurology, Faculty of Medicine, Alkhairaat University, Palu, Central Sulawesi, Indonesia
¹⁰ Department of Biochemistry, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, Indonesia
¹¹ Indonesia Neuroscience Institute, Jakarta, Indonesia
¹² Department Neuroscience, University of Florida, Gainesville, Florida, USA

Ashari Bahar
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Muhammad Akbar
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Andi Kurnia Bintang
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Muhammad Nasrum Massi
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Rusdina Bte Ladju
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Agussalim Bukhari
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Jumraini Tammasse
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Wijoyo Halim
Roles: Data Curation, Formal Analysis, Investigation, Writing – Review & Editing

Gita Vita Soraya
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Irawan Satriotomo
Roles: Conceptualization, Formal Analysis, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

When the brain is deprived of oxygen and nutrients due to stenosis or arterial rupture, neurons in the affected area suffer irreversible damage and cellular death. MicroRNA has been shown to regulate target genes implicated in arterial hypertension, atherosclerosis, and diabetes mellitus, all of which influence the risk of ischemic stroke through inflammation, oxidative stress, cell proliferation, and apoptosis. The study aims to determine the changes in miRNA expression, namely miRNA-21, between acute ischemic stroke patients and controls and their relationship to proinflammatory cytokines, clinical severity, and outcome.

Methods

Serum samples from tertiary hospitals and controls were used to evaluate miRNA-21 expression as well as cytokines TNF-α, IL-10, ICAM-1, and CCL5 levels within 7 days of stroke onset. The 30-day clinical severity and outcome were assessed using the National Institute of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS), respectively.

Result

A total of 64 acute ischemic stroke patients and 22 age-matched controls were recruited, with median ages of 56 and 55.5 years old, respectively. There were more male subjects than females (35 to 29). A statistically significant difference was observed in miRNA-21 expression between patients and controls (p<0.001). This finding implies that miRNA-21 expression may have a contribution in acute stroke patients. This was followed by an increase in proinflammatory markers TNF-α, IL-10, ICAM-1, and CCL5. However, no association was found between miRNA-21 and any pro-inflammatory cytokine. There was no significant correlation between miRNA-21 or cytokines markers with clinical severity or prognosis.

Conclusion

Our study demonstrated increased miRNA-21 expression and proinflammatory cytokine expression in acute ischemic stroke patients relative to controls. However, this was not related to clinical severity or clinical outcomes.

Keywords

miRNA, acute ischemic stroke, proinflammatory cytokine, clinical outcome

Corresponding authors: Ashari Bahar, Irawan Satriotomo

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2024 Bahar A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Bahar A, Akbar M, Bintang AK et al. Expression of microRNA-21 in acute ischemic stroke: relationship with inflammatory cytokines, clinical severity, and clinical outcome [version 1; peer review: 2 approved with reservations]. F1000Research 2024, 13:1142 (https://doi.org/10.12688/f1000research.156542.1) First published: 07 Oct 2024, 13:1142 (https://doi.org/10.12688/f1000research.156542.1) Latest published: 07 Oct 2024, 13:1142 (https://doi.org/10.12688/f1000research.156542.1)

Introduction

Stroke is the world’s second leading cause of death and the major cause of long-term impairment in people. When the brain experiences a lack oxygen and nutrients, either due to blockage, constriction, or rupture of an artery, the neurons in the afflicted area suffer irreversible damage and cellular death, resulting in disability.¹ The global prevalence of all types of stroke is approximately 89.13 million cases, up 2.17% from 2010 to 2020. The largest prevalence occurs in Sub-Saharan Africa, Southeastern and Eastern USA, and Southeast Asia.^2,3 According to current data from 2018, around 7.6 million people in the United States have had a stroke, with the majority of those over the age of 20. While this prevalence is rising in both sexes,⁴ males are far more susceptible to the disease than females.⁵ In China, for example, ischemic stroke accounts for 70% of all stroke cases, while hemorrhagic stroke is less common.¹

