Non-Coding RNA Profile in the Progression of Carotid Atherosclerosis: A Systematic Review

Introduction

Methods

Results

Flow Diagram

All the phases in the systematic review process are shown in Figure 2. The research carried out on PubMed and Scopus yielded 80 and 187 studies, respectively. These 267 papers were merged into a non-redundant database and 190 papers remained. Then, by eliminating all studies not related to our research question and all studies that did not match PICOS criteria related to Study Design (exclusion for all types of reviews, meta-analysis, and conference papers), the number of studies was reduced to 104. Finally, by applying all the PICOS criteria, we obtained 49 studies to be included in the systematic review.

Synthesized Findings

This section outlines the principal findings pertinent to the research objective, as extracted from the 49 studies included in the analysis. All included articles were published within the last 15 years (2010–2024) and originate from research groups across various geographic regions. Specifically, 29 studies were conducted in Asia, with 25 originating from Chinese groups; 11 studies were from Europe, 4 from Russia, 4 from North America (United States and Canada), and 1 from South America. For each publication, significantly dysregulated non-coding RNAs (ncRNAs) are reported based on the statistical thresholds applied in the original studies.

The results are systematically organized into the following subsections, structured hierarchically according to the extent and stage of carotid atherosclerosis within the studied populations. Consequently, ncRNAs identified as dysregulated in multiple comparative contexts may appear in more than one subsection.

All ncRNAs included in this synthesis were identified in human-derived samples, specifically carotid artery tissue, peripheral blood cells, or serum. For each ncRNA, the direction of dysregulation (i.e., upregulation or downregulation) is indicated, along with the proposed molecular mechanism, when reported.

1. ncRNA associated with carotid atherosclerosis risk factors

Although the exact causes or risk factors of carotid atherosclerosis are incompletely determined, certain conditions, traits, or habits may predispose to the development of the disease. The principal recognized risk factors of carotid atherosclerosis are systemic inflammation, high cholesterol and LDL and/or low high-density lipoproteins (HDL) levels in the blood, hypertension, tobacco smoke, diabetes mellitus, obesity, and physical inactivity [19]. The identification of ncRNA signatures associated with these risk factors may offer novel biomarkers for early diagnosis, risk stratification, and preventive intervention. A detailed overview of dysregulated ncRNAs linked to these pathogenic determinants is summarized in Table 1.

The initiation of carotid atherosclerosis involves a series of intricate processes, with inflammatory processes playing a key role in the development and progression of plaque. Adhesion and migration of circulating monocytes through vascular walls, based on the activation of adhesion molecules and chemokines, are key events in the initiation of atherosclerotic plaque [20]. Beside the classical inflammatory biomarkers of carotid atherosclerosis risk prediction - such as proinflammatory cytokines TNF-alpha and IL-6, high-sensitivity C-reactive protein (hs-CRP) and lipoprotein associated phospholipase a2 (Lp PLA 2) [21] – Yang and coauthors identified hsa-miR-520b as a novel indicator, significantly downregulated in carotid atherosclerotic plaque versus adjacent non-atherosclerotic tissue [22]. Notably, authors demonstrated that hsa-miR-520b directly interacts with the 3’UTR of NFkB-subunit Rel A transcript, thereby attenuating the expression of adhesion molecules VCAM-1 and ICAM-1 in monocyte and consequently limiting monocyte-endothelial cell adhesion [22].

