WO2023134553A1

WO2023134553A1 - Plasma exosome circrna marker and application thereof

Info

Publication number: WO2023134553A1
Application number: PCT/CN2023/070832
Authority: WO
Inventors: 谢博洽; 刘晓艳; 苏丕雄; 袁雯; 高杰; 张文谦; 张叶萍; 贾彦熊; 杨敏福; 王丽
Original assignee: 首都医科大学附属北京朝阳医院
Priority date: 2022-01-11
Filing date: 2023-01-06
Publication date: 2023-07-20
Also published as: CN114182010B; CN114182010A

Abstract

The present invention relates to the technical field of molecular diagnostics, and relates in particular to a plasma exosome circRNA marker and application thereof. The plasma exosome circRNA marker is hsa_circ_001558, and a nucleotide sequence of hsa_circ_001558 is represented by SEQ ID NO: 1. The present invention further discloses an application of the plasma exosome circRNA marker in the preparation of a marker for detecting an acute myocardial infarction and a diagnostic product. The marker exosome hsa_circ_001558 provided by the present invention can be used as a diagnostic marker for an acute myocardial infarction.

Description

A plasma exosome circRNA marker and its application

technical field

The invention relates to the technical field of molecular diagnosis, in particular to a plasma exosome circRNA marker and its application.

Background technique

At present, the number of cardiovascular diseases in my country is as high as 290 million, and the fatality rate ranks first, higher than that of tumors and other diseases, accounting for more than 40% of the residents' disease deaths. Acute myocardial infarction (AMI) is one of the most common cardiovascular diseases, which seriously threatens human health. Acute myocardial infarction is myocardial necrosis caused by acute and persistent coronary ischemia and hypoxia. Clinically, there are often severe and persistent substernal pains, which cannot be completely relieved by rest and nitrates, accompanied by increased serum myocardial enzyme activity and progressive ECG changes, which may be complicated by arrhythmia, shock or heart failure, often life-threatening.

Traditional circulating biomarkers such as cTnT play an important role in the diagnosis and prognosis of AMI. cTnT has high sensitivity, but since cTnT is also easily detected in non-AMI patients (such as heart failure or pulmonary embolism patients), So its specificity is relatively low. Therefore, exploring the pathogenesis of AMI and finding more specific biomarkers is the key to improving the treatment and prognosis of AMI patients.

Contents of the invention

The first purpose of the present invention is to provide a plasma exosomal circRNA marker, which can be used as a detection biomarker for acute myocardial infarction, and has higher specificity than traditional circulating biomarkers, The detection is quick and convenient, and the cost is low. The present invention also provides the application of plasma exosome circRNA markers.

The present invention provides a plasma exosome circRNA marker, the plasma exosome circRNA marker is hsa_circ_0001558, and the nucleotide sequence of the hsa_circ_0001558 is shown in SEQ ID NO:1.

The present invention provides an application of a plasma exosome circRNA marker as a detection marker for acute myocardial infarction.

The present invention provides a diagnostic product including plasma exosomal circRNA markers for detecting acute myocardial infarction.

Preferably, the diagnostic product is a chip, preparation or kit.

The study found that the relative expression level of exosome hsa_circ_0001558 in plasma exosomes of AMI patients was significantly higher than that of NCCP (non-cardiac chest pain) patients, which was 4.45 times that of NCCP patients. The area under the ROC curve for the diagnosis of AMI was 0.793; the expression of exosome hsa_circ_0001558 gradually increased in patients with NST-AMI (non-ST-segment elevation acute myocardial infarction) and ST-AMI (ST-segment elevation acute myocardial infarction) High, the relative expression in NST-AMI patients was 2.80 times that of NCCP patients, and the area under the ROC curve for diagnosing NST-AMI was 0.72; the relative expression in ST-AMI patients was 5.27 times that of NCCP patients, and its diagnosis of ST The area under the ROC curve of -AMI is 0.831.

In summary, the present invention has the following advantages:

(1) Compared with traditional markers, the marker exosome hsa_circ_0001558 provided by the present invention has higher sensitivity for detecting AMI, the area under the ROC curve for diagnosing AMI is 0.793, and the area under the ROC curve for diagnosing NST-AMI is 0.72 , the area under the ROC curve for the diagnosis of ST-AMI is 0.831, the specificity is stronger, the detection is faster and more convenient, and the cost is lower.

