Application of NLRC3 in the preparation of products for diagnosis, screening or evaluation of coronary atherosclerotic heart disease

Abstract

This invention belongs to the field of biomedicine and discloses the use of NLRC3 in the preparation of products for diagnosing, screening, or evaluating coronary atherosclerotic heart disease. It provides a novel marker, NLRC3, for detecting coronary heart disease. The marker exhibits excellent stability, making it convenient and less invasive for the diagnosis of coronary heart disease while avoiding radiation risks. NLRC3 expression is significantly downregulated in human carotid artery plaques compared with normal carotid arteries; NLRC3 expression in peripheral blood mononuclear cells of patients with coronary heart disease is significantly lower than that in patients with normal coronary arteries; and NLRC3 expression in the plasma of patients with coronary heart disease is significantly lower than that in patients with normal coronary arteries. The receiver operating characteristic (ROC) curve analysis showed an AUC value as high as 0.926. When the diagnostic threshold for NLRC3 relative expression was set at 0.012, the sensitivity reached 85% and the specificity reached 80%. The accuracy of screening for coronary heart disease in patients with chest pain was 84.4%, demonstrating excellent clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of NLRC3 in preparing products for diagnosing, screening or evaluating coronary atherosclerotic heart disease. Background Art

[0002] Coronary atherosclerotic heart disease (CAD) is a cardiovascular disease caused by the formation of atherosclerotic plaques within the coronary arteries. Its pathological basis primarily involves damage to the arterial endothelium, inflammatory responses, and abnormal lipid metabolism. Recent studies have demonstrated that chronic inflammation plays a key role in the development and progression of CAD, particularly in the formation of atherosclerosis. During the atherosclerotic process, endothelial cell damage leads to increased vascular permeability, promoting the deposition of lipids such as low-density lipoprotein (LDL) within the vessel wall, forming lipid streaks that subsequently develop into atherosclerotic plaques. Furthermore, the infiltration and activation of inflammatory cells exacerbate this process, promoting smooth muscle cell proliferation and matrix remodeling, leading to the formation of unstable plaques that may, in some cases, rupture and cause acute coronary syndromes. With changing lifestyles and an increase in cardiovascular risk factors, the incidence of CAD is increasing annually, particularly among younger individuals. Early identification and intervention of CAD can significantly reduce the risk of heart attack and related complications, thereby improving patients' quality of life. In clinical practice, coronary angiography, a commonly used technique for CAD diagnosis, is not suitable for routine screening due to its invasive nature, difficulty in performing the procedure, and high cost. Therefore, circulating biomarkers, with their convenience, speed, and easy-to-interpret results, have gained importance in the clinical diagnosis of CAD.

[0003] Nucleotide-binding domain-like receptors (NLRs) are a family of cytoplasmic pattern recognition receptors that play a key role in identifying pathogens and monitoring changes in body homeostasis. NLRs are crucial for the regulation of inflammatory and immune-related diseases by regulating downstream signaling pathways. NLRC3 is a new member of the NLR family, discovered in Jurkat T cells and widely present in mammalian tissues. Studies have shown that NLRC3 negatively regulates immune inflammatory responses by interacting with TRAF6, reducing its ubiquitination levels, and inhibiting NF-κB activation. NLRC3-deficient mice exhibit enhanced inflammatory responses after endotoxin stimulation, confirming its immunomodulatory role. Rajendra et al. revealed that NLRC3 expression is downregulated in colorectal cancer tissues and inhibits tumor cell proliferation through the PI3K-PKB-mTOR signaling pathway. However, NLRC3 has not been studied in coronary atherosclerotic heart disease, and its function and role remain unclear. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of the aforementioned prior art, the primary objective of the present invention is to provide the use of NLRC3 in the preparation of products for diagnosing, screening, or assessing coronary atherosclerotic heart disease. This invention utilizes NLRC3 as a genetic marker for the timely, specific, and sensitive diagnosis, screening, or assessment of coronary atherosclerotic heart disease, enabling patients to identify their disease risk early in the disease's development and take appropriate preventive and treatment measures tailored to their risk.

[0005] The purpose of the present invention is achieved through the following solutions:

[0006] In a first aspect, the present invention provides a use of NLRC3 in the preparation of a product for diagnosing, screening or evaluating coronary atherosclerotic heart disease (CHD).