Metabolic disorders such as diabetes and obesity are characterized by persistent low-grade inflammation, predisposing thus to carotid atherosclerosis and correlated cardiovascular diseases. The primary characteristic trait of diabetics is hyperglycemia, which exerts many cellular effects driving toward endothelial dysfunction and enhanced inflammation [23]. Wang et al. demonstrated that miRNA-29c expression in carotid plaque was decreased in patients with diabetes as compared with the non-diabetic patients, proposing that it could regulate the phenotypic transformation of VSMCs, leading to VSMC proliferation and migration [24]. Obesity may origin from excessive caloric intake along with a sedentary lifestyle, resulting in increased metabolically active subcutaneous adipose tissue, responsible for the secretion of inflammatory adipokines and contributing to atherosclerosis progression [25]. MiRNAs belonging to let-7 family have been demonstrated to exert an atheroprotective function, by modulating VSMC and endothelial cell activation and inflammation, and suppressing vascular inflammation mediators such as interleukin (IL)-6, IL-1β and NFkB [26,27]. Elevated serum levels of let-7d-5p have been detected in overweight patients with carotid atherosclerosis relative to lean counterparts and healthy controls, although this upregulation is absent in obese atherosclerotic individuals, possibly due to advanced systemic inflammation impairing let-7d-5p homeostasis [28].

Hypercholesterolemia, particularly elevated LDL-cholesterol (LDL-C), remains the most important risk factor for carotid atherosclerosis. Mandolini et al. reported increased hsa-miR-33b and hsa-miR-758, in carotid plaques from hypercholesterolemic patients (LDL-C > 4.14 mmol/L), compared to the normocholesterolemic carotid atheroma [29], while Tanashyan et al. found both strands of hsa-miR-33a upregulated in blood of patients with an LDL-C ≥ 1.8 mmol/L presenting carotid plaque [30]. Interestingly, these miRNAs directly target ATP Binding Cassette Subfamily A Member 1 (ABCA1), required for cholesterol efflux and, thus, essential for maintaining cellular cholesterol homeostasis [31,32].

In accordance with the recommendations of the American College of Cardiology/American Heart Association [33] and with the European Society of Hypertension and European Society of Cardiology [34], essential hypertension is defined as diastolic blood pressure ≥ 90 mmHg or systolic blood pressure ≥ 140 mmHg, or the use of antihypertensive medications. Hypertension damages endothelium by increasing the hemodynamic pressure on endothelium and may increase the permeability of arterial walls for lipoproteins. Zhang et al. identified five circRNAs upregulated in hypertensive individuals with carotid atherosclerosis relative to normotensive controls [35]. Conversely, Qian et al. described three circRNAs with reduced expression in hypertensive patients with carotid plaques, among which circ-0127342 was specifically diminished in hypertensives with plaques versus those without, suggesting its potential as a disease-specific biomarker [36].

2. ncRNA correlated with carotid Intima- Media Thickness (cIMT)

In the primary prevention setting, the presence of increased cIMT is validated a marker of subclinical atherosclerosis and, as such, predictor of carotid plaque formation and atherosclerosis [37]. Thus, identification of ncRNAs modulated in early atherogenesis is pivotal for timely intervention. Relevant biomarkers discovered in the present systematic review are presented in Table 2. Minin and collaborators found significantly higher expressions of hsa-miR-145-5p and hsa-miR-let7c in the serum of patients with carotid plaques relative to those with increased cIMT alone, suggesting that these miRNAs could be used for distinguishing the early phase from a more advanced stage of carotid atherosclerosis [38]. A subsequent study demonstrated the significantly higher expression of lncRNA SOX2-OT in serum of subjects with increased cIMT compared to healthy individuals, which was proposed as a valuable clinic marker of atherosclerosis, with diagnostic metrics including an AUC of 0.921, sensitivity of 87.4%, and specificity of 82.2% [39]. Yan et al. described a progressive upregulation of hsa_circ_0043621 in serum from healthy individuals to those with cIMT and ultimately with plaques, suggesting a linear correlation with disease severity [40]. Notably, all these studies defined increased cIMT as 1.0 < cIMT < 1.5 mm, while the presence of plaque is indicated by a cIMT value higher than 1.5 mm or a focal structure that encroached into the arterial lumen measuring at least 0.5 mm or 50% of the surrounding cIMT [38,40].

