Circulating microRNAs and ischemic stroke: From blood biomarkers to pathogenesis: A systematic review

Eligibility criteria

Literature search

The PRISMA 2020 flow diagram is shown in Figure 2, which shows the studies that were used in this systematic review. A total of 1,617 articles were identified through electronic searches, including 531 from Web of Science, 482 from PubMed, and 604 from Scopus. After removing the duplicates, the titles and abstracts of the remaining articles (n = 1088) were used to determine their eligibility. Of the 118 articles assessed in full text, 109 were excluded for not meeting the eligibility criteria. Forty-four were excluded due to an inappropriate study design, and in 36 studies, blood samples other than plasma were used. Ultimately, nine articles were included in the review.

Quality assessment

The quality and validity assessment of the NOS checklist suggested that six studies were of high quality and three studies were of moderate quality. In all nine studies, the definition of “case” groups was adequate. In one study, there was no data on the comparison of case and control groups by age and gender. In six studies, information was provided on the comparison of the case and control groups according to additional criteria.

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Figure 2:

Flow diagram of search and selection of studies in the systematic review.

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Study design and methods

The design and methods of the included studies^[6-14] are summarized in Table 1. Eight studies were conducted in China, and the remaining one in Germany. In six studies, a single miRNA was investigated in both the case and control groups. In the remaining studies, the expression levels of two, three, and five miRNAs were examined. In all studies, case groups were diagnosed using neuroimaging methods: MRI, CT, or magnetic resonance angiography. The number of patients with ischemic stroke ranges from 30 to 200, whereas the number of controls ranges from 21 to a maximum of 112. In seven studies, the number of male patients with stroke predominated over females, while two studies did not report sex characteristics. Plasma was used as the blood sample in all studies. Peripheral blood was collected from patients within the first 24 h after the onset of neurological symptoms in five studies, within the first 6 h and within 72 h in only one study. In one study, blood collection was performed during the acute phase without specifying the exact timing. Multiple post-stroke time points (24 h, 1 week, 4 weeks, 28 weeks, and 48 weeks) for blood sampling were reported in only one study.

Plasma circulating miRNAs

In all included studies, miRNA expression in plasma was analyzed using quantitative real-time polymerase chain reaction. A total of 16 miRNAs were investigated. The regulation of nine miRNAs was upregulated in the blood plasma (miR-487b, miR-153, miR-125a-5p, miR-125b-5p, miR-143-3p, miR-222, miR-218, miR-185, and miR-128), while the expression of the remaining seven miRNAs (miR-130a, miR-378, miR-126, miR-99b, miR-21, miR-24, and miR-335) was downregulated compared to the control group.