(2) The marker exosome hsa_circ_0001558 provided by the present invention has a high expression level in AMI patients, and can be used as a diagnostic marker for acute myocardial infarction. Compared with AMI, exosome hsa_circ_0001119 has higher expression level and stronger specificity.

Description of drawings:

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

Figure 1 is a TEM image of exosomes (bar, 200nm) extracted from plasma in an embodiment of the present invention;

Figure 2 is a particle size distribution diagram of plasma exosomes in an embodiment of the present invention;

Fig. 3 is the Western blot analysis figure of exosome marker protein CD63 and HSP70 in the embodiment of the present invention;

Fig. 4 is the volcano graph and histogram of differentially expressed circRNAs in the embodiment of the present invention;

Fig. 5 is a small sample size verification result figure in the embodiment of the present invention;

Fig. 6 is a large sample size verification result figure in the embodiment of the present invention;

Fig. 7 is the expression change and ROC curve of exosome hsa_circ_0001558 in NST-AMI and ST-AMI in the embodiment of the present invention.

Detailed ways

It should be pointed out that the following detailed description is exemplary and intended to provide further explanation to the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It should be noted that the terminology used here is only for describing specific implementations, and is not intended to limit the exemplary implementations according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this description, it indicates that there are Features, steps, operations, devices, components and/or combinations thereof.

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Apparently, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example

1. The main reagents and manufacturers of the experiment are as follows:

Trizol was purchased from Invitrogen; Transcriptor First Strand cDNA synthesis kit (first strand cDNA synthesis kit) was purchased from Roche; Select SYBR master mix was purchased from ABI; PBS was purchased from Biolegend; 10% SDS (dodecyl sulfonic acid Sodium), 10% ammonium persulfate, 1.0MTris-HCl (pH=6.8), 30% acrylamide, 10X blocking washing buffer, 10X electrophoresis buffer, 10X electrophoresis buffer were purchased from Beijing Pulilai Gene Technology Co., Ltd. ; DEPC water, 1.5MTris-HCl (pH=8.8), Western protein lysate, and BCA protein concentration assay kit were purchased from Biyun Tiansheng Technology Co., Ltd.; TEMED (tetramethylethylenediamine) was purchased from Scientific Research Special; NC The membrane was purchased from Hyclone; Difco ^TM Skim Milk was purchased from BD; CD63 antibody, HSP70 antibody, and GAPDH antibody were purchased from abcam; goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from Beijing Ligaotai Technology Company; exosome extraction The kit (Cat. No. 217184), exosomal RNA extraction kit (Cat. No. 76064) was purchased from QIAGEN; the reverse transcription kit was purchased from Promega; the fluorescent quantitative PCR kit was purchased from Thermo Fisher; 18S RNA was purchased from Aoke Dingsheng Biotech Technology Co., Ltd.

2. Experimental method

2.1 Patient specimen collection

Patients hospitalized in Beijing Chaoyang Hospital Affiliated to Capital Medical University and Baotou Central Hospital in Inner Mongolia from October 2016 to March 2018 were selected, including 120 patients with AMI (acute myocardial infarction) and 83 patients with NCCP (non-cardiac chest pain). Record the patient's gender, age, smoking history, drinking history, SBP (systolic blood pressure), DBP (diastolic blood pressure), TC (total cholesterol), TG (triglycerides), HDL (high-density lipoprotein), LDL (low-density lipoprotein) lipoprotein) and other clinical data.

Inclusion criteria: acute myocardial infarction (diagnostic criteria based on 2015 ESC/AHA/ACC guidelines): ischemic symptoms, elevated expression of cTnT (troponin) and CK-MB (creatine kinase isoenzyme), pathological Q wave in electrocardiogram. All AMI patients were diagnosed for the first time. Patients with non-cardiac chest pain: admitted to hospital with chest pain, coronary angiography was negative, and the final diagnosis was cardiac neurosis, arteriosclerosis, reflux esophagitis, and hyperlipidemia.

Exclusion criteria: AMI combined with other diseases such as diabetes, hypothyroidism, bronchial asthma, chronic kidney disease, tumors, etc.

