Application of interferon-induced transmembrane protein 1 in diagnosis of acute coronary syndrome

Abstract

The application belongs to the technical field of biology and specifically relates to application of interferon-induced transmembrane protein 1 in diagnosis of acute coronary syndrome. The application discloses that IFITM1 is a new type of serum marker and has potential value in diagnosis of acute coronary syndrome (ACS). First, it is found through ELISA technology detection that the level of IFITM1 in serum of ACS patients is significantly higher than that of healthy people and coronary heart disease patients. Second, the level of IFITM1 is related to the severity of coronary heart disease, and the result of ROC analysis shows that the AUC values of IFITM1 in diagnosis of CAD and ACS are 0.9530 and 0.9932 respectively, indicating that IFITM1 has high diagnostic efficiency and can effectively distinguish ACS patients from other groups. The discovery of IFITM1 provides a new auxiliary diagnosis method for clinic and is helpful for early identification of ACS patients, so that treatment measures can be taken in time and the prognosis of patients can be improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of interferon-induced transmembrane protein 1 in the diagnosis of acute coronary syndrome. Background Technology

[0002] Acute coronary syndrome (ACS) is a clinical syndrome caused by the rupture or erosion of atherosclerotic plaques in the coronary arteries, leading to thrombus formation and resulting in acute or subacute myocardial ischemia and hypoxia. ACS is characterized by rapid onset, rapid progression, and poor prognosis, and is one of the major cardiovascular diseases threatening human health. Therefore, early diagnosis and risk assessment of ACS are of great significance for improving patient outcomes.

[0003] In clinical practice, the diagnosis of ACS requires comprehensive consideration of the patient's clinical symptoms (such as chest pain), electrocardiogram results, cardiac enzyme levels, and coronary angiography findings. Currently, there is no specific diagnostic indicator for ACS. Troponin I (cTnI) is a widely recognized important serum marker for diagnosing and differentiating ACS; it rises in the early stages (2-6 hours) after the onset of chest pain. However, elevated cTnI can also be seen in other diseases, such as viral myocarditis, heart failure, and chronic renal failure. While D-dimer and C-reactive protein are associated with inflammation and stress responses, they lack cardiac specificity due to their involvement in multiple pathophysiological processes. Coronary angiography is considered the authoritative method for diagnosing coronary artery stenosis; however, as an invasive procedure, it is expensive and requires sophisticated techniques and equipment, which limits its acceptance and widespread application among patients. Therefore, exploring a highly specific and sensitive blood test indicator for the early identification of ACS is of paramount importance.

[0004] In recent years, the application of serum biomarkers in the diagnosis and risk assessment of acute coronary syndrome (ACS) has received widespread attention. Studies have shown that traditional myocardial injury biomarkers, such as troponin and creatine kinase isoenzymes, have limitations in the early diagnosis of ACS. Currently, several studies have identified some promising novel serum biomarkers for ACS, such as heart-type fatty acid-binding protein (H-FABP), growth differentiation factor-15 (GDF-15), and angiotensin-converting enzyme 2 (ACE2). These biomarkers show significantly elevated expression levels in the serum of ACS patients and are correlated with disease severity, potentially becoming new indicators for the auxiliary diagnosis and prognostic assessment of ACS. However, the clinical application value of these biomarkers still requires further validation.

[0005] Interferon-inducible transmembrane protein 1 (IFITM1) is a member of the interferon-inducible transmembrane protein family. It not only possesses antiviral functions but is also upregulated in various tumors. Currently, existing technologies have not found a clinical link between IFITM1 and acute coronary syndrome (ACS). Summary of the Invention

[0006] This invention aims to provide a novel biomarker for acute coronary syndrome (ACS) to address the insufficient sensitivity and specificity of existing ACS diagnostic techniques. By detecting and analyzing serum protein levels in patients, this invention found that IFITM1 is elevated in the serum of ACS patients. Therefore, this invention provides a new serum biomarker, IFITM1, with high specificity and sensitivity, enabling accurate diagnosis of ACS in its early stages and reducing misdiagnosis and missed diagnosis.