[0007] Furthermore, the expression level of NLRC3 in patients with coronary heart disease was significantly lower than that in patients with normal coronary arteries.

[0008] Furthermore, the expression level of NLRC3 in carotid plaques is significantly lower than that in normal carotid arteries. By comparing the results of NLRC3 immunofluorescence in situ detection in human carotid plaques and normal carotid arteries, the present invention found that the expression level of NLRC3 in carotid plaques is significantly lower than that in normal carotid arteries.

[0009] Furthermore, the expression level of NLRC3 in peripheral blood single cells of patients with coronary heart disease was significantly lower than that in patients with normal coronary arteries.

[0010] Furthermore, the expression level of NLRC3 in the plasma of patients with coronary heart disease was significantly lower than that in those with normal coronary arteries.

[0011] Furthermore, the present invention revealed through correlation analysis that the expression level of NLRC3 is significantly correlated with the coronary artery stenosis score index (Gensini score) of patients with coronary heart disease.

[0012] Furthermore, ROC curve analysis showed that NLRC3 had a high diagnostic value, with an AUC value of 0.926; when the diagnostic threshold of NLRC3 relative expression was set at 0.012, the sensitivity of diagnosing CAD was 85% and the specificity was 80%.

[0013] Furthermore, by detecting the expression level of NLRC3, it was further used as a screening tool for coronary heart disease, and compared with the results of coronary angiography, the accuracy was as high as 84.4%.

[0014] Based on this, the present invention proposes for the first time a biomarker NLRC3 for diagnosing coronary heart disease, provides a method for diagnosing coronary heart disease based on the circulating marker NLRC3, and proposes the application of NLRC3 in the preparation of products for diagnosing, screening or evaluating coronary atherosclerotic heart disease.

[0015] Furthermore, the products include chips, test kits, test strips, or high-throughput sequencing platforms. High-throughput sequencing platforms are specialized diagnostic tools, and products for detecting NLRC3 gene expression can be used on these platforms to detect NLRC3 gene expression. With the advancement of high-throughput sequencing technology, constructing a gene expression profile for an individual has become a highly convenient task. By comparing the gene expression profiles of diseased patients with those of healthy individuals, it is easy to analyze which gene abnormalities are associated with the disease. Therefore, determining through high-throughput sequencing that NLRC3 gene abnormalities are associated with coronary atherosclerotic heart disease also constitutes a use of the NLRC3 gene and is also within the scope of protection of the present invention.

[0016] Furthermore, the product can diagnose coronary heart disease by detecting the expression of the NLRC3 gene in a sample.

[0017] In a second aspect, the present invention also provides the use of a product for detecting NLRC3 gene expression in the preparation of a product for diagnosing, screening or evaluating coronary atherosclerotic heart disease.

[0018] Furthermore, the products for detecting NLRC3 gene expression include: products for detecting NLRC3 gene expression levels through RT-PCR, fluorescent quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform.

[0019] Furthermore, the product for detecting the expression level of the NLRC3 gene by RT-PCR includes at least a pair of primers for specifically amplifying the NLRC3 gene.

[0020] Furthermore, the product for detecting the expression level of the NLRC3 gene by fluorescent quantitative PCR includes at least a pair of primers for specifically amplifying the NLRC3 gene.

[0021] Furthermore, the product for detecting the expression level of the NLRC3 gene by immunoassay comprises at least an antibody that specifically binds to the NLRC3 protein.

[0022] Furthermore, the product for detecting the expression level of the NLRC3 gene by in situ hybridization at least includes a probe that hybridizes with the nucleic acid sequence of the NLRC3 gene.

[0023] Furthermore, the product for detecting the expression level of the NLRC3 gene by chip includes at least a protein chip and a gene chip; wherein the protein chip includes an antibody that specifically binds to the NLRC3 protein, and the gene chip includes a probe that hybridizes with the nucleic acid sequence of the NLRC3 gene.

[0024] Furthermore, sources of the NLRC3 gene and its expression products for diagnosing coronary heart disease include, but are not limited to, blood, tissue fluid, urine, saliva, cerebrospinal fluid, and other body fluids from which genomic DNA can be obtained. In a specific embodiment of the present invention, the source of the NLRC3 gene and its expression products for diagnosing coronary heart disease is blood.

