Abstract
Background/Aim: Atherosclerosis is a chronic and progressive pathological condition marked by the accumulation of lipids, fibrous materials, and inflammatory cells, within the arterial walls. MicroRNAs (miRNAs) are single-stranded, evolutionarily conserved, non-coding small RNAs, that play a pivotal role in controlling various pathophysiological cellular functions and molecular signalling cascades associated with the development of atherosclerosis. Additionally, dysregulation in cholesterol and lipid metabolism is known to increase susceptibility to atherosclerosis. In this study, we aimed to determine the changes in serum levels of miRNA-199a-5p, examine its relationship with LDL cholesterol, and investigate its diagnostic value in patients diagnosed with atherosclerosis. Materials and Methods: MiRNA-199a-5p expression analysis was conducted using PCR on serum samples from 20 patients diagnosed with atherosclerosis and 26 completely healthy, voluntary control subjects. The blood biochemical analysis values for all groups participating in the study were obtained from their records. Results: The data analysis revealed significant up-regulation of miRNA-199a-5p in the serum of the patient group. Additionally, miRNA-199a-5p expression levels positively correlated with LDL cholesterol levels. Conclusion: miRNA-199a-5p can be considered a reliable biomarker in patients with atherosclerosis, potentially informing and guiding future therapeutic approaches. Additionally, a significant relationship was found between lipid metabolism and miRNA-199a-5p in atherosclerosis.
Atherosclerosis (AS) is a process characterized by plaque accumulation in the vascular system due to disturbances in lipid metabolism and lipid oxidation, leading to cerebrovascular events, myocardial infarction, coronary artery diseases (CAD), and peripheral vascular pathologies (1). This pathological condition is recognized as a primary phase in the pathogenesis of cardiovascular disorders (2). With a multitude of established risk factors, such as hypertension, obesity, hypercholesterolemia, diabetes mellitus, and smoking disrupting endothelial homeostasis and precipitating endothelial dysfunction, the development of atherosclerosis becomes inevitable (3).
Cholesterol, a crucial substance for organisms, can damage cells and form the molecular basis for many diseases, including atherosclerosis, when it accumulates excessively. Therefore, to maintain cholesterol homeostasis, especially in peripheral cells, the efflux of cholesterol is essential for eliminating excess cholesterol from these cells (4).
Cholesterol is carried by lipoproteins, which facilitate both the distribution of cholesterol to cells and tissues via low-density lipoproteins (LDL) and the removal of cholesterol from cells and tissues via high-density lipoproteins (HDL) (5, 6). However, in the transportation of cholesterol, LDLs are considered the classical antagonists of the circulatory system due to their tendency to bind to the connective tissue in the sub-intimal layers of arteries (7). Particularly, modified LDL-cholesterol (LDL-C) accumulating in arterial walls increases individual susceptibility to atherosclerosis and its complications, which are leading contributors to morbidity and mortality in cardiovascular diseases (CVD) (8). It is known that imbalances, such as elevated LDL-C levels and reduced HDL-C levels, contribute to cellular cholesterol accumulation, thereby promoting the development of atherosclerosis (6).
Thus, the primary initiating event in AS involves the activation of endothelial cells (ECs) due to excessive retention of LDL-C in the subendothelial matrix, leading to the recruitment of monocytes to the area to clear the accumulated cholesterol (8). Clinical and experimental studies have provided definitive evidence of an etiological role between cholesterol accumulation and inflammation (9). Ongoing inflammatory and hemodynamic assaults on the atherosclerotic lesion can eventually lead to local endothelial dysfunction or compromise, triggering thrombus formation, which can result in myocardial infarction and ischemic stroke. However, the disease may remain asymptomatic and undetectable until thrombus formation occurs (10). Thus, it is essential to diagnose AS early and explore new ways to better understand how atherosclerosis develops. It is equally important to identify reliable biomarkers for diagnosis and to create targeted treatments for each stage of the disease, as this will be key in preventing atherosclerosis (11). In this context, regulatory microRNAs (miRNAs/miRs) have garnered special interest as key components that modulate various pathomechanisms involved in the development of atherosclerotic plaque.
