Application of ALDH1A1 and AMIGO2 genes in the preparation of atrial fibrillation diagnostic and intervention products

Abstract

This invention discloses the application of ALDH1A1 and AMIGO2 genes in the preparation of diagnostic and interventional products for atrial fibrillation (AF). Peripheral blood leukocytes were collected from AF patients, and peripheral blood leukocytes from individuals of different ages without AF were collected as controls. The expression levels of ALDH1A1 and AMIGO2 in peripheral blood leukocytes of AF patients and controls were detected by RT-qPCR. The results showed that the expression levels of AMIGO2 and ALDH1A1 in the AF patient group were significantly lower than those in the control group, suggesting that ALDH1A1 and AMIGO2 may be protective factors or intervention targets for AF. Functional analogs of ALDH1A1 and AMIGO2 hold promise for the preparation of drugs to prevent or treat AF. Furthermore, ROC curve analysis showed that the AUC areas of both AMIGO2 and ALDH1A1 genes were greater than 0.75, indicating that AMIGO2 and ALDH1A1 have high diagnostic value for AF and can be used alone or in combination for the diagnosis of AF, potentially becoming biomarkers for AF.

Description

Technical Field

[0001] This invention relates to the field of atrial fibrillation diagnosis and intervention products, and particularly to the application of ALDH1A1 and AMIGO2 genes in the preparation of atrial fibrillation diagnosis and intervention products. Background Technology

[0002] Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. my country has the highest prevalence of AF, with approximately 8 million patients according to the latest national survey. Of these, 36% are unaware of their condition. Due to hemodynamic changes, AF patients are prone to thrombosis if not treated promptly and effectively, leading to serious complications such as myocardial infarction, heart failure, and stroke, resulting in high rates of disability and death and placing a heavy burden on society and families. The prevalence of AF increases with age, reaching 5% in people aged 60-70 and as high as 8% in those over 80. Therefore, advanced age is a major risk factor for AF. With the increasing aging of my country's population, the incidence and prevalence of AF will continue to rise. However, many patients have asymptomatic AF. Some patients with paroxysmal AF experience intermittent episodes without any complaints, but the absence of symptoms does not mean the absence of harm. These patients often present with stroke as their first symptom, and some are diagnosed with coronary enlargement and heart failure. Therefore, actively carrying out early diagnosis and treatment of atrial fibrillation is of great significance in reducing stroke and disability caused by atrial fibrillation.

[0003] Currently, screening methods for atrial fibrillation (AF) mainly include pulse palpation, blood pressure measurement, smart bracelets, wearable ECG monitoring devices, electrocardiograms (ECGs), and Holter monitoring. Pulse palpation and blood pressure measurement require experience and have low sensitivity; smart bracelets and wearable ECG monitoring devices are expensive; ECGs and Holter monitoring require medical institutions, which is inconvenient and can easily lead to missed diagnoses in patients with infrequent paroxysmal AF. Blood biomarkers are widely used for early diagnosis and screening due to their simple sampling and convenient testing, but specific biomarkers for AF are still lacking. Furthermore, although the treatment of AF has made significant progress with the continuous development and promotion of new technologies such as radiofrequency ablation and left atrial appendage occlusion, specific drugs are still lacking. Existing anticoagulants and rhythm control drugs only alleviate symptoms and reduce complications. Therefore, to effectively improve the diagnostic rate of AF and enrich intervention methods to reduce its harm, clinicians urgently need to find serum biomarkers and intervention targets for AF.

[0004] Aldehyde dehydrogenase 1 (ALDH1A1) is highly expressed in tumor stem cells and serves as a biomarker for breast, prostate, colon, and lung cancers, playing a crucial role in promoting tumor angiogenesis and metastasis, and in the acquisition of resistance to anticancer drugs. Furthermore, ALDH1A1 regulates retinoic acid (RA) biosynthesis and RA signaling in acute promyelocytic leukemia. Recently, ALDH1A1 has been found to be associated with cardiac development and protective effects after myocardial infarction. However, the role of ALDH1A1 in atrial fibrillation (AF) has not been reported. This invention proposes that ALDH1A1 may serve as a biomarker or intervention target for the diagnosis of AF, and that ALDH1A1 antagonists or their functional analogs hold promise for the development of drugs to treat AF.

