Study on the mechanism and clinical value of miR-210-3p and miR-582-5p in acute myocardial infarction by targeting MXD1

Abstract

Background

Acute myocardial infarction (AMI) occurs rapidly and is a major cause of death in patients with coronary artery disease (CAD). To explore new biomarkers of AMI and its pathogenesis.

Methods

Mining key miRNAs (miR-210-3p, miR-582-5p) and gene (MXD1) using the GEO and starbase databases. The expression of miRNAs and related genes was detected by qPCR. The clinical value of miR-210-3p/miR-582-5p was evaluated by constructing ROC curves. The targeted regulation of MXD1 by miR-210-3p and miR-582-5p was demonstrated by the dual-luciferase assay and transfection of mimics. The levels of cTnI, MDA, and Fe²⁺ were measured using kits. Overexpressing miR-210-3p, miR-582-5p, and MXD1 to explore their roles in hypoxia-induced cardiomyocyte injury.

Results

MiR-210-3p and miR-582-5p were deficient in AMI patients and hypoxic cardiomyocytes, while MXD1 was high-expressed. MiR-210-3p combined with miR-582-5p could well distinguish ST elevation-AMI and non-ST elevation-AMI patients from CAD patients, with AUC of 0.865 (0.806–0.923) and 0.813 (0.744–0.881), respectively. The mimics of miR-210-3p and miR-582-5p significantly reduced luciferase activity in cells transfected with the wild-type luciferase vector of MXD1 and inhibited MXD1 expression, which was promoted by their inhibitors. Overexpression of miR-210-3p/miR-582-5p saved hypoxia-induced ferroptosis, which was reversed by co-transfection of the MXD1 plasmid.

Conclusion

MiR-210-3p and miR-582-5p are good classifiers for AMI patients and CAD patients. They can negatively regulate the expression of MXD1 by directly targeting its mRNA, inhibiting ferroptosis of hypoxic cardiomyocytes, and may be effective predictors of CAD progression to AMI.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13019-025-03518-3.

Introduction

Acute myocardial infarction (AMI), also called myocardial infarction, is mainly due to the insufficient blood supply of the heart, myocardial hypoxia, and excessive oxygen consumption of the heart caused by myocardial necrosis [1]. AMI has a high morbidity and mortality rate worldwide and is more common in middle-aged and elderly people and high-risk groups such as heart disease, smoking, diabetes, and hyperlipidemia. AMI can be divided into ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (non-STEMI) based on electrocardiographic findings [2].

Coronary artery disease (CAD), such as coronary atherosclerosis, is the most common cause of AMI. The degree of coronary arteriosclerosis varies, and mild lesions have no obvious effect on the heart and no obvious symptoms. More severe lesions may cause narrowing of the lumen. The blood supply to the heart muscle is inadequate during hard work or emotional agitation, leading to symptoms such as angina, irregular heartbeat, and heart failure [3]. A myocardial infarction will occur when a larger branch of a coronary artery is completely or almost completely blocked and the corresponding myocardium dies due to lack of blood supply [4].

Ferroptosis is considered to be an important form of cardiomyocyte death in myocardial ischemia/reperfusion (IR) injury, characterized mainly by iron-dependent lipid peroxidation accumulation [5]. Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) are the end products of lipid peroxidation, and their content is positively correlated with ferroptosis [6]. Long-chain acyl-CoA (CoA) synthase 4 (ACSL4) is an enzyme that esterifies CoA to a specific polyunsaturated fatty acid, and catalyzes the biosynthesis of arachidonic acyl-CoA, contributing to the execution of ferroptosis by triggering phospholipid peroxidation [7]. Glutathione peroxidase 4 (GPX4) is a selenoprotein antioxidant enzyme that is considered to be an important regulator of ferroptosis. GPX4 can be used as a catalyst for glutathione (GSH) to clear intracellular reactive oxygen species (ROS) and reduce ferroptosis reaction [8]. Studies have shown that extensive iron death occurs in hypoxic cardiomyocytes and myocardial tissue of AMI mice [9, 10]. Epigallocatechin gallate (EGCG) can inhibit ferroptosis through the miR-450b-5p/ACSL4 axis to reduce myocardial ischemic injury in AMI mice [10]. LncRNA AC005332.7 inhibits ferroptosis in myocardial cells of AMI mice by regulating the miR-331-3p/CCND2 axis, thereby alleviating myocardial damage [11].

