CN114182010B

CN114182010B - Plasma exosome circRNA marker and application thereof

Info

Publication number: CN114182010B
Application number: CN202210028321.7A
Authority: CN
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: Beijing Chaoyang Hospital
Current assignee: Beijing Chaoyang Hospital
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2022-06-07
Anticipated expiration: 2042-01-11
Also published as: CN114182010A; WO2023134553A1

Abstract

The invention relates to the technical field of molecular diagnosis, in particular to a plasma exosome circRNA marker and application thereof. The plasma exosome circRNA marker is hsa _ circ _001558, and the hsa _ circ _001558 nucleotide sequence is shown in SEQ ID NO: 1 is shown. The plasma exosome circRNA marker is applied as an acute myocardial infarction detection marker. The marker exosome hsa _ circ _001558 provided by the invention has a very high expression level in an AMI patient, can be used as a diagnosis marker of acute myocardial infarction, and has a higher expression level and stronger specificity in AMI compared with exosomes hsa _ circ _0001535, hsa _ circ _0003270, hsa _ circ _0000972 and hsa _ circ _ 0001119.

Description

Plasma exosome circRNA marker and application thereof

Technical Field

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

Background

At present, the number of cardiovascular diseases in China is as high as 2.9 hundred million, the death rate is the first place, and is higher than that of tumors and other diseases, and accounts for more than 40 percent of the death rate of resident diseases. Acute Myocardial Infarction (AMI) is one of the most common cardiovascular diseases, seriously threatening human health. Acute myocardial infarction is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary artery. Clinically, severe and persistent poststernal pain, rest and incomplete relief of nitrate medicines are caused, and the increased activity of serum myocardial enzyme and progressive electrocardiogram change are accompanied, so that arrhythmia, shock or heart failure can occur, and the life can be threatened.

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

Disclosure of Invention

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

The invention provides a plasma exosome circRNA marker, which is hsa _ circ _0001558, wherein the nucleotide sequence of hsa _ circ _0001558 is shown as SEQ ID NO: 1 is shown.

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

The invention provides a diagnostic product for detecting acute myocardial infarction, which comprises a plasma exosome circRNA marker.

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

The relative expression level of the exosome hsa _ circ _0001558 in the plasma exosome of the AMI patient is found to be significantly higher than that in the plasma exosome of the NCCP (non-cardiogenic chest pain) patient, and is 4.45 times higher than that in the plasma exosome of the NCCP patient, and the area under the ROC curve for diagnosing AMI is 0.793; exosome hsa _ circ _0001558 was progressively expressed in NST-AMI (non-ST elevated acute myocardial infarction), ST-AMI (ST elevated acute myocardial infarction) patients, with relative expression in NST-AMI patients 2.80 times that of NCCP patients, with an area under the ROC curve diagnosing NST-AMI of 0.72; relative expression in ST-AMI patients was 5.27 times that of NCCP patients, and the area under the ROC curve for diagnosis of ST-AMI was 0.831.

In summary, the invention has the following advantages:

(1) compared with the traditional marker, the marker exosome hsa _ circ _0001558 provided by the invention has higher sensitivity in AMI detection, the area under the ROC curve for diagnosing AMI is 0.793, the area under the ROC curve for diagnosing NST-AMI is 0.72, the area under the ROC curve for diagnosing ST-AMI is 0.831, the specificity is stronger, the detection is quicker and more convenient, and the cost is lower.

(2) The marker exosome hsa _ circ _0001558 provided by the invention has a very high expression level in an AMI patient, can be used as a diagnosis marker of acute myocardial infarction, and has a higher expression level and stronger specificity in AMI compared with exosome hsa _ circ _0001535, exosome hsa _ circ _0003270, exosome hsa _ circ _0000972 and exosome hsa _ circ _ 0001119.