[0005] The Ig-like domain 2 adhesion molecule (AMIGO2), a member of the AMIGO family, is a transmembrane mucin containing a six-amino acid repeat sequence. Mutations in the AMIGO2 gene may lead to first and second branchial arch syndrome. Studies have found that AMIGO2 plays an important role in the development of retinal cell dendrites, and its high expression can enhance the adhesion ability of gastric adenocarcinoma cells, degrade the extracellular matrix, and damage the basement membrane, thereby promoting the development and progression of gastric cancer. Furthermore, high expression of AMIGO2 protein is closely related to the development and progression of prostate cancer, oral cancer, cervical cancer, and liver cancer, suggesting it may be a potential target for tumor therapy. Recent findings also indicate that AMIGO2 plays an important role in protecting cardiac function after myocardial ischemia. However, the role of AMIGO2 in atrial fibrillation has not been reported. This invention proposes that AMIGO2 may serve as a biomarker or intervention target for the diagnosis of atrial fibrillation, and that AMIGO2 antagonists or their functional analogs hold promise for the development of drugs to treat atrial fibrillation. Summary of the Invention

[0006] The purpose of this invention is to provide the application of ALDH1A1 and AMIGO2 genes in the preparation of atrial fibrillation diagnostic and intervention products, so as to solve the problems mentioned in the background art and facilitate promotion.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] Application of ALDH1A1 and AMIGO2 genes in the preparation of atrial fibrillation diagnostic and intervention products, wherein the ALDH1A1 and AMIGO2 genes can serve as biomarkers for atrial fibrillation diagnosis.

[0009] Furthermore, the diagnostic method involves extracting peripheral blood leukocytes from patients with atrial fibrillation and performing RT-qPCR detection, and then comparing the mRNA levels of ALDH1A1 and AMIGO2 genes in the peripheral blood leukocytes of patients with atrial fibrillation with those of normal individuals.

[0010] Furthermore, compared with normal individuals, patients with atrial fibrillation showed significantly reduced mRNA levels of the ALDH1A1 and AMIGO2 genes in peripheral blood leukocytes.

[0011] A product for diagnosing atrial fibrillation, the product comprising a detection kit for detecting the expression levels of the ALDH1A1 and AMIGO2 genes and their expression products.

[0012] Furthermore, the detection kit includes a protein antibody against ALDH1A1 or AMIGO2.

[0013] Furthermore, the detection kit includes nucleic acid probes with detectable labels, including chromophores, chemiluminescent groups, fluorescent groups, isotopes, or enzymes.

[0014] A drug for treating atrial fibrillation includes agents that can promote or alter the expression of the ALDH1A1 or AMIGO2 genes.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention relates to the application of ALDH1A1 and AMIGO2 genes in the preparation of diagnostic and interventional products for atrial fibrillation (AF). Peripheral blood leukocytes were collected from AF patients, and peripheral blood leukocytes from individuals of different ages without AF were collected as controls. RT-qPCR was used to detect the expression of ALDH1A1 and AMIGO2 genes. The results showed that the expression levels of AMIGO2 and ALDH1A1 in the AF patient group were significantly lower than those in the control group, suggesting that ALDH1A1 and AMIGO2 may be protective factors or intervention targets for AF. Functional analogs of ALDH1A1 and AMIGO2 hold promise for the development of drugs to prevent or treat AF. Furthermore, ROC curve analysis showed that the AUC areas of both AMIGO2 and ALDH1A1 genes were greater than 0.75, indicating that AMIGO2 and ALDH1A1 have high diagnostic value for AF and can be used alone or in combination for the diagnosis of AF, potentially becoming biomarkers for AF. Attached Figure Description

[0017] Figure 1 To illustrate the application of the ALDH1A1 and AMIGO2 genes in the preparation of atrial fibrillation diagnostic and intervention products, a schematic diagram and ROC curve of ALDH1A1 expression level in peripheral blood leukocytes are shown.