Recent investigations in ischemic stroke have highlighted the importance of microRNAs (miRNA), which are small noncoding RNA molecules (16-29 nucleotides) that act as post-transcriptional regulators of gene expression in mammalian cells. MiRNAs can modulate “base pairing to complementary sequences” in messenger RNA (mRNA), resulting in reduced gene activity via “translational repression.” These molecules play critical roles in the most fundamental biological processes, including cell cycle control, cell metabolism, apoptosis, and immune response.^4–6 MiRNAs have been linked to the regulation of target genes involved with arterial hypertension, atherosclerosis, and diabetes. These genes can regulate inflammation, oxidative stress, cell proliferation, and apoptosis, all of which may contribute to the development of ischemic stroke.^7–9 MiRNAs have also been linked to stroke risk.⁸ The high prevalence of ischemic stroke emphasizes the need to detect early warning signs of risk factors in order to choose an immediate and effective treatment option against the disease process.

We chose miRNA-21 as a marker in this study for a variety of reasons. First, in ischemic stroke patients, miRNA-21 is highly dysregulated relative to healthy controls. Studies have found that miRNA-21 levels are significantly reduced in the peripheral blood of ischemic stroke patients.⁵ Second, miRNA-21 protects against ischemic injury. It can bind to the IL-6R gene and suppress IL-6R expression,⁵ reducing endothelial cell damage following oxygen-glucose deprivation (OGD), suggesting that miRNA-21 has neuroprotective properties. Third, miRNA-21 levels are associated with stroke severity and outcomes. According to several studies, increased miRNA-21 expression correlated to better treatment response and disease-free survival.⁶ Fourth, miRNA-21 remains stable and detectable in blood samples.⁹ All of this evidence leads to miRNA-21 as a prognostic biomarker, making it a viable candidate for non-invasive liquid biopsy approaches that are easier to use clinically than tissue-based markers.

Inflammation has a critical role in stroke pathogenesis. Neurovascular units become dysfunctional after stroke due to a lack of oxygen and nutrients. During ischemia, the brain releases glutamate and produces reactive oxygen species (ROS), which cause oxidative stress and microglial activation. This may influence the release of inflammatory mediators.¹⁰ Following a stroke, serum TNF-α levels increase within 6 hours and remain high for 10 days.¹¹ Similarly, CSF levels of interleukin-1β (IL-1β) increase and peak 2-3 days post-stroke. Interleukin-1β damages neurons, glia, and blood vessels, leading to ischemia and excitotoxic brain injuries. CCL5, a member of the circulating chemokine family, is produced by a variety of cells, including T lymphocytes, platelets, endothelial cells, and glial cells. It can bind to chemokine receptors, attracting white blood cells to the injured area and letting them migrate along the inner lining of blood vessels, causing arterial damage.¹² T-cell recruitment and activation in the damaged brain tissue may result in further ischemia injury.¹³ Furthermore, intercellular adhesion molecule-1 (ICAM-1) is a type I transmembrane protein that secretes soluble ICAM-1, which can be detected in a variety of body fluids.¹⁴ Previous studies have shown that ICAM-1 levels are elevated in conditions such as atherosclerosis, cardiovascular diseases, metabolic vascular neuropathy, and acute ischemic stroke. Similarly, IL-6 levels are elevated in the CSF of severe stroke patients, with peak levels recorded on days 2 and 4, or in some investigations, on days 3 and 7.^15–19 In contrast, transforming growth factor-β (TGF-β) and IL-10 are anti-inflammatory cytokines that reduce inflammation following ischemic stroke by inhibiting proinflammatory cytokines. These pro- and anti-inflammatory agents predict and influence the prognosis of ischemic injury; hence, the balance of beneficial and detrimental effects of cytokines is significantly influenced by the brain’s biochemical and physiological condition.^20,21 Surprisingly, miRNAs and proinflammatory cytokines interact both directly and indirectly. For example, miR-124, miR-9, and miR-219 were reported to be lower in acute ischemic stroke (AIS), which may contribute to neuroinflammation and brain damage by reducing MMP-9 levels, a pro-inflammatory marker.²²

Given the potential role of miRNAs and inflammatory cytokines in stroke mechanisms, as well as their potential interactions with inflammatory pathways, we hypothesize that these factors may influence patient clinical severity and prognosis. This study will compare the expression of miRNA-21 and proinflammatory cytokines (TNF-α, IL-10, CCL5, and ICAM-1) between ischemic stroke patients and controls, as well as their relationship with clinical severity and outcomes.