The dysregulation of microRNAs (miRNAs) is an important sign of AMI. Studies have shown that high levels of circulating miR-106b and miR-137 are not only effective indicators of early myocardial ischemia in CAD patients [12], but also represent a more severe degree of ischemia in the disease [13], indicating that they are valuable biomarkers for AMI. Here, we explored the mechanism of miR-210-3p and miR-582-5p in hypoxia-induced ferroptosis of cardiomyocytes by regulating MXD1, and explored their predictive value in predicting the occurrence of AMI, hoping to provide new ideas for the treatment and prevention of AMI.

Materials and methods

Bioinformatics

To screen the novel miRNAs or targets involving in AMI, the AMI-related GSE168856 and GSE61145 datasets were selected in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). GSE168856 is a miRNA expression dataset obtained through quantitative PCR analysis of the serum samples from healthy controls and STEMI/non-STEMI patients. GSE61145 is a dataset of gene expression based on STEMI patients and healthy controls. The GEO2R function in the database was used for differential expression analysis. The downstream targets of miRNAs were predicted using the starbase database (https://rnasysu.com/encori/index.php).

Cell culture and hypoxia model

Human cardiomyocytes (AC16) were purchased from SUNNCELL Bio (China) and cultured at 37℃ with 5% CO₂ using the same company’s special medium (SNLM-483). Referring to the published research [10, 14], AC16 cardiomyocytes were cultured in an anaerobic incubator containing 5% CO₂, 1% O_2, and 94% N₂ for 18 h, using a serum-free medium instead of a complete medium, to construct the hypoxic injury model.

Clinical samples

Based on the sample size calculation, 67 patients with STEMI (confidence = 95%, power = 80%, difference = 15, population variance = 900) and 72 patients with non-STEMI (confidence = 95%, power = 80%, difference = 15, population variance = 1000) were included in this retrospective study, and 63 healthy volunteers and 70 CAD patients (no AMI), matched by age and gender, were used as controls. The baseline information of volunteers was shown in Supplementary Table 1. The peripheral blood samples from these participants who underwent physical examinations or received medical treatment in Shengli Oilfield Central Hospital from 2016 to 2018 were well preserved. Before the experiment began, all the volunteers signed an informed consent form. This study was approved by the Ethics Committee of Shengli Oilfield Central Hospital.

Quantitative Real-time PCR (qPCR)

Total RNA was isolated using TRIzol reagent (Invitrogen, USA). Quantitative PCR was performed on a CFX96 Real-Time PCR apparatus (BIO-RAD, USA) using the BeyoFast™ SYBR Green One-Step qRT-PCR Kit (Beyotime, CHN). MiRNAs and genes were normalized using U6 and GAPDH, respectively, and their relative fold changes of expression were calculated using the 2^ΔΔCt method.

Relevant experimental kits

The secretion level of cardiac troponin I (cTnI) and the intracellular levels of MDA and Fe²⁺ were detected using the Human cTnI ELISA Kit (MULTI SCIENCES, CHN), lipid oxidation (MDA) Assay Kit (Beyotime) and Iron Assay Kit (Abcam, UK), respectively.

Evaluation of the clinical value of MiRNAs

The predictive value of serum miR-210-3p and miR-582-5p was evaluated using GraphPad Prism software to construct the receiver operating characteristic (ROC) curves for predicting AMI occurrence. The area under the curve (AUC) was used to measure prediction accuracy. SPSS software was used to analyze each factor by binary logistic regression.

Dual-luciferase report assay

The miR-210-3p/miR-582-5p mimics (Genepharma, CHN) and wild-type or mutant luciferase vectors of MXD1 (Genecreate, CHN) were co-transfected into cells. After 48 h, the luciferase activity of cells was measured by the Firefly Luciferase Reporter Gene Assay Kit (Beyotime).

