Objective: To investigate the expression of LncRNA uc063iod.1 in patients with different diseases and to explore the correlation between expression level of LncRNA uc063iod.1 and Coronary Artery Disease (CAD). Method: Plasma samples were prepared using the blood extracted from total 101 patients undergoing coronary artery angiogram (63 CAD patients and 38 non CAD patients). The expression level of LncRNA uc063iod.1 was detected using quantitative real time polymerase chain reaction (qRT-PCR) and the relationship between expression level of LncRNA uc063iod.1 and CAD was explored. The clinicopathological features of patients with CAD were also discussed in our study. Results: The expression of LncRNA uc063iod.1 was significantly increased in the plasma of patients with CAD (p < 0.05). AUC was 0.701 (95%CI: 0.598 - 0.804), and the sensitivity and specificity were 77.8% and 57.9% respectively. Conclusion: Upregulation of LncRNA uc063iod.1 in plasma can be used as a biomarker for the diagnosis of CAD.
Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the world. An estimated 17.7 million people died from CVDs in 2015, representing 31% of all global deaths. Of these deaths, an estimated 7.4 million were due to Coronary artery Disease (CAD). Atherosclerotic disease and its thrombotic complication are the common manifestation of CVDs that leads to the development of CAD; if untreated, it progresses into Myocardial Infarction and Heart Failure [
Modern advancement in genome-wide analyses has identified large number of long non-coding RNAs (LncRNAs), which are transcribed in human genome. LncRNAs are non-protein coding RNA, which are 200 to 10,000 nucleotides in length [
LncRNA uc063iod.1 is human equivalent of LncRNAE330013P06 (E33), which is also named as Cardiac Mesoderm Enhancer-associated Noncoding RNA (CARMN) or MIR143HG [
In this study, the expression pattern of LncRNA uc063iod.1 in plasma of the patients with CAD and a variety of other diseases were detected by qRT-PCR. And we tested the potential correlations between the plasma LncRNA uc063iod.1 levels and clinicopathological features in patients with CAD. The possibility of LncRNA uc063iod.1 to serve as a biomarker for the diagnosis of CAD was discussed.
Plasma samples were prepared using the blood extracted from total 101 patients undergoing coronary artery angiogram (63 CAD patients and 38 Non-CAD patients) in Zhongda Hospital, Southeast University, Nanjing PR China, between May 2017 and October 2017. The study was approved by ethics committee of medical faculty of the Zhongda Hospital, and written informed consent was taken from all included patients or their families in accordance with the Declaration of Helsinki [
1) Case: Patients with >50% stenosis of at least one of the major coronary arteries (left main coronary trunk, anterior descending branch, circumflex artery and right coronary artery) confirmed by two independent intervention cardiologist during percutaneous coronary artery angiogram.
2) Control: No stenosis in any coronary arteries confirmed by two independent interventional cardiologists.
3) Undergone percutaneous coronary artery angiogram for the first time.
4) Age >50 yrs to <75 yrs.
1) Patients with severe valvular disease, malignant tumors, other systemic disease like renal failure and hepatic disease.
2) Patients who rejects to take part in the study.
3) Patients with serious acute infections in recent 4 weeks periods.
10 ml of arterial blood were collected in EDTA tube from radial artery, after the insertion of the arterial catheter before the administration of anticoagulants. Plasma were collected after centrifugation at 2000 rpm at 4˚C for 10 mins and stored at −80˚C before use.
Total RNA was extracted from plasma using TRIzol reagent (Invitrogen, USA) and cDNA synthesis was carried out by 1 μl of RNA, using 5xPrimeScriptRT Master Mix (Takara Biomed Co. Ltd., Beijing) 4 μl, in total volume of 20 μl reaction mixture. SYBR Green Real-Time PCR Master Mixes (Nanjing VazymeBioTech Co. Ltd.) was used to prepare PCR reaction system. The following primers were used in PCR reactions: 5’-TCTTTCTCACAGGCCGCATT-3’ (forward) and 5’-TGATTTCTCCACGGTCAGGC-3’ (reverse) for LncRNA uc063iod.1; 5’-GCACCGTCAAGGCTGAGAAC-3’ (forward) and 5’-TGGTGAAGACGCCAGTGGA-3’ (reverse) for GAPDH. The primers used were all synthesized by Genscript USA Inc. PCR reaction conditions were: denaturation at 95˚C for 1 minute, followed by 40 cycles of denaturation 95˚C for 5 seconds annealing at 60˚C for 31 seconds. Data were analysed using 2−∆∆CT methods, and relative expression level of LncRNA uc063iod.1 was normalized to endogenous control GAPDH. Each sample was independently tested for three times.