3. Differentially expressed ncRNA in carotid atherosclerosis versus healthy controls

From this study, several classes of ncRNA have resulted differentially expressed in individuals with carotid atherosclerosis with respect to non-atherosclerotic subjects, and summarized in Table 3, with miRNAs representing the predominant class. Among the upregulated miRNA in carotid atherosclerosis patients compared to healthy control, hsa-miR-33a and hsa-miR-33b had been identified by three distinct studies, both in serum and in carotid tissue (41–43). Notably, this subgroup of miRNA directly targets 3’ UTR of ABCA1 transcript, thus regulating the cholesterol uptake. hsa-miR-148a-3p, another direct regulator of ABCA1 expression, was found upregulated in plasma of carotid stenosis patients [41]. In contrast, miRNA-148b expression resulted significantly lower in carotid atherosclerosis [44]. Conflicting evidence surrounds hsa-miR-21: two studies reported its upregulation [45,46], whereas a third study observed a lower expression of both the hsa-miR-21-5p and the -3p strand [43]. Additional upregulated circulating miRNAs in carotid atherosclerosis group included hsa-miR-146a [47], let-7d-5p[28], hsa-miR-200c [42], hsa-miR-135a, hsa-miR-137, hsa-miR-149, and hsa-miR-219a [48]. Higher expression of hsa-miR-127-3p [49], hsa-miR-19b, hsa-miR-22 and hsa-miR-143 [46] was found in carotid tissue from atherosclerotic patients when compared to non-atherosclerotic subjects.

Conversely, several miRNAs were consistently downregulated in carotid atherosclerosis. Two different studies reported a significative reduction in serum level of hsa-miR-126 in carotid atherosclerosis patients [43,48], which has been demonstrated to directly target cycloxigenase-2 (COX2) 3’ UTR. Significantly lower levels of hsa-miR-320b [50], hsa-miR-638 [51], hsa-miR-145 [52], hsa-miR-216b [53], hsa-miR-223, hsa-miR-101, hsa-miR-577 and hsa-miR-384 [48] were found in serum of atherosclerotic patients whit respect to healthy control. Additionally, downregulated expression levels of hsa-miR-210 [54], hsa-miR-1, hsa-miR-29b and let-7f [46] were found in carotid plaque compared to normal carotid tissue.

Several lncRNAs exhibited altered expression in carotid atherosclerosis, thus representing valid biomarkers for diagnosis. Circulating lncRNA AC078850.1 was upregulated and shown to interact with Hypoxia Inducible Factor 1 (HIF-1) transcription factor, forming a ribonucleoprotein which promotes ROS production, Nod-like receptor protein 3 (NLRP3) inflammasome activation and foam cell formation in macrophages [55]. Similarly, the lncRNA MIAT, involved in the promotion of inflammation in macrophages and plaque progression, resulted upregulated in carotid plaque than in normal control artery [56]. Another lncRNA found upregulated both in serum and in carotid plaque from atherosclerotic patients and associated with VSMCs migration and proliferation is LINC01123 [57]. Mechanistically, LINC01123 directly binds and sequesters hsa-miR-1277-5p, which is responsible for regulating the expression of transcription factor Krueppel-like factor 5 (KLF5) [57]. Consistently, authors also found that expression level of hsa-miR-1277-5p itself was downregulated in serum form affected individuals [57]. Lou et al. reported the downregulation of SENCR, alongside the reciprocal upregulation of its target hsa-miR-126a, in carotid plaque with respect to healthy arteries [58]. Similarly, downregulation of lncRNA FGF7-5 and lncRNA GLRX3, and the concomitant upregulation of its target miRNA, hsa-miR-2681-5p, was observed in serum of atherosclerotic patients compared to those from healthy volunteers [59]. Additional dysregulated lncRNAs in carotid atherosclerosis subjects resulting from this research included CCAT2 [53]and HOXC-AS1 [60].

