|Home | About | Journals | Submit | Contact Us | Français|
Aim: To explore the relationship between lipometabolism-related microRNAs (miRNAs) in peripheral blood mononuclear cells (PBMCs) and the presence of coronary artery disease (CAD).
Methods: In the present study, 161 stable CAD patients and 149 health controls were enrolled. The expression levels of seven miRNAs (miR-21, miR-24, miR-29a, miR-33a, miR-34a, miR-103a, and miR-122) in PBMCs were qualified by quantitative real-time polymerase chain reaction (qRT-PCR). The miRNA markers that showed significant difference between the two groups were used for further analysis. The risk of miRNA contributing to the presence of CAD was estimated by univariate and multivariate logistic regression models. The area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy.
Results: The expression levels of miR-24, miR-33a, miR-103a, and miR-122 in PBMCs were significantly increased in CAD patients compared with controls and were significantly correlated with blood lipids in both CAD patients and controls. The increased levels of miR-24 (adjusted OR = 1.32, 95% CI 1.07–1.62, P = 0.009), miR-33a (adjusted OR = 1.57, 95% CI 1.35–1.81, P < 0.001), miR-103a (adjusted OR = 1.01, 95% CI 1.01–1.02, P < 0.001), and miR-122 (adjusted OR = 1.03, 95% CI 1.01–1.04, P < 0.001) were associated with risk of CAD. We identified a miRNA panel (miR-24, miR-33, miR-103a, and miR-122) that provided a high diagnostic accuracy of CAD (AUC= 0.911, 95% CI 0.880–0.942).
Conclusion: The increased expression levels of miR-24, miR-33a, miR-103a, and miR-122 in PBMCs are associated with risk of CAD. A panel of the four miRNAs has considerable clinical value in diagnosing stable CAD.
Coronary artery disease (CAD) is currently a major cause of mortality and morbidity in both men and women in many countries, including China1). Atherosclerosis, which is the pathogenetic basis of CAD, is characterized by accumulation of lipid in the arterial wall resulting in narrowing of the vessel lumen2). Disorders of lipid homeostasis are common risk factors for cardiovascular disease, and dyslipidemia has been confirmed to play an important role in the development and progression of CAD3). Reductions in the levels of atherogenic lipoproteins such as low-density lipoprotein cholesterol (LDLC) are associated with substantially decreased risk for CAD4).
MicroRNAs (miRNAs) are a class of highly conserved, noncoding small RNAs that usually comprise 19–24 nucleotides. These RNAs play a vital role in the regulation of gene expression on the post-transcriptional level by inhibiting the translation of protein from mRNA or by inducing mRNA degradation5). In recent years, accumulating data from in vivo and in vitro studies have identified that several miRNAs, including miR-33, miR-24, miR-34a, miR-103a, and miR-122, play crucial roles in regulating lipid metabolism. Several key genes involved in cholesterol synthesis/transport and fatty acid metabolism were found to be regulated by miR-33. Experiments performed in both normal and high-fat diet-fed mice demonstrated that inhibition of miR-33 results in an increase in circulating HDLC6). Ng R et al. report that the expression of miR-24 is significantly increased in the livers of high-fat diet-treated mice and in isolated human hepatocytes incubated with fatty acid. Knockdown of miR-24 in these mice resulted in impaired hepatic lipid accumulation and reduced plasma triglycerides7). MiR-34a is considered to be involved in nonalcoholic fatty liver disease (NAFLD). MiR-34a inhibits very low-density lipoprotein secretion and promotes liver steatosis and hypolipidemia in a hepatocyte nuclear factor 4α (HNF4α)-dependent manner8). Trajkovski et al. demonstrated that miR-103 silencing improved insulin sensitivity in adipose tissue of mice and demonstrated an association between miR-103 and body weight. The results suggest that miR-103 could increase lipolysis and elevate circulating free fatty acid levels by repression of caveolin-19). Another miRNA, miR-122, was also found to have prominent effects on lipid metabolism10).
