Application of plasma exosome miR-106b-5p in differential diagnosis of large atherosclerotic cerebral infarction and cardiac embolic cerebral infarction

Abstract

The invention relates to the field of biomarkers, in particular to application of a plasma exosome miR-106b-5p in differential diagnosis of large atherosclerotic cerebral infarction and cardiac embolic cerebral infarction. The nucleotide sequence of the miR-106b-5p is 5 '-AUCUGCACUGUCAGCACUUUA-3', and the nucleotide sequence of the miR-106b-5p is 5 '-AUCUGCACUGUCA-3'. Experiments find that miR-106b-5p has different expressions in plasma exosomes of patients with great atherosclerosis type cerebral infarction, patients with cardiogenic embolic type cerebral infarction and healthy and normal people, can be used as a marker for differential diagnosis of great atherosclerosis type cerebral infarction and cardiogenic embolic type cerebral infarction, and has specificity and sensitivity. The invention provides a new marker for differential diagnosis of large atherosclerotic cerebral infarction and cardiogenic embolic cerebral infarction.

Description

Technical Field

[0001] The present invention relates to the field of biomarkers, and in particular to the application of plasma exosome miR-106b-5p in the differential diagnosis of large artery atherosclerotic cerebral infarction and cardioembolic cerebral infarction. Background Art

[0002] Ischemic stroke (IS), also known as cerebral infarction, is one of the common diseases of the nervous system that seriously affects human health and life safety. It is a clinical syndrome caused by various reasons that cause cerebral blood supply disorders, resulting in local brain tissue ischemia, hypoxia or even necrosis, and the rapid emergence of corresponding neurological deficits. It presents the characteristics of high morbidity, high disability rate, high mortality rate and complex complications, namely "three highs and one more". The causes of IS are complex. It is of great clinical significance to clarify the causes of patients and adopt individualized diagnosis and treatment plans for different causes to improve the treatment, prognosis and recurrence prevention of IS.

[0003] There is no internationally accepted disease diagnostic standard. The Trial of Org 10172 in Acute Stroke Treatment (TOAST) is the most commonly used stroke disease type in foreign countries. Ischemic stroke can be divided into five types: large artery atherosclerotic stroke (LAA), small artery occlusion stroke (SAO), cardioembolism (CE), acute stroke of other causes (SOE) and stroke of undetermined etiology (SUE). Among them, LAA and CE are the two most common IS. Although TOAST etiology classification is the most widely used classification method in clinical practice, it also has obvious limitations. Because this method mainly relies on the patient's clinical manifestations and imaging examinations, including magnetic resonance angiography, CT angiography, cardiac ultrasound and other examinations. On the one hand, the improvement of these examinations requires corresponding instruments and time, especially for areas with scarce medical resources or patients with contraindications to relevant examinations, thus limiting the application of TOAST classification. More importantly, in clinical practice, we found that even when relevant examinations such as cerebrovascular imaging and cardiac ultrasound are available, some patients still cannot be accurately classified into etiological subtypes. For example, patients have evidence of atrial fibrillation and large artery stenosis at the same time, and the specific cause of the disease cannot be determined, which will also lead to an excessively high proportion of IS of unknown cause. In the case of unclear etiology, it is impossible to formulate personalized treatment plans and subsequent targeted secondary prevention strategies for the cause, which has never affected the treatment effect and prognosis of patients. At present, there is no clinically specific serological biological marker that distinguishes LAA stroke from CE stroke. Therefore, finding a convenient and accurate diagnostic marker can achieve timely and accurate diagnosis of IS patients, so as to meet the clinical needs for rapid and accurate treatment of IS patients, which is of great clinical significance.

[0004] Exosomes are active vesicles that can be secreted by various types of cells and play an important role in intercellular communication. Exosomes contain a variety of bioactive molecules, including proteins, lipids, mRNAs, and non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). In addition, plasma exosomal miRNAs are more stable than miRNAs. At the same time, circulating exosomes are increasingly attracting attention as biomarkers for the diagnosis and prognosis of IS due to their advantages such as being non-invasive, stable, and maintaining biological activity.

[0005] At the same time, more and more studies have shown that miRNAs play an important role in the occurrence and development of IS. However, although many studies have focused on the different expressions and significance of miRNAs in IS patients, the results of these studies are very heterogeneous. We preliminarily screened several miRNAs molecules that have been repeatedly reported to be closely related to the occurrence of IS by reviewing and sorting out the literature. Using the IS cases we collected for verification, it was found that only miR-106b-5p was stably expressed in the plasma exosomes of IS patients. Further analysis found that compared with CE-type IS patients, the expression level of plasma exosome miR-106b-5p in LAA-type IS patients was significantly increased.