Finally, Yan et al. identified three circRNA, specifically circ-0043621, circ-0051995 and circ-123388, upregulated in peripheral blood mononuclear cells (PBMC) from carotid atherosclerosis patients with respect to healthy subjects [40]. Among them, circ-0043621 was shown to directly bind hsa-miR-223-3p and negatively regulate its expression. Thus, authors found that hsa-miR-223-3p expression was significantly downregulated [40], corroborating earlier findings of reduced hsa-miR-223-3p levels in carotid atherosclerosis [48].

4. ncRNA and carotid plaque features

Atherosclerotic involvement of the carotid artery displays considerable histopathological and biological heterogeneity. Carotid plaques do not exhibit uniform structural or molecular characteristics, and such differences are largely governed by distinct epigenetic mechanisms, primarily mediated by ncRNAs. Table 4 provides a comprehensive overview of ncRNAs exhibiting differential expression profiles associated with specific plaque phenotypes identified through this systematic review.

The plaque core markedly differs from the adjacent proximal region. In addition to the accumulated atheromatous material, the plaque zone is enriched in macrophages compared to the adjacent region, which is predominantly composed of VSMCs [61]. Accordingly, these two distinct regions exhibit unique ncRNA expression profile. Raju and collaborators identified a group of upregulated miRNAs in plaque - namely hsa-miR-146a, hsa-miR-155, let-7a, hsa-miR-200b, hsa-miR-223 and hsa-miR-181b- with respect to the corresponding marginal zone, from macrophage-derived extracellular vesicles [61]. In contrast, Yan et al. reported a lower expression level of hsa-miR-223 in plaque relative to surrounding tissue, where its expression is modulated by circ-0043621 [40]. Indeed, the latter is found upregulated in plaque region with respect to the marginal zone, positively regulating its downstream target NLRP3, required for atherogenesis [40]. Another upregulated marker in carotid plaques compared to common carotid region is hsa-miR-148a-3p, whose function is to negatively regulate the ABCA1 expression, a key transporter involved in cholesterol efflux [41].

Carotid plaques present histological and phenotypical differences which ultimately influence clinical outcomes. One key anatomical parameter is the degree of stenosis, angiographically quantified as:

% Stenosis = (1 − A_stenosis/A_open) × 100

where A_stenosis and A_open are the cross-sectional areas of the stenosis throat and open lumen, respectively [62].

According to the North America Symptomatic Carotid Endarterectomy Trial (NASCET) criteria, stenosis is classified as mild (<30%), moderate (30–69%), or severe (70–99%), with 100% indicating complete occlusion [63]. Huang et al. reported progressive increase in hsa-miR-146a in PBMC from patients with increasing stenosis severity [47]. Another research group found that patients with advanced stenosis exhibited decreased levels of circulating hsa-miR-126-5p and -3p, hsa-miR-21-5p and -3p and hsa-miR-29a-3p, along with elevated levels of hsa-miR-33a (both -5p and -3p strands) compared to subjects with moderate carotid stenosis [64]. Stenosis progression, defined as the temporal increase in arterial narrowing due to plaque growth, is associated with heightened risk of ischemic events [65]. Dolz et al. analyzed serum miRNA profile in patients over a two-year follow-up and identified increased expression of hsa-miR-199b-3p, hsa-miR-130a-3p, and hsa-miR-24-3p in individuals exhibiting stenosis progression, suggesting their potential role as prognostic biomarkers [66].

From a clinical perspective, plaques are often classified as “stable” or “unstable,” reflecting their rupture potential. Stable plaques exhibit low rupture risk, whereas unstable (or ruptured) plaques are prone to embolization, increasing the likelihood of cerebrovascular events [67]. These phenotypes are mainly due to their biochemical composition. Histological analyses distinguish vulnerable from stable plaques based on structural features: vulnerable plaques possess large lipid-rich cores, reduced calcification, and thin fibrous caps (<200 µm), whereas stable plaques exhibit higher calcific content, denser fibrous tissue, and thicker caps (>200 µm), conferring mechanical stability [68,69].