[0025] Furthermore, the above-mentioned product was used to detect the expression level of NLRC3 in peripheral blood single cells of patients with coronary heart disease and those with normal coronary arteries for diagnosis. The results showed that the expression level of NLRC3 in patients with coronary heart disease was significantly reduced.

[0026] In a third aspect, the present invention also provides the use of NLRC3 in preparing a fluorescent quantitative PCR kit for diagnosing coronary heart disease.

[0027] Furthermore, the kit includes at least a pair of primers for specifically amplifying the NLRC3 gene, the sequences of which are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0028] Forward primer 5′-TGAAGGCGTTTGGTGTAGACC-3′ (SEQ ID NO. 1);

[0029] Reverse primer: 5′-CGATGCCAACGTCTCCTC-3′ (SEQ ID NO. 2).

[0030] In a fourth aspect, the present invention provides a fluorescent quantitative PCR kit for diagnosing coronary heart disease.

[0031] Furthermore, the kit includes at least a pair of primers for specifically amplifying the NLRC3 gene, the sequences of which are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0032] Furthermore, the kit also includes GAPDH primer sequences, SYBR Green fluorescent dye, etc. to ensure the accuracy and reliability of the experiment. Furthermore, the kit is used to diagnose coronary heart disease, aiming to provide an efficient and accurate molecular detection method.

[0033] The GAPDH primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0034] Forward primer 5′-ATGACATCAAGAAGGTGGTG-3′ (SEQ ID NO. 7);

[0035] Reverse primer: 5′-CATACCAGGAAATGAGCTTG-3′ (SEQ ID NO. 8).

[0036] The present invention provides a new marker NLRC3 for detecting coronary heart disease. The marker has excellent stability, brings convenience and low trauma to the diagnosis of coronary heart disease, and avoids radiation risks. The present invention found that NLRC3 expression in human carotid artery plaques was significantly downregulated compared with that in normal carotid arteries; RT-qPCR detection in peripheral blood mononuclear cells (PBMCs) of patients with coronary heart disease and normal coronary arteries revealed that NLRC3 expression was significantly reduced in PBMCs of patients with coronary heart disease; in patients with coronary heart disease, NLRC3 expression was significantly correlated with the coronary artery stenosis score (Gensini score); ROC curve analysis found that the marker exhibited significant value, with an AUC value as high as 0.926 (95% confidence interval: 0.889-0.963); when the diagnostic threshold of NLRC3 relative expression was set at 0.012, the sensitivity of the marker reached 85% and the specificity was 80%; the accuracy of screening for coronary heart disease patients in chest pain patients was 84.4%; these results demonstrate that NLRC3 has good diagnostic value; ELISA was used to detect NLRC3 expression in the plasma of patients with coronary heart disease and those with normal coronary arteries, and it was found that plasma NLRC3 levels were significantly reduced in patients with coronary heart disease. The present invention provides the use of NLRC3 in the preparation of products for diagnosing, screening, or evaluating coronary atherosclerotic heart disease. The above-mentioned excellent performance indicators indicate that the NLRC3 marker has excellent clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 Figure 3 is the expression of NLRC3 in carotid plaques and normal carotid arteries.

[0039] Figure 2 Amplification products were detected by agarose gel electrophoresis.

[0040] Figure 3 The expression of NLRC3 in peripheral blood mononuclear cells of patients with coronary atherosclerotic heart disease and patients with normal coronary arteries was investigated.

[0041] Figure 4 To investigate the relationship between NLRC3 expression and Gensini score in patients with coronary atherosclerotic heart disease.

[0042] Figure 5 The clinical diagnostic value of NLRC3 in coronary atherosclerotic heart disease.

[0043] Figure 6 The expression of NLRC3 in the plasma of patients with coronary atherosclerotic heart disease. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the materials involved in the following examples can be obtained from commercial channels. The methods described are conventional methods unless otherwise specified.

[0045] Example 1: Immunofluorescence in situ detection of NLRC3 expression in carotid plaque cores and normal carotid arteries in patients with coronary atherosclerotic heart disease (CHD)

[0046] 1. Research subjects

[0047] Carotid artery plaques and adjacent normal intimal tissue were collected from patients diagnosed with coronary heart disease in the emergency department of Meizhou People's Hospital. All patients signed informed consent.

