
==== Front
J Cardiothorac Surg
J Cardiothorac Surg
Journal of Cardiothoracic Surgery
1749-8090
BioMed Central London

39307907
2998
10.1186/s13019-024-02998-z
Research
Correlation analysis between the expression of serum microRNA-665 and the degree of coronary artery stenosis and major adverse cardiovascular events in patients with acute myocardial infarction
Wang Chen 1
Yang Jie 2
Shi Yujie 1
Liu Lining 1
Fu Yujie Yujiefu_zjk@163.com

1
1 Department of Pharmacy, Zhangjiakou First Hospital, No. 6, Jianguo Road, Qiaodong District, Zhangjiakou, 075000 China
2 https://ror.org/052vn2478 grid.415912.a 0000 0004 4903 149X Department of Cardiovascular, Shijiazhuang City Luancheng People’s Hospital, Shijiazhuang, 051430 China
23 9 2024
23 9 2024
2024
19 54330 4 2024
13 8 2024
© The Author(s) 2024
2024
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Background

The purpose of this study was to explore the expression of miR-665 in acute myocardial infarction (AMI) and evaluate its significance in the diagnosis and prognosis of AMI.

Methods

100 patients with AMI were selected as the study group and 80 healthy subjects were chosen as the control group. The levels of miR-665 were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in the two groups. The diagnostic value of miR-665 expression level in AMI was analyzed by the receiver operator characteristic (ROC) curve. Kaplan-Meier curve and Cox regression were used to evaluate the predictive value of miR-665 for major adverse cardiovascular events (MACEs) in patients with AMI within 30 days after percutaneous coronary intervention (PCI).

Results

The serum miR-665 level of the study group was significantly lower than that of the control group. The level of miR-665 was significantly correlated with clinical indicators of patients with AMI. ROC curve showed that miR-665 has a high diagnostic value for AMI. Survival analysis showed that Gensini score and miR-665 were independent risk factors for the occurrence of MACEs within 30 days after PCI in patients with AMI.

Conclusions

Abnormal decrease of serum miR-665 expression level in patients with AMI may increase the risk of MACEs occurrence after PCI.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13019-024-02998-z.

Keywords

Acute myocardial infarction
MicroRNA-665
Gensini score
Major adverse cardiovascular events
Hebei Medical Science Research Project20241321 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

In recent years, due to changes in diet structure and the intensification of population aging, the incidence of circulatory system diseases, mainly coronary artery disease (CAD), is increasing rapidly [1]. Acute myocardial infarction is a type of CAD. Its pathological essence is coronary atherosclerosis complicated with coronary artery thrombosis, which causes different degrees of occlusion of the diseased blood vessels, leading to myocardial tissue necrosis caused by long-term imbalance of oxygen supply and demand in the coronary artery [2, 3]. AMI is an acute disease with a high mortality and disability. At present, the key to the treatment of AMI lies in accurate early diagnosis and timely revascularization, which not only reduces damage to myocardial cells, but also greatly reduces the mortality rate of patients [4]. Recently, scientists have found multiple susceptible sites and segments related to AMI through large-scale genome analysis, indicating that AMI is caused by the interaction of environment and heredity [5]. Therefore, it is of great significance to find the diagnostic and prognostic markers related to AMI.