Identifying vulnerable plaques before they become unstable is critical for preventing major cardiovascular events. Numerous miRNAs have been associated with plaque vulnerability, which may serve as potential biomarkers. Wang et collaborators examined the expression level of hsa-miR-124 in serum of patients with acute cerebral infraction, demonstrating a significantly higher expression in patients with vulnerable carotid plaque compared to the those with stable lesions [70]. Similarly, Huang et al. reported an increased PBMC expression of hsa-miR-146a in vulnerable plaque patients [47]. Yang et al. found a panel of upregulated circulating miRNAs – specifically hsa-miR-23a-5p, hsa-miR-320a, hsa-miR-2110 and hsa-miR-134-5p - in vulnerable versus stable plaque, while also noting lower expression of hsa-miR-4439 in the vulnerable group [71]. Other miRNAs downregulated in subjects with vulnerable carotid plaque include hsa-miR-532-3p [72] and hsa-miR-320b [50].

Regarding calcification, although controversial, the prevailing hypothesis suggests that highly calcified plaques are generally more stable and less symptomatic than non-calcified ones of similar size [73]. Katano et al. found that hsa-miR-4530, hsa-miR-133b and hsa-miR-1-3p were more highly expressed in low-calcified plaques, with respect to high-calcified plaques with similar degree of stenosis [74]. Vasuri et al. compared calcification subtypes and reported significantly higher expression of hsa-miR-30a-5p and hsa-miR-30d in protruding nodular calcifications versus calcific cores [75].

Furthermore, molecular profiling of carotid plaques might predict the progression of the pathology and help identify patients at high risk of cerebral events, who could benefit from earlier surgical revascularization. Numerous studies investigated the different expression levels of ncRNAs by comparing stable and unstable plaques. Reportedly, unstable plaques exhibited higher level of hsa-miR-200c [42], hsa-miR-127-3p [49] and hsa-miR-330-5p [76] with respect to stable plaque. Conversely, Eken et al. observed decreased levels of hsa-miR-210 and hsa-miR-21 in ruptured plaque when compared to stable plaque [54], while Zhu and coworkers identified lower serum level of hsa-miR-126 and hsa-miR-223 in patients with unstable carotid plaque with respect to subject with stable lesion [48]. Badacz et al. analyzed miRNA profiles in different plaque groups, based on plaque echogenicity. Plaque echogenicity is an important characteristic used to assess plaque composition and stability, which refers to the ability of an atherosclerotic plaque to reflect ultrasound waves during imaging, such as carotid or intravascular ultrasound. Patients with hyperechogenic plaque showed significantly higher serum levels of hsa-miR-134-5p, hsa-miR-34a-5p and hsa-miR-375 when compared to patients with moderately echogenic carotid plaque. Interestingly, these miRNAs resulted further reduced in hypoechogenic group compared to the moderately echogenic one, suggesting a progressive downregulation of these markers with the decrease in echogenicity [77]. Conversely, the serum level of hsa-miR-133b was higher in hypoechogenic plaque group with respect to moderately echogenic group[77]. Conversely, hsa-miR-133b levels were higher in hypoechogenic plaques. Notably, hsa-miR-16-5p was the only miRNA significantly upregulated in hyperechogenic versus hypoechogenic groups [77]. Additionally, hsa-miR-1-3p and hsa-miR-16-5p were both elevated in patients with ulcerated plaques[77].

Beyond miRNAs, other ncRNA classes—particularly long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs)—also show distinct expression patterns between stable and unstable plaques. MIAT and CCAT2 lncRNAs, previously mentioned as upregulated in carotid plaque group with respect to healthy control, were further elevated in unstable plaques [53,56]. Bao et al. identified 488 lncRNAs and 91 circRNAs differentially expressed in stable versus unstable plaques. Among them, ENST00000430222, ENST00000602895, circ-013041 and circ-025902 were upregulated in unstable group, while ENST00000631338, MSTRG18183, circ-054182 and circ-037511 were downregulated [78]. Finally, unstable plaque was characterized by higher level of circulating circRNA-0006896 [79], as well as of circ-0001523, circ-0008950 and circ-0000571, but by reduced expression of circ-0001946 and circ-0000745, compared to stable plaque [80].

