DU Ying， YANG Sheng Hua， LI Sha， CUI Chuan Jue， ZHANG Yan， ZHU Cheng Gang，GUO Yuan Lin， WU Na Qiong， GAO Ying， SUN Jing， DONG Qian， LIU Geng， and LI Jian Jun

Division of Dyslipidemia， State Key Laboratory of Cardiovascular Disease， Fuwai Hospital， National Center for Cardiovascular Disease， CAMS and PUMC， Beijing 100037， China

?

Original Article

Circulating MicroRNAs as Novel Diagnostic Biomarkers for Very Early-onset （≤40 years） Coronary Artery Disease*

DU Ying?， YANG Sheng Hua?， LI Sha， CUI Chuan Jue， ZHANG Yan， ZHU Cheng Gang，GUO Yuan Lin， WU Na Qiong， GAO Ying， SUN Jing， DONG Qian， LIU Geng， and LI Jian Jun#

Division of Dyslipidemia， State Key Laboratory of Cardiovascular Disease， Fuwai Hospital， National Center for Cardiovascular Disease， CAMS and PUMC， Beijing 100037， China

Abstract

Objective Very early-onset coronary artery disease （CAD） is a great challenge in cardiovascular medicine throughout the world， especially regarding its early diagnosis. This study explored whether circulating microRNAs （miRNAs） could be used as potential biomarkers for patients with very early-onset CAD.

Methods We performed an initial screening of miRNA expression using RNA isolated from 20 patients with angiographically documented very early-onset CAD and 20 age- and sex-matched normal controls. For further confirmation， we prospectively examined the miRNAs selected from 40 patients with very early-onset CAD and 40 angiography-normal controls.

Results A total of 22 overexpressed miRNAs and 22 underexpressed miRNAs were detected in the initial screening. RT-qPCR analysis of the miRNAs obtained from the initial screening revealed that four miRNAs including miR-196-5p， miR-3163-3p， miR-145-3p， and miR-190a-5p exhibited significantly decreased expression in patients compared with that in controls （P＜0.05）. The areas under the receiver operating characteristic curve for these miRNAs were 0.824 （95% CI， 0.731-0.917； P＜0.001）， 0.758 （95% CI， 0.651-0.864； P＜0.001）， 0.753 （95% CI， 0.643-0.863； P＜0.001）， and 0.782 （95% CI， 0.680-0.884；P＜0.001）， respectively， in the validation set.

Conclusion To our know ledge， this is an advanced study to report about four serum miRNAs（miR-196-5p， miR-3163-3p， miR-145-3p， and miR-190a-5p） that could be used as novel biomarkers for the diagnosis of very early-onset CAD.

MicroRNA； Biomarker； Early-onset coronary artery disease

Coronary artery disease （CAD） is a major worldwide public health problem and the single largest cause of mortality. CAD，

INTRODUCTION

especially early-onset CAD， is also a huge burden for both developing and developed countries. By definition， onset of CAD at a young age （at or before 55 years in men or 65 years in women） is diagnosed as early-onset CAD. Early-onset CAD is different fromCAD in certain aspects， such as a higher incidence of smoking， dyslipidemia， and family history in younger patients than in older patients［1-3］. Early-onset CAD also significantly affects the quality of life of patients and leads to morbidity and premature death. It has also been reported that early-onset CAD may result in worse outcomes for the patients and society than those by CAD［3］. Nevertheless， information regarding the effective predictors of this complication is still scarce.

Over the past two decades， remarkable changes in the diagnosis， treatment， and prognosis of cardiovascular disease have led to significant improvement in the diagnosis， treatment， and prognosis of early-onset CAD［4］. However， early-onset CAD is a great challenge in cardiovascular medicine throughout the world， especially regarding its early diagnosis. Therefore， development of innovative biomarkers may be of great clinical interest.

MicroRNAs （miRNAs） are a class of endogenous，noncoding， single-stranded short RNAs of approximately 22 nucleotides in length， which negatively regulate target gene expression at the post-transcriptional level by binding to the 3' untranslated regions of mRNAs［5-6］. They have been identified as key regulators of several physiological and pathophysiological processes of mammalian cardiovascular development and diseases［5，7］. For instance， miR-26 participates in the regulation of cardiovascular repair［8-9］and miR-181 family plays an important role in vascular inflammation［10］.