MiRNAs are non-coding, single-stranded RNAs, composed of 19-24 nucleotides, capable of modulating a significant portion of the genome post-transcriptionally (12). Evidence indicates that cellular processes, including cell death, migration, proliferation, invasion, and differentiation are controlled by miRNAs (13). In studies conducted for this purpose, it has been reported that the cardiovascular system exhibits a high degree of sensitivity to alterations in miRNA levels, and that the dysregulation of miRNA expression in tissues is directly associated with CVD (14).
Research has demonstrated that miRNAs play a pivotal role in regulating plasma LDL-C levels through their modulation of critical metabolic pathways, including very low-density lipoprotein (VLDL) secretion, cholesterol biosynthesis, and LDL-receptor (LDLR) activity. They may also serve as reliable and specific markers, particularly for early diagnosis (13). Additionally, genome-wide association studies (GWAS) meta-analysis data suggest that variations in miRNA levels can lead to irregular blood lipid levels, potentially increasing the risk of cardiometabolic diseases in individuals (15).
MiRNA-199a, known as a primary regulator of hypoxia-induced pathways, is a cluster of miRNAs whose precursor gene is located on chromosomes 1 and 19. It is processed into two forms, miR-199a-5p and miR-199a-3p (16, 17). Among these mature forms, miRNA-199a-5p has garnered increased attention in relation to CVH (18). Research has demonstrated that this miRNA is sensitive to low oxygen levels, involved in endothelial angiogenesis and integrity in various pathological conditions, and plays a critical role in regulating various metabolic diseases by influencing key genes in atherosclerosis (13, 19).
The modulation of cholesterol and lipoprotein metabolism is crucial in the pathogenesis of atherosclerosis. The role of Silent Information Regulator 1 (SIRT1) in regulating cholesterol and lipoprotein metabolism, fundamental to the pathogenesis of atherosclerosis, has been confirmed as a target gene of miRNA-199a-5p (20, 21). Additionally, it has been suggested that SIRT1 may alleviate apoptosis and endothelial cell dysfunction in atherosclerosis (Figure 1) (4, 22).
The impact of miRNA-199a-5p on SIRT1 (a member of the sirtuin protein family) and the potential interaction mechanisms of SIRT1 with key genes involved in atherosclerosis and lipid metabolism.
As a major health concern, atherosclerosis is predominantly diagnosed using active screening methodologies (1, 9). Given the critical nature of preventing severe cardiovascular events, there is an imperative need for the development and implementation of more sophisticated diagnostic approaches. At this juncture, miRNAs are increasingly recognized as pivotal biomarkers for the early diagnosis of CVDs due to their critical regulatory functions in the biological pathways implicated in CVD pathogenesis (11).
This study is designed considering the limited research on miRNA-199a-5p expression levels in patients diagnosed with atherosclerosis. To our knowledge, there are no studies in the literature specifically addressing the relationship between miRNA-199a-5p, and blood lipid levels in atherosclerosis. We also believe that the use of miRNA-based tests can enable more accurate diagnosis and monitoring in the early stages of the disease, facilitating the timely implementation of treatment strategies. Therefore, our study also aimed to provide insights for future investigations into the potential use of miRNA-199a-5p, which is implicated in the pathophysiology of atherosclerosis, as a therapeutic agent or biomarker.
Materials and Methods
Study population. This present case-control investigation encompassed (n=20) patients who underwent detailed clinical examinations at the Yeditepe University Hospital Cardiovascular Clinic. The study protocol has been approved (Approval No: 2024-11-Hitit University) and conducted in accordance with the ethical principles of the Declaration of Helsinki.