Upregulated Plasma miRNAs and Their Potential Roles in Stroke Pathophysiology

A key mechanism by which miR-487b may exert its effects is through the modulation of angiogenesis, a critical process in tissue repair and vascular remodeling following ischemic injury. In vitro studies using human umbilical vein endothelial cells (HUVECs) have demonstrated that miR-487b overexpression significantly promotes endothelial cell proliferation, migration, invasion, and capillary-like tube formation, all of which are essential steps in the angiogenic cascade.^[6] These findings suggest that miR-487b may play a pro-angiogenic role, potentially enhancing vascular regeneration in ischemic tissues. Furthermore, miR-487b is thought to influence the expression of angiogenesis-related genes and signaling pathways, such as vascular endothelial growth factor (VEGF) and components of the PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. Understanding the precise molecular targets and regulatory networks of miR-487b could provide valuable insights into its therapeutic potential in promoting angiogenesis and improving outcomes after acute ischemic stroke. In addition to its proangiogenic role, miR-487b has been implicated in promoting vascular injury under pathological conditions. It has been reported to contribute to cell death and loss of vascular wall integrity through suppression of insulin receptor substrate 1, an anti-apoptotic signaling molecule, particularly in the context of hypertension-related vascular disease.^[16] miR-153 has been identified as a potential regulator of neuronal survival mechanisms following ischemic injury. Specifically, overexpression of miR-153 in cortical neurons leads to a significant increase in the activation of mTOR downstream effectors, suggesting that miR-153 may function as an activator of the mTOR signaling pathway. Given that the mTOR complexes (mTORC1 and mTORC2) are central mediators of cell metabolism, growth, and survival through integration of intracellular energy status, oxygen availability, amino acid levels, and extracellular growth factor signals, the modulation of mTOR activity by miR-153 may have important implications in the cellular response to ischemic stress. By enhancing mTOR signaling, miR-153 could contribute to neuroprotective processes, including inhibition of apoptosis, promotion of neuronal recovery, and metabolic adaptation during cerebral ischemia.^[17] miR-143-3p plays a crucial role in maintaining vascular homeostasis through intercellular communication between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). It is a key regulatory molecule that contributes to the coordination of cellular responses required for vascular function and adaptation. In ECs, miR-143 contributes to vessel stabilization by repressing important target genes such as hexokinase II and integrin b8, thereby modulating endothelial metabolism and angiogenic capacity. By downregulating these proangiogenic and metabolic factors, miR-143 promotes a quiescent endothelial state, which is important for vessel maturation and stability. Simultaneously, in VSMCs, miR-143 supports a contractile and differentiated phenotype, which is essential for maintaining vascular tone and structural integrity. It achieves this by targeting signaling molecules involved in VSMC dedifferentiation and proliferation, such as ELK-1 (ETS Like-1 protein) and KLF4 (Kruppel-like factor 4), thus preventing phenotypic switching toward a synthetic, proliferative state often associated with vascular pathology. Given the importance of vascular remodeling, stabilization, and regeneration following ischemic injury, elevated levels of miR-143 after stroke may significantly influence endothelial function, angiogenesis, and structural repair of the vasculature in the post-stroke period.^[18,19] Furthermore, dysregulation of miR-143 may contribute to impaired revascularization or pathological remodeling, making it a potential biomarker and therapeutic target for enhancing vascular recovery and improving neurological outcomes in patients with acute ischemic stroke. miR-125b has emerged as a potential regulator of neuronal function in the context of cerebral ischemia. A significant downregulation of miR-125b was observed during the early reperfusion period following ischemic stroke, mirroring changes in motor function assessments over the first 48 h.^[20] This temporal association suggests that miR-125b may be involved in post-ischemic neuroplasticity and functional recovery, highlighting its potential as a target for miRNA-based therapies. Moreover, miR-125b-5p has been shown to regulate synaptic morphology and function, contribute to neuronal differentiation, and influence cytoskeletal organization – processes that are critically disrupted during and after ischemic injury.