2.2 Blood collection and separation of plasma

20 mL of fasting venous blood was collected from the patient on the morning after admission with a blood collection needle and an anticoagulant tube (including EDTA), mixed well and left to stand at 4°C for 3-4 hours. Centrifuge at 3000rpm and 4°C for 10min, the supernatant is plasma, filter with a 0.2μm filter, aliquot 1mL/tube and store at -80°C.

2.3 Extraction of exosomes from plasma

Use the exosome extraction kit to isolate exosomes from plasma as follows:

(1) Take out the frozen plasma from the -80°C refrigerator, melt it in a metal bath at 37°C, centrifuge at 10,000g for 15min to get the supernatant, and transfer 4mL of plasma to a new 15mL centrifuge tube;

(2) Add 1 volume of XBP buffer and gently invert the tube 5 times, incubate at room temperature; then put the mixture into the column and centrifuge at 500g for 1 min, pour off the waste liquid and put the column back into the centrifuge tube;

(3) Add 10mL of XWP buffer and centrifuge at 3000g for 5min, and transfer the column to a new centrifuge tube;

(4) Finally, add 1mL of XE buffer eluent to the column, incubate for 1min, collect the filtrate after centrifuging at 500g for 5min, add it to the column again, incubate for 1min again, and centrifuge at 3000g for 5min;

(5) Collect the filtrate (ie exosomes) into a new RNase-free EP tube and store at -80°C.

2.4 Size analysis of exosomes

Add 1 mL of phosphate buffer saline (PBS) to the exosome stock solution and mix well. Measurements were performed using the Nanoparticle Tracking Analyzer Zeta View-Particle Metrix, injecting the sample into the sample cell. Open the software to adjust the brightness and focus, adjust the field of view, observe, and use the software to analyze the particle size according to the time and displacement of the particle trajectory.

2.5 TEM analysis of exosomes

(1) Add 50-100 μL of 2% paraformaldehyde solution to the extracted exosomes to obtain an exosome solution;

(2) Take 5-10 μL of exosome solution and drop it on the Formvar-carbon sample-loading copper grid, and place it at room temperature for 10 minutes;

(3) Then use PBS buffer to wash once;

(4) First use 50 μL of 1% glutaraldehyde droplets on the copper grid for 5 minutes, and then wash with 100 μL redistilled water 8 times for 2 minutes each time;

(5) Treat with 50 μL uranyl oxalate droplets (pH 7.0) for 5 min;

(6) Put the copper grid on ice, drop 50 μL of methylcellulose for 10 minutes, and air dry for 5-10 minutes;

(7) Put the copper mesh in the sample box, adjust the appropriate focus and brightness under 80kV and take pictures.

2.6 Extraction and quantification of exosomal proteins

Add 100 μL of protease inhibitor-containing lysate (Radio-Immunoprecipitation Assay, RIPA) to the isolated exosomes, lyse on ice for 5 min, centrifuge at 13,000 rpm and 4°C for 5 min, and discard the precipitate. The BCA protein concentration determination kit was used to extract the total protein of the cells and determine the protein concentration. The protein samples in this experiment were collected from three independent repeated exosome experiments. The detailed operation steps are as follows:

(1) Take 0.8mL protein standard preparation solution and add it to protein standard product (20mg BSA), fully mix and dissolve, and prepare a 25mg/mL protein standard solution;

(2) Take 20 μL protein standard solution and add it to 980 μL diluent to prepare 0.5 mg/ml protein standard solution;

(3) According to the number of samples, add BCA reagent A to BCA reagent B according to the volume of 50:1 to prepare an appropriate amount of BCA working liquid, and mix thoroughly;

(4) Add the standard substance into the standard well of the 96-well plate according to 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL, and add standard dilution solution to make up to 20 μL;

(5) Add 10 μL of the sample to be tested to the sample wells of the 96-well plate, add standard diluent to 20 μL, add 200 μL of BCA working solution to each well, and place at room temperature for 2 hours;

(6) Measure the absorbance value of the sample at a wavelength of 560nm, and calculate the protein concentration of the sample according to the standard curve.

After the protein is quantified, add 5X SDS gel loading buffer, boil at 100°C for 5 minutes to denature the protein, complete the protein extraction step, and store the sample in a -80°C ultra-low temperature refrigerator.