[0007] The first aspect of the present invention aims to provide the application of IFITM1 as a biomarker in the diagnosis of acute coronary syndrome.

[0008] The second aspect of the present invention aims to provide the application of reagents for detecting IFITM1 in the preparation of products for diagnosing acute coronary syndrome.

[0009] The third aspect of this invention aims to provide a method for constructing a model for acute coronary syndrome.

[0010] The fourth aspect of the present invention is to provide a system for the diagnosis of acute coronary syndrome.

[0011] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0012] In a first aspect, the invention provides the use of IFITM1 (UniProt ID: P13164) as a biomarker in the diagnosis of acute coronary syndrome.

[0013] In some embodiments of the present invention, the acute coronary syndrome refers to a group of clinical syndromes based on the pathological basis of rupture or invasion of atherosclerotic plaques in the coronary arteries, leading to complete or incomplete occlusive thrombosis. These include acute ST-segment elevation myocardial infarction, acute non-ST-segment elevation myocardial infarction, and unstable angina. The emphasis is on the acute and subacute clinical manifestations that occur after rupture or invasion of atherosclerotic plaques.

[0014] The second aspect of the present invention aims to provide the application of reagents for detecting IFITM1 in the preparation of products for diagnosing acute coronary syndrome.

[0015] In some embodiments of the present invention, the reagents include reagents for detecting interferon transmembrane protein 1 at the protein level or RNA level.

[0016] In some embodiments of the present invention, the reagent for detecting interferon transmembrane protein 1 at the protein level is selected from reagents of one or more detection methods from the group consisting of: chemiluminescence, immunofluorescence, protein chip, proteometry, immunohistochemistry, patch tracing based on labeling technology, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

[0017] In some embodiments of the present invention, the reagent for detecting interferon transmembrane protein 1 at the RNA level is selected from reagents of one or more detection methods from the group consisting of: high-throughput RNA sequencing, RNA-in situ hybridization, digital PCR, and quantitative real-time PCR.

[0018] In some embodiments of the present invention, the product includes a test kit, a test chip, or a test strip.

[0019] In some embodiments of the present invention, the test sample for the product is a serum, plasma, or tissue sample.

[0020] In some embodiments of the present invention, the test subjects of the product include humans.

[0021] A third aspect of this invention aims to provide a method for constructing a model for the diagnosis of acute coronary syndrome, comprising the following steps:

[0022] Models were constructed using interferon-induced transmembrane protein 1 expression levels.

[0023] In some embodiments of the present invention, the model construction algorithm includes at least one of logistic regression, linear discriminant analysis, support vector machine, random forest, and recursive partitioning tree.

[0024] A fourth aspect of the present invention provides a system for diagnosing acute coronary syndrome, the system comprising a computing device for diagnosing acute coronary syndrome based on the test results of any one of a1) to a7).

[0025] In some embodiments of the present invention, the detection results include protein level results or RNA level results.

[0026] In some embodiments of the present invention, the system further includes any one or more of the following:

[0027] 1) Detection result collection device, also known as detection result input device, may specifically be one or more of the following: mouse, keyboard, touch screen display, one or more buttons, one or more switches, one or more triggers, etc.

[0028] 2) Diagnostic result output device, also known as diagnostic result display device, can specifically be one or more of the following: liquid crystal display (LCD), light-emitting diode (LED) display, plasma display, projection display, touch screen display, etc.

[0029] 3) Diagnostic result sending device, which can send the results of distinguishing whether the subject is in a strong or weak risk group to an information communication terminal device that can be viewed by the patient or medical staff.