^[21] miR-125a-5p has emerged as a key regulator of microglial cell fate in the context of cerebral ischemia. Its expression is significantly upregulated in microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) conditions in vitro, as well as in the brains of rats following middle cerebral artery occlusion (MCAO). This elevated expression is associated with increased microglial apoptosis, suggesting a detrimental role for miR-125a-5p during ischemic injury. Experimental evidence indicates that downregulation of miR-125a-5p reduces microglial cell death, thereby exerting a protective effect against ischemia-induced injury. The pro-apoptotic function of miR-125a-5p is mediated through its direct targeting of insulin-like growth factor binding protein 3 (IGFBP3), a protein known for its role in promoting cell survival. Notably, silencing IGFBP3 abolishes the anti-apoptotic effect observed with miR-125a-5p inhibition, confirming the functional significance of the miR-125a-5p–IGFBP3 axis. Beyond its influence on apoptosis, miR-125a-5p may also contribute to the neuroinflammatory response by modulating microglial activity, including the release of pro-inflammatory cytokines and regulation of phagocytic behavior. These actions may amplify neuronal damage and hinder post-ischemic recovery. Collectively, these findings suggest that miR-125a-5p plays a critical role in ischemia-induced microglial dysfunction and represents a potential therapeutic target for attenuating neuroinflammation and limiting secondary brain injury following stroke.^[22] miR-222 is involved in various physiological and pathological processes within the cardiovascular system, including vascular remodeling and atherosclerosis. Notably, reduced miR-222 expression has been observed at sites of atherosclerotic plaque rupture, suggesting its association with plaque stability.^[23] In experimental models, suppression of miR-222 in the carotid arteries inhibited VSMC proliferation and reduced neointimal lesion formation following angioplasty. These findings indicate that miR-222 promotes VSMC proliferation and neointimal hyperplasia, processes that contribute to vascular injury and may influence stroke pathogenesis by affecting plaque vulnerability and post-ischemic vascular repair.^[24] miR-218-5p has been implicated in the regulation of endothelial cell survival and vascular inflammation. A reduction in miR-218-5p levels exacerbates injury to HUVECs, particularly in the context of downregulated small nucleolar RNA SNHG12. This downregulation suppresses endothelial proliferation, promotes apoptosis, and enhances the inflammatory response, thereby contributing to the development of atherosclerosis.^[25] Given the critical role of endothelial dysfunction and inflammation in stroke pathogenesis, miR-218-5p may serve as a protective factor in ischemic cerebrovascular injury. miR-185 has been shown to play a regulatory role in vascular homeostasis and the development of atherosclerosis, a major risk factor for ischemic stroke. One of its key molecular targets is stromal interaction molecule 1 (STIM1), a calcium sensor within the endoplasmic reticulum. Downregulation of miR-185 leads to increased STIM1 expression, which promotes vascular cell proliferation, migration, and invasion, thereby accelerating atherosclerotic plaque progression. The miR-185/STIM1 axis thus represents a potential mechanism linking vascular remodeling with ischemic stroke pathogenesis.^[26] miR-128-3p has been identified as a critical regulator of neuronal survival in the context of cerebral ischemia. It exerts its effects primarily by targeting p38a (Mapk14), a well-known pro-apoptotic protein kinase involved in cellular stress responses, inflammation, and programmed cell death. Although p38a mRNA levels remain unchanged following ischemic injury, its protein expression is significantly reduced, indicating that miR-128-3p mediates a post-transcriptional regulatory mechanism rather than transcriptional silencing. Mechanistically, miR-128-3p binds directly to the 3'UTR of p38a mRNA, thereby repressing its translation into protein. This targeted suppression results in decreased p38a-mediated activation of downstream apoptotic pathways, including caspase activation and mitochondrial dysfunction, both of which are central to ischemia-induced neuronal death. Experimental studies have demonstrated that overexpression of miR-128-3p significantly reduces infarct volume, attenuates neuronal apoptosis, and improves functional neurological outcomes in animal models of acute ischemic stroke.^[27] Conversely, inhibition of miR-128-3p leads to increased p38a protein levels, greater neuronal loss, and exacerbation of ischemic damage, further supporting its neuroprotective role. These findings highlight miR-128-3p as a promising therapeutic target for reducing neuronal injury and improving outcomes in patients with ischemic stroke, particularly through its selective suppression of stress-related pro-apoptotic pathways.