Methods

Study design, participants, and clinical assessment

This case-control study was conducted at the Wahidin Sudirohusodo General Hospital, Makassar, Indonesia, from January to May 2024. Physical examination and imaging using non-contrast computed tomography (CT) scans and/or magnetic resonance imaging (MRI) were used to identify subjects with acute ischemic stroke. Inclusion criteria are acute ischemic stroke less than 7 days of onset, age 18-80 years old, with no history of coronary artery disease, malignancy, or peripheral arterial disease. Infection and myocardial infarction during follow-up were both considered exclusion criteria. In addition, age-matched controls were chosen. Written permission to conduct the studies for the purposes of this research was obtained by all subjects, who were fully informed about the purposes of this research and how their data would be used and stored. Ethical approval was obtained from the Ethics Committee of Faculty of Medicine, University of Hasanuddin (98/UN4.6.4.6.5.31/PP36/2024) on February 21st, 2024. We obtained the patients’ serum within 7 days of onset. The clinical severity was evaluated using the National Institute of Health Stroke Scale (NIHSS), and the clinical outcome was determined using the modified Rankin Scale (mRS) 30 days after onset.

Laboratory analysis

Subjects were recruited sequentially from the Neurology Inpatient Unit Wahidin Sudirohusodo General Hospital in Makassar, Indonesia until the sample size was sufficient. A total of 5 mL of whole blood samples were collected from each individual in EDTA-containing tubes, centrifuged, separated into serum, and stored at -80°C for analysis. To determine the expression of miRNA-21, we used Homo sapiens hsa-miR-21-3p mature miRNA taken from microRNA database (https://mirbase.org), which has the following sequences: 46-CAACACCAGUCGAUGGGCUGU-66. DNA was isolated from the whole blood sample using a preamplification kit (miScript preAmp, Cat. No. #331452, Qiagen, Germany), and expression was measured using DNA complementary amplification with reverse transcriptase-polymerase chain reaction (PCR) and real-time PCR.

First-Strand cDNA Synthesis Protocol

The miRCURY LNA RT Kit (catalog number 339340) was used for first-strand cDNA synthesis. The template RNA samples were diluted to 5 ng/μl using nuclease-free water. The reverse transcription reactions were prepared on ice by mixing 2 μl of 5x miRCURY SYBR^® Green RT Reaction Buffer + 4.5 μl of RNase-free water + 1 μl of 10x miRCURY RT Enzyme Mix + 0.5 μl of UniSp6 RNA spike-in (optional) and 2 μl of Template RNA (5 ng/μl) up to a total reaction volume of 10 μl. Samples were incubated at 42°C for 60 minutes, followed by heat inactivation at 95°C for 5 minutes, and immediate cooling to 4°C for real-time PCR procedure.

Real-Time Quantitative PCR Protocol for miRNA

Dilution of cDNA was performed at 1:60 by adding 590 μl RNase-free water to the 10 μl RT reaction. A reaction mix was made using 2x miRCURY SYBR Green Master Mix. A 10 μl reaction was added to PCR tubes and placed in the real-time cycler. The PCR protocol consisted of 2 minutes heat activation at 95°C, followed by 2 steps of cycling, 10 seconds of denaturation at 95°C, and 60 seconds annealing/extension at 56°C, all at a rapid ramp rate. The Roche LightCycler 480 was used capture The SYBR Green fluorescence data, with 45 cycles each step and melting curve analysis done between 60 and 95°C. Raw Cq and CT values were then retrieved for analyzed.

Enzyme-Linked Immunosorbent Assay (ELISA) protocols

All inflammatory cytokines were analyzed with the enzyme-linked immunosorbent assay (ELISA) technique in accordance with the manufacturers’ instructions outline below. All laboratory values were assessed at the Hasanuddin University Medical Research Center laboratory.