Besides their intracellular functions， recent studies demonstrate that miRNAs can be increasingly found in the systemic circulation of both animals and humans［11］. They can be released by cells and circulate in the blood［12］. Unlike miRNAs in tissues， circulating miRNAs are extremely stable and exist in an RNase-resistant form［4，13］. Multiple freeze-thaw cycles or prolonged room temperature incubation w ill not affect isolation of circulating miRNAs［4］. Since the levels of circulating miRNAs may significantly vary in different pathological conditions，such as cancer， liver injury， and hepatitis， they have been suggested as great potential biomarkers for diagnosis of these diseases［14-15］. Furthermore，previous studies have shown that miR-423-5p may be used as a clinical diagnostic biomarker for heart failure［16］， and miR-29a has been identified as a potential biomarker for myocardial remodeling assessment in hypertrophic cardiomyopathy［17］. More importantly， much evidence has demonstrated that circulating m iRNAs can be useful diagnostic and prognostic markers in CAD［18-22］， especially in acute coronary syndrome （ACS）［19，23-28］. Nonetheless，whether circulating miRNAs can be useful as diagnostic and prognostic markers in early-onset CAD， especially in very early-onset CAD， remains unknown.

Therefore， the purpose of this study is to search for distinctive miRNA profiles in the serum of patients with angiographically documented very early-onset CAD in comparison with angiographynormal controls. The potential diagnostic significance of circulating miRNAs has been challenged by the ROC curve with the aim of identifying putative specific very early-onset CAD-associated miRNA signatures.

MATERIALS AND METHODS

Study Population

Our study complied with the Declaration of Helsinki and was approved by the hospital ethics review board （Fuwai Hospital & National Center for Cardiovascular Diseases， Beijing， China）. Informed written consents were obtained from all patients enrolled in this analysis. A total of 60 patients aged below 40 years with angiographically documented stable CAD and 60 age- and sex-matched controls were enrolled. Based on our previous studies， we diagnosed CAD by the criterion of the presence of coronary lesion ≥50% in at least one major epicardial artery segment assessed by coronary angiography［29-30］. On the basis of general exclusion criteria， patients with the following conditions were excluded from the study： ACS， heart failure，significant hematologic disorders， thyroid disorders，infectious or systematic inflammatory diseases，severe liver and/or renal insufficiency， and malignant disease. In parallel， all the controls were examined by coronary angiography to rule out CAD.

Study Flow

The study flow was shown in Figure 1. A two-phase case-control study was designed to identify serum miRNAs as potential biomarkers for very early-onset CAD. An miScript miRNA PCR Array analysis was performed to screen and select miRNAs that showed significant changes in pooled serum samples between patients with very early-onset CAD（n=20） and controls （n=20）. Differentially expressed miRNAs were exam ined in the same cohort that was used for screening （training set）. We then detectedthose altered miRNAs in a larger cohort including 40 patients with very early-onset CAD and 40 controls（validation set）. Finally， ROC curve analysis was used to confirm the diagnostic value of these identified miRNAs.

Blood Collection and RNA Isolation

Serum samples obtained from blood collected from the cubital vein from all patients after a 12-h overnight fasting were collected in EDTA-anticoagulated tubes and stored at -80 °C until analysis. Total miRNAs were isolated by using an miRNeasy Serum/Plasma Kit （Qiagen， California，USA）， following the manufacturer's instruction. Briefly， each sample was supplemented （after addition of QIAzol） with 3.5 μL miRNeasy Serum/Plasma Spike-In Control （1.6 × 108copies/μL working solution） （Qiagen， California， USA）. Reverse transcription （RT） reactions were performed using the miScript II RT Kit （Qiagen， California， USA） using 4 μL miScript Hispec buffer， 2 μL miScript nucleric mix， RNase free water， 2 μL miScript reverse transriptase mix， and 12 μL RNA template.

miRNA Expression Profiling

miRNA expression profiles were obtained by using an miScript miRNA PCR Array （MISH-3106Z，Qiagen， California， USA）， following the manufacturer's instruction. Data were analyzed by a website tool provided by Qiagen （http：// pcrdataanalysis.sabiosciences.com/m irna）.

Detection and Quantification of m iRNAs by Quantitative RT-PCR

A total of 5 μL the product of RT reactions was diluted in a ratio of 1：10 and then used for detecting miRNA expression by RT-qPCR using miScript SYBR? Green PCR Kit （Qiagen， California， USA）. Each PCR reaction was carried out in a volume of 25 μL containing 2 μL of cDNA， 0.1 μmol/L of each primer，and 2 × SYBR Green PCR Master mix （Qiagen）. At the end of the PCR cycles， melting curve analyses were performed. Values were normalized to cel-miR-39 and were expressed as 2-［CT（miRNA）-CT（cel-miR-39）］. The criteria for selecting miRNAs are relatively high expression level in both the controls and the very early-onset CAD group， at least 10-fold changes， and the first six greatest fold changes.