Description of the drawings:

in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

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

FIG. 2 is a particle size distribution diagram of plasma exosomes in an example of the invention;

FIG. 3 is a Western blot analysis chart of exosome marker proteins CD63 and HSP70 in the example of the present invention;

FIG. 4 is a volcano and histogram of differentially expressed circRNAs in an example of the invention;

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

FIG. 6 is a graph of large sample size validation results in an embodiment of the present invention;

FIG. 7 is a graph of the change in expression of exosome hsa _ circ _0001558 in NST-AMI and ST-AMI and ROC in an example of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an", and "the" include plural forms as well, unless the context clearly indicates otherwise, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples

The first, experiment main reagent and manufacturer name are as follows:

trizol is available from Lnvitrogen; transcriptor First Strand cDNA Synthesis kit (First Strand cDNA Synthesis kit) purchased from Roche; select SYBR master mix available from ABIA company; PBS from Biolegend; 10% SDS (sodium dodecyl sulfate), 10% ammonium persulfate, 1.0MTris-HCl (pH 6.8), 30% acrylamide, 10 × blocking wash buffer, 10 × electrophoresis buffer, 10 × electrotransfer buffer were purchased from beijing prilley gene technology ltd; DEPC water, 1.5MTris-HCl (pH 8.8), Western protein lysate, and BCA protein concentration assay kit were purchased from bi yun sky biotechnology limited; TEMED (tetramethylethylenediamine) was purchased from Sciencific Research Special; NC membrane purchased from Hyclone; difco^TMSkim Milk from BD; CD63 antibody, HSP70 antibody, GAPDH antibody were purchased from abcam; the goat anti-rabbit secondary antibody and the goat anti-mouse secondary antibody are purchased from Beijing Gaotai science and technology company; the exosome extraction kit (cat # 217184) and the exosome RNA extraction kit (cat # 76064) are purchased from QIAGEN; the reverse transcription kit is purchased from Promega; fluorescent quantitative PCR kit purchased from Thermo Fisher; 18S RNA was purchased from Okkensheng Biotech limited.

Second, Experimental methods

2.1 patient specimen Collection

Patients admitted to the Beijing Kogyang Hospital and the Central Hospital of inner Mongolia Baotou City, affiliated to the headquarters medical university at 10 to 2018 in 2016, 120 patients with AMI (acute myocardial infarction) and 83 patients with NCCP (non-cardiogenic chest pain). Clinical data such as sex, 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 lipoprotein) and the like of the patient are recorded.

And (3) inclusion standard: acute myocardial infarction (criteria for diagnosis are based on 2015ESC/AHA/ACC guidelines): ischemic symptoms, elevated cTnT (troponin) and CK-MB (creatine kinase isoenzyme) expression, and electrocardiogram pathological Q wave. All AMI patients were first diagnosed. Patients with non-cardiogenic chest pain: chest pain admission and coronary angiography are negative, and the diagnosis is finally cardioneurosis, arteriosclerosis, reflux esophagitis and hyperlipidemia.

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

2.2 blood sampling and plasma separation

20mL of fasting venous blood of the morning of the next admission of the patient is collected by a blood taking needle and an anti-coagulation tube (containing EDTA), and is evenly mixed and then stands for 3 to 4 hours at the temperature of 4 ℃. Centrifuging at 3000rpm and 4 deg.C for 10min to obtain supernatant as plasma, filtering with 0.2 μm filter, packaging with 1 mL/tube, and storing at-80 deg.C.

2.3 plasma extraction of exosomes

Exosomes were isolated from plasma using an exosome extraction kit by the following method:

(1) taking out the frozen plasma from a refrigerator at minus 80 ℃, melting the plasma in a metal bath at 37 ℃, centrifuging the plasma for 15min at 10000g, taking supernatant, and taking 4mL of plasma into a new 15mL centrifuge tube;

(2) adding 1 volume of XBP buffer solution, slightly inverting the tube for 5 times, and incubating at room temperature; then placing the mixed solution into a column, centrifuging for 1min at 500g, pouring off waste liquid, and placing the column into the centrifuge tube again;

(3) adding 10mLXWP buffer, centrifuging at 3000g for 5min, and transferring the column to a new centrifuge tube;

(4) adding 1mLXE buffer eluate into the column, incubating for 1min, centrifuging at 500g for 5min, collecting filtrate, adding to the column again, incubating for 1min, and centrifuging at 3000g for 5 min;

(5) the filtrate (i.e., exosomes) was collected into a new rnase-free EP tube and stored at-80 ℃.