Western blotting

The RIPA cell lysate (Solarbio, CHN) was used to extract total protein whose concentration was determined by the BCA Kit (Solarbio). The total protein was isolated using a 10% polyacrylamide separation glue and then electrically transferred to the PVDF membrane. The isolated proteins were incubated in 5% skim milk at room temperature for 2 h and then incubated in the primary antibody at 4℃ overnight. The next day, the PVDF membrane was transferred to the secondary antibody and incubated for 1.5 h, and the enhanced ECL chemiluminescence detection Kit (Vazyme, CHN) was used for development.

Statistical analysis

All experiments in this study were independently repeated three or more times. The experimental data were statistically analyzed using GraphPad Prism software. Student’s t-test was used for experimental data between two groups, and one-way ANOVA was used for data from three groups or more. p value less than 0.05 is statistically significant.

Results

MiR-210-3p and miR-582-5p in AMI

We used the GSE168856 dataset to obtain the differentially expressed miRNAs in the serum of patients with STEMI or non-STEMI (compared to healthy individuals). MiR-210-3p and miR-582-5p are the intersections of these miRNAs (Fig. 1A). Data from the GSE168856 dataset displayed an under-expression of miR-210-3p and miR-582-5p in the serum of patients with STEMI or non-STEMI (Fig. 1B-E).

Fig. 1.

Expressions of miR-210-3p and miR-582-5p in AMI. (A) Intersection of miRNAs differentially expressed in STEMI and non-STEMI patients, respectively, compared with healthy volunteers. Expressions of miR-210-3p (B and C) and miR-582-5p (D and E) in the GSE168856 dataset. (F) The secretion level of cTnI in cardiomyocytes. (G) Expressions of miR-210-3p and miR-582-5p in the hypoxic cardiomyocytes. Expressions of miR-210-3p (H) and miR-582-5p (I) in AMI patients. **p < 0.01; ***p < 0.001

In the hypoxia cell model, the cTnI level in the culture medium was significantly increased (Fig. 1F), indicating that the membrane integrity of cardiomyocytes was impaired by anoxia stress. Hypoxia led to downregulation of miR-210-3p and miR-582-5p in cardiomyocytes (Fig. 1G). The low expression of miR-210-3p and miR-582-5p in AMI was confirmed by qPCR on the clinical samples (Fig. 1H and I).

MiR-210-3p and miR-582-5p restrained hypoxia-induced ferroptosis

Transfection of miR-210-3p and miR-582-5p mimics obviously increased their intracellular levels (Fig. 2A and B). MDA level and ACSL4 expression were significantly up-regulated in hypoxic cardiomyocytes, but GPX4 expression was down-regulated (Fig. 2C-E), indicating that hypoxia promoted lipid peroxidation in cardiomyocytes. Moreover, hypoxia led to a significant increase of free Fe²⁺ in the cells (Fig. 2F), indicating that the cardiomyocytes initiated the ferroptosis program. Importantly, transfection of miR-210-3p and miR-582-5p mimics both reversed these changes in cells, saving the occurrence of ferroptosis, and suggesting that these two miRNAs are potential protective factors for myocardial ischemia.

Fig. 2.

Roles of miR-210-3p and miR-582-5p in hypoxia-induced ferroptosis. The mimics of miR-210-3p (A) and miR-582-5p (B). Effects of hypoxia and miR-210-3p/miR-582-5p mimics on MDA level (C), ACSL4 and GPX4 expressions (D and E), and Fe²⁺ level (F). **p < 0.01; ***p < 0.001