Statistical Analyses

Data are presented as mean±SEM for serum miRNA levels or mean±SD for other variables. The differences in clinical parameters between the groups were analyzed using independent sample t-test and χ2tests wherever appropriate. The receiver operating characteristic （ROC） curve was generated and the area under the curve （AUC） was calculated to evaluate the specificity and sensitivity of prediction of very early-onset CAD for each serum miRNA. Pearson's correlation analysis was used to investigate the correlation of relative expression of miRNAs with Gensini score， and log-normalization was used for variables with positively skewed distribution. A P value of ＜0.05 was considered as statistically significant. Statistical analysis was performed using SPSS， version 22.0， software （SPSS Inc.， Chicago， IL， USA）.

RESULTS

Baseline Clinical Characteristics

A total of 60 patients （56 men and 4 women）with angiographically documented stable very early-onset CAD and 60 control subjects （53 men and 7 women） were included in the present study. The baseline characteristics of patients enrolled in the training set and validation set are shown in Table 1.

miScript miRNA PCR Array Analysis of Serum miRNAs in Patients with Very Early-onset CAD

We first performed an miScript miRNA PCR Array analysis to screen and select candidate miRNAs. miRNAs in this analysis were divided into three groups according to the expression changes between patients with very early-onset CAD andcontrols（Figure 2A；the red circles denote upregulated miRNAs，the green ones denote downregulated miRNAs，and the black ones denote unchanged miRNAs in patients with very early-onset CAD）.Of the 372 miRNAs scanned，we identified 22 upregulatedm iRNAs and 22 downregulated miRNAs in patients very early-onset CAD compared with angiography-normal controls（Table 2）.Among the downregulated miRNAs，we focused on six miRNAs（miR-196b-5p，miR-29c-5p，miR-203a-3p，miR3613-3p，miR-145-3p，andmiR-190a-5p）from alteredm iRNAs，ofwhich the relative expression level was relatively high in either the control or the very early-onset CAD group and showed the largest fold changes（Figure 2B）.

Confirmation of Serum miRNA Profiling Data by RT-PCR Analysis

We next employed RT-qPCR assay to confirm the expression of the six candidate miRNAs selected from the miScript miRNA PCR Array analysis.The levels of the six miRNAs were detected in a set of individual serum samples including 20 patients with very early-onset CAD and 20 controls（derivation set），which were the same cohort used in miScript miRNA PCR Array screening phase.We found that four miRNAs including miR-196a-5p，miR-3613-3p，miR-145-3p，and miR-190a-5p were markedly decreased in patients with very early-onset CAD compared to that in control subjects（P＜0.05）（Figure 3）.Expression levels of the four altered miRNAs were then further confirmed in a new cohort sample set（validation set）composed of 40 patients with very early-onset CAD and 40 age-and sex-matched controls.Consistent with the results from the derivation set，serum levels of miR-196a-5p，miR-3613-3p，miR-145-3p，and miR-190a-5p were significantly lower in patients with very early-onset CAD than those in the control subjects（Figure 4）.

Table 1.Baseline Characteristics of the Study Population

Receiver Operating Characteristic Curve Analysis

To evaluate the usefulness of the four altered circulating miRNA as potential biomarkers for early-onset CAD， an ROC curve analysis was performed. The AUC associated with each circulating miRNA was used to confirm its diagnostic value. The diagnostic values of these four miRNAs were evaluated in the validation set. Their AUCs were 0.824， 0.758， 0.753， and 0.782， respectively （Figure 5，Table 3）. These results imply that the four serum miRNAscan represent as suitable biomarkers for distinguishing patients w ith very early-onset CAD from other subjects.

Table 2. Differentially Expressed m icroRNAs in VECAD Patients Compared w ith Controls

Table 3. Areas under the Receiver Operating Characteristic Curve and Predictive Value of Four Candidate miRNAs

DISCUSSION

Very early-onset CAD is a great challenge as well as a serious issue in the society in developing and developed countries throughout the world，especially in China. Hence， the exploration of markers for predicting or diagnosing very early-onset CAD may be extremely important for current clinical practice. Therefore， we investigated whether an miRNA could be a biomarker for detecting very early-onset CAD using a Chinese Han case-control model. The results strongly indicated that expression levels of miR-196a-5p， miR-3613-3p， miR-145-3p，and miR-190a-5p were significantly low in patients with very early-onset CAD compared to those in control subjects and they exhibited a great ability to distinguish patients with very early-onset CAD from other subjects. Hence， our study provides novel information with regard to the relationship between the diagnosis of very early-onset CAD and miRNAs，which may have important implications for very early-onset CAD.