[0030] The beneficial effects of this invention are:

[0031] This invention, through bioinformatics analysis and experimental verification, reveals the potential value of IFITM1 as a novel serum biomarker in the diagnosis of acute coronary syndrome (ACS). First, the discovery of IFITM1 improves the sensitivity and specificity of ACS diagnosis. ELISA testing showed that serum IFITM1 levels in ACS patients were significantly higher than in healthy individuals and patients with coronary artery disease (CAD). Specifically, the average IFITM1 level in the ACS group was 4.56 ng / ml, significantly higher than the 2.43 ng / ml in the CAD group and the 1.37 ng / ml in the Control group. Second, IFITM1 levels are correlated with the severity of CAD, aiding in patient assessment. ROC analysis showed that the AUC values ​​of IFITM1 in diagnosing CAD and ACS were 0.9530 and 0.9932, respectively, indicating that IFITM1 has high diagnostic efficacy and can effectively distinguish ACS patients from other groups. The discovery of IFITM1 provides a new auxiliary diagnostic tool for clinicians, helping to identify ACS patients early, thereby enabling timely treatment and improving patient prognosis. The implementation of this invention not only helps to improve the diagnostic accuracy of ACS, but also has important significance for reducing the risk of cardiovascular events and improving the quality of life of patients. Attached Figure Description

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0033] Figure 1 The expression results of the IFITM1 gene in datasets GSE28829, GSE163154, and GSE43292 are shown.

[0034] Figure 2 The results of quantitative real-time PCR detection of IFITM1 gene expression in human carotid atherosclerotic plaques and adjacent tissues.

[0035] Figure 3Results of measuring the level of IFITM1 protein in peripheral blood serum of healthy individuals (Control), patients with coronary artery disease (CAD), and patients with acute coronary syndrome (ACS) using ELISA technology.

[0036] Figure 4 The ROC curve represents the diagnostic efficacy of peripheral blood serum IFITM1 in patients with CAD and ACS. Detailed Implementation

[0037] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0038] The experimental materials used in this invention are as follows:

[0039] The reagents used in this invention include: Trizol (Invitrogen) and PrimeScript. TM RT reagent Kit with gDNA Eraser (Takara), TB Premix Ex TagTMII (Takara), IFITM1 and internal reference gene amplification primers, ELISA kit (Wuhan Feien Biotechnology); Consumables used in this invention include: pipette tips, enzyme-free EP tubes, PCR eight-tube sets, etc.

[0040] The experimental instruments used in this invention include: a spectrophotometer (Thermo Fisher), an enzyme-linked immunosorbent assay (ELISA) reader (Thermo Fisher), and a real-time PCR instrument (ABI).

[0041] Example 1: Discovery of IFITM1 serum biomarkers

[0042] 1. Bioinformatics Analysis

[0043] Dataset acquisition: Datasets GSE28829, GSE163154 and GSE43292 related to atherosclerotic plaques were downloaded from the public database GEO.

[0044] Data Analysis: The GSE28829 dataset, containing early and late-stage atherosclerotic plaque tissue, was analyzed. The analysis revealed that IFITM1 gene expression was significantly higher in late-stage atherosclerotic plaques at an earlier stage. Figure 1(A). Analysis of the GSE163154 dataset, which contains both hemorrhagic and non-hemorrhagic plaque tissue, revealed that IFITM1 gene expression was significantly higher in hemorrhagic plaques than in non-hemorrhagic plaques. Figure 1 Similarly, in the dataset GSE43292, which includes atherosclerotic plaques and their adjacent tissues, the expression level of the IFITM1 gene in the plaques was significantly higher than that in their adjacent tissues. Figure 1 (C)

[0045] 2. Expression of the IFITM1 gene in atherosclerotic plaques and adjacent tissues

[0046] Sample Collection: Samples were collected from 12 patients with carotid artery stenosis who underwent carotid endarterectomy. The samples were divided into carotid plaque tissue and adjacent tissue. Participants were treated at Meizhou People's Hospital, and all participants signed informed consent forms.

