CN114182010B - A plasma exosomal circRNA marker and its application - Google Patents

Abstract

The invention relates to the technical field of molecular diagnosis, in particular to a plasma exosome circRNA marker and its application. The plasma exosomal circRNA marker is hsa_circ_001558, and the nucleotide sequence of hsa_circ_001558 is shown in SEQ ID NO: 1. The application of the plasma exosomal circRNA marker as a marker for detecting acute myocardial infarction. The marker exosome hsa_circ_001558 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. high, the specificity is stronger.

Description

A plasma exosomal 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 death of residents. 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 ischemia and hypoxia of coronary artery. Clinically, there is often severe and persistent retrosternal pain, which cannot be completely relieved by rest and nitrates, accompanied by increased serum myocardial enzyme activity and progressive electrocardiographic changes, and can 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 prognostic assessment of AMI. cTnT has high sensitivity, but because 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 are the keys to improve the treatment and prognosis of AMI patients.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a plasma exosomal circRNA marker, which can be used as a biomarker for the detection of 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 invention provides the application of a plasma exosome circRNA marker as a marker for detecting acute myocardial infarction.

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

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' plasma exosomes. The area under the ROC curve for diagnosing AMI was 0.793; the expression of exosome hsa_circ_0001558 gradually increased in NST-AMI (non-ST-segment elevation acute myocardial infarction) and ST-AMI (ST-segment elevation acute myocardial infarction) patients High, the relative expression in NST-AMI patients was 2.80 times that of NCCP patients, and the area under the ROC curve for the diagnosis of 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 for -AMI was 0.831.

To sum up, 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, and its area under the ROC curve for diagnosing AMI is 0.793, and its 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, with stronger specificity, faster and more convenient detection, and lower cost.

(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 the exosome hsa_circ_0001119, the expression level is higher and the specificity is stronger.

Description of drawings:

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

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

Fig. 2 is the particle size distribution diagram of plasma exosomes in the embodiment of the present invention;

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

Figure 4 is a volcano plot and a bar graph of differentially expressed circRNAs in the embodiment of the present invention;

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

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

Figure 7 is a graph showing the expression changes and ROC curves of exosome hsa_circ_0001558 in NST-AMI and ST-AMI in the embodiment of the present invention.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, 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 herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, the singular also includes the plural unless the context clearly dictates otherwise, and it should also be understood that when the terms "comprising" and/or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and/or combinations thereof.

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example

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

Trizol was purchased from Lnvitrogen; Transcriptor First Strand cDNA synthesis kit was purchased from Roche; Select SYBR master mix was purchased from ABI; PBS was purchased from Biolegend; 10% SDS (dodecylsulfonic acid) sodium), 10% ammonium persulfate, 1.0M Tris-HCl (pH=6.8), 30% acrylamide, 10X blocking wash buffer, 10X electrophoresis buffer, 10X electrotransfer buffer were purchased from Beijing Prily Gene Technology Co., Ltd. ; DEPC water, 1.5MTris-HCl (pH=8.8), Western protein lysate, and BCA protein concentration assay kit were purchased from Biyunsheng Technology Co., Ltd.; TEMED (tetramethylethylenediamine) was purchased from Scientific ResearchSpecial; NC membrane Purchased from Hyclone; Difco ^TM Skim Milk was purchased from BD; CD63 antibody, HSP70 antibody, GAPDH antibody were purchased from abcam company; goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from Beijing Ligaotai Technology Co., Ltd.; exosome extraction reagent Kit (Cat. No. 217184) and Exosome RNA Extraction Kit (Cat. No. 76064) were purchased from QIAGEN; reverse transcription kit was purchased from Promega; fluorescence quantitative PCR kit was purchased from Thermo Fisher; 18S RNA was purchased from Aoke Dingsheng Biotechnology Ltd.