The TNF-α assay utilized the human TNF-α ELISA Kit (BMS223-4, Invitrogen, Austria). After allocating 50 μL of buffer, either standards, controls, or samples were added into the well (100 μL). After mixing and washing, TNF-α biotin conjugate solution was added to the well for 1-hour incubation, washed and followed by 30 minutes incubation with 100 μL 1x streptavidin-HRP solution. After washing, 100 μL of Stabilized Chromogen was applied and incubated in the dark for 30 minutes. The experiment was terminated with 100 μL stop solution and read at 450 nm.

IL-10 was measured using the human IL-10 ELISA Kit (BMS215-2, Invitrogen, Austria). Add 100 μL of blank assay buffer and 50 μL sample assay buffer to the corresponding wells. Add 50 μL of sample to each well, then 50 μL of biotin-conjugate and incubate for 2 hours. After washing, 100 μL streptavidin-HRP was added to each well, followed by an hour of incubation and washing. Incubate 100 μL TMB substrate for 10 minutes. Then, add 100 μL of stop solution and read at 450 nm.

The CCL5 assay (Human CCL5 (RANTES) ELISA Kit Cat. No. 440807, BioLegend USA) was initiated by adding of 50 μL wash buffer, 50 μL standard or sample, followed by 2 hour incubation. After washing, add 100 μL detection antibody solution and incubate for 1 hour. Wash again, add 100 μL of Avidin-HRP and incubate for 30 minutes. After washing, add 100 μL substrate solution and incubate for 10 minutes in the dark. Terminate with 100 μL stop solution and measure absorbance at 450 nm and 570 nm.

For the ICAM-1 sandwich ELISA (Human ICAM-1/CD54 ELISA Kit Cat. No. E-EL-H6114, Elabscience, USA): 100 μL of standards or samples were incubated in wells for 90 minutes. After discarding, add 100 μL of biotinylated detection antibody solution and incubate for 60 minutes. Then, wash and add 100 μL HRP conjugate working solution for another 30 minutes of incubation. After washing, add 90 μL of substrate reagent, incubate for 15 minutes, finish with 50 μL of stop solution, and read at 450 nm.

Statistical analysis

The data were analyzed using IBM SPSS version 27 (https://www.ibm.com/products/spss-statistics#110). The levels of miRNA expression and proinflammatory cytokines were compared between patients and controls using the t-test and the Mann-Whitney test. Spearman correlation analysis was utilized to examine the relationship between miRNA expression and the pro-inflammatory cytokine levels, as well as clinical severity and clinical outcomes. Data are presented as means ± standard deviation (SD). A p-value < 0.05 was considered significant.

Results

In this study, we recruited 64 patients for the acute ischemic stroke case group and 22 age-matched subjects for the control group, with a median age of 56 and 55.5 years old, respectively. Table 1 illustrates the similarity in median age between cases and controls due to sample matching. Table 2 displays gender characteristics, with 35 males (54.7%) and 29 females (45.3%). The control group had an equal number of males and females (n = 11). Table 3 lists the individuals’ risk factors, with hypertension and smoking being the most common among men.

Table 1. Demographic characteristics (age) of ischemic stroke patients (cases) and controls.

Age (years)	Median	Min-Max
Case (n=64)	56	34-80
Control (n=22)	55.5	34-73

Table 2. Demographic characteristics (gender) of ischemic stroke patients (cases) and controls.

Gender	Case n (%)	Control n (%)	Total
Males	35 (54.7%)	11 (50%)	46 (53.5%)
Females	29 (45.3%)	11 (50%)	40 (46.5%)
Total	64	22	86

Table 3. Demographic risk factors of ischemic stroke patients.

