Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance, hyperglycemia, and progressive β-cell dysfunction. Atherosclerosis, a major macrovascular complication of T2DM, remains a leading cause of cardiovascular morbidity and mortality in diabetic populations worldwide (1). Subclinical atherosclerosis (SA), which refers to the early, asymptomatic stages of arterial wall thickening and plaque development, frequently precedes overt cardiovascular events (2). The early identification of SA is crucial for initiating preventive strategies and improving long-term outcomes in high-risk individuals.

Carotid intima-media thickness (CIMT) is a validated surrogate marker for early atherosclerotic changes and is widely used in clinical research to assess subclinical vascular damage (3). However, the requirement for imaging equipment and trained personnel limits its routine use in primary care settings. Hence, there is a growing interest in identifying accessible, non-invasive molecular biomarkers that can reliably predict early atherosclerotic changes, particularly in asymptomatic diabetic individuals.

MicroRNAs (miRNAs) are small, endogenous, non-coding RNA molecules that regulate gene expression post-transcriptionally. They have been implicated in various biological processes, including endothelial function, inflammation, lipid metabolism, and vascular remodeling—all of which are central to atherogenesis (4,5). Circulating miRNAs are stable in plasma and can be detected using sensitive molecular techniques, making them attractive candidates as diagnostic biomarkers (6). In particular, miR-126 and miR-146a have been shown to play protective roles in endothelial homeostasis, while miR-21 is often associated with pro-inflammatory pathways and vascular injury (7–9).

Emerging evidence suggests that specific miRNA signatures may differ between individuals with and without subclinical vascular disease, especially in the context of diabetes (10). However, there is limited data evaluating the utility of these biomarkers in predicting SA among T2DM patients in routine clinical practice. This study aims to evaluate the diagnostic potential of selected circulating miRNAs—miR-126, miR-146a, and miR-21—in identifying subclinical atherosclerosis among individuals with T2DM, using CIMT as a comparative reference.

Study Design and Participants

A total of 90 individuals aged 40 to 65 years were recruited and divided into three equal groups: Group A included 30 patients with T2DM and subclinical atherosclerosis; Group B included 30 patients with T2DM but without subclinical atherosclerosis; and Group C comprised 30 age- and sex-matched healthy controls. Patients with a history of cardiovascular disease, active infection, malignancy, autoimmune disorders, or chronic kidney disease were excluded from the study. The diagnosis of T2DM was based on American Diabetes Association (ADA) criteria.

Assessment of Subclinical Atherosclerosis

Subclinical atherosclerosis was evaluated by measuring carotid intima-media thickness (CIMT) using high-resolution B-mode ultrasonography (7.5 MHz linear probe). Measurements were taken at three points on the common carotid artery, 1 cm proximal to the bifurcation, on both sides. A CIMT value >0.75 mm was considered indicative of subclinical atherosclerosis.

Blood Collection and RNA Isolation

Fasting venous blood samples (5 mL) were collected in EDTA tubes. Plasma was separated by centrifugation at 3000 rpm for 10 minutes and stored at -80°C until further analysis. Total RNA, including small RNA fractions, was isolated using a commercial miRNA extraction kit according to the manufacturer’s protocol. RNA concentration and purity were assessed using a NanoDrop spectrophotometer.

Quantification of MicroRNAs

The expression levels of miR-126, miR-146a, and miR-21 were quantified using real-time reverse transcription polymerase chain reaction (RT-qPCR). cDNA synthesis was performed using specific stem-loop primers. RT-qPCR was conducted using SYBR Green Master Mix on a StepOnePlus™ Real-Time PCR System. U6 small nuclear RNA was used as an internal control. The relative expression levels were calculated using the 2^-ΔΔCt method.

Statistical Analysis

Data were analyzed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for group comparisons. Receiver Operating Characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of miRNAs. A p-value < 0.05 was considered statistically significant.

A total of 90 participants were included in the study, evenly distributed across three groups: Group A (T2DM with subclinical atherosclerosis), Group B (T2DM without subclinical atherosclerosis), and Group C (healthy controls). The mean age of participants was 54.3 ± 6.1 years, with no statistically significant difference between groups (p = 0.45). The gender distribution was also comparable (p = 0.68).

Carotid Intima-Media Thickness (CIMT)

The mean CIMT was significantly higher in Group A (0.87 ± 0.09 mm) compared to Group B (0.63 ± 0.05 mm) and Group C (0.58 ± 0.06 mm) (p < 0.001). This confirms the presence of subclinical atherosclerosis in Group A (Table 1).