MicroRNAs (miRNAs) are a kind of endogenous non-coding small RNAs with highly conservative characteristics, which play an important roles in regulating proliferation, development, differentiation, and inflammation [6]. MiRNAs have been proved to be regulators of key pathways such as cell adhesion and inflammation in the pathological process of coronary atherosclerosis. They are involved in the pathogenesis of atherosclerosis by regulating lipid metabolism, oxidative stress, and endothelial function [7]. A study shown that the expression level of miR-208b in peripheral blood of patients with AMI was as high as 1600 times higher than that of healthy people [8]. Another study found that miR-1 is a risk factor affecting the prognosis of AMI, which can be used to predict the prognosis of AMI [9]. MiR-665 is located in gene 14q32.2, and its abnormal expression is associated with various diseases, such as tumor cell migration and invasion, myocardial cell inflammation and fibrosis [10]. It was found that the expression of miR-665 was decreased in patients with CAD, and the ectopic expression of miR-665 could obviously inhibit the growth of vascular smooth muscle cells [11]. Wang et al. reported that overexpression of miR-665 could regulate the expression of transforming growth factor β receptor 1 (TGFBR1) and inhibit the proliferation of vascular smooth muscle cells, thus alleviate the progress of atherosclerosis [12]. However, the research data on miR-665 in AMI population are still very few.

This study aimed to investigate the correlation between the level of miR-665 and the severity of coronary artery stenosis and poor prognosis in patients with AMI, and to provide valuable research data for finding biomarkers of AMI.

Methods

Subject inclusion and exclusion criteria

This study retrospectively analyzed the relevant information of 180 participants. Among them, 100 patients with AMI who underwent PCI admitted to the cardiology department of this hospital were selected as the study objects. The relevant indexes after PCI are summarized in Supplementary Table 1. Inclusion criteria: 1). It confirms to the diagnostic criteria published in the 2012 ESC/AHA/ACC guidelines [13]; 2). Coronary angiography was carried out and confirmed. 3). First onset. The exclusion criteria are as follows: 1). Combined with cardiomyopathy and other cardiovascular diseases; 2). Patients with history of chronic heart failure and myocardial infarction; 3). Complicated with liver and kidney dysfunction, mental illness, malignant tumor; 4). Previous history of vascular reconstruction or coronary artery transplantation. In addition, 80 healthy people with age and sex matching were selected as the control group during the same period. The control group were adults from the physical examination department who had no history of cardiovascular disease, and the results of electrocardiogram and stress echocardiography showed no suspicion of coronary artery disease. Patients or their immediate family members sign informed consent. This study conforms to the Helsinki Declaration and is implemented after being reviewed and approved by the Ethics Committee of Zhangjiakou First Hospital.

Sample collection

8mL fasting venous blood was gathered from subjects after enrollment, centrifuged for 15 min at a rate of 3000 r/min at 4℃, and the serum was separated and stored in a -80℃ refrigerator for later use. The AMI patients were sub-grouped according to the Gensini score obtained by coronary angiography. Gensini scores were performed on patients based on coronary angiography results by two experienced cardiologists who were unaware of the patient’s medical history and laboratory test results. Gensini scoring criteria: 0 for no coronary artery stenosis, scored 1 for 0 ~ < 26% coronary artery stenosis, scored 2 for 26% ~<51%, scored 4 for 51% ~<76%, scored 8 for 76% ~<91%, scored 16 for 91% ~<100%, and scored 32 for 100% coronary artery stenosis. The Gensini score for each AMI patient was the sum of each branch: 0–25 point is classified as mild, 26–49 is moderate, and ≥ 50 is severe.

Detection of serum miR-665 expression level

The expression level of miR-665 in serum was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Serum samples were collected and total RNA was extracted from serum by adding TRIzol reagent. The purity of RNA samples was detected by spectrophotometer, and qualified RNA specimens with absorbance of 1.8–2.1 were selected, and cDNA was synthesized from 1 µg total RNA by reverse transcription kit. After the reaction, the amplified cDNA was used as the PCR reaction template. RT-qPCR was carried out with ABI 7900 RT-qPCR and SYBR Green I fluorescence quantitative kit. All the reactions were performed using 2 multiple Wells. U6 was specified as the internal reference, the relative miR-665 expression was calculated according to the 2−△△Ct method, where △Ct = CtmiR−665-CtU6.