Risk factors	Male (%)	Female (%)
Diabetes Mellitus	4 (11.4)	7 (24.1)
Hypertension	18 (51.4)	18 (62.1)
Heart disease	3 (8.6)	3 (10.3)
Smoking	13 (37.1)	3 (10.3)

In addition, Table 4 shows a significant difference in miRNA-21 expression between acute ischemic stroke patients and controls (5.01 ± 9.27 versus 0.21 ± 0.38, p < 0.001). Table 5 shows the serum levels of each proinflammatory cytokine in both ischemic stroke patients and controls. Stroke patients have significantly higher serum levels of TNF-α than controls (5.04 ± 3.82 versus 3.79 ± 0.88, p < 0.05, respectively). In patients, IL-10 inflammatory cytokines levels are three times greater than in controls (1.42 ± 2.71 versus 0.39 ± 0.29, p < 0.001, respectively). Stroke patients also exhibited considerably higher serum levels of CCL5 (1973.28 ± 1281.41 versus 1434.15 ± 173.18, p < 0.001) and ICAM-1 (18.26 ± 8.74 versus 13.17 ± 3.62, p < 0.001).

Table 4. Comparison between miRNA-21 expression in ischemic stroke patients with control subjects.

microRNA		Median (min-max)	Mean (SD)	p
miRNA-21	Case (n=48)	1.85 (0-48.88)	5.01 ± 9.27	< 0.001
miRNA-21	Control (n=14)	0.01 (0.01-1.00)	0.21 ± 0.38	< 0.001

Table 5. Description of TNF- α, IL-10, CCL5, and ICAM-1 serum level between ischemic stroke patients and control subjects.

Biomarker		Median (Min-Max)	Mean (SD)	p
TNF-α	Case (n=64)	4.13 (0.00-16.80)	5.04 ± 3.82	< 0.05
(pg/mL)	Control (n=7)	4.43 (2.44-5.15)	3.79 ± 0.88
IL-10	Case (n=64)	0.5165 (0-18.824)	1.42 ± 2.71	< 0.001
(pg/mL)	Control (n=16)	0.5 (0-0.7)	0.39 ± 0.29
CCL5	Case (n=63)	1442.98 (423.93-4986.67)	1973.28 ± 1281.41	< 0.001
(pg/mL)	Control (n=21)	1693.63 (1158.19-1757.05)	1434.15 ± 173.18
ICAM-1	Case (n=63)	15.85 (6.48-56.90)	18.26 ±8.74	< 0.001
(ng/mL)	Control (n=22)	13.31 (6.18-17.82)	13.17 ±3.62

According to the Spearman test analysis, we found no significant correlation between miRNA-21 with TNF-α (r = 0.013, p = 0.931), IL-10 (r = 0.276, p = 0.058), CCL5 (r = 0.033, p = 0.824), and ICAM-1 (r = 0.054, p = 0.717) inflammatory cytokines (Table 6). There was also no link between miRNA-21 and either NIHSS (r = 0.229, p = 0.118) or mRS (r = -0.022, p = 0.88) scores (Table 7).

Table 6. Correlation between miRNA-21 expression with cytokines TNF-α, IL-10, CCL5, and ICAM-1 in acute ischemic stroke patients.

	TNF-α		IL-10		CCL5		ICAM-1
	p	r	p	r	p	r	p	r
miRNA-21 (n =48)	0.931	0.013	0.058	0.276	0.824	0.033	0.717	0.054

Table 7. Correlation between miRNA-21 expression, TNF-α, IL-10, CCL5, and ICAM-1 with severity and clinical outcome in acute ischemic stroke patients.

	NIHSS		mRS
	p	r	p	r
miRNA-21	0.118	0.229	0.88	-0.022
TNF-α	0.961	0.006	0.383	-0.111
IL-10	0.793	0.033	0.689	0.051
CCL5	0.832	0.027	0.314	-0.129
ICAM-1	0.573	-0.072	0.557	0.075