Expression of Circulating MicroRNAs

Expression levels of miR-126 and miR-146a were significantly downregulated in Group A compared to the other two groups, while miR-21 was markedly upregulated. The relative fold changes (mean ± SD) for miR-126 were 1.9 ± 0.6 (Group A), 3.5 ± 0.8 (Group B), and 4.1 ± 0.9 (Group C). For miR-146a, the values were 2.2 ± 0.7, 3.9 ± 1.0, and 4.3 ± 0.6 respectively. Conversely, miR-21 expression was highest in Group A (5.6 ± 1.2), followed by Group B (3.4 ± 0.9) and Group C (2.1 ± 0.5) (Table 2). All intergroup differences were statistically significant (p < 0.001).

Diagnostic Performance

Receiver Operating Characteristic (ROC) analysis showed that miR-126 had the highest diagnostic accuracy for identifying subclinical atherosclerosis, with an area under the curve (AUC) of 0.89. miR-146a and miR-21 also showed good diagnostic values, with AUCs of 0.85 and 0.82, respectively (Table 3).

Table 1. Comparison of Carotid Intima-Media Thickness (CIMT) Among Groups

*Statistically significant difference (p < 0.05)

CIMT: Carotid Intima-Media Thickness; SA: Subclinical Atherosclerosis

Table 2. Relative Expression Levels of Circulating miRNAs in Study Groups

*Statistically significant difference (p < 0.05)

Values expressed as mean ± standard deviation

Table 3. ROC Curve Analysis of miRNAs for Diagnosing Subclinical Atherosclerosis

AUC: Area Under Curve; CI: Confidence Interval

This study explored the diagnostic utility of circulating microRNAs—miR-126, miR-146a, and miR-21—in detecting subclinical atherosclerosis (SA) among patients with Type 2 Diabetes Mellitus (T2DM). The findings demonstrate that T2DM patients with early atherosclerotic changes exhibit a distinct miRNA expression profile, marked by significant downregulation of miR-126 and miR-146a and upregulation of miR-21. These results are consistent with previous reports linking miRNAs to vascular inflammation, endothelial dysfunction, and atherogenesis in metabolic disorders (1–3).

Subclinical atherosclerosis often remains undetected in diabetic individuals due to its asymptomatic nature, but it significantly elevates the risk of future cardiovascular events (4). CIMT, used in this study as a gold-standard imaging technique for detecting SA, has been previously validated as a surrogate marker for cardiovascular risk assessment in diabetic populations (5,6). However, the need for skilled personnel and equipment limits its applicability in resource-constrained settings, highlighting the importance of identifying circulating biomarkers such as miRNAs for early detection.

miR-126 plays a pivotal role in maintaining endothelial integrity and promoting vascular repair by targeting negative regulators of the PI3K/Akt signaling pathway (7,8). Its marked reduction in T2DM patients with SA, as observed in this study, aligns with evidence suggesting that miR-126 downregulation is associated with endothelial dysfunction and impaired angiogenesis (9,10). Similarly, miR-146a is known to act as a negative regulator of pro-inflammatory pathways via inhibition of NF-κB signaling, and its reduced expression has been correlated with increased vascular inflammation in diabetic atherosclerosis (11,12).

On the contrary, miR-21 has been widely recognized for its role in promoting smooth muscle cell proliferation and inflammatory cytokine production, both of which are critical in atheromatous plaque development (13). Its elevated levels in the SA group support previous findings that associate miR-21 with vascular remodeling and metabolic stress responses in diabetic individuals (14,15).

The ROC curve analysis further substantiates the clinical utility of these miRNAs, with miR-126 showing the highest diagnostic accuracy (AUC = 0.89), followed closely by miR-146a and miR-21. These values are comparable to earlier studies assessing miRNA-based diagnostics for cardiovascular risk stratification in T2DM (8,10). Importantly, the use of a panel of miRNAs rather than a single marker may enhance diagnostic sensitivity and specificity, as each miRNA reflects a different facet of the atherosclerotic process—endothelial damage, inflammation, and vascular remodeling.

Nevertheless, this study has some limitations. The sample size was modest, and the cross-sectional design limits causal inference. Furthermore, while miRNAs were quantified using RT-qPCR, future studies should consider validating these findings using high-throughput sequencing technologies to uncover additional candidate markers. Longitudinal studies are also warranted to assess the prognostic value of miRNA signatures over time and their potential to guide therapeutic interventions.

This study highlights the potential of circulating microRNAs—miR-126, miR-146a, and miR-21—as non-invasive biomarkers for early detection of subclinical atherosclerosis in patients with Type 2 Diabetes Mellitus. Their distinct expression patterns correlate significantly with carotid intima-media thickness, underscoring their diagnostic value. Incorporating miRNA profiling into routine screening could aid in timely risk stratification and intervention to prevent cardiovascular complications in diabetic individuals.

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