5. Symptomatic vs asymptomatic patients

As previously stated, carotid plaques may remain stable and asymptomatic for extended periods or may gradually worse and evolve into unstable lesion. Unstable plaques can release atheromatous material or thrombi into the cerebral circulation, leading to clinical manifestations such as TIA, transient monocular vision loss ipsilateral to the affected artery (amaurosis fugax), or minor/non-disabling ipsilateral strokes [41]. The evaluation of ncRNAs expression profile in symptomatic versus asymptomatic patients offers valuable insights into the molecular pathways underlying symptom onset and provides potential prognostic biomarkers for cerebrovascular risk. revealed a wide array of dysregulated microRNAs (miRNAs) distinguishing symptomatic from asymptomatic individuals (Table 5).

Recently, Tanashyan et al. analyzed the expression of a total of 24 miRNAs in plasma from carotid atherosclerosis patients, reporting a significant upregulation of hsa-miR-200c-3p, hsa-miR-106b-5p and hsa-miR-494-5p in symptomatic subjects, alongside a marked reduction of hsa-miR-183-3p, hsa-miR-126-5p and hsa-miR-216-3p [81]. In a microarray-based study assessing 742 miRNAs, Eken et al. identified hsa-miR-210 as the sole miRNA significantly downregulated in symptomatic patients [54]. Mechanistically, authors proposed hsa-miR-210 as a direct negative regulator of Adenomatous Polyposis Coli (APC) protein, affecting the canonical Wnt signaling in VSMCs and promoting atherosclerosis progression. Moreover, another study documented a significative increase in hsa-miR-29c expression in carotid artery of symptomatic patients undergoing carotid endarterectomy (CEA) [24]. Interestingly, this finding was corroborated into specimens from stroke patients (see following subsection, [24]). Badacz et al. analyzed organ-specific circulating microRNAs – namely cardiac, skeletal muscle, brain, liver and pancreas related miRNAs – in serum from symptomatic and asymptomatic subjects, reporting a significative upregulation of brain-derived miRNAs in symptomatic subjects, specifically hsa-miR-124-3p and hsa-miR-134-5p, both inversely correlated with plaque stability [77]. Grosse and colleagues identified hsa-miR-92a as the only miRNA significantly increased in plasma from symptomatic compared to asymptomatic individuals [82]. Other upregulated miRNAs in symptomatic patients are hsa-miR-148a-3p and hsa-miR-33a-5p [41], with the latter also being upregulated in patients with high-grade stenosis [64], likely suggesting a possible positive correlation between hsa-miR-33a-5p expression and carotid atherosclerosis severity. Several others reported miRNAs were found to be downregulated in carotid tissue from symptomatic patients, including hsa-miR-21, hsa-miR-143 [46], hsa-miR-484, hsa-miR-942 and hsa-miR-1287 [83].

6. Association of ncRNAs with ischemic stroke

Stroke remains a massive public health problem with an increasing need for better strategies to prevent and treat disease in our aging society. Although some ischemic events are caused by reduced blood flow (hypoperfusion), the majority are attributable to embolic mechanism linked to atherosclerotic plaque rupture or acute carotid occlusion, leading to downstream thrombus migration to distal regions of the brain [84].