[0006] In the present invention, we take plasma exosome miRNA as the starting point, based on the differential expression of plasma exosome miRNA in patients with different subtypes of ischemic stroke, in order to find biomarkers that can distinguish LAA-type stroke and CE-type IS patients, which account for the highest proportion in clinical practice. Through a large number of literature reviews and repeated experimental explorations, the present invention optimizes the plasma exosome purification method and solves the problem of obtaining high-quality exosomes from trace plasma. At the same time, this study identified exosomes by Western blot detection of plasma exosome surface marker proteins, and preliminarily verified that exosome surface marker proteins were expressed in the extract. It further verified the feasibility of this method as a method for separating and extracting exosomes, and the separated exosome particles were sufficient for the study of miRNA.

[0007] The present invention explored plasma exosomes based on the extraction of high-quality plasma exosomes, and used real-time fluorescence PCR to detect the expression of miR-106-5p in the LAA type IS group, CE type IS group and the healthy control group, to evaluate whether it could be used as a biomarker for the etiology of cerebral infarction, providing a new idea for the research on the differential diagnosis of large artery atherosclerotic cerebral infarction and cardiogenic embolism cerebral infarction. Summary of the invention

[0008] The purpose of the present invention is to provide an application of plasma exosome miR-106b-5p in the differential diagnosis of large artery atherosclerotic cerebral infarction and cardioembolic cerebral infarction.

[0009] To achieve the above object, the present invention adopts the following technical solution: Use of plasma exosomal miR-106b-5p in the preparation of a kit for differential diagnosis of large artery atherosclerotic cerebral infarction and cardioembolic cerebral infarction, wherein the nucleotide sequence of the miR-106b-5p is 5'-AUCUGCACUGUCAGCACUUUA-3'.

[0010] Furthermore, the above kit is a real-time fluorescence quantitative PCR kit.

[0011] Furthermore, the above-mentioned real-time fluorescence quantitative PCR kit includes a primer pair for determining the expression level of miR-106b-5p in plasma exosomes.

[0012] Furthermore, the nucleotide sequences of the primer pair are: miR-106b-5p-RT: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATCTGC-3', miR-106b-5p-F: 5'-GCGCGTAAAGTGCTGACAGT-3'.

[0013] Furthermore, the above-mentioned kit also includes exosome separation reagents, exosome purification reagents, exosome identification reagents, miRNA extraction reagents, miRNA reverse transcription reagents and real-time fluorescence quantitative PCR reaction reagents.

[0014] The significant advantages of the present invention are: Exosomes are small double-layer membrane vesicles with a diameter of about 30-100nm. They can be secreted by various tissues and cells and exist in body fluids such as serum, cerebrospinal fluid, saliva and urine. They contain multiple components such as microRNA and proteins from host cells, and are important media for information exchange between cells. They are also ideal objects for the study of non-invasive disease diagnostic markers. The present invention adopts real-time fluorescence quantitative polymerase chain reaction (PCR) technology based on exosomes in high molecular weight polymer precipitation solution to analyze the expression and difference of plasma exosome miR-106b-5p in plasma exosomes of patients with large artery atherosclerotic cerebral infarction, patients with cardiogenic embolism cerebral infarction and healthy normal people, and explores the possibility of plasma exosome miR-106b-5p as a marker in the differential diagnosis of large artery atherosclerotic cerebral infarction and cardiogenic embolism cerebral infarction. The results showed that the expression level of plasma exosome miR-106b-5p in patients with LAA type IS was significantly higher than that in patients with CE type IS and healthy normal people, and the expression level of plasma exosome miR-106b-5p in patients with CE type IS was significantly lower than that in healthy normal people; when plasma exosome miR-106b-5p was used to differentiate between LAA type IS and CE type IS, the AUC under the ROC curve was 0.75, and the sensitivity and specificity were 72.73% and 71.43%, respectively. Therefore, the present invention can provide a scientific reference basis for the differential diagnosis of large artery atherosclerosis type cerebral infarction and cardioembolism type cerebral infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : A, Expression levels of plasma exosomal miR-106b-5p in patients with LAA type IS, patients with CE type IS and healthy controls; B, Plasma exosomal miR-106b-5p can differentiate between large artery atherosclerotic cerebral infarction and cardioembolic cerebral infarction. DETAILED DESCRIPTION

[0016] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0017] Embodiment 1: S1: General Information Patients with large artery atherosclerotic cerebral infarction (79 cases) and cardioembolic cerebral infarction (46 cases) were admitted to the Department of Neurology of the First Affiliated Hospital of Fujian Medical University from May 2019 to July 2023. The diagnosis was in accordance with the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke in China (2014)" and was confirmed by detailed neurological examinations and cranial CT or MRI imaging examinations. The control group was healthy normal people (62 cases) who underwent physical examinations at the First Affiliated Hospital of Fujian Medical University during the same period. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Fujian Medical University. All samples were obtained with the consent of the research subjects and signed informed consent.