[0047] RNA extraction from human carotid artery exfoliated tissue: Take appropriate amounts of tissue, cut them into small pieces, add liquid nitrogen and grind them into powder. Transfer the powder to an EP tube containing 1 mL Trizol and vortex for 2 h to ensure complete tissue lysis. Centrifuge the sample at 11000g for 10 min at 4°C. Transfer the supernatant to a new EP tube containing 200 μL chloroform. After vigorous vortexing, let it stand at room temperature for 3 min. Centrifuge at 11000g for 15 min at 4°C. Transfer the colorless aqueous phase (RNA solution) to a new EP tube. Add 500 μL isopropanol and mix. Let it stand at room temperature for 10 min. Centrifuge at 11000g for 10 min at 4°C. Discard the supernatant and add 75% ethanol. Centrifuge at 7500g for 5 min at 4°C. Discard the supernatant. Air dry at room temperature for 3–5 min. Add 25 μL of enzyme-free water to dissolve the total RNA and determine the concentration.

[0048] RNA reverse transcription: Add 2 μL of 5×gDNA Eraser Buffer and 1 μL of gDNA Eraser to a 200 μL PCR tube, then add 1 μg of RNA to each tube, and finally add enzyme-free water to adjust the volume to 10 μL. Gently tap, centrifuge briefly, and incubate at 42°C for 2 min in a PCR instrument to remove genomic DNA. Add 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5×PrimeScript Buffer 2 (for Real Time), and 4 μL of enzyme-free water to the above reaction solution. Gently tap, centrifuge briefly, and synthesize cDNA in a PCR instrument following these steps: 37°C for 15 min, 85°C for 5 s.

[0049] Quantitative real-time PCR detection: Add 1 μL cDNA, 1 μL amplification primer, 3 μL enzyme-free water, and 5 μL LTB to a 200 μL PCR tube. Premix Ex Tag TM II. In the PCR instrument, the following steps are performed sequentially: pre-denaturation: 95℃, 30s; PCR reaction: 95℃, 5s, 60℃, 34s, with 40 cycles of PCR reaction. After generating the fluorescence curve values, the relative expression level of the IFITM1 gene is calculated according to the formula 2^-△△Ct, where △Ct=Ct(target gene)-Ct(internal reference gene).

[0050] The specific primer sequences are as follows:

[0051] IFITM1 forward primer: 5'-CTTGAACTGGTGCTGTCTGG-3' (SEQ ID NO: 1);

[0052] IFITM1 reverse primer: 5'-AATCAGGGCCCAGATGTTCA-3' (SEQ ID NO: 2);

[0053] GAPDH forward primer: 5'-CTCCTCCACCTTTGACGC-3' (SEQ ID NO: 3);

[0054] GAPDH reverse primer: 5'-CCACCACCCTGTTGCTGT-3' (SEQ ID NO: 4).

[0055] Gene expression detection: The expression level of the IFITM1 gene in carotid artery tissue was detected at the mRNA level using quantitative real-time PCR. Results showed ( Figure 2 The expression level of the IFITM1 gene in atherosclerotic plaque tissue was significantly higher than that in its neighboring tissue.

[0056] These results indicate that IFITM1 is highly expressed in plaque tissue and is associated with plaque stability. Ruptured and hemorrhagic plaques exhibit even higher levels of IFITM1 expression.

[0057] Example 2: Validation of serum biomarkers

[0058] To verify whether the IFITM1 protein can serve as a peripheral blood serum biomarker for ACS, this embodiment collected peripheral blood serum samples from 32 healthy individuals (Control), 135 patients with coronary artery disease (CAD), and 50 patients with ACS.

[0059] Disease differentiation criteria:

[0060] CAD is chronic atherosclerosis, and patients typically present with stable angina. Treatment primarily involves medication and lifestyle modifications.

[0061] ACS is an acute event caused by plaque rupture. Patients present with acute ST-segment elevation myocardial infarction, acute non-ST-segment elevation myocardial infarction, and unstable angina. Treatment for ACS requires emergency management such as PCI or thrombolytic therapy.

[0062] There were no statistically significant differences in the baseline clinical data among the three groups of subjects (P>0.05) (Table 1). Laboratory test results of the subjects are shown in Table 2.

[0063] Table 1. Clinical baseline data of subjects in Control, CAD, and ACS programs.