Downregulated Plasma miRNAs and Their Potential Roles in Stroke Pathophysiology

Besides increased regulation, reduced miRNA levels in blood plasma have been identified as potential biomarkers for early acute stroke detection [Table 1].

miR-126 is a key regulator of endothelial cell responses to growth factors such as VEGF and fibroblast growth factor, promoting angiogenesis primarily through activation of the MAPK signaling pathway. It exerts its pro-angiogenic effects by targeting Spred-1, a negative regulator of Raf phosphorylation and ERK activation. Suppression of Spred-1 by miR-126 enhances endothelial cell proliferation, migration, and cytoskeletal reorganization, all of which are essential for vascular repair following ischemic injury. Deficiency of miR-126 leads to impaired endothelial function and vascular abnormalities that closely resemble phenotypes observed with disrupted MAPK signaling, underscoring its critical role in post-stroke vascular remodeling.^[28] Moreover, experimental models demonstrate that miR-126 restoration promotes revascularization and supports neurovascular integrity, highlighting its potential as a therapeutic target in ischemic stroke. miR-130a has been shown to exert neuroprotective effects in ischemic stroke models by modulating the PTEN/PI3K/AKT (Phosphatase and Tensin Homolog/Phosphatidylinositol 3-Kinase/Protein Kinase B) signaling pathway signaling pathway, which plays a central role in regulating cell survival, apoptosis, and oxidative stress responses. Its expression is significantly downregulated following oxygen-glucose deprivation/reperfusion (OGD/R) in PC12 cells and in the MCAO model in rats. Restoration of miR-130a levels improves cell viability, reduces apoptosis and oxidative damage, and leads to a marked decrease in infarct volume and neurological deficits. Mechanistically, miR-130a exerts its effects by directly targeting PTEN, a negative regulator of the PI3K/AKT pathway, thereby promoting AKT activation and enhancing neuronal survival after ischemic injury.^[29] miR-99b has been shown to play a protective role in ischemic brain injury by enhancing neuronal cell survival and reducing apoptosis. In an in vitro OGD/R model, expression of miR-99b was significantly upregulated in SH-SY5Y neuroblastoma cells, suggesting a potential adaptive response to ischemic stress. Functional studies demonstrated that overexpression of miR-99b promotes cell viability, inhibits apoptotic cell death, and contributes to the preservation of neuronal integrity under ischemic conditions. Mechanistically, miR-99b directly targets the insulin-like growth factor 1 receptor (IGF1R), a critical mediator of neuronal survival and anti-apoptotic signaling. This interaction was confirmed using a luciferase reporter assay, supporting the specificity of miR-99b binding to the 3'UTR of IGF1R mRNA and its role in regulating downstream signaling pathways. Suppression of IGF1R expression by miR-99b may help to fine-tune IGF1-mediated responses, preventing excessive or maladaptive activation of survival signaling cascades during reperfusion.^[12] These findings suggest that miR-99b contributes to neuroprotection by modulating IGF1R-dependent signaling, and its upregulation may represent an endogenous mechanism aimed at limiting ischemia-induced neuronal injury. Further investigation into the role of miR-99b in vivo may clarify its potential as a therapeutic target for stroke intervention. miR-21 plays a critical role in regulating the phenotype of VSMCs, which is essential for vascular remodeling and stabilization following ischemic stroke. Its expression is rapidly induced by transforming growth factor-beta and bone morphogenetic protein signaling through a Sma and Mad related proteins (SMAD)-dependent, post-transcriptional mechanism, which facilitates the processing of primary miR-21 transcripts (pri-miR-21) through enhanced interaction with the DROSHA (Drosha Ribonuclease III) microprocessor complex. Functionally, miR-21 downregulates programmed cell death protein 4, a known inhibitor of contractile gene expression, thereby promoting a contractile, differentiated VSMC phenotype and suppressing proliferation and phenotypic switching associated with vascular pathology.^[30] This shift toward a contractile state contributes to vessel wall stabilization and may prevent maladaptive remodeling processes such as neointima formation or vascular stiffening. These findings suggest that miR-21 not only supports structural repair after stroke but may also serve as a potential target for therapeutic modulation of VSMC behavior in cerebrovascular disease. miR-24 plays a neuroprotective role in ischemic brain injury by modulating microglia/macrophage polarization. In a rat model of MCAO, overexpression of miR-24 reduced infarct volume and neurological deficits, while its inhibition exacerbated ischemic damage. miR-24 suppresses pro-inflammatory M1 polarization and promotes anti-inflammatory M2 polarization in both in vivo brain tissue and BV-2 microglial cells. Mechanistically, miR-24 targets Clcn3, a chloride channel protein, to regulate microglial phenotype.^[31] These findings suggest that miR-24 contributes to post-stroke recovery by shifting microglial activation toward a protective, anti-inflammatory state. miR-335 is downregulated in the acute phase of ischemic stroke and plays a key role in regulating neuronal survival and stress granule (SG) formation. In a rat MCAO model, restoration of miR-335 expression promoted SG assembly, reduced infarct volume, and suppressed apoptosis. Mechanistically, miR-335 directly targets Rho-associated protein kinase-2 (ROCK2), whose inhibition contributes to enhanced SG formation and decreased neuronal death.^[32] These findings suggest that miR-335 exerts neuroprotective effects during ischemic injury by modulating the ROCK2 pathway and cellular stress responses. miR-378 has been identified as a neuroprotective factor in ischemic stroke, with its expression markedly reduced in both the peri-infarct region of MCAO mice and OGD-treated neuronal cells. Overexpression of miR-378 enhances cell viability and reduces apoptosis by directly targeting the 3'-UTR of Caspase-3 mRNA, thereby suppressing its expression and activity. In vivo, miR-378 agomir treatment led to decreased cleaved-caspase-3 levels, reduced infarct volume, and mitigated neuronal death following MCAO.^[33] These findings suggest that miR-378 protects against ischemic injury by inhibiting caspase-3–mediated apoptosis, making it a promising therapeutic target.