Study end point and follow-up

According to the prognosis of patients within 30 days after therapy, the patients were divided into two groups: good prognosis group (n = 26) and poor prognosis group (n = 74). The poor prognosis was defined as the occurrence of major adverse cardiovascular events (MACEs) within 30 days after surgery, such as sudden cardiac death, thrombosis, hospitalization for heart failure, atrial ventricular block, and angina recurrence. The good prognosis group was defined as no such MACEs within 30 days after surgery.

Statistical analysis

SPSS 22.0 software was used for data processing. The Kolmogorov-Smirnov test was used to evaluate the normality of the data. The measurement data conforming to normal distribution were expressed as mean ± standard deviation (SD), and t-test was used for inter-group comparison. Counting data was represented by n, and Chi-square test was used for comparison between groups. The correlation between miR-665 expression level and clinical indicators was examined by Pearson method. The diagnostic value of serum miR-665 in AMI was evaluated by receiver operator characteristic (ROC) curve. The patients were divided into miR-665 high expression group and miR-665 low expression group according to the mean level of miR-665. Kaplan-Meier method was applied to draw the survival curve, and the Log-rank test was used to compare the prognosis differences between the two groups. P < 0.05 means significant differences.

Results

Comparison of baseline data between the two groups

The general data of these two groups are summarized in Table 1. There were no significant differences in gender, age, body mass index (BMI), total cholesterol (TC), smoking history and drinking history in two groups (P > 0.05). There were significant differences in systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and cardiac troponin I (cTnI) between the two groups (P < 0.05).

Table 1 Comparison of subjects’ baseline data

Characteristics	Control group (n = 80)	AMI group (n = 100)	P	
Sex (Male/Female)	45/35	58/42	0.480	
Age (Years)	58.64 ± 7.08	59.27 ± 6.36	0.530	
BMI (kg/m2)	22.15 ± 3.16	22.73 ± 2.97	0.446	
Smoking history (n, %)	38 (47.5%)	49 (49.0%)	0.493	
Drinking history (n, %)	43 (53.8%)	55 (55.0%)	0.459	
Hypertension (n, %)	/	50 (50.0%)	/	
Diabetes (n, %)	/	22 (22.0%)	/	
Dyslipidemia (n, %)	/	48 (48.0%)	/	
Gensini score	/	25.56 ± 17.24	/	
SBP (mmHg)	121.62 ± 10.79	136.61 ± 13.16	< 0.001	
DBP (mmHg)	74.49 ± 6.85	85.83 ± 6.91	< 0.001	
FBG (mmol/L)	4.77 ± 0.92	5.81 ± 2.20	< 0.001	
TC (mmol/L)	4.17 ± 0.81	4.36 ± 0.69	0.090	
TG (mmol/L)	1.41 ± 0.52	1.88 ± 0.76	< 0.001	
LDL-C (mmol/L)	2.43 ± 0.74	2.93 ± 0.98	< 0.001	
HDL-C (mmol/L)	1.67 ± 0.41	1.41 ± 0.70	0.003	
cTnI (ng/mL)	0.02 ± 0.01	8.96 ± 4.61	< 0.001	
Medication (n, %)				
β-receptor blockers	/	71 (71%)	/	
Statins	/	96 (96%)	/	
Antiplatelet drugs	/	97 (97%)	/	
ACEI/ARB	/	69 (69%)	/	
Calcium antagonists	/	19 (19%)	/	
Abbreviations: AMI: acute myocardial infarction; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; FBG: fasting blood glucose; TC: total cholesterol; TG: triglyceride; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; cTnI: cardiac troponin I. P < 0.05 means a significant difference

The expression level of serum miR-665 and its value in clinical diagnosis of AMI

The serum level of miR-665 was determined by RT-qPCR, and the results revealed that the level of miR-665 in patients with AMI was significantly lower than that in the control group (Fig. 1A, P < 0.001). Further, the ROC curve was established to evaluate the diagnostic significance of miR-665 in AMI. Figure 1B showed that the area under the curve (AUC) value of this curve is 0.909, and the sensitivity and specificity are 82.5% and 88.0%, respectively, which indicats that the diagnostic accuracy of miR-665 for AMI is high.