[0064]

[0065] Table 2 Laboratory test results of subjects with Control, CAD, and ACS

[0066]

[0067] ELISA test results:

[0068] 1) Sample preparation and dilution: Take an appropriate amount of serum and dilute it with sample diluent at a ratio of 1:2.

[0069] 2) Sample addition: Add 100 μL of standard and diluted serum sample to the corresponding well and incubate at 37°C for 90 minutes;

[0070] 3) Washing: Discard the supernatant and wash the plate twice with 1x washing buffer;

[0071] 4) Add biotin-labeled antibody: Add 100 μL of biotin-labeled antibody working solution to the well and incubate at 37°C for 60 minutes;

[0072] 5) Washing: Discard the supernatant and wash the plate three times with 1x washing buffer;

[0073] 6) Add HRP-streptavidin conjugate (SABC): Add 100 μL of SABC working solution to the well and incubate at 37°C for 30 minutes;

[0074] 7) Washing: Discard the supernatant and wash the plate 5 times with 1x washing buffer;

[0075] 8) Add TMB substrate: Add 90 μL of TMB to the well and incubate at 37°C in the dark for 10-20 minutes;

[0076] 9) Termination of reaction: Add 50 μL of termination solution to each well, measure the OD value at 450 nm using an ELISA reader, plot the standard curve and calculate the sample concentration.

[0077] The level of IFITM1 protein in the serum samples was assessed using ELISA technology. The results showed ( Figure 3 The average levels of IFITM1 in the Control, CAD, and ACS groups were 1.37, 2.43, and 4.56 ng / ml, respectively, with significant differences between each pair. The ACS group represents a severe type of coronary artery disease, manifested as unstable angina or myocardial infarction, while the CAD group represents stable coronary artery disease with a relatively milder condition.

[0078] ELISA results showed that IFITM1 levels were significantly higher in the ACS group than in the CAD group, suggesting that IFITM1 may be an indicator of the severity of coronary artery disease. Higher levels may indicate more unstable coronary plaques and a greater risk of acute cardiovascular events.

[0079] Diagnostic efficacy assessment:

[0080] The efficacy of serum IFITM1 protein in the clinical diagnosis of CAD and ACS was evaluated using receiver operating characteristic (ROC) curve analysis. The results showed that... Figure 4 Compared with healthy controls, the AUC values ​​of IFITM1 were 0.9530 and 0.9932, respectively, and the use of IFITM1 protein as a clinical diagnostic biomarker for ACS has higher diagnostic efficacy.

[0081] Based on the above bioinformatics analysis and experimental verification, this invention found that the IFITM1 gene is highly expressed in atherosclerotic plaques and is associated with plaque stability. The serum level of IFITM1 in the peripheral blood of ACS patients is significantly higher than that in Control and CAD patients, and the protein encoded by this gene can effectively diagnose ACS patients. Therefore, IFITM1 has the potential to serve as a biomarker for ACS, providing a new auxiliary means for the diagnosis of ACS.

Claims

1. Application of reagents for detecting interferon-induced transmembrane protein 1 in the preparation of products for diagnosing coronary artery disease; The test samples for the product are serum, plasma, or tissue samples.

2. The application according to claim 1, characterized in that: The reagents include those for detecting interferon-induced transmembrane protein 1 at the protein or RNA level.

3. The application according to claim 2, characterized in that: The reagents for detecting interferon-induced transmembrane protein 1 at the protein level are selected from one or more of the following detection methods: chemiluminescence, immunofluorescence, protein chip, proteometry, immunohistochemistry, patch tracing based on labeling technology, Western blotting, and enzyme-linked immunosorbent assay (ELISA). The reagents for detecting interferon-induced transmembrane protein 1 at the RNA level are selected from one or more of the following detection methods: high-throughput RNA sequencing, RNA-in situ hybridization, digital PCR, and quantitative real-time PCR.

4. The application according to claim 1, characterized in that: The products include test kits, test chips, or test strips.

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