2.7 Western Blot

2.7.1 The preparation of SDS polyacrylamide separation gel and base glue is as follows:

The composition of the separating gel (concentration 10%): ultrapure water 4.0mL, 30% acrylamide 3.3mL, 1.5MTris-HCl (pH=8.8) 2.5mL, 10% SDS 0.1mL, 10% ammonium persulfate 0.1mL, TEMED 0.004mL;

The composition of the stacking gel (concentration 5%): ultrapure water 4.1mL, 30% acrylamide 1.0mL, 1.0MTris-HCl (pH=6.8) 0.75mL, 10% SDS 0.06mL, 10% ammonium persulfate 0.06mL, TEMED 0.006mL.

2.7.2 Western blot

(1) Add 1/5 volume of 5× loading buffer to the supernatant (plasma), cook at 95°C for 5 minutes;

(2) After cooking, take 20 μL and add it to 10% SDS polyacrylamide gel for electrophoresis;

(3) After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane;

(4) Block with 5% BSA for 1 hour;

(5) Add CD63 antibody (abcam company, 1:1000), HSP70 antibody (abcam company, 1:3000) or GAPDH antibody (abcam company, 1:5000), incubate overnight at 4°C, TBST (Tris salt wash buffer ) washed 3 times, 10min each time;

(6) Then add goat anti-rabbit secondary antibody (Beijing Ligaotai Technology Company, 1:15000) or goat anti-mouse secondary antibody (Beijing Ligaotai Technology Company, 1:15000), incubate in the greenhouse for 1 hour, wash 3 times with TBST, Expose for 10 minutes each time.

2.8 Extraction of total RNA from exosomes

Use the exosome extraction kit to extract exosome RNA, the steps are as follows:

(1) Add 600 μL of lysate to the extracted exosome stock solution, incubate at room temperature for 2 minutes, centrifuge at 3000 g for 5 minutes, and take the supernatant;

(2) Add chloroform, separate layers, take the upper aqueous phase and add 1.5 times the volume of 100% ethanol, and mix up and down with a pipette;

(3) After mixing, add in batches to the column provided by the kit, centrifuge at 8000g for 15s, discard the waste liquid, and recover the column;

(4) Add the buffer solution of the kit to the column, centrifuge to wash the column, then add 80% ethanol to the column and centrifuge at 8000g for 2min;

(5) After centrifugation, transfer the column to a new RNase-free EP tube, and dry the filter membrane on the column;

(6) Then add 14 μL of eluent, centrifuge at 8000 g for 1 min, throw away the column, collect the filtrate, and store it in a -80 ° C refrigerator;

(7) The RNA concentration was measured with Nano Drop 2000 (Thermo Scientific, USA), and the RNA amount was >4 μg; the RNA concentration was 50ng/μL-500ng/μL; the RNA absorbance 260/280 was between 1.8-2.1.

2.9 High-throughput sequencing

Firstly, exosomal RNA undergoes pre-treatments such as purification, and then reverse-transcribes to form cDNA, ends are repaired, adapters are added, and PCR amplification is used to build a library. After passing the quality inspection, it is sequenced on the machine. After obtaining the original data, first filter the data, remove adapter sequences and low-quality read lengths, then evaluate the sequencing quality to obtain high-quality data, and compare the high-quality data with reference data to obtain BAM files.

2.10 Reverse transcription

The reverse transcription of RNA is carried out with a reverse transcription kit using a two-step method, and the specific steps are as follows:

(1) RNA (100ng) x μL, OligoT15 (0.5 μg) 1 μL, DEPC·H ₂ O 9-x μL, the total volume is 10 μL;

(2) React at 70°C for 5 minutes to open the RNA secondary structure; place it on ice immediately, then add 5×AMV Buffer 4μL, 10mM dNTP mixture 2μL, Mg ²⁺ 2μL, RNasin (40U/μL) 1μL, AMV 1μL, total The volume is 10 μL, react at 42°C for 60 minutes, react at 99°C for 2 minutes, keep the temperature at 4°C, and store the product at -20°C.

2.11 Real-time quantitative PCR

The level of circRNA was detected using PowerUp ^TM SYBR ^TM Green Master Mix Fluorescence Quantitative Kit and ABI 7500 PCR instrument. Take 4 μL of 10-fold diluted cDNA as a template, set up 3 parallel wells for each sample, and use 18S RNA as an internal reference gene.