[0018] Figure 2This invention demonstrates the application of ALDH1A1 and AMIGO2 genes in the preparation of atrial fibrillation diagnostic and intervention products. The diagram shows the expression level of AMIGO2 in peripheral blood leukocytes and the ROC curve. Detailed Implementation

[0019] according to Figures 1 to 2 As shown, the ALDH1A1 and AMIGO2 genes are used in the preparation of atrial fibrillation diagnostic and intervention products. The ALDH1A1 and AMIGO2 genes can be used as biomarkers for the diagnosis of atrial fibrillation.

[0020] The diagnostic method involves extracting peripheral blood leukocytes from patients with atrial fibrillation (AF) and performing RT-qPCR detection. The mRNA expression levels of ALDH1A1 and AMIGO2 genes in the peripheral blood leukocytes of AF patients were compared with those of normal individuals. It was found that the expression levels of ALDH1A1 and AMIGO2 genes in the peripheral blood leukocytes of AF patients were significantly reduced. Subsequently, ROC curve analysis of ALDH1A1 and AMIGO2 genes showed that the AUC area was greater than 0.75, indicating that they have high diagnostic value for AF.

[0021] The experimental steps are as follows:

[0022] Peripheral blood leukocyte extraction includes the following steps:

[0023] Step 1: Collect 3-5 ml of venous blood, anticoagulate with EDTA, and centrifuge at 2500 rpm for 10 min;

[0024] Step 2: Carefully aspirate the supernatant plasma and aliquot it into three 0.5ml centrifuge tubes;

[0025] Step 3: Add 3 times the volume of lysed blood to the blood cells, shake well, and incubate on ice for 15 minutes;

[0026] Step 4: Centrifuge at 2500 rpm for 10 min, then discard the supernatant;

[0027] Step 5: Add 10ml of blood lysate, shake well, and incubate on ice for 15 minutes;

[0028] Step 6: Centrifuge at 3000 rpm for 10 minutes, then discard the supernatant;

[0029] Step 7: Invert the centrifuge tube to remove any remaining liquid;

[0030] Step 8: Obtain white blood cells and freeze them at -80°C to avoid repeated freeze-thaw cycles.

[0031] The interval between blood collection and leukocyte separation should not exceed 2 hours at room temperature and 5 hours at 4°C to prevent leukocyte autolysis.

[0032] RT-qPCR detection of ALDH1A1 and AMIGO2 expression:

[0033] Primer design and synthesis

[0034]

[0035] RNA extraction includes the following steps:

[0036] Step 1: After thawing the frozen white blood cells, add 1 ml of sterile PBS to resuspend them, and centrifuge at 4°C and 1000 rpm for 1 min.

[0037] Step 2: After discarding the supernatant, add 1 ml of TRIzol to lyse the cells and mix by pipetting.

[0038] Step 3: Let stand at room temperature for 5 minutes, then transfer the supernatant to a 1.5 ml EP tube;

[0039] Step 4: Centrifuge at 12,000 rpm for 5 min, take the supernatant, add chloroform, shake to mix, and let stand at room temperature for 15 min to allow it to separate into phases naturally;

[0040] Step 5: Centrifuge at 12,000 rpm for 15 min at 4℃. The sample will separate into three layers: a yellow organic layer, a middle layer, and an upper layer of colorless aqueous phase. The RNA is mainly in the aqueous phase.

[0041] Step 6: Carefully aspirate the upper aqueous phase into a new 1.5 ml EP tube, add an equal volume of ice-cold isopropanol, and incubate at -20°C for 1 hour;

[0042] Step 7: Centrifuge at 12,000 rpm for 10 minutes at 4℃;

[0043] Step 8: Discard the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water), gently shake the EP tube to suspend the precipitate;

[0044] Step 9: Centrifuge at 8,000 rpm for 5 minutes at 4℃, discard the supernatant, and air dry at room temperature for 5-10 minutes;

[0045] Step 10: Dissolve the RNA precipitate in 50 μl of DEPC water, detect the RNA concentration using a spectrophotometer, and store at -80℃ for later use.