Discussion

This study aimed to compare the expressions of miRNA-21 and inflammatory cytokines (TNF-α, IL-10, CCL5, and ICAM-1) in acute ischemic stroke patients and controls, analyzing their relationship with clinical outcomes and severity. A recent study has demonstrated that miRNAs have a role in the pathological process of ischemic injury via mechanisms that change inflammatory signaling by targeting cytokine production in immune cells.¹⁶ MicroRNA-21 is involved in a wide range of biological processes, including proliferation, apoptosis, and inflammation.²³ In this study, we observed a significantly difference in miRNA-21 expression between acute ischemic stroke patients and controls (Table 5). This is consistent with Zhou J’s (2014) experiments, which showed that miRNA-21 and miRNA-24 levels in cultured N2A cells increased 3.3 and 4.9 times, respectively, during the 24-hour recovery period and 3 hours after oxygen-glucose deprivation (OGD). Furthermore, the difference in miRNA-21 and miRNA-24 levels between acute cerebral infarction and controls was statistically significant and positively associated, indicating that release occurred during or after the ischemia event, elucidating their mechanisms in acute cerebral ischemia as plasma markers. This suggests that miRNA-21 and miRNA-24 may operate as protective and anti-apoptotic agents in cerebral ischemia, making them potential therapeutic agents for stroke.^23,24

Our data findings support the concept that miRNA-21 regulates inflammatory processes during ischemic stroke, most likely by modulating gene expression associated with inflammatory responses, immune cell migration, and atherosclerotic plaque formation.^25–28 MicroRNA-21 has emerged as a potential therapeutic target for inflammation-related disorders.²⁹ Zhou and Zhang (2014) discovered miRNA-21 as a possible early-stage marker of acute cerebral infarction. Inhibiting miRNA-21 has been proposed as a therapeutic strategy for controlling endothelial cell dysfunction and vascular inflammation.^27,30 Zhan et al. (2023) found that miRNA-21 protects against ischemic stroke by inhibiting IL-6R. These collective findings provide evidence for miRNA-21’s involvement in neuroprotection and its potential as a therapeutic target in neurological disorders.

In this study, we discovered that acute stroke patients had significantly higher levels of all proinflammatory cytokines than controls. We found that the TNF-α level in the stroke patients is greater compared to healthy subjects. This finding is similar to a recent study, which reported that patients with acute ischemic stroke had significantly greater levels of TNF-α in both cerebrospinal fluid (CSF) and serum within 24 hours after stroke onset compared to healthy controls.³¹ A previous study reveals that TNF-α, a proinflammatory protein present throughout the body, may contribute to ischemic damage in cerebral tissue.³² Using TNF-α for prognosis yielded varied outcomes. Although earlier research has indicated that levels of TNF-α in brain tissue may remain high for 24 hours following ischemic injury and may be linked to the severity of the injury,³¹ we observed no relationship between TNF-α levels and the severity of symptoms or outcomes in the patients we evaluated.

In several animal stroke models, IL-10 mRNA and protein levels, as well as IL-10R mRNA levels, were elevated on microglia and astrocytes in the ischemic penumbra. Low IL-10 levels are associated with increased inflammatory responses and poorer stroke outcomes.³³ Indeed, in transgenic mice overexpressing IL-10, infarct size decreased and apoptosis was reduced 4 days after an ischemic stroke.³⁴ Furthermore, overexpression of IL-10 increased the neuroprotective benefits of mesenchymal stem cell transplantation by controlling inflammation, hence promoting neuron survival following acute ischemia.^33–35 Our findings are consistent with previous studies that have demonstrated higher levels of IL-10 in ischemic stroke patients compared to healthy subjects.^36,37 Vila et al. (2003) found that lower plasma IL-10 concentrations (<6 pg/mL) were associated with clinical deterioration in acute ischemic stroke patients.³⁷ Increased IL-10 levels in female patients were associated with poorer outcomes after ischemic stroke, although they were not the only independent predictor.³⁸ This suggests that IL-10 effects may be influenced by other factors such as gender, age, and stroke severity.