To date, circulating miRNAs have been considered as promising novel biomarkers for cardiovascular diseases, particularly CAD11). Circulating monocytes are important cells that participate in practically all stages of atherogenesis12). Various cross-sectional and interventional studies in different patient cohorts demonstrated monocyte subset-specific interactions with different lipoprotein subclasses13). Considering the crucial role of hyperlipidemia and circulating monocytes in the development of CAD, dysregulation of lipometabolism-related miRNAs in peripheral blood mononuclear cells (PBMCs) may also be related to the presence of CAD. In addition, PBMCs circulating throughout the body maintain close contact with various body organs and may be considered as an appropriate source for disease related miRNA expression signature14). Prior studies demonstrated that plasma/serum levels of some miRNAs increase or decrease in CAD patients; however, expression level of lipometabolism-related miRNAs in PBMCs have not been evaluated.
In this study, a panel of seven miRNAs (miR-21, miR-24, miR-29a, miR-33a, miR-34a, miR-103a, and miR-122) which have been report to be associated with lipid metabolism both in patients and animal models15, 16) was selected. To explore the clinical value including risk and diagnostic accuracy for the presence of CAD, the expression level of the seven lipometabolism-related miRNAs in PBMCs were measured in CAD patients and healthy controls. The study focused on these miRNAs because they have been report to be associated with lipid metabolism both in patients and animal models.
A total of 161 stable CAD patients (40–75-year old) were consecutively recruited from outpatients who admitted to the Xuanwu Hospital, Capital Medical during March–July 2015. Diagnosis of CAD was confirmed by coronary angiography17). Coronary angiograms were evaluated independently by two operators, who made visual estimation of luminal narrowing in multiple segments based on a modified form of the AHA/ACC classification of the coronary tree. Using these data, significant CAD was defined as at least one major epicardial vessel with > 50% stenosis, assessed by quantitative coronary angiography. Depending on the extent of epicardial coronary artery involvement, CAD patients were divided into single-, double-, and triple-vessel disease subgroups in accordance with the Coronary Artery Surgery Study classification. The exclusion criteria for CAD patients were as follows: previous history of acute myocardial infarction (AMI), elevated cardiac troponin I (cTnI) or creatine kinase (CK-MB) levels, impaired left ventricular ejection fraction (LVEF) (≤ 45%), cardiac arrhythmias and congestive heart failure. We simultaneously recruited 149 health control subjects without any evidence of CAD or dyslipidemia from those who take routine physical examination in the hospital. Cases and controls were frequency matched according to age (± 3 years), gender, smoking, and drinking. All individuals had no cardiomyopathy or congenital heart, severe hepatic and renal dysfunction, inflammatory and malignant disease, bleeding disorders, and any major operation within the previous month. Subjects with a history of using lipid lowering (statin agents) or antidiabetic and antihypertensive drugs were also excluded. This study was approved by the university ethical committees (approval number: 2015SY27) and informed consent was obtained from each participant.
A structured questionnaire was used to collect information on demographic data, environmental exposure and medical histories. Current cigarette smokers were defined as those who smoked ≥ 1 cigarette/day. Alcohol use was defined as intake of wine/beer/cider/spirits ≥ 1 time per week. Physical activity was defined as walking or riding 15 min/day, or doing sports or physical exercise > 2 h/week, or lifting or carrying heavy objects at work daily18).
Following an overnight fast, venous blood samples were collected in EDTA tubes (Sarstedt, Nümbrecht, Germany) from each subject. Blood samples were immediately centrifuged and plasma was preserved at −80°C. PBMCs were subsequently isolated by Ficoll-Hypaque density gradient centrifugation. Immediately after, 5–8 × 106 cells per ml of Trizol (Invitrogen, USA) were stored at −80°C until use19).
Total cholesterol (TC), triglycerides (TG), and high-density lipoprotein-cholesterol (HDLC) were measured using standard laboratory methods (Hitachi autoanalyzer 7060; Hitachi, Japan). Low-density lipoprotein-cholesterol (LDLC) was calculated using the Friedewald method. Fasting serum glucose levels were measured by the glucose oxidase method. Glycated hemoglobin (HbAlc) was estimated by high-pressure liquid chromatography method (Tosoh Corporation, Tokyo, Japan).
Total RNA was extracted from PBMCs using Trizol according to the manufacturer's specifications. The yield of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and the integrity was evaluated using agarose gel electrophoresis stained with ethidium bromide. RNA electrophoresis and the A260/280 ratio (ranging from 1.80 to 2.22) indicated that both integrity and purity of the isolated RNA samples were acceptable.