Comparison of the diagnostic potential of circulating miRNAs versus neuroimaging techniques

Neuroimaging remains a cornerstone in the diagnosis of acute ischemic stroke, enabling rapid assessment of infarct size, vascular occlusion, and tissue viability. Advanced techniques such as diffusion-weighted imaging and perfusion-weighted imaging provide critical information for therapeutic decision-making within narrow time windows.^[34] Circulating miRNAs have emerged as promising minimally invasive biomarkers for the early diagnosis of acute ischemic stroke, offering the potential for rapid detection through blood-based assays. Unlike neuroimaging techniques such as MRI, which require specialized equipment and may be limited by contraindications or delayed availability in emergency settings, circulating miRNAs can be measured quickly and repeatedly, including in pre-hospital or resource-limited environments. While MRI remains the gold standard for anatomical localization and confirmation of infarction – with high sensitivity and specificity, particularly in the acute phase – it may not detect very early or small ischemic lesions. In contrast, circulating miRNAs may reflect molecular changes that occur before structural abnormalities visible on imaging. Therefore, although circulating miRNAs cannot replace neuroimaging, they may serve as a valuable complementary tool, particularly for early triage and diagnosis when imaging is unavailable or inconclusive.

Diagnostic cost comparison

MRI remains the gold standard for anatomical and diagnostic assessment in acute ischemic stroke. However, direct cost– utility analysis in a Spanish hospital revealed that MRI yields an average direct cost of €5,693 per patient – comparable to CT scan (mean cost €5,831) – with only marginal quality-adjusted life-year (QALY) improvement, resulting in an incremental cost-effectiveness ratio of €11,869/QALY.^[35] By contrast, assays for circulating miRNAs are substantially lower-cost: Reverse transcription quantitative polymerase chain reaction profiling through commercial platforms incurs approximately $0.53–$0.82 per miRNA per sample, with individual panels costing $250–400.^[36] Moreover, such assays can be performed in standard molecular laboratories without the need for specialized imaging infrastructure, offering faster turnaround times and wider accessibility – particularly in settings where MRI availability is limited. These data support the cost-effective and practical potential of circulating miRNA assessment as a complement to, or triage tool for, MRI in stroke care. The development and implementation of strategies aimed at removing barriers to accessibility and improving the quality of services are crucial for ensuring the health and well-being of the population.^[37] Despite their promise as diagnostic and prognostic biomarkers for ischemic stroke, the clinical utility of miRNAs requires further investigation.

Thus, we reviewed data from nine independent studies. This study is subject to several limitations. The included studies varied in sample sizes, which may affect the comparability of findings. In many cases, the number of patients analyzed was relatively small, limiting the statistical power and generalizability of the results.