2.4 exosome particle size analysis

1mL of Phosphate Buffer (PBS) was added to the exosome stock solution and mixed well. The measurements were performed using a nanoparticle tracking analyzer Zeta View-Particle Meter, and the samples were injected into the sample cell. And opening software to adjust the brightness and the focal length, adjusting the visual field, observing, and analyzing the particle size of the particles according to the time and the displacement of the motion trail of the particles by using the software.

2.5 exosome Transmission Electron microscopy analysis

(1) Adding 50-100 mu L of 2% paraformaldehyde solution into the extracted exosomes to obtain an exosome solution;

(2) dripping 5-10 μ L of exosome solution on Formvar-carbon sample-carrying copper net, and standing at room temperature for 10 min;

(3) then washing with PBS buffer solution for 1 time;

(4) dripping 50 μ L of 1% glutaraldehyde solution on copper net for 5min, and washing with 100 μ L redistilled water for 2min each time for 8 times;

(5) treating with 50 μ L of uranyl oxalate droplets (pH 7.0) for 5 min;

(6) placing the copper mesh on ice, dripping 50 μ L methylcellulose solution for 10min, and air drying for 5-10 min;

(7) the copper mesh was placed in the sample box and the appropriate focus and brightness was adjusted at 80kV and photographed.

2.6 extraction and quantification of exosome proteins

To the isolated exosomes, 100. mu.L of a lysis solution (Radio-ionization Assay, RIPA) containing a protease inhibitor was added, lysed on ice for 5min, centrifuged at 13000rpm at 4 ℃ for 5min, and the precipitate was discarded. And extracting the total cell protein by using a BCA protein concentration determination kit and determining the protein concentration, wherein the experimental protein sample is collected and completed for 3 independent repeated exosome experiments. The detailed operation steps are as follows:

(1) adding 0.8mL of protein standard preparation solution into a protein standard substance (20mg BSA), and fully mixing and dissolving to prepare a 25mg/mL protein standard solution;

(2) adding 20 mu L of protein standard solution into 980 mu L of diluent to prepare 0.5mg/ml protein standard solution;

(3) adding the BCA reagent A into the BCA reagent B according to the number of samples and the volume of 50:1 to prepare an appropriate amount of BCA working liquid, and fully mixing;

(4) adding standard substance into standard substance well of 96-well plate according to 0 μ L, 1 μ L, 2 μ L, 4 μ L, 8 μ L, 12 μ L, 16 μ L, and 20 μ L, adding standard substance diluent to make up to 20 μ L;

(5) respectively adding 10 mu L of samples to be detected into sample holes of a 96-well plate, adding standard substance diluent to 20 mu L, adding 200 mu L of BCA working solution into each hole, and standing at room temperature for 2 h;

(6) the absorbance of the sample was measured at a wavelength of 560nm and the protein concentration of the sample was calculated from the standard curve.

After the protein is quantified, adding 5X SDS gel sample adding buffer solution, boiling at 100 ℃ for 5min to denature the protein, finishing the protein extraction step, and storing the sample in an ultra-low temperature refrigerator at-80 ℃.

2.7 Western Blot (Western Blot)

2.7.1 preparation of SDS polyacrylamide gel and base layer gel as follows:

composition of the separation gel (10% strength): 4.0mL of ultrapure water, 3.3mL of 30% acrylamide, 2.5mL of 1.5m tris-HCl (pH 8.8), 0.1mL of 10% SDS, 0.1mL of 10% ammonium persulfate, and 0.004mL of TEMED;

composition of lamination glue (concentration 5%): 4.1mL of ultrapure water, 1.0mL of 30% acrylamide, 0.75mL of 1.0MTris-HCl (pH 6.8), 0.06mL of 10% SDS, 0.06mL of 10% ammonium persulfate, and 0.006mL of TEMED.