Clinical value of miR-210-3p and miR-582-5p

Because of the differences in the expression of miR-210-3p and miR-582-5p, we hypothesize that they may serve as biomarkers of AMI. The ROC curves were constructed using the serum miR-210-3p and miR-582-5p from CAD and AMI patients. The AUC of miR-210-3p and miR-582-5p in distinguishing CAD and STEMI patients was 0.750 (0.668–0.831) and 0.763 (0.683–0.843), respectively (Fig. 3A and B), and the AUC of their combination in predicting the occurrence STEMI was 0.865 (0.806–0.923) (Fig. 3C). The AUC of their univariate prediction of non-STEMI occurrence was 0.751 (0.670–0.832) and 0.686 (0.599–0.773), respectively (Fig. 3D and E), and the AUC of their joint prediction was 0.813 (0.744–0.881) (Fig. 3F). In addition, the multivariate binary logistic regression analysis showed that miR-210-3p and miR-582-5p were independent influencing factors of STEMI and non-STEMI (p < 0.001) (Table 1). These results indicate that miR-210-3p combined with miR-582-5p is a good classifier between CAD patients and AMI patients, and may be potential protective factors for AMI.

Fig. 3.

Clinical value of miR-210-3p/miR-582-5p. The ROC curves of miR-210-3p, miR-582-5p, and their combination in predicting STEMI (A-C) and non-STEMI (D-F)

Table 1.

Multivariate binary logistic regression analysis

Factors	STEMI				non-STEMI
	p value	OR	95% CI		p value	OR	95% CI
			Lower	Upper			Lower	Upper
Gender	0.683	1.204	0.495	2.93	0.358	1.475	0.644	3.379
Age	0.901	1.057	0.438	2.555	0.386	1.432	0.636	3.221
BMI	0.236	1.76	0.691	4.482	0.727	1.163	0.498	2.714
Smoking history	0.744	1.158	0.48	2.792	0.181	1.758	0.769	4.019
Diabetes mellitus	0.711	1.183	0.486	2.881	0.578	1.26	0.558	2.843
miR-210-3p	< 0.001	0.658	0.553	0.784	< 0.001	0.657	0.554	0.779
miR-582-5p	< 0.001	0.631	0.519	0.767	< 0.001	0.754	0.647	0.879

Notes: STEMI, ST elevation myocardial infarction; non-STEMI, non-ST elevation myocardial infarction; OR, odds ratio; CI, confidence interval; BMI, body mass index

Moreover, serum cTnI is the “gold standard” for diagnosing AMI in clinical practice. Our analysis shows that its AUC in predicting the occurrence of STEMI and non-STEMI is 0.976 (0.949-1.000) and 0.974 (0.946-1.000), respectively (Supplementary Fig. 1A-B). A correlation analysis displays that the expressions of miR-210-3p and miR-582-5p present a negative correlation with cTnI levels in AMI patients (Supplementary Fig. 1C-D). These evidences further illustrate the clinical value of serum miR-210-3p/miR-582-5p.

Negative regulation of MXD1 by miR-210-3p and miR-582-5p

For the further exploration of the mechanism, we used the starbase database to predict the downstream target genes of miR-210-3p and miR-582-5p respectively. Among them, MXD1 was their common target, and the GSE61145 dataset showed its high expression in the serum of STEMI patients (Fig. 4A and B). Figure 4C shows the interaction sites of miR-210-3p or miR-582-5p with MXD1 mRNA. The interaction between them was verified by the dual-luciferase report assay. Both miR-210-3p and miR-582-5p mimics significantly reduced the cellular luciferase activity transfected with wild-type MXD1 vector, but had little effect on cells transfected with mutant vector (Fig. 4D and E). Transfection of miR-210-3p or miR-582-5p mimics inhibited the expression of MXD1 which was further decreased by the co-transfection of these two mimics (Fig. 4F and G). These findings manifest that miR-210-3p and miR-582-5p synergistically negatively regulate the expression of MXD1 by targeting its mRNA.

Fig. 4.