The expression profiles of ncRNAs can vary substantially during cerebrovascular disease. Table 6 outlines the dysregulated ncRNAs in individuals with carotid atherosclerosis-associated cerebrovascular events, emerging from this systematic research. Recently, authors investigated the expression of circulating ncRNAs in patients with carotid artery- related atherosclerotic cerebrovascular events. Particularly, a total of 225 patients were classified into low-risk, medium-high risk of cerebrovascular event groups and control group which includes healthy subjects without carotid plaque. They reported elevated hsa-miR-874 levels in the medium-high risk group, while circSCMH1—a circular RNA implicated in neural repair and known to interact with hsa-miR-874 [85,86]—was significantly downregulated in the same group [85,86].

In a separate study involving 167 stroke patients and 157 healthy subjects, researchers observed higher serum levels of hsa-miR-21 and decreased levels of hsa-miR-221 in stroke cohort [45]. Interestingly, the downregulation of hsa-miR-221 was independently confirmed by two additional studies. Specifically, Bazan et al. observed reduced levels of hsa-miR-221 and hsa-miR-222 in carotid tissues from “urgent” patients—those undergoing CEA within five days of a neurological event—compared to asymptomatic or symptomatic individuals [87]. A subsequent study by the same group demonstrated the downregulation of hsa-miR-221 in a different cohort of urgent subjects with carotid atherosclerosis, also demonstrating a negative regulation operated by circR-284 [88]. Consistently, the expression level of circR-284 resulted higher in serum from urgent subjects with carotid atherosclerosis[88]. Wang and colleagues studied the expression level of has-miR-29c in a cohort of carotid plaque specimens collected from patients with or without cerebral events, reporting an upregulation of hsa-miR-29c in the cerebral stroke group compared with the non-cerebral stroke group [24]. Lastly, Luque and collaborators identified a downregulation of hsa-miR-638 in serum from atherosclerosis stroke patients versus non- atherosclerotic controls, proposing it as a potential biomarker for primary and secondary ischemic stroke risk prediction [51].

7. Interaction network analysis

7.1. miRNA-target interaction network

Since the measurements of ncRNAs were not obtained using quantitative methods in the studies included in this systematic review, the comparison of data by meta-analyses was not performed. Therefore, to improve the interpretation of the results without changing the methodology and nature of this systematic review, a functional analysis of identified ncRNAs was performed by interaction network analysis. Uncovering the molecular mechanisms underlying carotid atherosclerosis requires the integration of regulatory and functional data. In this context, miRNA–target interaction networks represent a powerful approach to reveal how post-transcriptional regulation may influence key pathogenic pathways.

Thus, we focused specifically on the 51 circulating miRNAs associated with carotid atherosclerosis, resulting from the analysis of included articles. To prioritize miRNAs with functional relevance to carotid atherosclerosis, we matched their validated targets (via the miRWalk database) with a curated list of protein-coding genes known to be associated with carotid atherosclerosis, obtained from GeneCards and MSigDB. This intersection led to the identification of 13 miRNAs with high-confidence interactions with atherosclerosis-related genes. These miRNAs were subjected to Gene Ontology enrichment analysis and network-based visualization to explore their regulatory roles.

Five interaction networks were generated using Cytoscape, each corresponding to a distinct biological area involved in atherogenesis: inflammation, oxidative stress, vascular response, lipid metabolism and plaque formation. Each network revealed both hub miRNAs with broad targeting capabilities and more specialized miRNAs with selective pathway involvement (Figure 3).

The network focused on inflammation and immune response (Figure 3A) illustrates the complex interactions between selected miRNAs and their target genes involved in inflammatory signaling. hsa-miR-145-5p, hsa-miR-320a-5p, and hsa-miR-137-5p emerged as key regulators, showing high connectivity with pro-inflammatory targets. Notably, miR-124-3p and miR-106b-5p are linked to multiple genes involved in interleukin-10 regulation, suggesting their potential contribution to the resolution of inflammation.