2. Experimental method

2.1 Patient Specimen Collection

Patients who were 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). Gender, age, smoking history, drinking history, SBP (systolic blood pressure), DBP (diastolic blood pressure), TC (total cholesterol), TG (triglyceride), HDL (high density lipoprotein), LDL (low density) were recorded. 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), and pathological Q wave on ECG. All AMI patients were diagnosed for the first time. Patients with non-cardiogenic chest pain: chest pain was admitted to hospital, coronary angiography was negative, and the final diagnosis was cardiac neurosis, arteriosclerosis, reflux esophagitis, and hyperlipidemia.

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

2.2 Blood collection and plasma separation

20 mL of fasting venous blood was collected from the patient on the morning of the next day after admission with a blood collection needle and an anticoagulant tube (containing EDTA). Centrifuge at 3000rpm and 4°C for 10min, and the supernatant is plasma, which is filtered with a 0.2 μm filter, aliquoted in 1mL/tube and stored at -80°C.

2.3 Extraction of exosomes from plasma

Exosomes were isolated from plasma using an exosome extraction kit as follows:

(1) Take out the frozen plasma from the -80°C refrigerator, thaw it in a metal bath at 37°C, centrifuge at 10,000 g for 15 min to take the supernatant, and take 4 mL of plasma into a new 15 mL 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 out the waste liquid and put the column back into the centrifuge tube;

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

(4) Finally, add 1 mL of XE buffer eluate to the column, incubate for 1 min, collect the filtrate after centrifugation at 500g for 5min, re-add it to the column, incubate again for 1min, 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 Exosome particle size analysis

1 mL of phosphate buffered saline (PBS) was added to the exosome stock solution and mixed. Measurements were performed using the Zeta View-Particle Metrix, a nanoparticle tracking analyzer, with the sample injected 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's 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-loaded 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 mesh for 5 minutes, and then wash with 100 μL of re-distilled water for 8 times for 2 minutes each time;

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

(6) Put the copper mesh on ice again with 50 μL methylcellulose droplets for 10 minutes, and air dry for 5-10 minutes;

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

2.6 Extraction and quantification of exosomal proteins

100 μL of lysate containing protease inhibitor (Radio-Immunoprecipitation Assay, RIPA) was added to the isolated exosomes, lysed on ice for 5 min, centrifuged at 13000 rpm and 4 °C for 5 min, and the pellet was discarded. The BCA protein concentration assay kit was used to extract the total cell protein 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 0.8 mL of protein standard preparation solution and add it to the protein standard product (20 mg BSA), mix and dissolve thoroughly, and prepare a protein standard solution of 25 mg/mL;

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

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

(4) Add the standard to the standard wells 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 the 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 the standard dilution solution 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 560 nm, and calculate the protein concentration of the sample according to the standard curve.

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

2.7 Western Blot (Western Blot)

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

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

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

2.7.2 Western blotting

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

(2) After boiling, add 20 μL to a 10% SDS polyacrylamide gel for electrophoresis;

(3) Transfer the protein to a polyvinylidene fluoride (PVDF) membrane after electrophoresis;

(4) 5%BSA closed for 1h;

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

(6) After adding goat anti-rabbit secondary antibody (Beijing Ligaotai Technology Co., Ltd., 1:15000) or goat anti-mouse secondary antibody (Beijing Ligaotai Technology Co., Ltd., 1:15000), incubate for 1 h in the greenhouse, wash 3 times with TBST, Exposure every 10min.

2.8 Extraction of total RNA from exosomes

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

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

(2) adding chloroform, layering, taking the upper water phase, adding 1.5 times the volume of 100% ethanol, and mixing up and down with a pipette;

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

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

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

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

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

2.9 High-throughput sequencing

First, the exosomal RNA was purified and other preliminary treatments, and then reverse transcribed to form cDNA, end repaired with adapters, PCR amplification and other steps to build a library, and then sequenced on the machine after passing the quality inspection. After obtaining the original data, the data must first be filtered to remove the adapter sequences and low-quality reads, and then the sequencing quality should be evaluated to obtain high-quality data, and the high-quality data and reference data should be compared. BAM file.