[0048] 2. Experimental Methods

[0049] 2.1 Sample fixation

[0050] The carotid intima of CAD patients undergoing carotid endarterectomy was collected, and the plaque-containing intima and normal intima were separated, washed with normal saline, and fixed with 4% paraformaldehyde.

[0051] 2.1 Sample testing

[0052] (1) Sample preparation: First, place the tissue sections in environmentally friendly deparaffinization solutions I, II, and III, each for 10 minutes, to completely remove the paraffin. Then, treat with anhydrous ethanol I, II, and III for 5 minutes each, and finally wash with distilled water to prepare for the subsequent antigen retrieval step;

[0053] (2) Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 8.0) and perform antigen repair in a microwave oven. Follow the procedure of medium heat for 8 minutes, off heat for 8 minutes, and medium-low heat for 7 minutes. At the same time, be careful to prevent excessive evaporation of the buffer and avoid drying of the sections. After repair, let the sections cool naturally and wash them in PBS (pH 7.4) 3 times for 5 minutes each time to remove excess repair solution.

[0054] (3) Circle drawing and blocking: Shake the slide dry and use a histochemical pen to draw a circle around the tissue to prevent the loss of antibodies; add BSA to the circled area for blocking and incubate for 30 minutes;

[0055] (4) Primary antibody incubation: After removing the blocking solution, add NLRC3 rabbit polyclonal antibody (DF13411, Affinity Biosciences) to the slices, place the slices flat in a humidified chamber, and incubate overnight at 4°C to ensure effective binding of the antibody to the antigen;

[0056] (5) Secondary antibody incubation: Wash the slides three times in PBS (pH 7.4) for 5 minutes each time and spin dry. Add goat anti-rabbit secondary antibody (8889S, Cell Signaling Technology) to the circled area and incubate at room temperature for 60 minutes.

[0057] (6) Nuclear staining: Shake the slides dry, add DAPI staining solution to stain the nuclei, and incubate at room temperature for 10 minutes in the dark;

[0058] (7) Autofluorescence quenching: Shake the slide dry, add autofluorescence quencher, and incubate for 5 minutes, then rinse under running water for 10 minutes to reduce the interference of autofluorescence;

[0059] (8) Sealing and microscopic examination: The slides were washed three times in PBS (pH 7.4) for 5 minutes each time. After drying, they were sealed with anti-fluorescence quenching mounting medium to protect the fluorescence signal. The slides were placed under a confocal fluorescence microscope to collect images.

[0060] 3 Experimental results

[0061] Figure 1Immunofluorescence detection of NLRC3 expression in carotid artery plaques and normal carotid intima of patients with coronary heart disease. The results showed that the expression level of NLRC3 in carotid artery plaques was significantly lower than that in normal carotid arteries.

[0062] Example 2: NLRC3 expression in peripheral blood mononuclear cells (PBMCs) of patients with coronary atherosclerotic heart disease and patients with normal coronary arteries.

[0063] 1. Research subjects

[0064] All patients with coronary heart disease and normal coronary arteries signed informed consent.

[0065] All patients with coronary atherosclerotic heart disease (CAD) underwent coronary angiography for confirmation. The diagnosis of CAD followed the diagnostic criteria established by the American Heart Association (AHA) and the American College of Cardiology (ACC). Exclusion criteria included the following: ① cardiomyopathy, myocarditis, aortic dissection, various acute and chronic infectious diseases, pulmonary embolism, other non-coronary thrombotic diseases, and autoimmune diseases; ② malignant tumors, recent major trauma or surgery, severe liver and kidney damage, critical illness (such as cardiogenic shock), and patients using intraaortic balloon pump; ③ individuals who did not consent to participate in the study.

[0066] Normal coronary arteries were defined as patients whose coronary arteries were normal after coronary angiography. Patients with normal coronary arteries were matched to patients with CAD using age, sex, hypertension, and diabetes. Peripheral blood samples were collected from 80 patients with CAD and 80 patients with normal coronary arteries (NCA). Baseline data are shown in Table 1. No statistically significant differences in baseline clinical data were found between the two groups (all P values > 0.05).