Regulation of MXD1 by miR-210-3p/miR-582-5p. (A) The intersection of the targets of miR-210-3p/miR-582-5p and the high-expressed genes in STEMI patients. (B) The expression of MXD1 in the GSE61145 dataset. (C) The binding sequences between miR-210-3p/miR-582-5p and MXD1 mRNA, predicted in the starbase database. (D and E) The interaction between MXD1 mRNA and miR-210-3p/miR-582-5p was detected by the dual-luciferase report assay. (F and G) Regulation of MXD1 expression by miR-210-3p and miR-582-5p. *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant difference

Overexpression of MXD1 reversed the Inhibition of ferroptosis by miR-210-3p and miR-582-5p

MXD1 was abnormally high-expressed in hypoxic cardiomyocytes and AMI patients (Fig. 5A-D). The MXD1 overexpression plasmid (p-MXD1) was introduced to explore its role in AMI (Fig. 5E and F). Overexpression of MXD1 exacerbated the increase of MDA level and ACSL4 expression and the decrease of GPX4 expression induced by hypoxia (Fig. 5G-I), and further increased intracellular Fe²⁺ level (Fig. 5J), suggesting that MXD1 contributed to hypoxia-induced cardiomyocyte ferroptosis. In addition, overexpression of MXD1 reversed the inhibition of MDA, ACSL4, and Fe²⁺ and the promotion of GPX4 by miR-210-3p and miR-582-5p mimics in the hypoxia-damaged cell model (Fig. 5G-J), suggesting that miR-210-3p and miR-582-5p protected cardiomyocytes from hypoxia-induced ferroptosis by targeting MXD1.

Fig. 5.

Overexpression of MXD1 reversed the inhibition of ferroptosis by miR-210-3p/miR-582-5p. Expression of MXD1 in hypoxic cardiomyocytes (A-C) and AMI patients (D). (E-F) The overexpression plasmid of MXD1. pc, plasmid control. Effects of overexpression of MXD1, miR-210-3p/miR-582-5p on MDA level (G), ACSL4 and GPX4 expressions (H and I), and Fe²⁺ level (J) in hypoxic cardiomyocytes. *p < 0.05; **p < 0.01; ***p < 0.001

Discussion

AMI is a rapid-onset disease, and its treatment options usually include percutaneous coronary intervention, thrombolytic therapy, and surgical bypass surgery. However, once the golden treatment time is missed, it will bring irreversible harm to patients and even threaten their lives. Therefore, for AMI, effective prevention is greater than treatment.

MiRNAs are shown to have potential as biomarkers in a variety of human diseases, including AMI. The study by Guo et al. showed that the miR-124 levels in the peripheral blood of AMI patients were significantly higher than those of healthy controls. Moreover, they found that peripheral blood miR-124 peaked at 6 h after the onset of AMI, earlier than the peak of cTnI and creatine kinase isoenzyme MB (CK-MB). In addition, miR-124 distinguished AMI patients from healthy people with an AUC of 0.86 (0.815–0.937), suggesting that peripheral miR-124 is a strong candidate for the early diagnosis of AMI [15]. Another study showed that serum miRNA-203 levels were higher in STEMI patients than in controls, with a predicted AUC of 0.912 for STEMI. Moreover, their logistic regression analysis showed that miRNA-203 was an independent risk factor for STEMI (p < 0.05), suggesting that it may be a biomarker in the early prediction of STEMI [16].

To explore new biomarkers, we used bioinformatics to find miR-210-3p and miR-582-5p, which are closely related to AMI. MiR-210-3p and miR-582-5p were deficient in the serum of AMI patients, and their combination can effectively predict the occurrence of STEMI and non-STEMI, with AUC of 0.865 and 0.813, respectively. It is reported that upregulation of miR-210-3p can improve myocardial infarction and promote angiogenesis in mice by targeting EphrinA3 [17]. MiR-210 has been shown to improve cardiac function in AMI mice by inhibiting mitochondrial oxygen consumption and reducing ROS flux in cardiomyocytes during IR injury [18]. However, the role of miR-582-5p in AMI has not been reported. In addition to the predictive value of serum miR-582-5p for the occurrence of AMI, our cell experiments indicated that miR-582-5p was lowly expressed in hypoxia-induced cardiomyocytes. Its upregulation significantly reduced the MDA/Fe²⁺ level and ACSL4 expression and promoted the expression of GPX4, indicating that miR-582-5p can rescue cardiomyocyte injury caused by hypoxia by reducing ferroptosis.