(1) The reaction system is as follows: 10 μL of 2X Master Mix, 4 μL of H ₂ O, 1 μL of Primer F (10 μM), 1 μL of Primer R (10 μM), and 4 μL of cDNA.

(2) The reaction conditions are as follows:

(3) qRT-PCR primer sequences, the information is shown in Table 1 below.

Table 1 qRT-PCR primer sequence list

2.12 Statistical Analysis Methods

SPSS17.0 software was used for statistical analysis. Normally distributed data were expressed as mean ± standard deviation (X ± S), and non-normally distributed data were expressed as median (P25, P75). The t-test was used for the comparison between two groups of normally distributed data, the non-parametric test was used for the comparison of non-normally distributed data between two groups, and the chi-square test was used for the comparison of the data rates of the two groups. Differences in circRNA levels were determined by the Mann-Whitney U test. To assess the predictive value of selected circRNAs for AMI, we used ROC curves, and a P value < 0.05 was considered statistically significant.

3. Test results

1. Exosome identification

1.1 Observation of extracted exosomes by transmission electron microscope

Under the transmission electron microscope, it was observed that the extracted vesicles were uneven in size, round or almost round, with a lipid bilayer membrane, and the diameter was between 30-150 nm, which conformed to the morphological characteristics of exosomes, as shown in Figure 1.

1.2 Exosome particle size analysis

The particle size of the extracted vesicle particles is 30-200nm, and the main peak is around 100nm, as shown in Figure 2.

1.3 Western blot determination of exosome-specific molecular markers

The results of western blot experiments showed that the plasma exosomes of AMI patients and NCCP patients expressed specific marker proteins CD63 and HSP70, but not GAPDH, as shown in Figure 3, which further proved that the extracted vesicles were exosomes.

2. High-throughput sequencing

In order to clarify the expression of circRNA in plasma exosomes of patients with AMI, we screened 15 samples of NCCP and 15 samples of AMI (sequencing cohort) by high-throughput sequencing.

2.1 Clinical characteristics of the sequencing cohort

There was no significant difference in age and gender between the AMI and NCCP groups, but the levels of cTnT, CK-MB, and NT-proBNP (amino-terminal pro-brain natriuretic peptide) were significantly higher in the AMI group than in the NCCP group, which was consistent with the clinical characteristics of AMI patients. Table 2 shows the data of the patients in the NCCP group and the NCCP group.

Table 2 Basic clinical data of patients

2.2 High-throughput sequencing results

The results of high-throughput sequencing showed that the expression profile of circRNA in plasma exosomes of patients with AMI was significantly different from that of the control group. With |log2(Fold change)|>1, q-value<0.001 as the standard, AMI plasma was screened Among the 893 differentially expressed circRNAs in exosomes, 118 were up-regulated and 775 were down-regulated (as shown in Figure 4, A). These 107 differentially expressed circRNAs were annotated in circBase, as shown in Table 3.

Table 3 Differentially expressed circRNAs in AMIs annotated in circBase

2.3 Small sample size verification of differentially expressed circRNAs

In order to verify the results of high-throughput sequencing and consider the feasibility as a biomarker, we selected 5 circRNAs that were annotated in the circBase database, had high expression levels in the AMI group, and had a large increase factor among the up-regulated circRNAs. circRNAs (exosome hsa_circ_0001535, exosome hsa_circ_0003270, exosome hsa_circ_0000972, exosome hsa_circ_0001119 and exosome hsa_circ_0001558), as shown in panel B in Figure 4. The method of RT-PCR was verified in small samples.

2.3.1 Clinical characteristics of the small sample cohort

The small sample cohort selected 20 patients with NCCP and 20 patients with AMI, and the clinical data are shown in Table 4. There was no difference in age, sex, height, weight, etc. between the two groups; the proportion of hyperlipidemia, WBC (white blood cells), GLU (glucose), K ⁺ , AST (aspartate aminotransferase), ALT (alanine aminotransferase) , CRP (C-reactive protein), FT3 (serum free triiodothyronine), FT4 (serum free thyroxine), sTSH (sensitive thyroid-stimulating hormone), CK-MB, TNI (serum myocardial necrosis markers) levels The AMI group was significantly higher than the NCCP group; LVEF (%) (left ventricular ejection fraction), Na ⁺ levels were significantly lower in the AMI group than the NCCP group.