[0046] cDNA synthesis reaction system and conditions

[0047]

[0048] Mix gently and react at 37°C for 15 minutes, then at 85°C for 5 seconds, and gradually cool down to 4°C.

[0049] RT-qPCR reaction system and conditions

[0050]

[0051] Mix the reaction solution thoroughly, pre-denature at 95℃ for 30s; denature at 95℃ for 5s, anneal at 60℃ for 30-34s, for 40 cycles.

[0052] Data analysis: The Ct value of the target gene was normalized by standardizing the Ct value of the internal reference gene GAPDH, and then 2... -△△Ct The expression levels of ALDH1A1 and AMIGO2 mRNA were obtained by performing relative quantitative analysis of gene expression differences in the sample.

[0053] The main results obtained after the experiment are as follows:

[0054] (1) such as Figure 1 As shown, the expression level of ALDH1A1 in peripheral blood leukocytes of patients with atrial fibrillation was significantly lower than that in the control group. P =0.010), suggesting that ALDH1A1 may be a protective factor or intervention target for atrial fibrillation; subsequently, ROC curve analysis was used to analyze the diagnostic value of ALDH1A1 for atrial fibrillation, and the results showed that the area of ​​AUC was 0.8765, P =0.0071, indicating that the expression level of ALDH1A1 has high diagnostic value for atrial fibrillation and is expected to become a biomarker for the diagnosis of atrial fibrillation.

[0055] (2) such as Figure 2 As shown, the expression level of AMIGO2 in peripheral blood leukocytes of patients with atrial fibrillation was significantly lower than that in the control group. P =0.026) suggests that AMIGO2 may be a protective factor or intervention target for atrial fibrillation; subsequently, ROC curve analysis was used to analyze the diagnostic value of AMIGO2 for atrial fibrillation. The results showed that the area of ​​AUC was 0.7654, P=0.0576. The reason why the area of ​​AUC was relatively good but the P value was still greater than 0.05 may be due to the small sample size.

[0056] The ALDH1A1 and AMIGO2 genes were expressed at low levels in peripheral blood leukocytes of patients with atrial fibrillation. The AUC and P values ​​of ROC analysis were 0.8765 (0.0071) and 0.7654 (0.0576), respectively, indicating that ALDH1A11 and AMIGO2 have high diagnostic value for atrial fibrillation and can be used alone or in combination for the diagnosis of atrial fibrillation. These two genes are expected to become biomarkers for atrial fibrillation.

[0057] A product for diagnosing atrial fibrillation includes a detection kit for detecting the expression levels of the ALDH1A1 or AMIGO2 gene and its expression products. The detection kit includes protein antibodies against ALDH1A1 and AMIGO2, and includes nucleic acid probes with detectable labels, including chromophores, chemiluminescent groups, fluorescent groups, isotopes, or enzymes.

[0058] A drug for treating atrial fibrillation includes agents that can promote or alter the expression of the ALDH1A1 or AMIGO2 genes.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a kit for detecting the expression levels of ALDH1A1 and AMIGO2 genes in the preparation of atrial fibrillation diagnostic products, characterized in that... The ALDH1A1 and AMIGO2 genes can serve as biomarkers for the diagnosis of atrial fibrillation.

2. The application of the kit for detecting the expression levels of ALDH1A1 and AMIGO2 genes according to claim 1 in the preparation of atrial fibrillation diagnostic products, characterized in that, The diagnostic method involves extracting peripheral blood leukocytes from patients with atrial fibrillation and performing RT-qPCR detection, then comparing the mRNA levels of ALDH1A1 and AMIGO2 genes in the peripheral blood leukocytes of patients with those of healthy individuals.

3. The application of the kit for detecting the expression levels of ALDH1A1 and AMIGO2 genes according to claim 1 in the preparation of atrial fibrillation diagnostic products, characterized in that, Compared with normal individuals, patients with atrial fibrillation showed significantly reduced mRNA levels of the ALDH1A1 and AMIGO2 genes in peripheral blood leukocytes.

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