[0067] Table 1

[0068]

[0069] 2. PBMC Isolation and Total RNA Extraction

[0070] 2.1 PBMC Isolation

[0071] (1) Specimen collection: 3 mL of peripheral venous blood was collected from all patients using EDTA anticoagulant tubes upon admission to the emergency department;

[0072] (2) Cell separation: Peripheral venous blood was placed at room temperature, allowed to stand for 30 min, and then centrifuged at 3000 g for 15 min at room temperature, and the upper plasma layer was discarded;

[0073] (3) Blood sample dilution: After plasma separation, transfer the blood cells to a 15 mL centrifuge tube and add PBS in a ratio of 1:2 to form a cell suspension.

[0074] (4) Density gradient centrifugation: Add 5 mL of lymphocyte separation medium (Mede Pacific) to a new 15 mL centrifuge tube. Use a pipette to draw the cell suspension from the previous step and slowly add it to the surface of the lymphocyte separation medium. Centrifuge at 600 g and 20°C for 25 min.

[0075] (5) Cell collection and washing: Use a pipette to aspirate the milky white mononuclear cell layer (PBMC) in a circular shape and transfer it to another 15 mL centrifuge tube. Add 10 mL of PBS, mix the cells, and centrifuge at 250 g for 10 min at room temperature. Discard the supernatant, add 5 mL of PBS to resuspend the cells, and centrifuge at 250 g for 10 min at room temperature. Discard the supernatant, add 0.2 mL of PBS to resuspend the cells, and transfer the cell suspension to a 1.5 mL EP tube. Store at -80 °C for later use.

[0076] 2.2 Total RNA extraction

[0077] The total RNA of cells was extracted using the Trizo method. The specific steps are as follows:

[0078] (1) Cell lysis: Add 1 mL of Trizol reagent (65307-12-2, Invitrogen) to the EP tube containing PBMCs, vortex mix for 3 min to ensure that the cells are fully lysed, and let it stand for 10 min; add 200 μL of chloroform, invert and mix, let it stand for 3 min, and centrifuge at 12000g, 4°C for 15 min;

[0079] (2) RNA precipitation: aspirate the supernatant aqueous phase (about 0.5 mL) and transfer it to a new 1.5 mL EP tube. Add an equal volume of isopropanol to the volume of water, invert and mix thoroughly, and let it stand for 10 minutes to promote the formation of RNA precipitation. Centrifuge at 12000g, 4°C for 10 minutes.

[0080] (3) RNA rinsing and dissolution: Discard the supernatant and retain the white RNA precipitate at the bottom of the tube. Add 1 mL of pre-cooled 75% ethanol to wash the precipitate and centrifuge at 12,000 g at 4°C for 5 min. Aspirate the ethanol and dry the RNA precipitate at room temperature for 10 min. Depending on the size of the precipitate, add 30-50 μL of 56°C preheated DEPC water to dissolve the RNA.

[0081] (4) RNA quality control: Take 1.5 μL RNA sample and use a UV spectrophotometer (IMPLEN-NP80) to detect the concentration and purity. The quality of RNA is determined by measuring the A260 / 280 ratio. A ratio between 1.8 and 2.0 indicates that the RNA sample is of qualified quality.

[0082] 3. Reverse Transcription

[0083] Reverse transcription kit (TAKARA PrimeScript TM Perform reverse transcription using PrimeScript RT Master Mix. According to the kit instructions, prepare a 10 μL reverse transcription reaction system by adding 500 ng of total RNA template and 2 μL of buffer (5X PrimeScript RT Master Mix). If the volume is less than 10 μL, add nuclease-free water to the reaction volume and mix gently. After mixing, perform reverse transcription using a PCR instrument (ABI7500) to obtain cDNA template. Reverse transcription reaction conditions are as follows: 37°C for 15 minutes (reverse transcription reaction); 85°C for 5 seconds (reverse transcriptase inactivation reaction).

[0084] 4. Template Dilution

[0085] 90 μL of RNase-free water was added to the obtained 10 μL of cDNA template to dilute the cDNA template.