The number of patients was determined by power analysis. All patients were over 18 years of age and had pathologically confirmed atherosclerosis. The exclusion criteria were any systemic, chronic or autoimmune diseases, cancer, and acute or chronic infections. The control cohort comprised 26 serum specimens, each sourced from thoroughly screened, healthy volunteers. All participants were informed, and provided their signatures on consent forms. Patient demographic and clinical profiles were obtained from their medical records.
MiRNA isolation. Serum specimens were isolated through centrifugation (4,000 rpm/1,520 g force for 15 min) from the peripheral blood samples collected from both patients and control subjects. Once centrifugation was complete, the collected serum samples were transferred to sterilized tubes and immediately frozen at −80°C until further miRNA experiments could be conducted. The miRNAs were isolated from serum specimens, in adherence to the guidelines provided by the manufacturer of the kit, using the miReasy Kit (Qiagen, Hilden, Germany), and to assess the purity and concentration of the isolated miRNAs, a NanoDrop2000 (Thermo Scientific, Waltham, MA, USA) was used.
cDNA synthesis. cDNA synthesis was performed to generate first-strand cDNA using the miRCURY LNA RT Kit (Qiagen) following the manufacturer’s instructions. Qubit miRNA Assay Kit on the Qubit 3.0 Fluorometer (Thermo Scientific) was used to determine cDNA levels.
MiRNA expression analysis. After quantifying the sample concentrations, appropriate dilutions were performed, and expression levels of microRNA 199a-5p (miRCURY 199a-5p, Qiagen) were determined by PCR in Rotor-Gene (Rotor-Gene Q-Qiagen) using the miRCURY LNA SYBR Green PCR Kit (Qiagen Science, Germantown, MD, USA). The housekeeping assay RNU6-(Qiagen) was used as an internal control.
Statistical analysis. SAS (Statistical Analysis System) v9.4 software (SAS Institute, Cary, NC, USA) program was utilized for statistical analysis. The Chi-square test and the Student’s t-test were employed for the analysis of independent groups. The Kolmogorov-Smirnov test was conducted to evaluate whether the dependent and independent variables in the study followed a normal distribution. Pearson correlation analysis was performed to reveal the association between quantitative parameters. ROC analysis was used to evaluate the sensitivity and specificity of deltaCT. Throughout the research, a threshold for statistical significance was determined at p<0.05.
Results
Demographic profile of the study population. Demographic characteristics, including sex, smoking status, age, and weight, were analyzed comparatively between the patient and control cohorts (Table I). The differences between the groups did not reach statistical significance (p>0.05).
Demographic characteristics of the subjects.
Analysis of miRNA-199a-5p expression levels. A discernible statistical significance was identified in the expression levels of miRNA-199a-5p between the groups (Table II). Statistically significant elevated levels of expression were observed in the patient group compared to the control group.
Expression levels of miRNA-199a-5p in patient and control groups.
ROC analysis. In our study, sensitivity and specificity calculations for various cutoff values of deltaCT in predicting disease status were obtained and the optimum cutoff value of deltaCT is presented along with the sensitivity and specificity values in Table III. Furthermore, an empirical ROC curve corresponding to these findings was generated (Figure 2) using a nonparametric method via SAS software, yielding an AUC of 0.85 with a 95% confidence interval of 0.7342-0.9658 and a p-value less than 0.001. This curve, alongside the associated AUC, indicates that deltaCT possesses predictive capability in distinguishing patients with disease from normal subjects.
ROC curve; sensitivity and specificity calculations for various cutoff values of ΔCt.
This curve, alongside the associated area under the curve, indicates that deltaCT possesses predictive capability in distinguishing patients with disease from normal subjects.
Correlation analysis. There were positive and moderate statistically significant relationships between the dependent variable ΔCT and the independent variables cholesterol and LDL (respectively, r=0.3465, p=0.0183; r=0.31945, p=0.0305). However, the relationships between ΔCT and other variables were weak and not statistically significant. When examining the correlations among the independent variables, the highest correlation was found between Cholesterol and LDL (r=0.88146, p=0.001) (Table IV).