2.7.2 Western blot

(1) Adding 1/5 volume of 5 × loading buffer solution into the supernatant (blood plasma), and decocting at 95 deg.C for 5 min;

(2) after boiling, 20 mu L of the mixture is added into 10 percent SDS polyacrylamide gel for electrophoresis;

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

(4) blocking with 5% BSA for 1 h;

(5) adding CD63 antibody (abcam, 1: 1000), HSP70 antibody (abcam, 1: 3000) or GAPDH antibody (abcam, 1: 5000), incubating at 4 deg.C overnight, washing with TBST (Tris salt membrane washing buffer) for 3 times, each for 10 min;

(6) then adding goat anti-rabbit secondary antibody (Beijing Gaotai technology, 1:15000) or goat anti-mouse secondary antibody (Beijing Gaotai technology, 1:15000), incubating for 1h in greenhouse, washing with TBST for 3 times (10 min each time), and exposing.

2.8 exosome Total RNA extraction

The exosome extraction kit is used for extracting exosome RNA, and the steps are as follows:

(1) adding 600 μ L lysate into the obtained exosome stock solution, incubating at room temperature for 2min, centrifuging at 3000g for 5min, and collecting supernatant;

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

(3) mixing, adding into column provided by kit, centrifuging at 8000g for 15s, removing waste liquid, and recovering column;

(4) adding the buffer solution of the kit to the column, centrifuging to wash the column, adding 80% ethanol to the column, centrifuging at 8000g for 2 min;

(5) after centrifugation, the column was transferred to a new RNase-free EP tube and the filter membrane on the column was air dried;

(6) then adding 14 μ L of eluent, centrifuging at 8000g for 1min, discarding the column, collecting filtrate, and storing in a refrigerator at-80 deg.C;

(7) RNA concentration was determined with a Nano Drop 2000(Thermo Scientific, USA) with RNA amount > 4. mu.g; the RNA concentration is 50 ng/mu L-500 ng/mu L; the RNA absorbance 260/280 was between 1.8 and 2.1.

2.9 high throughput sequencing

Firstly, performing prophase treatment such as purification and the like on exosome RNA, then performing reverse transcription to form cDNA, performing terminal repair, adding a joint, performing PCR amplification and the like to build a library, and performing machine sequencing after quality inspection is qualified. After the original data are obtained, the data are firstly filtered, the connector sequences are removed, the low-quality read length is processed, the sequencing quality is evaluated, the high-quality data are obtained, the high-quality data and the reference data are compared, and the BAM file is obtained.

2.10 reverse transcription

The reverse transcription kit for RNA reverse transcription is carried out by adopting a two-step method, and the specific operation steps are as follows:

(1)RNA(100ng)xμL、OligoT15(0.5μg)1μL、DEPC·H₂o9-x mu L, and the total volume is 10 mu L;

(2) reacting at 70 ℃ for 5min, and opening the RNA secondary structure; immediately on ice, 5 × AMV Buffer 4 μ L, 10mM dNTP mix 2 μ L, Mg was added²⁺2 mu L, RNasin (40U/. mu.L) 1 mu L, AMV 1. mu.L, total volume of 10. mu.L, reaction at 42 ℃ for 60min, reaction at 99 ℃ for 2min, constant temperature of 4 ℃, and storage of the product at-20 ℃.

2.11 real-time quantitative PCR

Using PowerUp^TMSYBR^TMThe Green Master Mix fluorescent quantitative kit and the ABI 7500PCR instrument detect the level of the circRNA.mu.L of 10-fold diluted cDNA was used as template, 3 parallel wells were set for each sample, and 18S RNA was used as reference gene.

(1) The reaction system is as follows: 2X Master Mix 10. mu. L, H₂O 4μL、Primer F(10μM)1 μL、Primer R(10μM)1μL、cDNA 4μL。

(2) The reaction conditions were as follows:

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

TABLE 1 qRT-PCR primer sequence Listing

2.12 statistical analysis method

Statistical analysis was performed using SPSS17.0 software, with normal distribution data represented as mean. + -. standard deviation (X. + -. S) and non-normal distribution data represented as median (P25, P75). The normal distribution data adopts t test for comparison between two groups, the non-normal distribution data adopts non-parametric test for comparison between two groups, and the two groups of data rates adopt chi-square test for comparison. Differences in circRNA levels were determined by the Mann-Whitney U test. To assess the predictive value of the selected circrnas on AMI, we used ROC curves with differences of P value <0.05 as statistical significance.