[0086] 5. Real-time fluorescence quantitative PCR primer design and screening

[0087] 5.1 Primer design

[0088] Based on the NLRC3 transcript sequence (NM 178844.4), three pairs of NLRC3 primers and one pair of GAPDH primers were designed. The primers were synthesized by Sangon Biotech Co., Ltd. The specific primer sequences are shown in Table 2:

[0089] Table 2

[0090]

[0091] 5.2 Primer screening

[0092] ① Fluorescence quantitative PCR reaction analysis of primer specificity

[0093] Five patients' cDNA templates were randomly selected and fluorescent quantitative PCR was performed using the NLRC3 primers of group 1, group 2, and group 3. TM Premix Ex Taq TM II amplification kit for fluorescence quantitative PCR reaction, according to the kit instructions, prepare 10 μL of reaction buffer system. TM Premix Ex Taq TMFluorescent quantitative PCR was performed using a PCR amplification kit (II). According to the kit instructions, a 10 μL reaction buffer system (5 μL of TB Green buffer, 0.4 μL of forward primer, 0.4 μL of reverse primer, 0.2 μL of ROX reference dye, 2 μL of cDNA template, and 2 μL of deionized water) was prepared. Amplification conditions included one cycle of 95°C for 10 min (pre-denaturation) and 40 cycles of 95°C for 5 s and 60°C for 60 s (PCR). Amplification was performed using an ABI7500 fluorescent quantitative PCR instrument. The Ct values of the NLRC amplified by each primer were analyzed to assess primer specificity.

[0094] ② Verify primer specificity by agarose gel electrophoresis

[0095] Prepare 50 mL of 3% agarose gel with 1x TBE buffer, heat at 100°C for 2 min to dissolve the agarose, cool to 60°C, add 5 μL of GelRed indicator, and after the gel solidifies, add 1 μL of 10x DNA loading buffer to each tube of PCR product. Use DL500 DNA Marker as a reference, and perform electrophoresis at 100 V for 90 min. Image the gel using a high-sensitivity imaging system, identify PCR products, and evaluate primer specificity.

[0096] 6. Fluorescence quantitative PCR reaction to detect NLRC3 expression in samples

[0097] 6.1 Reaction system

[0098] Using TB Green TM Premix Ex Taq TM Perform quantitative PCR using the PCR amplification kit. Prepare 10 μL of reaction buffer according to the kit instructions. Refer to Table 3 for the specific preparation method. Vortex the reaction buffer and perform the amplification reaction using a real-time quantitative PCR instrument. Amplification conditions: 1 cycle at 95°C for 10 minutes (pre-denaturation); 40 cycles at 95°C for 5 seconds, 60°C for 60 seconds (PCR reaction).

[0099] Table 3 Reaction buffer system

[0100]

[0101] 6.2 Analysis of Fluorescence Quantitative PCR Reaction Results

[0102] GAPDH was used as the internal reference gene and 2 -△Ct The relative quantitative analysis of NLRC3 was performed by the ELISA method.

[0103] 7. Correlation Analysis: Coronary stenosis scores (modified Gensini score) were calculated based on coronary angiography results in patients with coronary artery disease. The scoring method is as follows: the extent and severity of proximal coronary artery lesions are scored for each lesion: 1 point for less than 50% luminal diameter stenosis; 2 points for 50%-74% stenosis; 3 points for 75%-99% stenosis; and 4 points for complete occlusion. The scores for each lesion in the proximal coronary circulation are summed to obtain a final overall score for the severity of coronary atherosclerosis (Gensini score).

[0104] 8 Experimental results

[0105] 8.1 Fluorescence quantitative PCR reaction and agarose gel electrophoresis analysis of primer specificity

[0106] The results of the amplification of 5 samples by different primer groups are shown in Table 4: the Ct value of NLRC3 amplified by primer group 1 was 25.2±0.5, the Ct value of NLRC3 amplified by primer group 2 was 25.2±0.6, and the Ct value of NLRC3 amplified by primer group 3 was 24.2±2.5. It can be seen that the efficiency of NLRC amplification by the three primer groups is good. Figure 2 As shown in the figure, the agarose gel electrophoresis results showed that the amplified product of group 1 had only one band, the amplified product of group 2 primers had a non-specific band in sample 5, and the amplified product of group 3 primers had non-specific bands in samples 1 / 2 / 4 / 5. Based on the above results, the primers of group 1 were more specific, and the primers of group 1 were selected for subsequent experiments.