Quantification was performed with a two-step reaction process: reverse transcription (RT) and PCR. Each RT reaction consisted of 1 µg RNA, 4 µl of miScript HiSpec Buffer, 2 µl of Nucleics Mix and 2 µl of miScript Reverse Transcriptase Mix (Qiagen, Germany), in a total volume of 20 µl. Reactions were performed in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 60 min at 37°C, followed by heat inactivation of RT for 5 min at 95 °C. The 20 µl RT reaction mix was then diluted 5 × in nuclease-free water and held at −20°C.
Real-time PCR was performed using LightCycler® 480 II Real-time PCR Instrument (Roche, Swiss) with 10 µl PCR reaction mixture that included 1 µl of cDNA, 5 µl of 2×LightCycler® 480 SYBR Green I Master (Roche, Swiss), 0.2 µl of universal primer (Qiagen, Germany), 0.2 µl of miRNA-specific primer and 3.6 µl of nuclease-free water. Reactions were incubated in a 384-well optical plate (Roche, Swiss) at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. The miRNA-specific primer sequences (Supplementary Table 1) were designed in the laboratory and synthesized by Generay Biotech (Generay, PRC) based on the miRNA sequ ences obtained from the miRBase database (Release 20.0).
All samples were performed in triplicate and the mean values of the triplicates were taken as the final result. The expression levels of miRNAs were normalized to RNU6B and were calculated using the 2−ΔCt method [ΔCt = mean Ct (miRNA of interest) − mean Ct (U6)].
The differences of demographic and clinical parameters between the CAD cases and healthy controls were evaluated using Student's t test or χ2 test. Logarithmic transformations were made if data were not normally distributed. Those miRNA markers that showed significant difference between the two groups were used for further analysis. For comparison of more than two groups, one-way ANOVA and LSD (least significant difference) tests were used as appropriate. The strength and direction of the relationship between miRNA markers and blood lipids were analyzed by the Spearman's correlation analysis. The risk of miRNAs contributed to the presence of CAD were estimated by computing odds ratios (ORs) and their 95% confidence intervals (95% CIs) from both univariate and multivariate logistic regression models, in which each miRNA expression level itself was used as a variable factor with or without adjustment for covariates. Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic accuracy of each miRNA. The area under the ROC curve (AUC) was used as diagnostic index. The diagnostic performance of the selected miRNA panel was further evaluated using the predicted probability of being diagnosed with CAD as a surrogate marker to construct ROC curve. A P value of less than 0.05 was considered statistical significance. The reported P values were two-tailed in all calculations. All statistical analyses were performed using SPSS 20.0 (IBM SPSS, Inc., Chicago, USA).
The demographic and clinical characteristics of the CAD patients and healthy controls were presented in Table 1. There was no significant difference between the two groups for a series of demographic parameters, including age, gender, smoking, alcohol use, and physical activity (P > 0.05). BMI, DBP, plasma levels of TC, TG, and LDLC, and the FBG level were higher in subjects with CAD (P < 0.05), whereas plasma levels of HDLC were found to be significantly lower in subjects with CAD than in controls (P < 0.05). By coronary angiography, 53 (32.9%) CAD cases had single-vessel disease, 76 (47.2%) had double-vessel disease and 32 (19.9%) had triple-vessel disease. The average LVEF (%) of the CAD patients was 63.41 ± 5.76.
The expression level of the seven selected miRNAs in the PBMCs of CAD patients and healthy controls were listed in Table 2. The expression levels of the miRNAs were logarithmic transformed for t-test. There are four miRNAs including miR-24, miR-33a, miR-103a, and miR-122 showed significant higher expression level among CAD patients than that among healthy controls (P < 0.001, Fig. 1). However, no significant difference was found for expression level of miR-21, miR-29a, and miR-34a between the two groups (P > 0.05). As the potential biomarker of CAD, the risk and clinical significance of miR-24, miR-33a, miR-103a, and miR-122 were evaluated in the further statistical analyses.
For the CAD patients, only the expression of miR-122 showed significant difference (F = 3.594, P = 0.030) among the three subgroups (single-, double-, and triple-vessel disease). The levels of miR-122 in patients with three-vessel disease were significantly higher than those with one- or two-vessel disease (66.06 ± 50.18 vs. 44.38 ± 28.67, P = 0.014 and 66.06 ± 50.18 vs. 46.47 ± 39.62, P = 0.018, respectively). However, there are no significant difference between patients with one-vessel and two-vessel disease (P = 0.765).