2.10 Reverse transcription

The reverse transcription of RNA is carried out with a reverse transcription kit using a two-step method. The specific operation 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) Reaction at 70°C for 5 minutes to open the RNA secondary structure; put 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 was 10 μL, the reaction was performed at 42°C for 60 minutes, at 99°C for 2 minutes, at a constant temperature of 4°C, and the product was stored 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 the template, set 3 parallel wells for each sample, and use 18S RNA as the 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 PrimerR (10 μM), and 4 μL of cDNA.

(2) 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

SPSS 17.0 software was used for statistical analysis, and the data with normal distribution was expressed as mean ± standard deviation (X ± S), and the data with non-normal distribution was expressed as median (P25, P75). The t-test was used for the comparison between the two groups of normally distributed data, the nonparametric test was used for the comparison of the non-normally distributed data between the two groups, and the chi-square test was used for the comparison of the data rates between the two groups. Differences in circRNA levels were determined by the Mann-Whitney U test. To evaluate the predictive value of selected circRNAs for AMI, we used ROC curves, with a P value < 0.05 as a statistically significant difference.

3. Test results

1. Exosome identification

1.1 Observation of the extracted exosomes by transmission electron microscope

Under the transmission electron microscope, it was observed that the extracted vesicles were of uneven size, round or quasi-circular, with a lipid bilayer membrane, and the diameter was between 30-150 nm, which was in line with 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 30-200 nm, and the main peak is about 100 nm, as shown in Figure 2.

1.3 Western blot determination of exosome-specific molecular markers

The results of western blotting 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

To clarify the expression of circRNAs in plasma exosomes from AMI patients, we screened 15 NCCP and 15 AMI (sequencing cohort) specimens 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. The levels of cTnT, CK-MB, and NT-proBNP (amino-terminal brain natriuretic peptide precursor) were significantly higher in the AMI group than in the NCCP group, which was in line with the clinical characteristics of AMI patients. The data of patients in group and NCCP group are shown in Table 2.

Table 2 Basic clinical data of patients

2.2 High-throughput sequencing results

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

Table 3 Differentially expressed circRNAs in AMI annotated in circBase

2.3 Validation of differentially expressed circRNAs with small sample size

In order to verify the results of high-throughput sequencing, and considering the feasibility of being used as biomarkers, we selected 5 of the up-regulated circRNAs that were annotated in the circBase database, were highly expressed in the AMI group, and had a larger fold increase. 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 RT-PCR method was validated in small samples.

2.3.1 Clinical characteristics of small sample cohort

The small sample cohort included 20 patients with NCCP and 20 patients with AMI. The clinical data are shown in Table 4. There were no differences in age, gender, 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 marker) levels The AMI group was significantly higher than the NCCP group; the levels of LVEF (%) (left ventricular ejection fraction) and Na ⁺ were significantly lower in the AMI group than in 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, and the expression of exosome hsa_circ_0003270 had no difference between the two groups of AMI patients and NCCP patients. The detection results of exosomal hsa_circ_0000972 and exosomal hsa_circ_0001558 were consistent with the sequencing results. The relative expression level of exosome hsa_circ_0001535 in the plasma exosomes of AMI patients (8.49 (5.34, 13.94)) was significantly higher than that of the NCCP group (5.45 (3.50, 7.57)) (P<0.05), which was 1.56 times that of the NCCP group. In this cohort, the area under the ROC curve for the diagnosis of AMI was 0.685 (as shown in panels A and D in Figure 5); the relative expression level of exosome hsa_circ_0000972 in AMI patients (15.7 (1.75, 21.71)) was higher than The NCCP group (3.43(1, 12.4)) was significantly higher (P<0.05), which was 4.58 times that of the NCCP group; the area under the ROC curve for the diagnosis of AMI in this cohort population was 0.683 (Figure 5, B and E in Figure 5). 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), which was It was 3.72 times that of the control group, and the area under the ROC curve for the diagnosis of AMI in this cohort was 0.790 (as shown in Figure C and F in Figure 5). Since exosomal hsa_circ_0001558 had the largest area under the ROC curve for diagnosing AMI among these three circRNAs, exosomal hsa_circ_0001558 was selected for further validation in large-scale samples.

