Long non‐coding RNA MALAT1 and its target microRNA‐125b associate with disease risk, severity, and major adverse cardiovascular event of coronary heart disease

Abstract Background This study aimed to explore the correlation of long non‐coding RNA metastasis‐associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) with microRNA (miR)‐125b and further investigated their associations with disease risk, severity, and prognosis of coronary heart disease (CHD). Methods Totally, 230 patients who underwent diagnostic coronary angiography were recruited; meanwhile, 140 of them were diagnosed as CHD and the remaining 90 non‐CHD patients served as controls. Plasma sample was collected from each participant for lncRNA MALAT1 and miR‐125b mRNA expression detection by reverse transcription‐quantitative polymerase chain reaction. The extent of coronary stenosis was evaluated by the Gensini score, and major adverse cardiovascular event (MACE) occurrence during the follow‐up was documented in CHD patients. Results Long non‐coding RNA metastasis‐associated lung adenocarcinoma transcript 1 relative expression was increased, but miR‐125b relative expression was decreased in CHD patients compared with controls. ROC curve exhibited that lncRNA MALAT1 and miR‐125b were of good value in differentiating CHD patients from controls, and further logistic regression analysis verified their independent correlation with CHD risk. Furthermore, lncRNA MALAT1 presented a closely negative correlation with miR‐125b in CHD patients, while it presented a weakly negative association with miR‐125b in controls. In CHD patients, lncRNA MALAT1 was positively correlated with Gensini score, total cholesterol, low‐density lipoprotein cholesterol, C‐reactive protein, tumor necrosis factor α, interleukin (IL)‐1β, IL‐6, IL‐17, and accumulating MACE occurrence; reversely, miR‐125b presented a opposite trend. Conclusion Long non‐coding RNA metastasis‐associated lung adenocarcinoma transcript 1 might be associated with increased CHD risk, severity, and accumulating MACE incidence via negative interaction with miR‐125b, suggesting their possible clinical application as biomarkers in the CHD screening and surveillance.


| INTRODUC TI ON
Coronary heart disease (CHD) remains the leading cause of morbidity and mortality globally, and the death rate of CHD has experienced a rise in the last decades considering the prevalence of obesity and lifestyle changes. 1,2 According to the global epidemiological data, it is estimated that there are approximately 93 million people suffering from CHD, which eventually contributes to 8.1 million deaths. 3 Pathologically, CHD is considered to be caused by atherosclerosis-induced arterial stenosis, and recent papers indicate that inflammation also plays an important role in the formation of atherosclerotic plaque, further leading to arterial stenosis. 4,5 Current clinical management, including arterial revascularization (such as percutaneous coronary intervention and coronary artery bypass grafting) and drug treatments (such as aspirin), represents great therapeutic advance for CHD patients; however, a portion of patients still suffer from unfavorable prognosis as the result of reoccurred coronary events. 5,6 Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) is recognized as a biomarker in various cancers, and recent studies indicate that it is also involved in the pathogenesis of atherosclerosis. [7][8][9][10] Mechanically, lncRNA MALAT1 silencing attenuates oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation and protects the endothelium from oxidative stress, further affecting the progression of atherosclerosis. 7 Additionally, another study indicates that lncRNA MALAT1 affects lipid metabolism disorder and is associated with inflammation response as well as the pathological progression of atherosclerosis via regulating ox-LDL-related macrophages. 8 Furthermore, lncRNA MALAT1 is reported to regulate cardiac inflammation and promote acute myocardial infarction via interaction of microRNA-125b (miR-125b), and miR-125b serves as a pathogenic mediator in multiple vascular diseases. [11][12][13] For example, miR-125b attenuates endothelin-1 expression, suppresses vascular endothelial-cadherin mRNA translation, and reduces endothelial permeability, involving the development of atherosclerosis. 13,14 Furthermore, the preliminary experiments of our study observed that lncRNA MALAT1 was upregulated but miR-125b was downregulated in CHD patients compared with controls. According to the aforementioned evidence and considering that the occurrence of CHD commonly had the pathophysiological basis of atherosclerosis accompanying with chronic inflammation and high lipid level, we hypothesized that lncRNA MALAT1 might be correlated with higher CHD risk, and presented correlation with increased level of coronary artery stenosis, lipid profile, and inflammation, contributing to enhanced severity and poor prognosis via interaction with miR-125b; however, to our best knowledge, there was no related research until now. [15][16][17] In the present study, we detected the correlation of lncRNA MALAT1 and miR-125b, and further investigated their potential in predicting CHD risk as well as their correlation with disease severity, level of lipid profile as well as inflammatory cytokines, and major adverse cardiovascular event (MACE) occurrence in CHD patients.

| Subjects
From February 2015 to September 2016, 230 patients were recruited in this study when they underwent diagnostic coronary angiography in our hospital due to unexplained chest pain or suspected CHD symptoms (such as angina, chest oppression, and short of breath). All recruited patients were older than 18 years and required to be in the absence of cardiomyopathy, congenital heart disease, severe liver or renal diseases, severe infection, sepsis, systemic autoimmune diseases (eg, rheumatoid arthritis), malignant hematological diseases, or tumors, and have no history of cardiovascular surgery (eg, coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty).
The pregnant or lactating women were not included in the study.
Through diagnostic coronary angiography, 140 of the 230 patients were diagnosed as CHD based on there was at least one major epicardial vessel with >50% stenosis and presenting with typical angina. The remaining 90 non-CHD patients were severed as control subjects in the analysis. This study was approved by the Institutional Review Board of our hospital. All participants provided the written informed consent.

