2.1 Study Population and Sample Collection
This part of the research content is complied with the Declaration of Helsinki and has got the permission from the Ethics Committee of Weifang People’s Hospital. All individuals recruited for this study have signed written informed consent. A total of 265 subjects were selected in this study, including 139 patients diagnosed with ACS by this hospital and 126 patients with simple chest pain but excluded ACS as the control group. The diagnostic criteria of ACS referred to the 2013 ACCF/AHA ST-Elevation myocardial infarction management guidelines [2], the 2014 ACC/AHA/AATS/PCNA/SCAI/STS focused update of the guideline for the diagnosis and management of patients with stable ischemic heart disease [17], the 2014 AHA/ACC guideline for the management of patients with Non-ST-Elevation acute coronary syndromes [18]. Subjects with systemic inflammatory, malignant carcinoma, kidney infirmities, or left ventricular systolic lesions were excluded from the study. Venous blood was taken from all subjects, and the supernatant was retained after centrifugation and stored in a refrigerator at −80 C for later use. The demographic features and clinicopathological characteristics of subjects were collected and summarized.
2.2 RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
TRIzol LS Reagent (ThermoFisher, USA, Catalog No.: 10296010) was used to extract total RNAs from serum samples. RNA was reverse transcribed into cDNA using a one-step miRNA Reverse Transcription Kit (Haigene, Harbin, China, Catalog No.: D1801). The above cDNA sample and Seven 2 × SYBR Green qPCR MasterMix (Seven BIOTECH, Beijing, China, Catalog No.: 330502) were mixed to detect the relative expression of miR-320a-3p in an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems). The relative expression level of miR-320a-3p was normalized by U6 using 2−ΔΔCt method. The primer sequences for qRT-PCR analysis are as follows: the forward primers of miR-320a-3p and U6 were 5′-AAAAGCTGGGTTGAGAGGGCGA-3′ and 5′-CTCGCTTCGGCAGCACA-3′, respectively. And the reverse primers of both were the universal primers of the kit.
2.3 Establishment and Treatment of ACS Animal Model
All the animal-related protocols involved in this study were implemented under the standards for the care and use of laboratory animals. The Animal Care and Use Committee of Weifang People’s Hospital supported and approved the experimental programs. Sprague–Dawley rats were selected to establish an ACS model to simulate the state of ACS in human body and detect the expression of miR-320a-3p according to the previous literature [11]. Briefly, rats were intraperitoneally injected with 1% pentobarbital (Sigma, USA, Catalog No.: P-3761) for deep anesthesia and connected to a ventilator. Through ECG monitoring, the skin was cut at the upper intercostal area of the apex beat, and the heart was exposed after opening the chest cavity. The position of the anterior descending branch of the left coronary artery was confirmed and ligation with appropriate strength was performed with a medical 6/0 suture. After modeling, the chest cavity was sutured, and the animals were injected with penicillin to prevent infection. In the control group, thoracic cavity was opened only without vascular ligation. Rats in the model group were randomly split into four groups, which were respectively injected with 2 μg of miR-320a-3p mimic, miR-320a-3p inhibitor and miR-NC for 7 consecutive days for in vivo transfection via the tail vein. Finally, blood was collected from the tail vein of the rats, and the serum was separated and stored in the −80 C refrigerator for later use.
2.4 Enzyme-Linked Immunosorbent Assay (ELISA)
The concentration of von Willebrand factor (vWF) (Haematologic Technologies, USA, Catalog No.: HCVWF-0190) and heart-type fatty acid-binding protein (H-FABP) (Oxis research, UK, Catalog No.: 11230) and the levels of inflammatory factors, such as IL-6 (ThermoFisher, USA, Catalog No.: 88-7066-86), TNF-α (ThermoFisher, USA, Catalog No.: 88-7346-76), and IL-1β (ThermoFisher, USA, Catalog No.: 88-7120-77), were detected by ELISA according to the product instructions. In short, the reagent is balanced at room temperature for 30 min. At the same time, the samples to be tested were defrosted naturally at room temperature, and then co-incubated with antibodies for 1 h. Subsequently, the antibodies were removed, and the substrate solution was added and incubated at room temperature for 30 min in the dark. Finally, the stop solution was added to terminate the reaction, and the absorbance value was detected at 450 nm in the microplate reader (Bio-Tek Instruments, Germany) and the corresponding concentration was calculated. Each experiment was repeated in triplicate.
2.5 Luciferase Reporter Gene Assay
Through the prediction of target genes, we found that miR-320a-3p had a complementary binding site with X-linked inhibitor of apoptosis protein (XIAP), and it was preliminarily speculated that XIAP was the target gene of miR-320a-3p, and luciferase reporter gene assay was used to verify this prediction. Specific steps were as follows: the human embryonic kidney cell line HEK-239T cells were obtained from American Type Culture Collection (ATCC, Manassas, Virginia, USA). HEK-239 T cells were cultured in DMEM supplemented with 10% FBS and 1% double antibiotics in a cell incubator at 37 C. The 3′-UTR sequence fragment of XIAP was cloned into pGL3 vector to construct wild-type (wild-type) reporter vector XIAP 3′-UTR-WT and mutant-type (mutant-type) reporter vector XIAP 3′-UTR-MUT. The HEK-239T cells were seeded into 24-well plate and cultured overnight until the cell density reached 60% or more. Subsequently, according to the product specification, the cells were co-transfected with the above-mentioned vector and miR-NC, miR-320a-3p mimic or miR-320a-3p inhibitor using Lipofectamine 3000, respectively. After 48 h of transfection, cells were collected, and the luciferase activity of each group was measured using the dual-luciferase reporter system (Promega). Renilla luciferase was chosen as a control gene.
2.6 Statistical Analysis
Data were presented as (mean ± SD). ROC curve was constructed to estimate the diagnostic value of miR-320a-3p in ACS. Student t test was used for comparison between two groups. One-way ANOVA was selected for multiple groups’ comparison, and chi-square test was selected for comparison between categorical variables. Multiple linear regression analysis was used to determine the independent effects of variables related to miR-320a-3p. Logistic regression analysis was used to evaluate the relationship between different variables and the occurrence of ACS. A P value less than 0.05 was considered to be significantly different.