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

2.4 Validation of differentially expressed circRNAs with large sample size

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 Clinical data of large sample cohort

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

Table 5 Clinical characteristics of the study population: Circular validation of exosomal circRNAs in large samples

2.4.2 Validation results of large sample cohort

The validation results of plasma exosome hsa_circ_0001558 in large sample size AMI and NCCP patients were consistent with the validation results in 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 diagnosing AMI is 0.793, as shown in Figure 6 (A is the expression level of hsa_circ_0001558 in plasma exosomes of AMI patients; B. The ROC curve of plasma exosome hsa_circ_0001558 for diagnosing AMI; **P<0.01).

AMI patients were further divided into ST-segment elevation AMI (ST-AMI) and non-ST-segment elevation AMI (NST-AMI). 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 its area under the ROC curve for diagnosing 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 its 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 Changes; Figure B is the ROC curve of exosome hsa_circ_0001558 in the diagnosis of NST-AMI; Figure C is the ROC curve of exosome hsa_circ_0001558 in 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 occlusion, resulting in 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. Existing studies mostly focus on the role and mechanism of miRNAs 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, which is a circular RNA molecule composed of exon or intron sequences. CircRNAs have no 5' and 3' ends and are not easily degraded by RNases, so they are more stable than linear RNAs. CircRNAs are abundant in the cytoplasm of eukaryotic cells and are tissue-, timing-, and disease-specific.

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

Next, we selected five exosomal circRNAs with annotations, high expression levels, and obvious increased folds in the circBase database, and verified them 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 diagnosing AMI was the highest. The area under the curve was the largest (shown in Figure 5), so it was validated in a larger sample size.

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

AMI can be divided into ST-AMI and NST-AMI myocardial infarction according to its electrocardiographic characteristics. Therefore, we further analyzed the expression level of exosomal hsa_circ_0001558 in ST-AMI and NST-AMI patients in a large sample size cohort. The results showed that the expression level of exosome hsa_circ_0001558 in NCCP, ST-AMI and NST-AMI plasma exosomes gradually increased, the area under the ROC curve for the diagnosis of NST-AMI was 0.72, and the ROC curve for the diagnosis of ST-AMI was 0.72. 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 circulating exosomes of AMI was significantly different from that of the control. 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, but not to limit 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: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

sequence listing

<110> Beijing Chaoyang Hospital Affiliated to Capital Medical University

<120> A plasma exosomal circRNA marker and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211>297

<212> DNA

<213> Artficial Sequence

<400> 1

GGGAAACTCT GTACCTGCTT CACAAAGTGT TGCTGCTTTG ACCAGTAAGA GAAGCTTAGT 60

CCTTATGCCA GAGAGTTCTG CAGAAGAAAT CACTGTTTGT CCTGAGACCC AGCTAAGTTC 120

CTCTGAAACT TTTGACCTTG AAAGAGAAGT CTCTCCAGGT AGCAGAGATA TCTTGGATGG 180

AGTCAGAATAATAATGGCAGATAAGGAGGTTGGTAACAAGGAAGATGCTGAGAAGGAAGT 240

AGCTATTTCT ACCTTCTCAT CCAGTAACCA GGTATCCTGC CCGCTATGTG ACCAATG 297

Claims (3)

1. Application of a plasma exosomal circRNA marker in the preparation of a marker for detection of acute myocardial infarction, the plasma exosomal circRNA marker is hsa_circ_0001558, and the nucleotide sequence of the hsa_circ_0001558 is shown in SEQID NO: 1 . 2. The application of a plasma exosomal circRNA marker in the preparation of a diagnostic product for acute myocardial infarction, the plasma exosomal circRNA marker is hsa_circ_0001558, and the nucleotide sequence of the hsa_circ_0001558 is shown in SEQID NO: 1 . 3. The diagnostic product according to claim 2, wherein the diagnostic product is a chip, a preparation or a kit.

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