| Clinical data collection
Clinical characteristics of patients were collected by interview before coronary angiography, including demographics (age, gender, and body mass index), smoke status, and medical histories (family history of CHD, hypertension, hyperlipidemia, hyperuricemia, and diabetes mellitus [DM]). Following the laboratory tests, the biochemical indexes were recorded, such as fasting blood glucose (FBG), serum creatinine, serum uric acid, triglyceride (TG), total cholesterol (TC), LDL cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and C-reactive protein (CRP). Additionally, by coronary angiography, the extent of coronary stenosis was evaluated by the Gensini score according to the previous study, 18 where a higher score indicated more severe coronary stenosis.

| Peripheral blood collection and detection
Peripheral blood samples of CHD patients and non-CHD patients (as controls) were extracted using anticoagulant tube before coronary angiography. After collection, the PB sample was centrifuged K E Y W O R D S coronary heart disease, Gensini score, long non-coding RNA MALAT1, major adverse cardiovascular event, microRNA 125b at 4°C and 1600 g for 10 minutes immediately; then, plasma was collected. Following that, the plasma was further centrifuged at

| RT-qPCR assay
The level of lncRNA MALAT1 and miR-125b in plasma was detected by RT-qPCR for all participants included. RNA extraction was conducted using QIAamp RNA Blood Mini Kit (Qiagen); subsequently, reverse transcription was performed with iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instruction. qPCR was performed with SYBR ® Green Realtime PCR Master Mix (Toyobo) according to the manufacturer's guidance. lncRNA MALAT1 and miR-125b were detected via qPCR, and qPCRs were performed in triplicate with lncRNA MALAT1 internal coefficient variation of 1.8% in CHD patients and 1.2% in controls as well as with miR-125b internal coefficient variation of 0.8% in CHD patients and 1.5% in controls. The relative expressions of lncRNA MALAT1 and miR-125b were calculated using the formula of 2 −ΔΔCt , with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for lncRNA MALAT1) and U6 (for miR-125b) used as the internal references. Detailed calculation process was as follows: (a) qPCR was performed in triplicate, and the average of lncRNA MALAT1 Ct, miR-125b Ct, GAPDH Ct, and U6 Ct in every sample was determined, respectively. (b) Calculations of ΔCt (Ct avg. lncRNA MALAT1 − Ct avg.

| Comparison of clinical characteristics between CHD patients and controls
The mean age was 62.6 ± 9.8 years for CHD patients and 58.3 ± 9.6 years for controls, respectively (

| Correlation of lncRNA MALAT1 and miR-125b with key biochemical indexes in CHD patients
In  Table 3). More detailed information about correlation of lncRNA MALAT1 and miR-125b with key biochemical indexes in CHD patients is listed in Table 3.

| Correlation of lncRNA MALAT1 and miR-125b with inflammatory cytokines in CHD patients
In CHD patients, lncRNA MALAT1 was positively associated with

| Correlation of lncRNA MALAT1 and miR-125b with accumulating MACE occurrence in CHD patients
According to the median value of lncRNA MALAT1, patients were categorized as lncRNA MALAT1-high patients and lncRNA MALAT1low patients, and lncRNA MALAT1-high expression was associated with increased accumulating MACE occurrence (P = .007) ( Figure 4A). Furthermore, based on the median value of miR-125b, patients were divided into miR-125b-high patients and miR-125blow patients, and miR-125b-high expression was correlated with reduced accumulating MACE occurrence (P = .002) ( Figure 4B).

| D ISCUSS I ON
In the present study, we found that (a) lncRNA MALAT1 was in- regarding the association of lncRNA MALAT1 and miR-125b with CHD risk, as well as their potential to predicting prognosis in CHD patients.

F I G U R E 2
Association of lncRNA MALAT1 with miR-125b in participants. Association of lncRNA MALAT1 with miR-125b in controls (A) and CHD patients (B). CHD, coronary heart disease; lncRNA MALAT1, long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1; miR-125b, microRNA 125b F I G U R E 3 Correlation of lncRNA MALAT1 and miR-125b with coronary stenosis severity in CHD patients. Correlation of lncRNA MALAT1 with Gensini score (A). Correlation of miR-125b with Gensini score (B). CHD, coronary heart disease; lncRNA MALAT1, long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1; miR-125b, microRNA 125b We conducted the present study, which observed that lncRNA MALAT1 expression was increased, while miR-125b expression was decreased in CHD patients compared with controls. Furthermore, lncRNA MALAT1 was an independent risk factor for CHD, while Subsequently, we further observed that lncRNA MALAT1 was positively but miR-125b was negatively associated with severity of coronary stenosis, hyperlipidemia, and systematic inflammation in CHD patients. The possible reasons might include that (a) according to the prior evidence, lncRNA MALAT1 was supposed to prevent the protective effect of miR-125b against the development of CHD such as atherosclerosis, vascular inflammation, and vascular calcification, which led to higher severity of CHD. 12 Abbreviations: CHD, coronary heart disease; IL, interleukin; lncRNA MALAT1, long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1; miR-125b, mircroRNA-125b; TNF, tumor necrosis factor.
patients; hence, lncRNA MALAT1 and miR-125b might be associated with MACE occurrence via interaction with these factors in CHD patients.
However, some limitations still existed in our study as follows: (a) Firstly, as 230 patients were from one single center, selection bias might exist, and more patients from multiple centers were needed to further verify the results in our study. (b) The follow-up duration was 36 months in our study; thus, the predictive value of lncRNA MALAT1/miR-125b for MACE incidence is needed to be investigated for a longer period in CHD patients. (c) The underlying mechanism of interaction between lncRNA MALAT1 and miR-125b was not included in our clinical study, which could be further explored in the future.
In summary, both lncRNA MALAT1 and miR-125b show potentials to be independent indicators for CHD risk and associate with disease severity and accumulating MACE occurrence in CHD patients, suggesting their possible clinical application as biomarkers in the CHD screening and surveillance.

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.