The Pearson correlation analysis results for quantitative variables used in the study.
Multivariate regression analysis. The parameter estimates obtained via multivariate stepwise regression are presented in Table V, showing that all independent variables significantly impact the dependent variable ΔCT (p<0.05). The model’s intercept is 3.24397, indicating that ΔCT will equal 3.24397 when all independent variables are zero.
Results of the multivariate stepwise selection method.
Based on the VIF and Tolerance values in the table, it can be concluded that the model does not exhibit multicollinearity.
According to the results, the variable with the greatest impact on ΔCT is Case. The independent variables in the multivariate regression model explain 39.75% of the total variance. The Case variable has the highest partial importance, with a partial R2 of 33.95%, followed by the Cholesterol variable with a partial R2 of 5.8%.
To obtain valid and accurate results from the multivariate linear regression model derived in the final step using the multivariate stepwise regression method, certain diagnostic plots are utilized to assess the model’s adequacy. The plots generated for this purpose are presented in Figure 3 and Figure 4.
The symmetry of the histogram of residuals in the distribution of the model’s standardized residuals indicates that the assumption of normality is satisfied and that the obtained model fits the data well.
Upon examining the plot of standardized residuals against leverage values for each observation, it is evident that there are no significant numbers of outliers or influential observations.
The histogram in Figure 3 assesses the normality of the model’s standardized residuals, a critical assumption in regression analysis. A symmetric distribution of residuals supports the fulfillment of the normality assumption, indicating that the model provides a good fit to the data.
The plot in Figure 4 allows for the investigation of outliers by displaying standardized residuals against leverage values for each observation. Standardized residuals exceeding ±2 is typically considered outliers. The leverage statistic identifies influential observations. Upon examining this plot, it is evident that there are no significant numbers of outliers or highly influential observations.
Discussion
Atherosclerosis represents the predominant variant of CVD’s and primarily involves the build-up of lipids and the inflammation of significant arterial vessels, which may culminate in clinical events like myocardial infarction (MI) and strokes (23).
Among a wide array of environmental and hereditary elements implicated in atherogenesis, elevated levels of LDL-cholesterol are deemed adequate to propel the advancement of this condition. Consequently, pathways governing plasma LDL-C levels and their inhibitors have been thoroughly investigated for conversion into effective treatments (24). It has been indicated that the risk of CVD because of atherosclerosis depends on cumulative exposure to LDL-C over time and independently on the area of accumulation. Accumulation of the same area at a younger age has resulted in a greater increase in risk compared to older ages, emphasizing the importance of optimal LDL-C control from early life (25).
Given that CVDs are primary contributors to the global disease burden and death rates, there is a medical imperative to identify new diagnostic biomarkers and develop therapeutic strategies to reduce the prevalence of cardiovascular disease (1, 10). Emerging research suggests that miRNAs hold promise as innovative biomarkers, offering considerable sensitivity for the early detection and contemporary management of CVDs. In addition to their presence in tissues, they are found in highly stable forms in body fluids (14). It has been reported that circulating miRNA expressions change in conditions, such as acute coronary syndrome (ACS), acute myocardial infarction (AMI), essential hypertension, atherosclerosis, and stroke (26).
Studies have shown that miRNA-199a regulates LDLR expression by directly targeting its 3′UTR, and in vivo silencing of miR-199a through antagomir leads to the restoration of genes involved in fatty acid metabolism (15, 27). Additionally, it has been demonstrated that miRNA-199a is found at elevated levels in various cell types during metabolic diseases, and these levels can provoke in vivo cardiac hypertrophy by suppressing autophagy in cardiomyocytes (2, 28).