The spearman correlation analysis showed that blood lipids are significantly associated with miR-24, miR-33a, miR-103a, and miR-122 in either CAD patients or health controls. As the results listed in Table 3, we found that miR-24 was positively correlated with TC (P < 0.05) and TG (P < 0.05), while miR-33a was negatively related to HDLC (P < 0.05). Moreover, expression level of miR-103a and miR-122 were found positively related to TC (P < 0.05). As an important risk factor of CAD, LDLC was significantly associated with miR-24, miR-103a, and miR-122 in CAD patients (P < 0.05).
Univariate logistic regression revealed that expression levels of the four miRNA markers were positively associated with the presence of CAD (P < 0.05) (Table 4). After corrected for possible confounding variables, these miRNAs remained significantly associated with CAD (P < 0.05) (Table 4). With a unit increase of miRNA-33a level, there was 1.57 (95% CI 1.35–1.81) times of greater risk of presenting CAD after adjustment for age, gender, smoking, drink, exercise and BMI, TC, TG, HDLC, LDLC, DBP, and GLU. With a unit increase of miRNA-24 level, there was 1.32 (95% CI 1.07, 1.62) times of greater risk of presenting CAD. To compared the magnitude of the associations between the miRNAs and CAD, the ORs* were calculated using standardized miRNA levels. Compared with the other three miRNAs (miR-24, miR-103a, and miR-122), miR-33a showed the highest level of association with CAD. Secondly, miR-122 showed a higher level of risk for the presence of CAD when comparing with miR-24 and miR-103a.
The diagnostic accuracy of miR-24, miR-33a, miR-103a, and miR-122, measured by AUC, was 0.722 (cutoff value: 2.10), 0.799 (cutoff value: 9.91), 0.687 (cutoff value: 18.59), and 0.739 (cutoff value: 25.85), respectively (P < 0.05). The corresponding sensitivity and specificity was presented in Fig. 2. As compared with the other three miRNAs, miR-33a is a more valuable biomarker for diagnosis for CAD.
A stepwise logistic regression model to estimate the risk of being diagnosed with CAD was applied. All the four miRNAs turned out to be significant predictors (P < 0.05). The predicted probability of being diagnosed with CAD from the logit model based on the four miRNA panel, logit (P = CAD) = −6.693 + 0.412 × miR-24 + 0.381 × miR-33a + 0.018 × miR-103a + 0.035 × miR-122 was used to construct the ROC curve. The diagnostic performance for the established miRNA panel was evaluated using ROC analysis. The AUC for the miRNA panel was 0.911 (95% CI 0.880–0.942, Fig. 3). The diagnostic performance for two pair and three pair of miRNA panel was also analyzed (Table 5), which may be useful for clinical usage with less number of miRNAs to predict CAD.
The present study examined the expression level of seven lipometabolism-related miRNAs in PBMCs to establish their potential role as CAD markers. We found that miR-24, miR-33a, miR-103a, and miR-122 average expression level in PBMCs were significantly higher in CAD patients than in healthy controls. Further, the increased levels of these four miRNAs were associated with CAD presence, independent of other cardiovascular risk factors. Our study also revealed that miR-24, miR-33a, miR-103a, and miR-122 in PBMCs were potential markers for diagnosing CAD. The miRNA panel with the four miRNAs from the multivariate logistic regression model demonstrated higher accuracy in the diagnosis of CAD than the separated miRNA.
MiR-33a is an intronic miRNA located within intro 16 of human SREBF-2, a master switch in controlling genes involved in cholesterol uptake and synthesis20). In a series of parallel yet independent studies, miR-33a was identified as a key post-transcriptional regulator of cellular cholesterol homeostasis21). In addition to the cholesterol transport genes ABCA1, ABCG1 and NPC1, Gerin I et al. showed that miR-33a binding sites are highly conserved in the 3′ UTR of Cpt1a, Crot, and Hadhb in various cell types22). Each of these genes plays a distinct role in the fatty acid oxidation pathway. We found that miR-33a expression in PBMCs negatively correlated to HDLC in CAD patients (r = −0.221, P = 0.005) and health controls (r = −0.161, P = 0.049). Compared with the other selected miRNAs, miR-33a showed the highest level of association with CAD (adjusted OR* = 7.84, 95% CI 4.06, 15.14) after adjustment for confounding variables. MiR-33a is also a valuable biomarker for diagnosis for CAD (AUC = 0.799). These results are consistent with previous reports on association between plasma miR-33a and CAD6, 20), indicating that similar regulation may also exist in PBMCs.
