LncRNA EBLN3P inhibits myocardial inflammation and apoptosis after acute myocardial infarction via targeting miR-675-3p

Study objects

This study received approval from the Tongji Hospital Branch Affiliated to Tongji University Medical Ethics Committee (No.: 20,220,012, Date of approval: 07-03-2022). Additional, and all participants provided written informed consent. Additionally, all procedures adhered strictly to the principles outlined in the Helsinki Declaration.

This study recruited 109 patients with AMI and 117 healthy controls from Tongji Hospital Branch Affiliated to Tongji University from January 2022 to June 2024. All AMI patients met the predefined inclusion and exclusion criteria. The inclusion criteria were as follows: Participants aged 27–80 years. For inclusion, the diagnosis of AMI must be confirmed either at admission or within 24 h post-admission. Clinical manifestations: Typical chest pain symptoms include a compressive, oppressive, or constrictive pain located behind the sternum, potentially radiating to the left upper limb, neck, jaw, or back, lasting more than 30 min, and not fully relieved by rest or sublingual nitroglycerin administration. Atypical symptoms may present as dyspnea, nausea, vomiting, fatigue, or palpitations, necessitating objective examinations to exclude other differential diagnoses. Auxiliary examinations: Electrocardiogram (ECG) demonstrated characteristic changes indicative of myocardial ischemia or necrosis. Myocardial injury markers reveal elevated levels of cardiac troponin I (cTnI) and creatine kinase isoenzyme (CK-MB), consistent with the dynamic evolution pattern associated with myocardial infarction. Imaging evidence: Echocardiography identifies segmental wall motion abnormalities, while coronary angiography confirms at least one major coronary artery stenosis of ≥70 % or complete occlusion. Conformity with universal definition: The case satisfies criterion outlined in the 2018 revised “Universal Definition of Myocardial Infarction”.

The exclusion criteria were as follows: patients with a history of coronary stent implantation or coronary artery bypass grafting; chest pain attributable to non-myocardial infarction causes, such as pleurisy, costochondritis, pneumothorax, and other conditions; individuals with known cardiac diseases other than AMI, including stable angina pectoris, hypertrophic cardiomyopathy, and other cardiomyopathies; cases where systemic diseases could confound the diagnosis, such as acute cerebrovascular events, severe infections, shock, and other critical illnesses; patients with a history of infectious diseases or stroke; and those with hematological, immune-related disorders, or malignant tumors.

Inclusion criteria for healthy controls were as follows: age and gender matched to the AMI group, no history of cardiovascular diseases (e.g., AMI, angina pectoris, heart failure), no severe infections, autoimmune diseases, or malignant tumors, and no recent use of anti-inflammatory or immunomodulatory drugs. Exclusion criteria included: those with a history of infectious diseases or stroke, those with abnormal liver or renal function, and those with habits such as smoking or heavy drinking that could potentially interfere with inflammatory markers.

Five milliliters of blood were drawn from the cubital vein of the upper limb into a containing separation gel (for serum) (Sanli Medical Technology, Hunan, China) tube immediately after diagnosis of AMI upon admission or within 24 h post-admission. The blood sample was centrifuged within 15 min (3000 rpm) of collection to separate the serum, and the following parameters were measured: blood lipid indices (total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG)), estimated glomerular filtration rate (eGFR), glycated hemoglobin (HbA1c), cardiac troponin I (cTnI), high-sensitivity C-reactive protein (hs-CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Selected inflammatory factors (e.g., TNF-α, IL-6) were quantified by ELISA on the same day, while the remaining serum was aliquoted and stored at −80 °C for subsequent analyses. RNA extraction was performed within one week of sample collection. The quality and concentration of the RNA were assessed using a NanoDrop spectrophotometer, with acceptable purity indicated by an A260/A280 ratio ranging from 1.8 to 2.0. Serum samples were maintained at −80 °C in a refrigerator to prevent repeated freeze-thaw cycles, thereby preserving the integrity and stability of RNA and proteins.