[0018] S2: Collection and pretreatment of plasma samples S2-1: Peripheral venous blood was drawn from patients with large artery atherosclerotic cerebral infarction, cardioembolic cerebral infarction and the control group before any treatment at admission. The blood was collected using medical EDTA anticoagulant tubes and centrifuged at 3000 rpm for 10 min. The venous blood samples were stratified into an upper plasma layer and a lower blood cell layer. The upper plasma was transferred to a cryopreservation tube using a pipette and stored in a -80°C refrigerator. S2-2: Take 0.2 mL of plasma sample frozen at -80°C, completely melt it into liquid at room temperature, and place it on ice; S2-3: Transfer the thawed plasma to a centrifuge tube, centrifuge at 3000 g for 10 min at 4°C to remove cell debris, aspirate all the supernatant and transfer it to a new centrifuge tube, centrifuge at 10000 g for 10 min at 4°C to remove other impurities, aspirate all the supernatant and transfer it to a new centrifuge tube to obtain the pre-treated plasma sample.

[0019] S3: Extraction of plasma exosomes S3-1: Add 1.5 mL of 1× PBS (pH 7.2-7.4) precooled at 4°C to the treated plasma sample, then add 0.5 mL of Blood PureExo Solution (BPS), mix thoroughly on a vortex oscillator for 1 min, and then place at 4°C for 2 h; S3-2: Take out the centrifuge tube containing the mixed solution, centrifuge it at 10000g for 60 min at 4°C, discard the supernatant, and resuspend the precipitate in 0.1mL 1×PBS (pH 7.2-7.4); S3-3: Transfer the resuspension to a new centrifuge tube, centrifuge at 12000g for 2 min at 4°C, discard the precipitate, and retain the supernatant, which is the crude exosome particle rich in exosome particles.

[0020] S4: Purification and identification of exosomes S4-1: The crude exosome particles were transferred into the upper chamber of the Exosome Purification Filter (EPF column), and centrifuged at 3000 g for 10 min at 4°C. The liquid at the bottom of the EPF column was collected. This liquid was the purified exosome particles. The purified exosomes were packaged into 50-100 μL portions and stored in a -80°C freezer for subsequent experiments.

[0021] S4-2: Take the purified exosome sample, add 5× protein loading buffer at a volume ratio of 4:1 and mix well, denature at 100℃ for 5 minutes, and detect the expression of exosome marker proteins TSG101 and CD63 in the sample by Western blot to ensure that the extracted plasma exosome sample meets the main characteristics of exosomes (TSG101 and CD63, common marker proteins of exosomes, are clearly expressed in the sample) and can be used for subsequent experiments.

[0022] S5: Extraction of miRNAs from exosomes S5-1: Take 200 μL of purified and identified exosome sample, add 5 times volume of TRlzon Reagent, shake and mix for 30 seconds to fully lyse it, and then leave it at room temperature for 5 minutes to completely separate the protein-nucleic acid complex; S5-2: Centrifuge at 4°C and 12000rpm for 5min, aspirate the supernatant and transfer it to a new centrifuge tube, add chloroform to the supernatant, add 200μL chloroform for every 1mL TRlzon Reagent, cover the tube, shake vigorously for 15s, and place at room temperature for 5min. Centrifuge at 4°C and 12000rpm for 15min, and the sample will be divided into three layers: red organic phase, middle layer, and colorless aqueous phase; S5-3: Transfer the upper colorless aqueous phase to a new centrifuge tube, add 1 / 3 volume of anhydrous ethanol, mix well, transfer to the adsorption column RM (Spin Columns RM) loaded in the collection tube, centrifuge at 12000rpm for 30s, discard the adsorption column RM after centrifugation, and retain the effluent; S5-4: Add 2 / 3 volume of anhydrous ethanol to the effluent, mix well, transfer to the spin columns RS in the collection tube, centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and put the spin columns RS back into the collection tube; S5-5: Add 700 μL Bufer RWT to the adsorption column RS and centrifuge at 12000 rpm for 30 seconds. Pour away the waste liquid in the collection tube and put the adsorption column RS back into the collection tube. S5-6: Add 500 μL Buffer RW2 to the adsorption column RS and centrifuge at 12000 rpm for 30 seconds. Pour away the waste liquid in the collection tube and put the adsorption column RS back into the collection tube. S5-7: repeat S5-6 once; S5-8: Centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column RS at room temperature for several minutes to dry thoroughly; S5-9: Place the adsorption column RS in a new RNase-free centrifuge tube, add 30-50 μL RNase-Free Water to the middle part of the adsorption column, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, collect the RNA solution, and detect the optical density of RNA at 260 nm and 280 nm using an ultra-micro UV spectrophotometer, A260 / A280=1.8~2.2.