Third, test results

1. Exosome identification

1.1 exosomes extracted by transmission electron microscope observation

Observed under transmission electron microscope, the extracted vesicles are of non-uniform size, are round or quasi-round, have lipid bilayer membranes, have diameters of 30-150nm, and conform to morphological characteristics of exosomes, as shown in fig. 1.

1.2 exosome particle size analysis

The extracted vesicle particles have a particle size of 30-200nm and a main peak of about 100nm, as shown in FIG. 2.

1.3 Western blot assay of exosome-specific molecular markers

The immunoblot experiment results show that the plasma exosomes of AMI patients and NCCP patients express the specific marker proteins CD63 and HSP70, but do not express GAPDH, as shown in fig. 3, further demonstrating that the extracted vesicles are exosomes.

2. High throughput sequencing

To clarify the expression of circRNA in plasma exosomes of AMI patients, samples of 15 NCCP and 15 AMI (sequencing cohort) were screened by high throughput sequencing.

2.1 sequencing cohort clinical features

The age and sex of AMI and NCCP groups are not obviously different, the levels of cTnT, CK-MB and NT-proBNP (amino-terminal pro-brain natriuretic peptide) are obviously increased in the AMI group compared with the NCCP group, the clinical characteristics of AMI patients are met, and the data of AMI and NCCP patients 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 profile of circRNAs in AMI patient plasma exosomes was significantly different compared to the control group, with 893 differentially expressed circRNAs were screened as being upregulated 118 and downregulated 775 (as shown in a-diagram in fig. 4) in AMI plasma exosomes as normalized by | log2(Fold change) | >1, q-value <0.001, and these differentially expressed circRNAs were annotated 107 in circBase as shown in table 3.

TABLE 3 differential expression of circRNA in AMI annotated in circBase

2.3 Small sample size validation of differentially expressed circRNAs

To validate the results of high throughput sequencing, and considering the feasibility as a biomarker, we selected 5 circRNAs (exosomes hsa _ circ _0001535, exosome hsa _ circ _0003270, exosome hsa _ circ _0000972, exosomes hsa _ circ _0001119 and exosomes hsa _ circ _0001558) with annotations in the circBase database expressed up-regulated, with high expression in the AMI group, with large fold-increase, as shown in panel B in fig. 4. The assay was performed in small sample size samples using RT-PCR.

2.3.1 Small sample cohort clinical features

A small cohort of samples was selected with 20 NCCP and 20 AMI patients and the clinical data are presented in table 4. The age, sex, height, weight and the like of the two groups are not different; proportion of hyperlipidemia, WBC (white blood cell), GLU (glucose), K⁺The levels of 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) were significantly higher in the AMI group than in the NCCP group; LVEF (%) (left ventricular ejection fraction), Na⁺The level of (c) in the AMI group was significantly lower than in the NCCP group.

Table 4 small sample queue clinical data table

2.3.2 Small sample size validation results

The results of RT-PCR validation showed that exosome hsa _ circ _0001119 was expressed in low amounts and was not detected in most samples, exosome hsa _ circ _0003270 was expressed differently between the two groups of AMI and NCCP patients, and exosome hsa _ circ _0001535, exosome hsa _ circ _0000972 and exosome hsa _ circ _0001558 were detected with the same 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 increased (P <0.05) compared to NCCP group (5.45(3.50, 7.57)) and was 1.56-fold higher than that of NCCP group, and the area under the ROC curve for diagnosing AMI was 0.685 in this cohort group (as shown in a and D in fig. 5); the relative expression level of exosome hsa _ circ _0000972 in AMI patients (15.7(1.75, 21.71)) was significantly elevated (P <0.05) compared to NCCP group (3.43(1, 12.4)) and was 4.58-fold higher than in NCCP group; the area under the ROC curve for its diagnostic AMI in this cohort population was 0.683 (as shown in FIG. 5, panels B and E); the relative expression level of exosome hsa _ circ _0001558 (27.62(20.47, 44.81)) in plasma exosomes of AMI patients was significantly increased (P <0.01) compared to NCCP group (7.42 (4.21,31.08)) by 3.72-fold compared to control group, with an area under the ROC curve for diagnosis of AMI of 0.790 in the present cohort population (as shown in fig. 5, panels C and F). Since the area under the ROC curve for exosome hsa _ circ _0001558 diagnostic AMI was largest among the three circRNAs, exosome hsa _ circ _0001558 was selected for further validation in large sample size samples.