RANTES (Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted) chemokines (C-C motif) ligand 5, or CCL5, is a kind of chemokine that plays an important role in stroke progression via angiogenesis and endothelial repair.^32,39 We observed that CCL5 levels were higher in ischemic stroke patients than in healthy subjects. We found no correlation between CCL5 levels and the severity of symptoms or outcomes in the stroke patients. Our findings are comparable with Pawluk et al. (2023), who discovered that when compared to healthy persons, this chemokine was elevated in less than 4.5 hours, 24 hours, and 7 days. Furthermore, they also determined the effect of CCL5 concentration on probability of patient death, finding that concentrations greater than 75 ng/mL resulted in a significant increase in stroke mortality. A similar relationship was also seen on the first day and during the onset of stroke, with CCL5 levels higher than 60 ng/mL and 100 ng/mL, respectively, making this chemokine an excellent biomarker for mortality during stroke progression.³² However, differences in CCL5 levels may vary depending on the type of stroke and the onset. CCL5 levels were shown to be different in ischemic and hemorrhagic stroke patients, with hemorrhagic stroke patients having lower levels than acute ischemic stroke patients.³² Julián-Villaverde (2022) discovered that patients suffering from either an ischemic or hemorrhagic stroke had different levels of CCL5. CCL5 levels were significantly lower in hemorrhagic stroke patients that persisted for at least 7 days. However, no association was found between CCL5 levels and the outcome of patients with hemorrhagic stroke.^32,39,40 Similarly, Pawluk et al. (2023) also found no significant correlations between CCL5 levels and mRS and NIHSS score after an acute ischemic stroke. Thus, assessing CCL5 levels upon admission can be used as a predictive biomarker for the progression of ischemic stroke in patients, as well as a promising diagnostic biomarker for distinguishing between different types of strokes.

We observed that ICAM-1 serum levels were elevated in ischemic stroke patients. Our findings are aligning with Wu et al. (2018), who discovered that ischemic stroke patients had considerably greater serum soluble ICAM-1 (sICAM-1) levels than healthy controls. Nielsen et al. (2020) also found that stroke patients had significantly higher ICAM-1 levels than controls within the first 8 hours of symptom onset but not beyond 72 hours. In contrast, Supanc et al. (2011) showed no significant difference in serum levels of soluble ICAM-1 between acute ischemic stroke patients and healthy controls. The disparities in these findings might be attributed to differences in the timing of sample collection, stroke subtypes, or other methodological factors. Overall, while some studies reported higher ICAM-1 levels in acute ischemic stroke, particularly in the early phase, the evidence is not entirely consistent across investigations. Further study may be required to elucidate the association between ICAM-1 levels and ischemic stroke, taking into consideration parameters such as stroke subtype and timing of assessment.^33,40,41

This study also evaluated the correlation between miRNA-21 and serum inflammatory cytokines TNF-α, IL-10, CCL5, and ICAM-1. We found no significant relationship between miRNA-21 and the four examined cytokines. Despite the lack of correlation in our study, other studies have demonstrated that miRNA-21 plays an important role in regulating inflammatory responses in the brain following an ischemic stroke, which is supported by studies showing its involvement in inflammation resolution and negative regulation of proinflammatory responses.²⁸ MicroRNA-21 is also implicated in attenuating inflammatory responses in cerebrovascular disease, indicating its potential role in modulating inflammation in the brain following ischemic events.³³ Understanding these complex regulatory networks is critical for determining miRNA’s functions in development and cerebrovascular disease progression. Future studies with large samples at a multi-research institution are required to evaluate the clinical application of miRNA-21 as a potential biomarker for ischemic stroke diagnosis.

Ethics and consent

Ethical approval was obtained from the Ethics Committee of Faculty of Medicine, University of Hasanuddin (98/UN4.6.4.6.5.31/PP36/2024) on February 21st, 2024. Written permission to conduct the studies for the purposes of this research was obtained by all subjects, who were fully informed about the purposes of this research and how their data would be used and stored.

Disclosures

The authors report no relevant disclosures.

Data availability

Figshare: Result of analysis samples re pcr - Gene Expression Results - Bar Chart aLL dATA.xlsxResult of Statistical Analysis.spv, we present the following article accordance with the STREGA guideline checklist DOI: https://doi.org/10.6084/m9.figshare.27146649.v1 ⁴²

The project contains the following underlying data:

• Result of analysis samples re PCR - Gene Expression Results - Bar Chart aLL dATA.xlsx
• Result of Statistical Analysis.spv

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

The author wants to acknowledge all the patients and all the supporting systems.

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