[0023] S6: Reverse transcription of miRNAs into cDNA S6-1: Add 4 μL 5×g DNA wiper MIX and 6 μL template RNA to an RNase Free centrifuge tube, gently pipette to mix, place in a PCR instrument, and set the program to 42°C for 2 min.

[0024] S6-2: The reverse transcription reaction system includes: 10 μL of the reaction product of S6-1, 1 μL of Stem-loop primer (2 μM), 2 μL of 10×RT Mix, 2 μL of HiScript II Enzyme Mix, and 20 μL of RNaseFree ddH2O. The reverse transcription reaction procedure is: 25℃ 5min, 50℃ 15min, 85℃ 5min. The cDNA reaction solution synthesized by reverse transcription is stored in a -20℃ refrigerator.

[0025] S7: Real-time fluorescence quantitative PCR detection of miR-106b-5p The real-time fluorescence quantitative PCR reaction system included: 2×miRNA Universal SYBR Green Master mix 10μL, primer miR-106b-5p-RT (10μM) 0.4μL, primer miR-106b-5p-F (10μM) 0.4μL, cDNA template 5μL, ddH2O 4.2μL. The real-time fluorescence quantitative PCR reaction program was: 95℃ 5min; 95℃ 10s, 60℃ 30s, 40 cycles; 95℃ 15s, 60℃ 60s, 95℃ 15s. miR-39-3p was used as an external reference, and universal downstream primers were used. The relative expression of miR-106b-5p was calculated using 2 -ΔΔCtThe nucleotide sequence of miR-106b-5p is 5'-AUCUGCACUGUCAGCACUUUA-3'; the nucleotide sequence of the primer miR-106b-5p-RT is 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATCTGC-3', and the nucleotide sequence of the primer miR-106b-5p-F is 5'-GCGCGTAAAGTGCTGACAGT-3'.

[0026] The results show that ( Figure 1 A), the expression level of plasma exosomal miR-106b-5p in LAA type IS patients was significantly higher than that in CE type IS patients and healthy subjects ( P The expression level of plasma exosomal miR-106b-5p in patients with CE type IS was significantly lower than that in healthy controls (P<0.05).

[0027] S8: Plasma exosomal miR-106b-5p in differential diagnosis of large artery atherosclerotic cerebral infarction and cardioembolic cerebral infarction The receiver operating characteristic curve (ROC) method was used for validation, and the relative expression level of miR-106b-5p in the plasma exosomes of LAA type IS patients and CE type IS patients was used to determine its ability to differentiate between LAA type IS patients and CE type IS patients.

[0028] The results show that ( Figure 1 B), plasma exosomal miR-106b-5p had a strong ability to differentiate LAA-type IS patients from CE-type IS patients, with an area under the ROC curve (AUC) of 0.75 (95% CI, 0.59-0.81); when the relative expression level of plasma exosomal miR-106b-5p was greater than or equal to 0.81 as the differential diagnosis threshold, its diagnostic sensitivity was 72.73% and its specificity was 71.43% (P<0.05).

[0029] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Use of plasma exosomal miR-106b-5p in the preparation of a kit for differential diagnosis of large artery atherosclerotic cerebral infarction and cardioembolic cerebral infarction, characterized in that: The nucleotide sequence of the miR-106b-5p is 5'-AUCUGCACUGUCAGCACUUUA-3'.

2. The kit according to claim 1, characterized in that: The kit is a real-time fluorescence quantitative PCR kit.

3. The kit according to claim 2, characterized in that: The real-time fluorescence quantitative PCR kit includes a primer pair for determining the expression level of miR-106b-5p in plasma exosomes.

4. The kit according to claim 3, characterized in that: The nucleotide sequences of the primer pair are: miR-106b-5p-RT: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATCTGC-3', miR-106b-5p-F: 5'-GCGCGTAAAGTGCTGACAGT-3'.

5. The kit according to claim 4, characterized in that: The kit also includes an exosome separation reagent, an exosome purification reagent, an exosome identification reagent, a miRNA extraction reagent, a miRNA reverse transcription reagent, and a real-time fluorescence quantitative PCR reaction reagent.

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