Early-onset CAD is well known to be a typical form of CAD，which occurs in men aged below 55 years and women aged below 65 years［31］. Early-onset CAD may cause heavier burden on patients and society than that by conventional CAD［32］. Several risk factors are involved in the onset and development of CAD and their contributions vary in people of different ages［3，33］. According to a study on 15，381 consecutive patients with ACS， the effects of cigarette smoking and family history of CAD were more dominant in patients with early-onset CAD compared to patients with conventional CAD［3］. Various studies have indicated that early-onset CAD exhibits a strong association with genetic factors， especially gene polymorphisms. Recently， the APOC4 rs1132899 polymorphism wasreported to be associated with an increased risk of early-onset CAD in Chinese Han people and the association was more significant among male subjects［34］. In the Genetics of Atherosclerotic Disease （GEA） study， the authors found that the rs2057482 T allele in HIF1A was associated with decreased risk of early-onset CAD compared to healthy controls and was also associated w ith certain metabolic parameters and cardiovascular risk factors［35］. They also found that the rs1024610 polymorphism of MCP-1 gene was associated with increased risk of developing early-onset CAD only in those patients without type 2 diabetes mellitus［36］. There were some factors associated with early-onset CAD that could be possible biomarkers for early-onset CAD. For instance， in Chinese Han people，high molecular weight （HMW） adiponectin and HMW-total adiponectin ratio showed stronger negative associations with severity of coronary atherosclerosis and this ratio can be an effective biomarker for predicting the risk of early-onset CAD in Chinese population［37］. Systemic capillary rarefaction and impaired microvascular endothelial function were observed in patients with early-onset CAD［38］. Therefore， these microvascular alterations in young adults can indicate an increased risk of developing CAD and could be a useful marker of subclinical atherosclerosis［38］. In another study，adding six additional biomarkers （hs-CRP， IL-6， RAGE，Lp-LPA2， adiponectin， and RANTES） to the conventional risk factors model moderately improved the prediction of premature CAD， it was also observed that hs-CRP and IL-6 had a significant contribution to the prediction of premature CAD［39］. Although there are various studies on early-onset CAD， there is currently no useful biomarker available for predicting very early-onset CAD. Hence， we chose very early-onset CAD as our principal target.

Several recent studies have indicated the potential role of circulating miRNA levels as valuable biomarkers for CAD. For instance， Fichtlscherer et al.［40］performed an miRNA profiling using RNA isolated from eight healthy volunteers and eight patients with stable CAD and found that circulating levels of miR-126， miR-17， miR-92a，inflammation-associated miR-155， and the smooth muscle-enriched miR-145 were significantly reduced in patients with stable CAD compared with healthy controls. These results were validated in a second cohort of 31 patients with documented CAD and 14 controls［40］. In another study［6］， the authors detected miR-122 and miR-370， which are already known to play a role in lipid metabolism， and hypothesized that they may be associated with CAD and hyperlipidemia. Consistent with their hypothesis，they found that increased plasma levels of miR-122 and miR-370 positively correlated with total cholesterol （TC）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C） levels in both patients with hyperlipidem ia and controls and were positively correlated w ith the severity of CAD quantified by the Gensini score［6］. Liu et al.［41］reported that four circulating miRNAs belonging to the miR-17-92 cluster were also found to be possible biomarkers for CAD. These authors also performed miRNA profiling analysis by using circulating miRNA samples from pooled plasma from 20 patients and 20 controls， similar to our study. They found that miR-17， miR-106b， miR-92a， and miR-18a from this miRNA cluster were significantly downregulated in patients with CAD and further validated these results in 50 healthy volunteers and 81 patients （including 20 controls and 20 patients using an miRNA profile analysis）［41］. However， all of the abovementioned studies focused on patients with CAD aged more than 55 years， whereas no such data have been reported with respect to very early-onset CAD. Therefore， it is necessary to explore new circulating miRNAs for diagnosis of very early-onset CAD by using very young patients with CAD.