Fig. 1 The expression level of miR-665 in the serum of patients with AMI and its clinical diagnostic value in AMI. A. The level of serum miR-665 was down-regulated in AMI patients. B. The AUC value of ROC curve was 0.909, and the sensitivity and specificity were 82.5% and 88.0%, respectively. ***P < 0.001 vs. Controls. Abbreviations: AMI, acute myocardial infarction; ROC, receiver operator characteristic; AUC, area under the curve

Expression of miR-665 in different degrees of coronary artery disease

According to the Gensini score, AMI patients were grouped into mild (n = 41), moderate (n = 39) and severe group (n = 20). The results showed that compared with the mild group, the level of miR-665 in the moderate and severe groups was significantly decreased. In addition, compared with the moderate group, the level of miR-665 in the severe group was lower (Fig. 2, P < 0.001). The above results indicated that the expression level of miR-665 decreased gradually with the severity of the disease.

Fig. 2 Expression levels of miR-665 under different Gensini scores. ***P < 0.001 vs. mild group. ###P < 0.001 vs. moderate group

Correlation between miR-665 level and clinical indicators in AMI patients

Pearson correlation coefficient was used to evaluate the correlation between miR-665 and clinical indicators in patients with AMI, and the results were shown in Table 2. Results suggested that SBP, DBP, TG, LDL-C, cTnI and Gensini were strongly negatively correlated with the level of miR-665 in patients with AMI (P < 0.001). There was a moderate negative correlation between FBG and miR-665 (P < 0.05). In addition, HDL-C was positively correlated with the level of miR-665 (P < 0.05).

Table 2 Correlation between miR-665 and clinical indicators

Clinical indicators	r	P	
SBP (mmHg)	-0.531	< 0.001	
DBP (mmHg)	-0.493	< 0.001	
FBG (mmol/L)	-0.325	0.026	
TC (mmol/L)	-0.201	0.087	
TG (mmol/L)	-0.608	< 0.001	
HDL-C (mmol/L)	0.396	0.011	
LDL-C (mmol/L)	-0.655	< 0.001	
cTnI (ng/mL)	-0.701	< 0.001	
Gensini score	-0.687	< 0.001	
Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; cTnI: cardiac troponin I. P < 0.05 means significant difference

Follow-up analysis

On the basis of the mean value of miR-665, patients with AMI were divided into miR-665 high expression group (n = 50) and miR-665 low expression group (n = 50). Six patients in the high miR-665 expression group developed MACEs within 30 days after PCI surgery, while 20 cases in the low miR-665 expression group had MACEs (Table 3, P < 0.01). As shown in Fig. 3, Kaplan-Meier curve showed that low expression of miR-665 increased the risk of poor prognosis in patients with AMI (P < 0.05). As shown in Table 4, Cox regression analysis showed that Gensini score (HR = 7.818, 95% CI = 1.821–33.574, P = 0.006) and miR-665 (HR = 0.245, 95% CI = 0.068–0.878, P = 0.030) were independent factors influencing the occurrence of MACEs in AMI patients within 30 days after surgery.

Table 3 The number of patients with AMI who developed MACEs within 30 days after surgery

MACEs	miR-665 high expression
(n = 50)	miR-665 low expression
(n = 50)	P	
Cardiogenic shock (n)	0	3		
Sudden cardiac death (n)	0	1		
Thrombosis (n)	1	4		
Heart failure (n)	2	2		
Atrial ventricular block (n)	2	5		
Angina recurrence (n)	1	5		
Total (n, %)	6	20	0.002	
Abbreviations: MACEs, major adverse cardiovascular events. P < 0.05 means significant differences

Fig. 3 Kaplan-Meier curve of MACEs incidence in AMI patients within 30 days after surgery. Abbreviations: MACEs, major adverse cardiovascular events; AMI, acute myocardial infarction

