Circulating exosomal long non‐coding RNAs in patients with acute myocardial infarction

Abstract Exosomes are attracting considerable interest in the cardiovascular field as the wide range of their functions is recognized in acute myocardial infarction (AMI). However, the regulatory role of exosomal long non‐coding RNAs (lncRNAs) in AMI remains largely unclear. Exosomes were isolated from the plasma of AMI patients and controls, and the sequencing profiles and twice qRT‐PCR validations of exosomal lncRNAs were performed. A total of 518 differentially expressed lncRNAs were detected over two‐fold change, and 6 kinds of lncRNAs were strikingly elevated in AMI patients with top fold change and were selected to perform subsequent validation. In the two validations, lncRNAs ENST00000556899.1 and ENST00000575985.1 were significantly up‐regulated in AMI patients compared with controls. ROC curve analysis revealed that circulating exosomal lncRNAs ENST00000556899.1 and ENST00000575985.1 yielded the area under the curve values of 0.661 and 0.751 for AMI, respectively. Moreover, ENST00000575985.1 showed more significant relationship with clinical parameters, including inflammatory biomarkers, prognostic indicators and myocardial damage markers. Multivariate logistic model exhibited positive association of ENST00000575985.1 with the risk of heart failure in AMI patients. In summary, our data demonstrated that circulating exosomal lncRNAs ENST00000556899.1 and ENST00000575985.1 are elevated in patients with AMI, functioning as potential biomarkers for predicting the prognosis of pateints with AMI.


| INTRODUC TI ON
Acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide, 1 resulting in sudden myocardial tissue ischaemia, and sudden cardiac death. 2 Thus, it is necessary to identify novel biomarkers for early diagnosis and prognosis prediction of AMI, as these may assist in providing valuable therapies.
Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts that are longer than 200 nucleotides. An increasing body of evidence suggested that lncRNAs play biologically fundamental roles in the occurrence and development of cardiovascular diseases. [3][4][5] LncRNAs are differently expressed and have been shown to be used as biomarkers for the progression of coronary artery disease, atrial fibrillation and heart failure. [6][7][8] In addition, lncRNAs might act as biomarkers in the early diagnosis of AMI and also help in predicting its outcome. [9][10][11] Compared with lncRNAs present in extracellular fluids of the body, those lncRNAs that are packaged in exosomes are highly stable. Exosomal lncRNAs have lipid bilayers and could protect them from enzymatic degradation of RNA enzymes in bodily fluids. Thus, exosomal lncRNAs have a relatively long and stable duration of expression in the cardiovascular system. Previous studies have shown that exosomal lncRNAs were involved in various diseases and can be used as biomarkers. [12][13][14] However, few reports have focused on the role of exosomal lncRNAs in AMI.
Hence, in the present study, we investigated the role of circulating exosomal lncRNAs in AMI patients and provide some potential biomarkers for diagnosis and prognosis prediction of AMI.

| Study patients
The present study compared the sequencing profiles of circulating exosomal lncRNA in AMI patients (n = 15) with controls (n = 15).
This was first validated in 20 AMI patients and 20 controls, and then second validation in 85 AMI patients and 48 controls. For sequencing profiles, the blood samples of every 5 AMI patients as well as the controls were pooled into one sample, and thus, '3 AMI' and '3 control' samples were profiled. All AMI patients enrolled in the present study had a heart attack within 12 hours from the time of admission, and revascularization was successfully performed in the emergency department before hospitalization.
AMI was defined based on clinical symptoms, typical changes in electrocardiogram (ECG), elevated cardiac biomarkers troponin-I (TnI) and creatine kinase MB (CKMB) given by the Universal Definition of myocardial infarction. 15 The controls included were of non-coronary chest pain patients (NCCP), that had chest pain, normal cardiac biomarkers and most importantly, no coronary stenosis as confirmed by angiography.
All patients were recruited from Beijing Chao-Yang Hospital Affiliated to Capital Medical University in China. Written informed consent was obtained and signed from all participants. This study was conducted in accordance with the Declaration of Helsinki, and the research protocol was approved by the Ethics Committee of Beijing Chao-Yang Hospital.

| Exosomal Isolation and Identification
The exosomes were isolated using a commercial kit (Qiagen Inc), following the manufacturer's instructions, and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting. Isolated exosomes were examined by TEM and NTA as described previously. 16,17 Western blotting of exosomal marker protein CD63 (Abcam) and heat shock protein 70 (HSP70) (Abcam) was done as previously described. 18

| Exosomal RNA Extraction, RNA Sequencing and Quantitative RT-PCR
The lncRNAs were extracted from exosomes by using a commercial kit (Qiagen Inc) according to the manufacturer's protocol. RNA sequencing procedure was done as described in the methods section of our previous study. 19 The relative expression levels of lncRNA were quantified using ViiA 7 Real-Time PCR System (Applied Biosystems) according to standard methods, and the forward and reverse primers included were listed in Table S1. The lncRNA IDs were searched in Ensembl Human GRCh37.p13.

| Laboratory measurements and echocardiography
Fasting venous blood samples were taken within the first 24 hours of admission. Blood samples for isolating the exosomes were collected by venipuncture into EDTA containing tubes. All patients received laboratory measurements including lipids, glucose, creatinine and other items. Echocardiography was performed within 72 hours after admission. The items measured included left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and others.

| Clinical conditions
Heart failure was defined by NT-BNP > 1000 pg/mL, or LVEDD > 55 mm, or LVEF < 40%. Major adverse cardiovascular events (MACE) included cardiac death, ventricular fibrillation and cardiac shock. Long-term hospitalized was defined as hospital time > 5 days.

| Statistical analysis
All analyses were performed using SPSS 24.0 software (IBM).
Continuous variables with normal distribution were expressed as mean ± standard deviation (SD) and compared by two-sample t test, while those with non-normal distributed were expressed as quartiles and compared by Mann-Whitney U test. Categorical variables were expressed as percentages and numbers, and compared using the chi-square test. Spearman's correlation coefficients were used to assess the relationships between variables. Receiver operating characteristic curves and areas under the curve (AUCs) were computed. All statistical tests were twotailed, and P-values of .05 were considered to be statistically significant.

| Sequencing profiles of circulating exosomal lncRNA in AMI patients
The clinical characteristics of sequencing samples are provided in Table S2. There were mostly well balanced between AMI patients and controls, except for the higher fasting glucose level in AMI patients. Given that the number of diabetics was the same in the two groups, the higher fasting glucose level in AMI patients was consid-

| First validation of circulating exosomal lncRNA
The first validation was performed in 20 AMI patients and 20 controls, and their clinical characteristics are shown in

| Second validation of circulating exosomal lncRNA
The second validation was performed in 85 AMI patients and 48 controls, and their clinical characteristics are shown in Table 1.
Their demographic characteristics were mostly well balanced between the two groups. In the second validation, lncRNA ENST00000556899.1 (P = .040, fold change = 2.49) ( Figure 4A) and lncRNA ENST00000575985.1 (P = .008, fold change = 3.14) ( Figure 4A) were shown to be significantly up-regulated in      should be conducted to verify these findings.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.