Table 4 Small sample cohort clinical data table

2.3.2 Small sample size verification results

The results of RT-PCR verification showed that the expression of exosome hsa_circ_0001119 was low and could not be detected in most samples. The detection results of body hsa_circ_0000972 and exosome hsa_circ_0001558 were consistent with the sequencing results. The relative expression level of exosome hsa_circ_0001535 in plasma exosomes of AMI patients (8.49 (5.34, 13.94)) was significantly higher than that of NCCP group (5.45 (3.50, 7.57)) (P<0.05), 1.56 times that of NCCP group, The area under the ROC curve for the diagnosis of AMI in this cohort was 0.685 (as shown in A and D in Figure 5); The NCCP group (3.43 (1, 12.4)) was significantly increased (P<0.05), which was 4.58 times that of the NCCP group; the area under the ROC curve for diagnosing AMI in this cohort population was 0.683 (as shown in Fig. 5 B and E shown in the figure); the relative expression level of exosome hsa_circ_0001558 in plasma exosomes of AMI patients (27.62 (20.47, 44.81)) was significantly higher than that of NCCP group (7.42 (4.21, 31.08)) (P<0.01), for 3.72 times that of the control group, the area under the ROC curve for diagnosing AMI in this cohort population was 0.790 (as shown in Figure C and F in Figure 5). Since the area under the ROC curve of the exosome hsa_circ_0001558 in the diagnosis of AMI was the largest among the three circRNAs, the exosome hsa_circ_0001558 was selected for further verification in a large sample size.

Among them, Figure A in Figure 5 shows the relative expression level of exosome hsa_circ_0001535 in AMI and NCCP; Figure B shows the relative expression level of exosome hsa_circ_0001558 in AMI and NCCP; Figure C shows the relative expression level of exosome hsa_circ_0000972 in AMI and NCCP The relative expression level in ; D is the ROC curve of exosome hsa_circ_0001535 for the diagnosis of AMI; E is the ROC curve of exosome hsa_circ_0000972 for the diagnosis of AMI; F is the ROC curve of exosome hsa_circ_0001558 for the diagnosis of AMI. *P<0.05, **P<0.01.

2.4 Large sample size verification of differentially expressed circRNAs

In order to validate the results of the small sample size cohort, we validated the level of exosomal hsa_circ_0001558 in a larger sample size.

2.4.1 Large sample size cohort clinical data

The clinical information of 48 NCCP and 85 AMI patients are shown in Table 5. The proportion of male patients in the AMI group, heart rate, proportion of hypertension, proportion of hyperlipidemia, WBC, HB, TG, GLU, AST, ALT, K ⁺ , DD, CRP, TT3, FT3, sTSH, CK-MB, TnI The level is higher than that of NCCP group, and the level of LVEF(%) and Na ⁺ is lower than that of NCCP group.

Table 5 Clinical characteristics of the study population: verification of exosomal circRNA circulation in large samples

2.4.2 Large sample cohort validation results

The results of plasma exosome hsa_circ_0001558 in AMI and NCCP patients with large sample size were consistent with those of small sample cohort. The relative expression level of exosome hsa_circ_0001558 in plasma exosomes of AMI patients (8.81 (4.87, 17.76)) was significantly higher than that of NCCP group (1.98 (0.74, 6.44)) (P<0.01), which was 4.45 times that of NCCP group , the area under the ROC curve for the diagnosis of AMI is 0.793, as shown in Figure 6 (A is the expression level of hsa_circ_0001558 in the plasma exosomes of AMI patients; B. is the ROC curve for the diagnosis of AMI by plasma exosomes hsa_circ_0001558; **P<0.01).

AMI patients were further divided into ST-segment elevation AMI (ST-AMI) and non-ST-segment elevation AMI (NST-AMI), and the relative expression of plasma exosome hsa_circ_0001558 in NST-AMI group (5.54(4, 11.96)) was significantly higher than that of the NCCP group (1.98(0.74,6.44)), which was 2.80 times that of the NCCP group, and the area under the ROC curve for the diagnosis of NST-AMI was 0.72; the relative expression in the ST-AMI group (10.44( 6.95, 22.93)) was 5.27 times that of the NCCP group, and the area under the ROC curve for the diagnosis of ST-AMI was 0.831, as shown in Figure 7 (Figure A shows the expression of exosome hsa_circ_0001 558 in NST-AMI and ST-AMI Change; B is the ROC curve of exosome hsa_circ_0001558 for the diagnosis of NST-AMI; C is the ROC curve of exosome hsa_circ_0001558 for the diagnosis of ST-AMI. *P<0.05, **P<0.01).