Wherein Panel A in FIG. 5 is the relative expression levels of exosome hsa _ circ _0001535 in AMI and NCCP; panel B is the relative expression level of exosome hsa _ circ _0001558 in AMI and NCCP; panel C is the relative expression level of exosome hsa _ circ _0000972 in AMI and NCCP; d is an ROC curve for diagnosing AMI by the exosome hsa _ circ _ 0001535; e is ROC curve of exosome hsa _ circ _0000972 diagnosis AMI; FIG. F is an ROC curve for exosome hsa _ circ _0001558 diagnosis AMI. P <0.05, P < 0.01.

2.4 validation of differentially expressed circRNAs in Large sample volumes

To validate the results of the small sample size cohort, we validated the level of exosome hsa _ circ _0001558 in larger sample sizes.

2.4.1 Large sample size cohort of clinical data

Clinical information for 48 NCCP and 85 AMI patients is presented in table 5. Proportion of Male patients in AMI group, Heart Rate, proportion of hypertension, proportion of hyperlipemia, WBC, HB, TG, GLU, AST, ALT, K⁺D-D, CRP, TT3, FT3, sTSH, CK-MB, TnI levels higher than the NCCP group, LVEF (%), Na⁺Is lower than in the NCCP group.

TABLE 5 clinical features of the study population exosome circRNA was validated cyclically in large samples

2.4.2 Large sample queue validation results

The results of plasma exosomes hsa _ circ _0001558 validated in large sample size AMI and NCCP patients were consistent with the small sample cohort validation results. The relative expression level of exosome hsa _ circ _0001558 in the plasma exosomes of AMI patients (8.81(4.87,17.76)) was significantly increased (P <0.01) compared to NCCP group (1.98(0.74,6.44)) by 4.45 times that of NCCP group, which was diagnostic for AMI with an area under the ROC curve of 0.793, as shown in fig. 6 (panel a is the expression level of hsa _ circ _0001558 in AMI patient plasma exosomes; panel b is the ROC graph of plasma exosome hsa _ circ _0001558 for AMI;. P < 0.01).

Further dividing AMI patients into ST-segment elevated AMI (ST-AMI) and non-ST-segment elevated AMI (NST-AMI), the relative expression of plasma exosome hsa _ circ _0001558 in the NST-AMI group (5.54(4,11.96)) was significantly increased over the NCCP group (1.98(0.74,6.44)) to 2.80-fold that of the NCCP group, which diagnosed NST-AMI with an area under the ROC curve of 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, with an area under the ROC curve diagnosing ST-AMI of 0.831, as shown in fig. 7 (panel a is the change in expression of exosome hsa _ circ _0001558 in NST-AMI and ST-AMI; panel B is the ROC curve diagnosing NST-AMI of exosome hsa _ circ _ 0001558; panel C is the ROC curve diagnosing ST-AMI of exosome hsa _ circ _ 0001558.