Table 4 Multivariate COX analysis

Items	Multivariate analysis	
HR	95% CI	P	
Sex (Male/Female)	0.593	0.195–1.798	0.356	
Age (Years)	0.712	0.226–2.178	0.418	
BMI (kg/m2)	0.951	0.315–2.869	0.929	
Smoking history (n, %)	1.377	0.339–5.588	0.655	
Drinking history (n, %)	0.644	0.223–1.858	0.416	
Hypertension (n, %)	1.133	0.307–4.187	0.851	
Diabetes (n, %)	0.799	0.157–4.054	0.786	
Dyslipidemia (n, %)	0.693	0.202–2.374	0.559	
SBP (mmHg)	2.901	0.934–9.015	0.067	
DBP (mmHg)	2.277	0.927–8.288	0.181	
TC (mmol/L)	1.412	0.410–4.869	0.584	
TG (mmol/L)	0.908	0.354–2.333	0.539	
HDL-C (mmol/L)	0.427	0.148–1.236	0.117	
LDL-C (mmol/L)	1.697	0.571–5.042	0.341	
FBG (mmol/L)	0.699	0.223–2.190	0.842	
Gensini score	7.818	1.821–33.574	0.006	
cTnI (ng/mL)	3.449	0.954–12.446	0.059	
MiR-665	0.245	0.068–0.878	0.030	
Abbreviations: AMI: acute myocardial infarction; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; FBG: fasting blood glucose; TC: total cholesterol; TG: triglyceride; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; cTnI: cardiac troponin I. P < 0.05 means a significant difference

Discussion

AMI is one of the most threatening cardiovascular emergencies. Due to its sudden onset, dangerous condition, rapid progression, high mortality and disability rate, it has become the main cause of endangering people’s health and life safety all over the world [14]. This is particularly prominent in developing countries with relatively backward economic and medical strength, so accurate and early diagnosis is crucial to control disease development, limit myocardial damage and protect heart function [15]. This study demonstrated that the expression level of serum miR-665 showed an obvious downward trend in patients with AMI, and further ROC curve analysis showed that miR-665 had the ability to distinguish AMI from healthy individuals. In addition, Pearson analysis indicated that the miR-665 level was significantly correlated with the degree of coronary artery stenosis in patients with AMI. In a 30-day follow-up analysis, it was found that the low expression of miR-665 was closely related to the poor prognosis in patients with AMI after PCI.

Recently, with the development of pathophysiology of the circulatory system, the application and influence of miRNA in cardiovascular diseases have been gradually revealed. At present, related studies have confirmed that miRNA may be involved in the pathogenesis of cardiovascular diseases such as CAD [16, 17]. As reported by Rawat et al., miRNA plays an indispensable role in promoting cell proliferation, differentiation and apoptosis, and it can supply a new direction for the clinical first-line evaluation of the severity of cardiomyopathy [18]. In terms of prognosis, some scholars have pointed out that circulating miR-1 level within 3 h of chest pain has potential diagnostic value for AMI and is an independent risk factor affecting the prognosis of AMI [19]. In terms of mechanism, Yu et al. reported that miR-133 can protect cardiomyocytes from invasion of myocardial infarction [20]. Therefore, circulating miRNAs have the potential to be powerful diagnostic or prognostic markers for a variety of cardiovascular diseases under different physiological and pathological conditions. In this study, it was learned that miR-665 is up-regulated in the serum of patients with AMI. As for miR-665, which is the focus of this study, some scholars have previously proposed that it is related to cardiovascular diseases such as heart failure, AMI and atherosclerosis, and it may become a circulating biomarker for the diagnosis of heart failure [10, 21]. We further verified this in this experiment. First of all, it was observed that serum miR-665 level in the AMI group was significantly lower than that in the healthy control group, which was consistent with the expected data analysis results. As far as the traditional study of general cardiovascular clinical data is concerned, blood pressure, blood glucose and blood lipids have been proved as risk factors for cardiovascular diseases in previous studies [22]. In this experiment, general data between the two groups were compared, and it was found that there were differences in blood pressure, lipids, blood sugar, Gensini score and cTnI between the two groups, which was consistent with previous studies [23, 24].