Cell culture and transfection

The human cardiomyocyte cell line AC16 (BeNa Culture Collection, BNCC339980) was obtained from the American Type Culture Collection (ATCC). The cells were maintained in DMEM medium (Solarbio, China), supplemented with 10–20 % fetal bovine serum (FBS) (Tianhang Biotechnology, China) and 1 % penicillin-streptomycin solution (double-antibiotic) to prevent microbial contamination. Cultivation was performed in a humidified incubator at 37 °C with 5 % CO₂. When the cells reached 80–90 % confluence in the culture flask, they were passaged at a specific ratio into new flasks for continued growth.

The hypoxia/reoxygenation (H/R) cell model was primarily utilized to simulate in vivo ischemia-reperfusion injury of tissues and organs. Based on previously reported methods [28], 29]. We replicated the conditions of AMI in vitro by subjecting AC16 cells to hypoxia/reoxygenation treatment. Specifically, well-growing AC16 cells were placed in a serum-free DMEM medium within a hypoxia chamber (Billups-Rothenberg, Inc.). The chamber was sealed tightly, and a gas mixture (95 % N₂, 5 % CO₂, with residual O₂ reduced to 1 %) was introduced to displace the oxygen, thereby establishing a hypoxic environment. The oxygen concentration was monitored by an oxygen sensor and maintained at≤1 %. Following an 18-h hypoxia period, the cells were transferred back to a standard incubator (37 °C, 5 % CO₂, 95 % air) for a 6-h reoxygenation phase. Cells maintained under normal culture conditions served as the control group.

AC16 cells were seeded in 96-well plates at a density of 2 × 10⁴ cells per well and cultured in an incubator at 37 °C with 5 % CO₂. The coding sequence of EBLN3P was cloned into the pcDNA 3.1 vector (GenePharma, China) to construct the overexpression plasmid pcDNA-EBLN3P. Cells were divided into several groups: control, H/R, H/R + pcDNA3.1, H/R + pcDNA3.1-EBLN3P, and co-expression groups including H/R + pcDNA3.1-EBLN3P + mimic-NC, H/R + pcDNA3.1-EBLN3P + miR-mimic. The miR-675-3p mimic and its corresponding negative control (mimic-NC) were synthesized by MedChemExpress (catalog numbers: HY-R02064, China). The miR-675-3p-mimic was used to regulate the expression level of miR-675-3p. The concentration of the pcDNA3.1-EBLN3P plasmid was 1 μg/μL. The concentration of miR-675-3p-mimic was 50 nM. The sequence of the miR-675-3p mimic and the sequence of the mimics-NC were as follows: miRNA-675-3p-mimics sense: CUG​UAU​GCC​CUC​ACC​GCU​CA; miRNA-675-3p-mimics antisense: AGC​GGU​GAG​GGC​AUA​CAG​UU; mimics-NC: UUG​UAC​UAC​ACA​AAA​GUA​CUG. Cell transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific, USA). After 6 h of co-cultivation, the culture medium was replaced with a fresh medium. Transfection efficiency was assessed using RT-qPCR to confirm that the transfection efficiency in AC16 cells exceeded 80 %.

Quantitative real-time PCR (RT-qPCR)