SPSS 20.0 software was used to analyse all data. The test of normality of data was checked by Shapiro-Wilk test. Independent t-test or Mann-Whitney U-test were used to assess the differences in the expression levels of genes between CAD and Non-CAD groups. Continuous variables were tested with student t test and 2 × 2 contingency table analysis of χ2 was used for categorical variables. Abnormally distributed data were processed using non-parametric Mann-Whitney U test. Kruskal-Wallis test was used to test the significance of expression of the gene with respect to the degree of lesion. The correlation between the expression levels of genes was performed by Spearman correlation coefficient. Finally, the Receiver Operating Characteristic (ROC) curve analysis was performed to analyse the sensitivity and specificity of the expression level of LncRNA uc063iod.1. The area under the ROC curve (AUC) was used to summarise the diagnostic accuracy of the genes. A value of p < 0.05 was considered statistically significant.
Case control study was designed; blood samples were collected from total 101 patients, which were divided into two groups, 63 patients with CADas Case and 38 Non-CAD as Control. Then we conducted a comparison analysis of clinicopathological characteristics of the study population. In this study we found that CAD patients were aged 64.17 ± 6.60 years, comparable with 61.88 ± 3.93 years in Non-CAD group. There was significance differences between CAD and Non-CAD in status of gender (p = 0.001), hypertension (p = 0.001), HDL (p = 0.042), WBC (p = 0.048), neutrophil (p = 0.027), monocyte (p = 0.027) and HbA1c (p = 0.028), whereas parameters like age, height, BMI, heart rate, diabetes mellitus II, smoking, trop I, LVEF, lymphocyte, TC, TG and LDL could not show any significance difference (
The expression of LncRNA uc063iod.1 in 63 CAD patients and 38 Non-CAD patients were detected by quantitative real-time polymerase chain reaction. As shown in
Characteristics | CAD | Non-CAD Control | p value | |
---|---|---|---|---|
AgeΩ (year) | N (Missing) | 63 (0) | 38 (0) | 0.055 |
Mean ± Std. | 64.17 ± 6.60 | 61.88 ± 3.93 | ||
Min/Max | 52/75 | 54/68 | ||
Median | 65 | 61.75 | ||
Range | 23 | 14 | ||
Gender# | Male | 44 (69.84%) | 14 (36.84%) | 0.001 |
Female | 19 (30.16%) | 24 (63.16%) | ||
Total (Missing) | 63 (0) | 38 (0) | ||
HeightΩ (cm) | N (Missing) | 63 (0) | 38 (0) | 0.060 |
Mean ± Std. | 167.25 ± 7.296 | 164.42 ± 7.176 | ||
Min/Max | 147/178 | 148/178 | ||
Median | 168 | 164 | ||
Range | 31 | 30 | ||
BMIΩ (kg/m2) | N(Missing) | 63 (0) | 38 (0) | 0.974 |
Mean ± Std. | 24.004 ± 2.587 | 23.98 ± 3.17 | ||
Min/Max | 18.29/30.11 | 17.85/32.34 | ||
Median | 23.66 | 23.29 | ||
Range | 11.82 | 14.49 | ||
Heart rateΩ (bpm) | N (Missing) | 63 (0) | 38 (0) | 0.415 |
Mean ± Std. | 77.21 ± 12.75 | 79.47 ± 14.64 | ||
Min/Max | 60/120 | 51/126 | ||
Median | 74 | 79 | ||
Range | 60 | 75 | ||
Hypertension# (mm of Hg) | Yes | 49 (77.78%) | 14 (36.84%) | 0.001 |