El Azzouzi et al. (27), have demonstrated an increase in miR-199a expression in various heart failure models. They confirmed that miR-199a is up-regulated in the hearts of mice subjected to excessive pressure due to transverse aortic constriction (TAC) (27). Additionally, the literature reveals that miR-199a may serve as a prognostic indicator for the manifestation of cardiovascular incidents in individuals diagnosed with Stable Coronary Artery Disease (29). D’Alessandra and colleagues, in their study of circulating miRNA expression profiles in the plasma of CAD patients, demonstrated that miR-199a is up-regulated in patients with both unstable and stable angina (30).
MiRNA-199a-5p is one of the most abundantly expressed microRNAs in the liver (4). The over-expression of miRNA-199-5p promotes hepatic lipid accumulation via the regulation of lipid synthesis and breakdown, while the inhibition of miR-199-5p significantly improves hepatic lipid accumulation and steatosis (31). Additionally, SIRT1 reduces plasma LDL by decreasing LDLR expression and activity in hepatocytes, thereby preventing and improving atherosclerosis. Notably, over-expression of miR-199a reduces endogenous SIRT1 by 50%, while knockdown of miR-199a increases its expression by 2.2-fold, highlighting the regulatory role of miR-199a on SIRT1 and its potential impact on lipid metabolism and atherosclerosis (32, 33).
It has been revealed that miRNA-199a-5p significantly increases in AMI, and its elevated levels hinder cell survival (28). Indeed, prior studies have demonstrated that suppressing miR-199a-5p has a protective effect on the cardiovascular system (4).
MiRNA-199a-5p, characterized as a proatherogenic miRNA due to its high expression positively associated with the development of AS, especially endothelial damage, has been indicated to exacerbate phenotypic abnormalities caused by ox-LDL, including proliferation, migration, and tube formation in endothelial cells (19). Tian et al. have shown that the concentration of miR-199a-5p in circulating blood obtained from individuals diagnosed with primary hypertension, a risk factor for atherosclerosis, is significantly higher than that in healthy individuals. Their study demonstrated that miR-199a-5p intensifies vascular endothelial impairment through the suppression of autophagy and the enhancement of apoptosis (17).
According to the results of our study, consistent with other research, we observed that miRNA-199a-5p expression levels were markedly higher in the cohort of individuals diagnosed with atherosclerosis compared to the control group, and we identified a positive correlation between miRNA-199a-5p and the high LDL levels in the patients.
Conclusion
According to our literature review, our study is the first to concurrently examine the relationship between miRNA-199a-5p and LDL in atherosclerosis to date. Considering the current increase in morbidity and mortality associated with atherosclerosis, there is a need for further research to elucidate the complex genetic pathways that contribute to the progression of this disease. Despite the limited size of our sample cohort, our study has identified miRNA-199a-5p as a promising candidate for use as a biomarker in atherosclerosis. Additionally, it may provide a foundation for future studies in this field to achieve a deeper understanding of the relationship between this miRNA and lipid metabolism through SIRT1 in AS. Our findings align with previous studies but underscore the necessity of conducting similar tests on a larger patient cohort to definitively establish miRNA-199a-5p as a reliable biomarker for atherosclerosis and to ascertain its potential as a diagnostic biomarker and therapeutic target in innovative treatment strategies.
Acknowledgements
The Authors would like to extend their sincere gratitude to their esteemed mentor, Professor Dr. Turgay Isbir, for his unwavering support.
Footnotes
Authors’ Contributions
All Authors contributed significantly to this study. The conceptualization of the study was carried out by A.T.C. and Z.B. The formal analysis and investigation were undertaken by F.T.A., Z.B., and O.A. while, F.T.A. and O.A. handled the methodology. Data curation was provided by A.T.C. Project management, visualization, and the original draft were prepared by Z.B. Finally, A.T.C., F.T.A., and O.A. collaborated in reviewing, and editing the manuscript.
Funding
The Authors declare that they received no funding for this study.
Conflicts of Interest
All Authors declare no conflicts of interest in relation to this study.
- Received July 29, 2024.
- Revision received August 9, 2024.
- Accepted August 19, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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