Total RNA was extracted from serum or cell samples using the Trizol (Invitrogen, China) reagent, followed by reverse transcription into cDNA using the First-Strand cDNA Synthesis kit (A5001, Promega, USA). PT-qPCR was conducted based on the standard SYBR-Green PCR kit (4,367,659, Applied Biosystems, China) protocol on ABI 7600 (Applied Biosystems, China). U6 and GAPDH were used as internal controls for miR-675-3p and EBLN3P, respectively. For PCR, the reaction conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 40 cycles consisting of denaturation at 95 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. The primer sequence was used in the RT-qPCR were as follows: EBLN3P forward, 5′-CAG​ACT​AAA​GGA​TCA​AGC​GAG​A-3′ and reverse 5′-ATC​AAT​TGC​CAC​AGG​TTG​AAG​A-3’; miR-675-3p forward 5′-GCC​GAG​CAT​CTT​ACC​GGA​CGT-3′ and reverse 5′-CTC​AAC​TGG​TGT​CGT​GGA-3’; U6 forward 5′-AAC​GCT​TCA​CGA​ATT​TGC​GT-3′ and reverse 5′-CTC​AAC​TGG​TGT​CGT​GGA-3’. GADPH forward: 5′-GGT​CGG​AGT​CAA​CGG​ATT​TG-3′ and reverse: 5′-ATG​AGC​CCC​AGC​CTT​CTC​CAT-3’. The relative mRNA expression levels of miR-675-3p and EBLN3P were quantified using the 2^−ΔΔCt method.

Enzyme-linked immunosorbent assay (ELISA) was used to measure the inflammatory factors

The concentrations of TNF-α, IL-1β, and IL-6 in serum were measured using ELISA. Serum samples and protein standards were diluted and added to the wells. Following the addition of the stop solution, the absorbance was read at 450 nm. All procedures were conducted strictly according to the kit (FineTest, China)) instructions. TNF-α: EH0302 (Range: 15.625–1000 pg/mL (1 pg=76mIU); Sensitivity: 9.375 pg/mL; Intra-assay CV (%): 5.82; Inter-assay CV (%):5.62). IL-1β: EH0185 (Range: 3.906–250 pg/mL (1 pg=140mIU); Sensitivity: 2.344 pg/mL; Intra-assay CV (%): 4.58; Inter-assay CV (%): 4.60). IL-6: EH0201 (Range: 4.688–300 pg/mL (1 pg=110mIU); Sensitivity: 2.813 pg/mL; Intra-assay CV (%): 4.91; Inter-assay CV (%): 4.63). The standard curve was prepared in accordance with the instructions of each kit. Each experiment was repeated three times.

Dual-luciferase reporter assay

Primers targeting the 3′-untranslated region (UTR) of the EBLN3P gene were designed using Primer Premier 5.0 and subsequently used for amplification via PCR. The resulting PCR products were inserted into the GLO plasmid (Promega, USA) to construct the wild-type EBLN3P vector, designated as EBLN3P-WT. Additionally, site-directed mutagenesis was conducted on the 3′-UTR sequence of EBLN3P to generate the mutant vector, EBLN3P-MUT. Perform sequencing to precisely identify the position of the mutation site within the sequence. These vectors, along with either the miR-675-3p mimic or the corresponding control, were co-transfected into AC16 cells using Lipofectamine 3000 (Invitrogen, USA). The plasmid DNA was transfected at a concentration of 0.5–2 μg per well. Following a 48-h incubation period, luciferase activity was assessed using a luciferase reporter gene assay kit (16,186, Thermo Fisher Scientific, USA). Luciferase activity was normalized to the co-transfected reference gene (e.g., Renilla luciferase) to account for variations in transfection efficiency and cell viability.