No | 14 (22.22%) | 24 (63.16%) | ||
Total (Missing) | 63 (0) | 38 (0) | ||
Diabetes mellitus type II# | Yes | 38 (60.31%) | 17 (40.74%) | 0.128 |
No | 25 (39.69%) | 21 (55.26%) | ||
Total (Missing) | 63 (0) | 38 (0) | ||
Smoking# | Yes | 19 (30.16%) | 10 (26.32%) | 0.679 |
No | 44 (69.84%) | 28 (73.68%) | ||
Total (Missing) | 63 (0) | 38 (0) | ||
TropIØ (ng/ml) | N (Missing) | 61 (2) | 36 (2) | 0.070 |
Mean ± Std. | 0.706 ± 3.09 | 0.14 ± 0.178 | ||
Min/Max | 0.1/19 | 00/0.1 | ||
Median | 0.01 | 0.01 | ||
Range | 19.01 | 0.1 |
HbA1CØ (%) | N (Missing) | 49 (14) | 24 (14) | 0.028 |
---|---|---|---|---|
Mean ± Std. | 0.706 ± 3.09 | 0.672 ± 3.83 | ||
Min/Max | 5.20/14.30 | 5.20/10.50 | ||
Median | 7.10 | 6.15 | ||
Range | 9.10 | 5.30 | ||
LVEFØ (%) | N (Missing) | 52 (11) | 33 (5) | 0.481 |
Mean ± Std. | 66.25 ± 7.17 | 67.57 ± 6.65 | ||
Min/Max | 52/80 | 55/80 | ||
Median | 67 | 68 | ||
Range | 28 | 25 | ||
WBCØ (109/L) | N (Missing) | 61 (2) | 37 (1) | 0.048 |
Mean ± Std. | 6.818 ± 2.268 | 5.9611 ± 1.812 | ||
Min/Max | 3.05/15.56 | 3.36/13.01 | ||
Median | 6.51 | 5.6200 | ||
Range | 12.51 | 9.65 | ||
NeutrophilØ (109/L) | N (Missing) | 61 (2) | 37 (1) | 0.027 |
Mean ± Std. | 4.624 ± 2.051 | 3.856 ± 1.638 | ||
Min/Max | 1.63/13.26 | 1.61/11.22 | ||
Median | 4.22 | 3.53 | ||
Range | 11.63 | 9.61 | ||
LymphocyteΩ (109/L) | N (Missing) | 61 (2) | 37 (1) | 0.986 |
Mean ± Std. | 1.633 ± 0.739 | 1.635 ± 0.561 | ||
Min/Max | 0.34/5.69 | 0.53/2.71 | ||
Median | 1.47 | 1.62 | ||
Range | 5.35 | 2.18 | ||
MonocyteØ (109/L) | N (Missing) | 61 (2) | 37 (1) | 0.027 |
Mean ± Std. | 0.411 ± 0.171 | 0.339 ± 0.126 | ||
Min/Max | 0.19/0.89 | 0.16/0.74 | ||
Median | 0.40 | 0.31 | ||
Range | 0.70 | 0.58 | ||
TCΩ (mmol/L) | N (Missing) | 58 (5) | 31 (7) | 0.646 |
Mean ± Std. | 4.355 ± 1.225 | 4.4729 ± 0.972 | ||
Min/Max | 2.16/8.86 | 2.89/6.94 | ||
Median | 4.25 | 4.57 | ||
Range | 6.70 | 4.05 | ||
TGØ (mmol/L) | N (Missing) | 58 (5) | 31 (7) | 0.288 |
Mean ± Std. | 1.768 ± 0.0677 | 1.554 ± 1.0732 | ||
Min/Max | 0.7/4.9 | 0.5/4.9 | ||
Median | 1.355 | 1.230 | ||
Range | 4.3 | 4.3 |
HDLΩ (mmol/L) | N (Missing) | 58 (5) | 31 (7) | 0.042 |
---|---|---|---|---|
Mean ± Std. | 1.088 ± 0.242 | 1.200 ± 0.25 | ||
Min/Max | 0.75/1.88 | 0.81/1.60 | ||
Median | 1.055 | 1.20 | ||
Range | 1.13 | 0.79 | ||
LDLΩ (mmol/L) | N (Missing) | 58 (5) | 31 (7) | 0.616 |
Mean ± Std. | 3.093 ± 1.259 | 2.58 ± 0.738 | ||
Min/Max | 1.10/6.52 | 1.41 | ||
Median | 2.81 | 4.24 | ||
Range | 5.42 | 2.83 |
BMI = Body mass index, bpm = beats per minute, TG = triglyceride, TC = total cholesterol, HDL = high density lipoprotein, LDL = low density lipoprotein, WBC = white blood cell, LVEF = left ventricular ejection fraction, TropI = troponin I, HbA1c = glycated haemoglobin. Data are presented as mean ± standard deviation (std.), Minimum/Maximum (Min/Max), Median and Range. N = number of patients. P < 0.05 was considered significant. ΩStudent T-test. #2 × 2 contingency table analysis of χ2. ØMann-Whitney U-test.