[0107] Table 4 Expression of NLRC amplified by different groups of primers

[0108]

[0109] 8.2 Detection of NLRC3 Expression in Patients with Coronary Heart Disease by Fluorescence Quantitative PCR

[0110] like Figure 3 As shown in Figure 2, the results of fluorescence quantitative PCR showed that the relative expression of NLRC3 in peripheral blood PBMC of patients with coronary heart disease (0.0067±0.0006) was significantly lower than that of the normal coronary artery group (0.0223±0.0014). The Student's t test was used to calculate the P value and found that the difference was statistically significant (P<0.001). Figure 4 As shown in the data, in patients with coronary heart disease, the Spearman test was used to calculate the correlation between NLRC3 expression and Gensini score. The results showed that NLRC3 expression was significantly correlated with Gensini score (r=-0.7933, P<0.001), suggesting that NLRC3 expression is associated with the degree of coronary artery stenosis in patients with coronary heart disease.

[0111] Example 3: Evaluation of the diagnostic value of NLRC3 in patients with coronary heart disease

[0112] like Figure 5 As shown, the diagnostic value of NLRC3 in patients with coronary artery disease was analyzed using a receiver operating characteristic (ROC) curve. The results showed that the AUC value of the ROC curve was 0.926, with a 95% confidence interval of 0.889-0.963 (P < 0.001). When the diagnostic threshold of NLRC3 relative expression was set at 0.012, the sensitivity for diagnosing coronary artery disease was 85% and the specificity was 80%, suggesting that NLRC3 has a good value as a circulating diagnostic marker for coronary artery disease.

[0113] Example 4: Verification of the accuracy of NLRC3 in diagnosing coronary heart disease.

[0114] 1. Research subjects and methods

[0115] Peripheral venous blood samples were collected from 32 patients admitted to the emergency department for chest pain. Baseline data are detailed in Table 5. Peripheral blood was collected from these patients upon emergency admission, and peripheral blood mononuclear cells (PBMCs) were isolated. Subsequently, the expression level of the NLRC3 gene was detected using fluorescent quantitative PCR to screen patients at risk for coronary artery disease. Furthermore, the NLRC3 expression results of these patients were compared with the results of subsequent coronary angiography to evaluate the accuracy and reliability of NLRC3 as a biomarker for diagnosing coronary artery disease. This approach is expected to provide new biomarkers for the clinical diagnosis of patients with chest pain and provide a scientific basis for the early identification and treatment of coronary artery disease.

[0116] Table 5

[0117]

[0118] 2. Blood collection method

[0119] All patients had 3 mL of peripheral venous blood collected upon admission to the emergency department.

[0120] 3. Peripheral Blood Mononuclear Cell Isolation and Total RNA Extraction

[0121] Peripheral blood mononuclear cells (PBMCs) were isolated from the patient's peripheral blood sample using density gradient centrifugation following the same procedure as in Example 2. Total RNA was then extracted from these isolated PBMCs using the same Trizol reagent extraction method as in Example 2.

[0122] 4. Accuracy of reverse transcription and qRT-PCR detection of NLRC3 in diagnosing coronary heart disease

[0123] The same experimental method and system as in Example 2 were used to perform reverse transcription and fluorescence quantitative PCR. -△Ct The method used GAPDH as an internal reference gene to perform relative quantitative analysis of NLRC3 gene expression levels. A diagnostic threshold of NLRC3 expression below 0.0012 was set. This result was compared with subsequent coronary angiography results in patients with chest pain to evaluate the accuracy of NLRC3 as a biomarker for diagnosing coronary artery disease.

[0124] 5. Experimental Results

[0125] The experimental results are detailed in Table 6. Using an NLRC3 expression level below 0.012 as the diagnostic threshold, 32 patients with chest pain were diagnosed. Based on this criterion, 23 patients were identified as having coronary artery disease, while 9 had chest pain symptoms caused by other diseases. Furthermore, coronary angiography revealed that 20 of these patients had coronary artery disease, while 12 had chest pain caused by other diseases.

[0126] Comparing NLRC3 relative expression levels as a diagnostic test with coronary angiography results revealed that the test accurately identified 19 "true positive" patients (coronary artery disease) and 8 "true negative" patients (those without coronary artery disease). Calculating the proportion of true positive and true negative results in the total number of cases tested yielded an accuracy (Acc) of 84.4%. This result demonstrates that NLRC3 expression levels have high accuracy and reliability as a diagnostic tool for coronary artery disease.

[0127] Table 6

[0128]

[0129] Example 5: A fluorescent quantitative PCR kit for diagnosing coronary heart disease

[0130] 1. Ingredients

[0131] NLRC3 and GAPDH primers (10 μM, sequences as shown in Table 7), SYBR Green buffer (2X), ROX reference dye (50X) and deionized water.