Apoptosis assay

Samples were collected and subjected to multiple washes with phosphate-buffered saline (PBS) to remove impurities. The cells were then resuspended, and stained with Annexin V-FITC and propidium iodide (Sigma-Aldrich, USA) to label cells with damaged membranes. FSC-A/FSC-H was used to define single-cell populations and exclude adherent cells. Quadrants were then set in the dual-parameter dot plot of Annexin V-FITC (FL1 channel) and PI (FL3 channel): the lower left quadrant represents live cells (Annexin V⁻/PI⁻), the lower right quadrant represents early apoptotic cells (Annexin V⁺/PI⁻), and the upper right quadrant represents late apoptotic/necrotic cells (Annexin V⁺/PI⁺). H9c2 cells were collected, washed twice with pre-cooled PBS, and adjusted to a concentration of (5 × 10⁵) cells/mL. A total of 100 μL of the cell suspension was mixed with 5 μL of Annexin V-FITC and 5 μL of PI, gently vortexed, and incubated in the dark at room temperature for 15 min. Subsequently, 400 μL of 1 × Binding Buffer was added, and the samples were immediately analyzed by flow cytometry. A total of 10,000 single-cell events were acquired per sample. The cells that received the staining process underwent analysis based on FACSCalibur flow cytometer (no. 342973, BD Biosciences, Pudong, Shanghai) by the use of CellQuest software. The final apoptotic cell count was determined as a total percentage of early apoptotic cells staining positive for Annexin V and negative for PI and late apoptotic cells positive for both Annexin V and PI. The cell apoptosis rate was calculated using the following formula: Cell apoptosis rate (%) = (apoptotic cell number/total cell number) × 100.

Statistical analysis

SPSS 16.0 and GraphPad Prism 7.0 were utilized for statistical analysis of data. Data were checked for normality via the Kolmogorov–Smirnov (K-S) normality test. Differences between two groups were calculated using the Mann–Whitney U test for non-normally distributed continuous variables, Student’s t-test for normally distributed continuous variables, and chi-square tests were employed to analyze inter-group differences. The diagnostic value of EBLN3P for AMI was assessed using receiver operating characteristic (ROC) curve analysis. Pearson correlation analysis was conducted to quantify the strength and direction of the linear relationship between two continuous variables. The cell experiment was independently repeated three times under identical conditions to ensure the reliability of the results. A p<0.05 was considered statistically significant.

Clinical features of study subjects

This study enrolled 109 patients with AMI and 117 healthy controls and compared the general clinical information and various characteristics between the two groups (Table 1). No significant differences were observed in age, sex, body mass index (BMI), smoking status, drinking, diastolic blood pressure (DBP), and HbA1c between the two groups (p>0.05). However, significant differences were noted in blood lipids (TC, LDL-C, HDL-C, TG), cardiac function (left ventricular ejection fraction (LVEF)), systolic blood pressure (SBP), eGFR, and cTnI, hs-CRP, TNF-α, IL-1β, IL-6 (p<0.05).

The expression of EBLN3P in AMI patients

The expression of EBLN3P was significantly decreased in AMI patients (Figure 1A). Additionally, the ROC curve analysis of EBLN3P demonstrated an area under the curve (AUC) of 0.881 (95%CI=0.8361–0.9253), with a sensitivity of 87.16 % and specificity of 78.63 % (p<0.0001) (Figure 1B), indicating that EBLN3P exhibited high diagnostic accuracy for distinguishing AMI patients from non-patients.

Figure 1:

LncRNA EBLN3P demonstrates potential predictive value for AMI. (A) LncRNA EBLN3P expression was downregulated in AMI (p<0.001). (B) ROC curve analysis of EBLN3P demonstrated an AUC of 0.881 (95%CI=0.8361–0.9253), with a sensitivity of 87.16 % and specificity of 78.63 % indicating that EBLN3P exhibited high diagnostic accuracy for distinguishing AMI patients from non-patients (p<0.0001). *** means p<0.001. Non-parametric tests and ROC curve analysis were used as statistical methods.

The correlation between EBLN3P and clinical features of AMI patients

We further investigated the correlation between EBLN3P levels and various clinical features (Table 2). Pearson correlation analysis revealed no significant association between EBLN3P levels and TC, LDL, DBP, or HbA1c (p>0.05). Conversely, EBLN3P levels exhibited a negative correlation with TG, SBP, cTnI, hs-CRP, TNF-α, IL-1β, IL-6, and SYNTAX scores.