In our present study， by using a miScript miRNA PCR Array technology， we found that the circulating miRNA profile was different between patients with very early-onset CAD aged below 40 years and ageand sex-matched controls. The serum miRNA profile revealed 22 upregulated miRNAs and 22 downregulated miRNAs in patients with very early-onset CAD compared with angiography-normal controls. Six miRNAs that exhibited the most significant fold changes were validated in the training cohort. The serum levels of four miRNAs were significantly altered in patients with very early-onset CAD. We then detected these four miRNAs in a larger validation cohort and found that all of the four miRNAs were significantly downregulated in patients with very early-onset CAD. Further ROC curve analysis indicated that these four miRNAs had good sensitivity and specificity for distinguishing patients with very early-onset CAD in both the training set and validation set. These four miRNAs were miR-196a-5p， miR-3613-3p，miR-145-3p， and miR-190a-5p. Among these four miRNAs， the serum level of miR-145-3p had been reported to be significantly downregulated inpatients w ith stable CAD compared with healthy controls， a finding consistent with our results［25，40］. However， there were no reports about the association between miR-196a-5p， miR-3613-3p， and miR-190a-5p with CAD. Thus， miR-196a-5p，miR-3613-3p， and miR-190a-5p could be more appropriate as biomarkers only for very early-onset CAD than miR-145-3p. Very few studies have reported about miR-196a-5p， miR-3613-3p， and miR-190a-5p， and hence studying the possible functions of these three miRNAs in cardiovascular disease could be very interesting. A previous study had reported that miR-765 and miR-149 could be used as diagnostic biomarkers for geriatric patients with CAD［42］. Nevertheless， no data are currently available about the diagnostic value of miRNAs in very early-onset CAD. In the present study， our results provided additional information that could have an important clinical implication for the prevention of CAD.

Additionally， we exam ined the relationship between the miRNAs （miR-196-5p， miR-3163-3p，miR-145-3p， and miR-190a-5p） and some of the laboratory parameters in 40 patients with very early-onset CAD （Supplementary Table 1 see the www.besjournal.com）. Unexpectedly， we failed to find a statistically significant relationship between the four selected miRNAs and TC， LDL-C， HDL-C， TG. Meanwhile， no statistically significant association of Gensini scores w ith the four miRNAs （miR-196-5p，miR-3163-3p， miR-145-3p， and miR-190a-5p） was found （Supplementary Table 1 see the www.besjournal.com）. We speculate that this unexpected result may be attributed to the small size of sample， and as of now we cannot identify the actual cause for this finding. Besides， it is unknown whether these miRNAs are just biomarkers. Thus，further study is needed.

The relatively small sample size of the study is the primary limitation of our work. Therefore， data from the current observation may require further validation using multicenter， large-scale studies. Because of the significantly less number of women with CAD in the very young age compared with men，due to the characteristic feature of this disease， and only 372 human miRNAs were allowed to be detected in our screening method， which fixed our examination protocol， other potentially important miRNAs could not be detected in such unique patients with very early-onset CAD. Furthermore， in the present study， we selected only six significantly altered miRNAs from the downregulated miRNAs and did not focus on the upregulated miRNAs due to the shortage of research fund. Thus， there may be other miRNAs that could also be useful as potential biomarkers for very early-onset CAD.

CONCLUSION

We successfully identified four miRNAs that showed different expression levels in patients with angiographically documented very early-onset CAD and normal controls， which may be useful as biomarkers for the diagnosis of very early-onset CAD. As novel invasive biomarkers， the established miRNAs could potentially decrease the underestimation of CAD and help to make risk stratification， although further studies are needed to confirm our finding.

DECLARATION OF INTEREST

The authors have no conflict of interests.

ACKNOWLEDGMENTS

We would like to thank all subjects who participated in the present study.

Accepted： August 5， 2016

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Biomed Environ Sci， 2016； 29（8）: 545-554 10.3967/bes2016.073 ISSN: 0895-3988 www.besjournal.com （full text） CN: 11-2816/Q Copyright ?2016 by China CDC

May 4， 2016；

*This work was partially supported by the National Natural Science Foundation of China （81070171， 81241121）； the Specialized Research Fund for the Doctoral Program of Higher Education of China （20111106110013）； the Capital Special Foundation of Clinical Application Research （Z121107001012015）； the Capital Health Development Fund （2011400302）； and the Beijing Natural Science Foundation （7131014）.

?The first two authors contributed equally to this study.

#Correspondence should be addressed to LI Jian Jun， MD， PhD， Tel： 86-10-88396077； Fax： 86-10-68331730； E-mail：lijianjun938@126.com

Biographical notes of the first authors： DU Ying， female， born in 1985， PhD in basic research of cardiovascular disease；YANG Sheng Hua， male， born in 1985， MD， specializing in disorders of lipid metabolism and cardiovascular disease.