AMI mostly occurs in coronary atherosclerotic stenosis, mainly caused by plaque rupture or thrombosis and secondary coronary artery obstruction, leading to myocardial cell apoptosis and myocardial necrosis. Studies have shown that exosome-mediated intercellular communication plays a role in AMI through multiple mechanisms. The basic information of cells can be directly obtained by analyzing the information substances in exosomes. Most of the existing studies have focused on the role and mechanism of miRNA in exosomes in the early diagnosis of AMI, transplantation therapy, and myocardial fibrosis and angiogenesis after AMI. circRNA is one of the important information substances in exosomes.

circRNA is a research hotspot in recent years, and it is a circular RNA molecule composed of exon or intron sequence. CircRNA has no 5' and 3' ends and is not easily degraded by RNases, so it is more stable than linear RNA. CircRNAs are abundantly present in the cytoplasm of eukaryotic cells and have tissue, temporal and disease specificity.

In this study, high-throughput sequencing was first found that the expression profile of circRNA in AMI plasma exosomes was significantly different from that in the control group. With |log2(Fold change)|>1, q-value<0.001 as the standard, AMI plasma was screened. Of the 893 differentially expressed circRNAs in exosomes, 118 were up-regulated and 775 were down-regulated, indicating that circulating exosomes are selective for the packaging of circRNAs during AMI and can be used as AMI biomarkers.

Next, we selected five exosomal circRNAs that were annotated in the circBase database, had high expression levels, and significantly increased folds, and were verified by RT-PCR in a small sample size. The results showed that the detection results of exosome hsa_circ_0001535, exosome hsa_circ_0000972 and exosome hsa_circ_0001558 were consistent with the sequencing results, and in the small sample cohort, exosome hsa_circ_0001558 had the highest increase in the AMI group, and the ROC for diagnosis of AMI The area under the curve was the largest (as shown in Figure 5), so it was validated in a larger sample size.

The verification results of exosome hsa_circ_0001558 in patients with a larger sample size are consistent with the verification results of a small sample size. Its expression in plasma exosomes of AMI patients is indeed significantly higher than that of the NCCP group, which is 4.45 times that of the NCCP group. Its diagnosis The area under the ROC curve of AMI is 0.793 (as shown in Figure 6).

According to its electrocardiographic features, AMI can be divided into ST-AMI and NST-AMI myocardial infarction. Therefore, we further analyzed the expression levels of exosomal hsa_circ_0001558 in ST-AMI and NST-AMI patients in a large sample size cohort. The results showed that the expression levels of exosome hsa_circ_0001558 in NCCP, ST-AMI and NST-AMI plasma exosomes gradually increased, and the area under the ROC curve for the diagnosis of NST-AMI was 0.72, and the area under the ROC curve for the diagnosis of ST-AMI The lower area is 0.831 (as shown in Figure 7). Exosome hsa_circ_0001558 is located on chromosome 5 with a length of 328nt and can be detected in tissues/cells such as platelets, K562 cells, and vascular endothelial cells.

The expression profile of circRNA in AMI circulating exosomes was significantly different from that of the control, and the expression level of exosome hsa_circ_0001558 in NCCP, ST-AMI and NST-AMI plasma exosomes gradually increased, which can be used as a diagnostic biomarker for AMI .

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims

An application of a plasma exosomal circRNA marker in the preparation of an acute myocardial infarction detection marker, the plasma exosomal circRNA marker is hsa_circ_0001558, and the nucleotide sequence of the hsa_circ_0001558 is shown in SEQ ID NO:1.
An application of a plasma exosome circRNA marker in the preparation of a diagnostic product for acute myocardial infarction, the plasma exosome circRNA marker is hsa_circ_0001558, and the nucleotide sequence of the hsa_circ_0001558 is shown in SEQ ID NO:1.
The diagnostic product according to claim 2, wherein the diagnostic product is a chip, a preparation or a kit.