Here, this study conducted a 30-day follow-up analysis of patients with AMI after PCI. From the Kaplan-Meier curve, it was found that the risk of poor prognosis in the group with high expression of miR-665 was significantly lower than that in the group with low expression of miR-665. Based on this, it is reasonable to speculate that miR-665 is significantly related to poor prognosis in patients with AMI. Further, Cox regression analysis showed that miR-665 and Gensini scores were independent risk factors for poor prognosis of AMI. Gensini score is usually used to assess the degree of CAD, and its good value has been confirmed in clinical practice [25]. Previous studies have confirmed that coronary artery disease with high degree of stenosis is more likely to develop into vascular occlusion and myocardial infarction, and the severity of coronary artery disease is one of the main predictors of cardiovascular adverse events and death risk [26]. In a study that included 102 AMI patients who underwent PCI, Wang et al. observed that the Gensini scores of patients with MACEs from hospitalization to 6 months after PCI were higher than that of the group without MACEs [27]. In this study, patients were divided into mild, moderate and severe disease groups according to Gensini scores. It was observed that the expression level of miR-665 decreased gradually with the deepening of severity, which further clarifying the correlation between miR-665 and the degree of coronary artery stenosis. More importantly, there was a significant negative correlation between miR-665 and Gensini scores in Pearson correlation analysis.

There are several limitations to this study. Firstly, single-center study and small sample size may lead to bias in experimental results. Therefore, multi-center experiments with larger sample size should be conducted in the future. Secondly, the short follow-up period does not represent a long-term situation. Finally, this study was an observational study, and the literature supporting miR-665 in the causes, mechanisms, plaque stability and treatment of acute myocardial infarction is insufficient, and its mechanism is still worthy of further study and verification.

In recent decades, researchers have been working to explore the mysteries of miRNAs and apply them to predict, diagnose, or treat diseases of the cardiovascular system. The death rate from AMI has not decreased in the past 10 years, which is also related to the lack of drugs available in clinical practice to prevent heart reperfusion injury. As a new type of biomarker, miRNA research in the cardiovascular field is still in its infancy and needs to be further explored. Studying the importance of miRNA in cardiovascular disease and understanding its mechanism of action will lead to better prevention and treatment of disease.

Conclusions

In summary, by analyzing the correlation between serum miR-665 level and clinical characteristics of patients with AMI, it was found that miR-665 level with relatively low expression in patients with AMI would present higher blood pressure, blood lipid, blood glucose and Gensini scores. In addition, low expression of miR-665 in patients with AMI increased the risk of poor prognosis 30 days after surgery. Therefore, it is speculated that monitoring the level of serum miR-665 can predict the occurrence of poor prognosis to some extent in the early stage of treatment of patients with AMI, which has a certain guiding role for clinical treatment and intervention.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

Not applicable.

Author contributions

Chen Wang, Jie Yang, Yujie Shi, Lining Liu made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, and draft of the manuscript. Yujie Fu revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

Funding

This study was funded by Hebei Medical Science Research Project (20241321).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Patients or their immediate family members sign informed consent, and this study conforms to the Declaration of Helsinki, and is implemented after review and approval by the Ethics Committee of Zhangjiakou First Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Abbreviations

AMI Acute myocardial infarction

RT-qPCR Reverse transcription quantitative polymerase chain reaction

ROC Receiver operator characteristic

MACEs Major adverse cardiovascular events

PCI Percutaneous coronary intervention

CAD Coronary artery disease

miRNAs MicroRNAs

TGFBR1 Transforming growth factor β receptor 1

BMI Body mass index

TC Total cholesterol

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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