It has been shown that miR-122 is over expressed in the liver of hyperlipidemia animals23). MiR-122 is located within exon 2 of the known noncoding RNA gene hcr (gi: 51212)23). Several genes involved in fatty acid synthesis and oxidation including SREBP, MTTP, KLF6 and CAT-1 were found to be regulated by miR-12210, 24, 25), and silencing of miR-122 resulted in decreased plasma levels of both TC and TG10, 24). Gao W et al. found that plasma level of miR-122 was increased in patients with hyperlipidemia, and positively correlated with TC, TG, and LDLC26). In the present study, we also found positive correlation between miR-122 and TC in ether CAD patients and health controls, and positive correlation between miR-122 and LDLC (r = 0.207, P = 0.009) in CAD group. Our observation that expression levels of miR-122 in PBMCs was significantly associated with CAD (adjusted OR = 1.03, 95% CI 1.01–1.04) is consistent with Gao's finding that the increased plasma level of miR-122 may be novel risk factors for CAD26).
Ng R et al. revealed an important role for the crosstalk between miR-24 and Insig1 in the control of hepatic lipid accumulation and hypertriglyceridemia in vivo7), suggesting that miR-24-evoked, Insig1-dependent inhibition of SREBP processing represents a candidate pathway to cause fatty liver and possibly hypertriglyceridemia. MiR-24 has been identified as a specific miRNA in early and advanced atherosclerosis plaque by the co-inertia approach27). Our data showed that miR-24 positive related to TC and TG in both CAD patients and health controls indicating miR-24 may be a risk factor for CAD.
The role of miR-103 is implicated in multiple mRNA regulations that are involved in cellular acetyl-CoA and lipid metabolism28). MiR-103 promotes 3T3-L1 cell adipogenesis through AKT/mTOR signal pathway with its target being MEF2D29). Bork-Jensen J et al. found that miR-103 levels were positively and independently associated with BMI, and that, a trend towards a positive association with plasma triglyceride levels30). In our study, miR-103a was found contributing to the presence of CAD (adjusted OR = 1.01, 95% CI 1.00–1.02). However, significant correlation was only found between miR-103a and TC when data was stratified by status of disease.
The spectrum of pathologies indicates that numerous metabolic factors like glucose, lipoproteids as well as various inflammatory and anti-inflammatory mediators lead to monocyte activation in the blood circulation31). Circulating peripheral blood cells, particularly PBMCs, have been proposed as cardiovascular disease (CVD) biomarkers11). They are easily accessible and its extraction is not invasive which facilitates its clinical testing. In recent years, there are quite a few studies focus on assessment of serum or plasma miRNAs used as biomarkers to detect and monitor CAD based on the rational that miRNAs released into circulation during the disease. PBMCs are main actors in atherosclerosis12). Our study measured lipometabolism-related miRNAs in PBMCs in CAD patients. Previous data showed that expressions of representative target genes of each miRNAs were changed in PBMCs (Supplementary Table 2) when diseases presented, indicating that these miRNAs may involve the pathogenesis of CAD by regulating lipometabolism gene. The present study may enable us to have better accuracy to identify their clinical value compared with those studies focus on plasma/serum miRNAs. However, well-designed prospective studies with larger sample sizes are required to validate our findings.
We found that the increased expression level of miR-24, miR-33a, miR-103a, and miR-122 in PBMCs were associated with CAD presence. The miRNA panel with the four miRNAs demonstrated high accuracy in the diagnosis of CAD. Lipometabolism-related miRNAs in PBMCs may emerge as valuable biomarkes for preventive, diagnostic, and therapeutic approaches for CAD.
This study was supported by the National Natural Science Foundation (81573214), the Beijing Municipal Natural Science Foundation (7162020), the Scientific Research Project of Beijing Municipal Educational Committee (KM201510025006), and the National Science and Technology Support Program (2012BAI37B03).
There was no conflict of interest in this study.