The mean expression level LncRNA uc063iod.1 in plasma of CAD patients found to be statistically significant with respect to numbers of coronary arteries involved, i.e. p < 0.01. The LncRNA uc063iod.1 expression is significantly increased in single vessel, bi vessel and three vessel disease than in non CAD patients. But there was no significant difference between the expression level of LncRNA uc063iod.1 with respect to degree of lesion in coronary arteries among CAD patients (
Plasma levels of LncRNA uc063iod.1 in patients with Stable Angina Pectoris, unstable Angina Pectoris and Acute Myocardial Infarction found to have significant difference with the control (p < 0.01). As shown in
Coromarker is already an established biomarker of CAD [
The correlation ship of the genes with clinical features of CAD and Non CAD
LncRNA uc063iod.1 | Coromarker | |
---|---|---|
r | p value | |
0.225 | 0.024 |
patients were calculated by bivariate spearman correlation analysis, which showed positive correlation ship between the expression levels of LncRNA uc063iod.1 with DM type II (r = 0.327, p = 0.009), hypertension (r = 0.296, p = 0.018) and negative correlation ship with Smoking (r = −0.363, p = 0.003) in CAD patients. There was negative correlation ship between the expression levels of LncRNA uc063iod.1 with diabetes mellitus II (r = −0.437, p = 0.006), hypertension (r = 0.413, p = 0.01) among Non CAD patients, shown in
Receiver Operating Characteristic (ROC) curve was used to determine the best positive plasma LncRNA uc063iod.1 reference values to improve its diagnostic value of CAD. The area under the ROC curve (AUC) of LncRNA uc063iod.1 for diagnosing CAD was 0.701 (95%CI: 0.598 - 0.804), with the sensitivity 77.8% and specificity 57.9% (
LncRNA uc063iod.1 | Correlation with | CAD | Non CAD control | ||
---|---|---|---|---|---|
r | p value | r | p value | ||
Smoking | −0.363 | 0.003 | 0.076 | 0.649 | |
DM II | 0.327 | 0.009 | −0.437 | 0.006 | |
Hypertension | 0.296 | 0.018 | −0.413 | 0.01 | |
TC | 0.202 | 0.129 | −0.188 | 0.311 | |
TG | 0.039 | 0.769 | −0.306 | 0.094 | |
HDL | 0.027 | 0.841 | 0.015 | 0.937 | |
LDL | 0.156 | 0.240 | −0.096 | 0.606 | |
HbA1c | −0.08 | 0.586 | −0.077 | 0.720 | |
Coromarker | Smoking | 0.074 | 0.563 | 0.005 | 0.974 |
DM II | 0.131 | 0.306 | −0.036 | 0.829 | |
Hypertension | 0.277 | 0.028 | −0.020 | 0.906 | |
TC | −0.20 | 0.880 | −0.355 | 0.050 | |
TG | 0.337 | 0.01 | −0.374 | 0.038 | |
HDL | −0.109 | 0.415 | −0.136 | 0.465 | |
LDL | 0.234 | 0.77 | −0.311 | 0.088 | |
HbA1c | 0.081 | 0.580 | −0.224 | 0.293 |
DM II = Diabetes Mellitus type II, TG = triglyceride, TC = total cholesterol, HDL = high density lipoprotein, LDL = low density lipoprotein, HbA1c = glycatedhemoglobin.
Coronary Artery Disease (CAD) is major cause of sudden cardiac death and it usually progresses to myocardial infarction if remain untreated. Among the individuals with CVDs, coronary artery disease accounts for 22% of early deaths and 15% of late death [
LncRNA uc063iod.1 being host gene of mir-143 and mir-145, is a crucial regulator of cardiac cell differentiation and homeostasis [
Coromarker is already an established biomarker of CAD. We had bivariate spearman correlation analysis between LncRNA uc063iod.1 and Coromarker which showed positive correlation. This result helps to provide supporting point for LncRNA uc063iod.1 to serve as biomarker for Coronary Artery Disease. Furthermore, we observed the positive correlation ship between the expression level of LncRNA uc063iod.1 with smoking, hypertension and diabetes mellitus type II in CAD patients, whereas negative correlation between the level of LncRNA uc063iod.1 with hypertension and diabetes mellitus in Non CAD patients. The underlying mechanism of these correlation is still unknown. However, there is a study indicated that the upregulation of the level of LncRNA uc063iod.1 in monocytes derived from diabetic patients [
The study was conducted in a Chinese Han Population who underwent coronary artery angiogram in Zhongda Hospital, Nanjing, China. Further studies expanded to different population groups and regions may ensure the true significance of the association between LncRNA uc063iod.1 and CAD. Selection bias might have affected our results. The sample size is relatively very small. Therefore, multicentre large number of studies are needed to be conducted for validation and consideration of LncRNA uc063iod.1 as biomarker of CAD. In conclusion, the expression for LncRNA uc063iod.1 was upregulated in the plasma of patients with CAD. However, the mechanism and pathway of the LncRNA uc063iod.1 in progression of CAD need further exploration.
The authors declare that there is no conflict of interest.
Rajan, V., Ma, G.S. and Jin, H. (2018) Expression of Circulating Plasma Long Non-Coding RNA uc063iod.1 and Its Relationship with Coronary Artery Disease. Open Journal of Internal Medicine, 8, 131-145. https://doi.org/10.4236/ojim.2018.82014