The impact of EBLN3P on cardiomyocyte injury

We examined the expression levels of EBLN3P under various treatment conditions and its effects on inflammatory factors and apoptosis in cardiomyocytes (Figure 2). Transfection with EBLN3P significantly restored its expression following H/R treatment (Figure 2A). H/R treatment markedly increased the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6. Transfection with pcDNA3.1 had no significant effect on these cytokine levels. In contrast, transfection with EBLN3P partially attenuated the increase in TNF-α, IL-1β, and IL-6 levels but did not fully restore them to control levels (Figure 2B–D). Transfection with EBLN3P significantly decreased the apoptosis rate after H/R treatment (Figure 2E).

Figure 2:

Effects of EBLN3P on inflammatory factors and apoptosis of cardiomyocytes. (A) H/R treatment can reduce the expression level of EBLN3P (p<0.001). (B-D) H/R treatment significantly increased levels of TNF-α, IL-1β, and IL-6 (p<0.001). (E) H/R treatment resulted in a significant increase in apoptosis rate (p<0.001). *** means p<0.001. Non-parametric tests were used as statistical methods.

EBLN3P targeted and regulated miR-675-3p

The potential target genes associated with EBLN3P were identified using the ENCORI database, revealing that miR-675-3p was a likely target gene of EBLN3P (Figure 3A). The luciferase reporter assay further validated the direct interaction between EBLN3P and miR-675-3p (Figure 3B). Specifically, transfection of the miR-675-3p mimic into the EBLN3P-WT group resulted in a significant reduction in luciferase activity. Conversely, no significant change in luciferase activity was observed in the EBLN3P-MUT group following miR-675-3p mimic transfection (Figure 3B). Additionally, miR-675-3p expression were significantly elevated in the serum of AMI patients (Figure 3C). Pearson correlation analysis revealed a negative correlation between miR-675-3p and EBLN3P levels in AMI (r = −0.5245, p<0.0001) (Figure 3D). In addition, H/R treatment resulted in a significant increase in the expression level of miR-675-3p. Transfecting EBLN3P partially attenuated this upregulation following H/R treatment (Figure 3E).

Figure 3:

Targeting relationship of EBLN3P-miR-675-3p. (A) EBLN3P and miR-675-3p target binding sites. (B) The dual-luciferase reporter assay was employed to validate the direct targeting interaction between EBLN3P and miR-675-3p. (C) Expression level of miR-675-3p was upregulated in patients with AMI (p<0.001). (D) The expression levels of EBLN3P and miR-675-3p exhibit a negative correlation (p<0.0001). (E) H/R treatment resulted in increased expression levels of miR-675-3p (p<0.001). *** Means p<0.001. Non-parametric tests and pearson correlation coefficient were employed as statistical methods.

A regulatory network where EBLN3P and miR-675-3p were interconnected

Under H/R conditions, transfecting cells with EBLN3P or co-expressing EBLN3P with mimic-NC significantly reduced the relative expression level of miR-675-3p. However, co-expression of EBLN3P with miR-mimic partially counteracted this effect on miR-675-3p expression (Figure 4A). Transfection with EBLN3P or co-expression of EBLN3P with mimic-NC also significantly decreased the levels of inflammatory factors TNF-α, IL-1β, and IL-6, while co-expression of EBLN3P with miR-mimic partially mitigated these reductions (Figure 4B–D). Additionally, transfection with EBLN3P or co-expression of EBLN3P with mimic-NC significantly reduced cell apoptosis, whereas co-expression of EBLN3P with miR-mimic partially alleviated this anti-apoptotic effect (Figure 4E).

Figure 4:

Effects of EBLN3P-miR-675-3p co-transfection on inflammatory factors and apoptosis of cardiomyocytes. (A) Verification of co-expression of miR-675-3p. (B-D) co-expression of EBLN3P+ miR-mimic could partially increased the effects of EBLN3P on the levels of TNF-α, IL-1β, and IL-6 (p<0.001). (E) Co-expression of EBLN3P+ miR-mimic could partially increase the effect of EBLN3P on apoptosis (p<0.001). *** Means p<0.001. Non-parametric tests were used as statistical methods.