AMI is mostly caused by atherosclerotic stenosis of the coronary arteries, mainly by plaque rupture or thrombosis and secondary coronary artery obstruction, resulting in apoptosis of myocardial cells and necrosis of the myocardium. Studies have shown that exosome-mediated intercellular information communication plays a role in AMI through a variety of mechanisms. The basic information of the cell can be directly obtained by analyzing the information substance in the exosome. Existing research mostly focuses on the role and mechanism of miRNA in exosome in early diagnosis of AMI, transplantation therapy, and myocardial fibrosis and angiogenesis after AMI. circRNA is one of the important informative substances in exosomes.

circRNA is a recent focus of research, circular RNA molecules consisting of exonic or intronic sequences. circRNA has no 5 'and 3' ends and is not readily degraded by rnases and is therefore more stable than linear RNA. circRNA is abundant in the cytoplasm of eukaryotic cells, with tissue, timing and disease specificity.

According to the research, the expression profile of circRNA in AMI plasma exosomes is found to be obviously different from that of a control group through high-throughput sequencing, 893 circRNAs which are differentially expressed in the AMI plasma exosomes are screened by taking | log2(Fold change) | >1 and q-value <0.001 as standards, the expression is up-regulated by 118 and the expression is down-regulated by 775, and the result shows that the circulating exosomes have selectivity on packaging of the circRNA during AMI and can be used as AMI biomarkers.

Next, we selected 5 exosomes circRNA with annotation in circBase database, high expression level and obvious fold increase, and verified by RT-PCR in small sample amount. The results show that the detection results for exosomes hsa _ circ _0001535, exosomes hsa _ circ _0000972 and exosomes hsa _ circ _0001558 are consistent with the sequencing results, and in the small sample queue, exosomes hsa _ circ _0001558 were the most elevated in the AMI group, with the largest product under the ROC curve for diagnosing AMI (as shown in figure 5), and were therefore validated in a larger sample size.

The exosome hsa _ circ _0001558 demonstrated results in larger sample size patients consistent with small sample size validation results, with significantly higher expression in AMI patient plasma exosomes than did the NCCP group, 4.45-fold higher than the NCCP group, with an area under the ROC curve for diagnosing AMI of 0.793 (as shown in figure 6).

AMI can be classified into ST-AMI and NST-AMI myocardial infarction according to its ECG characteristics. Therefore, we further analyzed the expression level of exosome 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 was gradually increased in NCCP, ST-AMI and NST-AMI plasma exosomes, with an area under the ROC curve for diagnosis of NST-AMI of 0.72 and an area under the ROC curve for diagnosis of ST-AMI of 0.831 (as shown in fig. 7). The exosome hsa _ circ _0001558 is located on chromosome 5, is 328nt long, and can be detected in tissues/cells such as platelets, K562 cells, vascular endothelial cells and the like.

The expression profile of circRNA in AMI circulating exosomes was significantly different compared to controls, and the progressively higher expression levels of exosome hsa _ circ _0001558 in NCCP, ST-AMI and NST-AMI plasma exosomes could serve as AMI diagnostic biomarkers.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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GGGAAACTCT GTACCTGCTT CACAAAGTGT TGCTGCTTTG ACCAGTAAGA GAAGCTTAGT 60

CCTTATGCCA GAGAGTTCTG CAGAAGAAAT CACTGTTTGT CCTGAGACCC AGCTAAGTTC 120

CTCTGAAACT TTTGACCTTG AAAGAGAAGT CTCTCCAGGT AGCAGAGATA TCTTGGATGG 180

AGTCAGAATA ATAATGGCAG ATAAGGAGGT TGGTAACAAG GAAGATGCTG AGAAGGAAGT 240

AGCTATTTCT ACCTTCTCAT CCAGTAACCA GGTATCCTGC CCGCTATGTG ACCAATG 297

Claims

1. The application of a plasma exosome circRNA marker in preparing an acute myocardial infarction detection marker, wherein the plasma exosome circRNA marker is hsa _ circ _0001558, and the nucleotide sequence of hsa _ circ _0001558 is shown in SEQ ID NO: 1 is shown.

2. The application of a plasma exosome circRNA marker in preparing a diagnostic product for acute myocardial infarction, wherein the plasma exosome circRNA marker is hsa _ circ _0001558, and the nucleotide sequence of hsa _ circ _0001558 is shown in SEQ ID NO: 1 is shown.

3. The diagnostic product of claim 2, wherein the diagnostic product is a chip, a formulation or a kit.