Cardiac troponin (cTn) is a protein that regulates myocardial muscle contraction and is composed of subunits of three different genes, including cardiac troponin T (cTnT), cTnI, and troponin C (TnC). cTnI and cTnT are free in cells or bind to cardiac structural proteins. When the cell membrane of cardiomyocytes is damaged due to ischemia or hypoxia, free cTnI, and cTnT can be rapidly released from the cells into the blood at first, and then bound cTnI and cTnT are gradually decomposed and slowly released into the circulating blood [19]. Therefore, blood cTnI and cTnT are important indicators for the clinical diagnosis of AMI. In addition, when myocardial tissue injury is severe, CK-MB will be released into the blood, which is also one of the important indicators of AMI laboratory examination [20]. Our clinical data analysis presented a close relationship between cTnI and miR-210-3p/miR-582-5p: the level of serum miR-210-3p/miR-582-5p showed a downward trend with the increase of cTnI. To explore their mechanism in AMI, we constructed a model of hypoxic cardiomyocytes. Hypoxia led to injury of the cell membrane of cardiomyocytes and increased secretion of cTnI. Hypoxia significantly decreased the miR-210-3p and miR-582-5p levels in cardiomyocytes and led to the upregulation of MXD1. MXD1 is a member of the MYC/MAX/MAD network that mediates cell proliferation, differentiation, and apoptosis. MXD1 is a potential tumor suppressor [21–23], and its mutation may play a role in human leukemia [24, 25]. The dual-luciferase report assay and overexpression experiments showed that miR-210-3p and miR-582-5p synergistically negatively regulated the expression of MXD1. Moreover, miR-210-3p/miR-582-5p rescued hypoxia-induced ferroptosis of cardiomyocytes, which was reversed by overexpression of MXD1.

CAD is an important risk factor for AMI. By monitoring the changes in related biomarkers and taking preventive measures in advance, AMI may be effectively prevented and the damage to patients can be reduced. In this study, we identified the potential of miR-210-3p and miR-582-5p as novel biomarkers to predict the occurrence of AMI. In the future, we will expand the clinical sample size to further enhance their credibility as biomarkers. In terms of mechanism, miR-210-3p and miR-582-5p are upstream regulators of MXD1, and they can jointly inhibit the hypoxia-induced ferroptosis process of cardiomyocytes by inhibiting the expression of MXD1, which will provide new ideas for understanding the pathogenesis of AMI.

Electronic supplementary material

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Abbreviations

ACSL4

Acyl-CoA (CoA) synthase 4

AMI

Acute myocardial infarction

AUC

Area under the curve

CAD

Coronary artery disease

CK-MB

Creatine kinase isoenzyme MB

cTn

Cardiac troponin

cTnI

Cardiac troponin I

cTnT

Cardiac troponin T

EGCG

Epigallocatechin gallate

GEO

Gene Expression Omnibus

GPX4

Glutathione peroxidase 4

GSH

Glutathione

IR

Ischemia/reperfusion

MDA

Malondialdehyde

miRNAs

microRNAs

non-STEMI

Non-ST-elevation myocardial infarction

ROC

Receiver operating characteristic

ROS

Reactive oxygen species

STEMI

ST-elevation myocardial infarction

TnC

Troponin C

4-HNE

4-hydroxynonenal

Author contributions

SF G contributed to the study conception and design. Material preparation, data collection and analysis were performed by SS L and J H. The first draft of the manuscript was written by SS L and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

No funding was received for conducting this study.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was performed in line with the principles of the Declaration of Helsinki. All protocols of this investigation were approved by the Ethics Committee of Shengli Oilfield Central Hospital (Date:2018.06.07/No.2018012). The participants’ right to be informed about the study was ensured and written informed consent was obtained from all individual participants included in the study.

Consent for publication

All patients provided written informed consent.

Competing interests

The authors declare no competing interests.