[0132] Table 7 Primer sequences

[0133]

[0134] 2. The reaction system preparation method is shown in Table 8

[0135] Table 8

[0136]

[0137] 3. Reaction Conditions

[0138] Pre-denaturation: 95°C for 10 min (1 cycle); PCR reaction: 95°C for 15 s, 60°C for 60 s (40 cycles).

[0139] 4. Results Analysis

[0140] Use 2 -△Ct The relative quantitative analysis of NLRC3 was performed using GAPDH as an internal reference.

[0141] Example 6: ELISA detection of NLRC3 expression in the plasma of patients with coronary heart disease and control patients

[0142] 1. Research subjects

[0143] Patients with coronary heart disease (100 cases) and patients with normal coronary arteries (92 cases) were sampled from all patients in Examples 2 and 4. Baseline data are shown in Table 9 (all P values > 0.05), and the two groups of patients were comparable.

[0144] Table 9

[0145]

[0146]

[0147] 2. Sample Type

[0148] All patients had 3 mL of peripheral venous blood collected for plasma separation upon admission to the emergency department.

[0149] 3. Detection Methods

[0150] The plasma NLRC3 concentration in patients with coronary artery disease and subjects with normal coronary arteries was measured using a human NLRC3 ELISA kit (AE30906HU, Abebio) according to the manufacturer's instructions. The specific steps are as follows:

[0151] ① Dissolve the standard and dilute the NLRC3 standard using the serial dilution method;

[0152] ② Add 100 μL of diluted standard and sample to the ELISA plate and incubate at 37°C for 2 h;

[0153] ③ Wash the plate three times using an automatic plate washer, add 100 μL of diluted biotinylated antibody to each well, and incubate at 37°C for 1 h;

[0154] ④ Wash the plate three times, add 100 μL of HRP-labeled streptavidin complex to each well, and incubate at 37°C for 1 hour;

[0155] ⑤ Wash the plate 5 times, add 100 μL of chromogenic substrate to each well, and incubate at 37°C for 20 min;

[0156] ⑥ Add 50 μL of stop buffer to each well and detect using a microplate reader at a wavelength of 450 nm.

[0157] 4. Experimental Results

[0158] like Figure 6 As shown in the results, the plasma NLRC3 level in patients with coronary heart disease (1.09±0.04 ng / mL) was significantly lower than that in patients with normal coronary arteries (2.30±0.07 ng / mL). The Student's t-test was used to calculate the P value and found that the difference was statistically significant (P<0.001).

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of products for detecting NLRC3 gene expression in the preparation of products for diagnosing, screening, or evaluating coronary atherosclerotic heart disease.

2. The use according to claim 1, characterized in that: The expression level of NLRC3 in patients with coronary heart disease was significantly lower than that in patients with normal coronary arteries.

3. The use according to claim 1, characterized in that: The expression level of NLRC3 in carotid artery plaques was significantly lower than that in normal carotid arteries.

4. The use according to claim 1, characterized in that: The products for detecting NLRC3 gene expression include: products for detecting NLRC3 gene expression levels through RT-PCR, fluorescent quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform.

5. The use according to claim 4, characterized in that: The product for detecting the expression level of the NLRC3 gene by RT-PCR includes at least a pair of primers that specifically amplify the NLRC3 gene; the product for detecting the expression level of the NLRC3 gene by fluorescent quantitative PCR includes at least a pair of primers that specifically amplify the NLRC3 gene; the product for detecting the expression level of the NLRC3 gene by immunoassay includes at least an antibody that specifically binds to the NLRC3 protein; the product for detecting the expression level of the NLRC3 gene by in situ hybridization includes at least a probe that hybridizes with the nucleic acid sequence of the NLRC3 gene; the product for detecting the expression level of the NLRC3 gene by chip includes a protein chip or a gene chip, the protein chip includes an antibody that specifically binds to the NLRC3 protein, and the gene chip includes a probe that hybridizes with the nucleic acid sequence of the NLRC3 gene.

6. Application of reagents for detecting NLRC3 in the preparation of fluorescent quantitative PCR kits for diagnosing coronary atherosclerotic heart disease.

7. The use according to claim 6, characterized in that: The kit includes at least a pair of primers for specifically amplifying the NLRC3 gene, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2.

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