The second network (Figure 3B) captures miRNA–target interactions related to oxidative stress and cellular responses to reactive oxygen species. Several miRNAs, including hsa-miR-124-3p, hsa-miR-320a-5p, and hsa-miR-145-5p, exhibit multiple connections with genes involved in antioxidant defense and redox homeostasis. For example, hsa-miR-320a-5p targets are clustered around detoxification processes related to glutathione metabolism, whereas hsa-miR-145-5p appears to regulate transcription factors activated by oxidative stimuli, highlighting their potential role in preserving redox balance within the vascular microenvironment.

Figure 3C shows miRNA-target interactions involved in endothelial function and vascular response. According to our analysis, hsa-miR-137-5p, hsa-miR-320a-5p and hsa-miR-2110 exhibit broad targeting profiles, whilst hsa-miR-126-3p and hsa-miR-21-5p are more specifically linked to genes involved in coagulation.

The fourth network (Figure 3D) reveals six miRNAs involved in key lipid-metabolism pathways. Among them, hsa-miR-4439 appears as a central node, targeting multiple genes involved in lipoprotein metabolism and cholesterol transport, suggesting its potential role in modulating plasma lipid levels and lipid deposition within the arterial wall.

Lastly, we analyzed miRNA–target interactions involved in foam cell differentiation, cholesterol accumulation, and plaque development. This network (Figure 3E) is enriched in genes related to cholesterol efflux, lipid transport, and macrophage-derived foam cell formation, with seven miRNAs participating in this regulatory scenario. Notably, hsa-miR-320a-5p stands out for its involvement across almost all identified biological processes, indicating a prominent regulatory role in atherogenesis.

7.2. lncRNA and circRNA-target interaction network

The analysis of the targets of other ncRNAs (namely lncRNAs and circRNAs) proved to be more challenging, resulting in a less comprehensive and more approximate network due to the limited availability of target annotations for these two classes of ncRNAs. Firstly, the databases used—lncTarD 2.0 for lncRNAs and circAtlas 3.0 for circRNAs—did not provide experimentally validated targets but rather computationally predicted interactions generated by specific algorithms. Secondly, many of the molecules identified through the systematic search were not found in these databases. In fact, among the 13 lncRNAs identified, targets could be retrieved for only 8, whereas among the 21 circRNAs detected, predicted targets were available for only 5 of them.

Figure 4 shows the analyzed lncRNAs together with the three most relevant circRNAs (hsa_circ_0008950, hsa_circ_0001946, and hsa_circ_0001523), which appeared to act as molecular sponges for miRNAs that were also independently retrieved through the systematic search, thereby reinforcing the consistency of the network with the bioinformatic screening. Among these, hsa-miR-4439 was a particularly prominent marker, also identified in the previous miRNA–target network analysis and implicated in all five functional areas considered. Another miRNA, hsa-miR-145-5p, was found to interact with one circRNA and two distinct lncRNAs. In the previous analysis, this miRNA was associated with processes related to inflammation and immune response, oxidative stress, endothelial and vascular regulation, as well as plaque formation and foam cell development.

Regarding lncRNAs, MIAT emerged as a central hub, showing extensive connections with numerous miRNAs (e.g., hsa-miR-133a-3p, hsa-miR-149-5p, hsa-miR-145-5p, already mentioned among the circulating miRNAs identified from the reviewed articles) and several downstream protein-coding targets, including CDH2, VEGFC, and STAT3, which are known to play roles in endothelial function, angiogenesis, and inflammatory signaling. Similarly, CCAT2, SOX2-OT, and ENST00000602895 exhibited relevant interactions with key regulatory miRNAs and downstream targets such as MTOR, AKT1, HIF1A, PIK3CA, CTNNB1, and VEGFA, suggesting their potential contribution to vascular remodeling and cell proliferation pathways.

Overall, this integrative analysis supports the hypothesis that the interplay between lncRNAs, circRNAs, and miRNAs may coordinate multiple pathogenic mechanisms underlying carotid atherosclerosis, including vascular inflammation, endothelial dysfunction, and smooth muscle cell proliferation.

Discussion

Limitations

Conclusion

List of Abbreviations

References

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