CN114250294A

CN114250294A - Marker for diagnosis and treatment of cerebral apoplexy

Info

Publication number: CN114250294A
Application number: CN202111587626.3A
Authority: CN
Inventors: 李伟荣; 王斌红; 贾艳焕; 那龙; 史静
Original assignee: Taiyuan Psychiatric Hospital
Current assignee: Taiyuan Psychiatric Hospital
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-03-29

Abstract

The invention relates to a marker which can be used for diagnosing and treating cerebral apoplexy, and the marker comprises two or three of DAAM2, ECHDC3 and FOLR 3. Compared with a normal control group, the expression levels of the three markers are obviously increased in the cerebral apoplexy patient. The invention provides a pharmaceutical composition for treating cerebral apoplexy. The invention also provides a system or apparatus for diagnosing whether a subject has stroke or predicting whether a subject is at risk for stroke. The marker provided by the invention has the advantages of high sensitivity, good specificity and good application prospect.

Description

Marker for diagnosis and treatment of cerebral apoplexy

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a marker for diagnosing and treating cerebral apoplexy.

Background

Stroke is a blood circulation disorder disease of the brain which suddenly starts, and is one of the main diseases which endanger human health. Stroke has become the leading cause of disability and the third cause of death worldwide, and two thirds of patients are concentrated in developing countries in the population who are fatal to it. The cerebral apoplexy comprises ischemic cerebral apoplexy and hemorrhagic cerebral apoplexy, and research shows that the disease proportion of the ischemic cerebral apoplexy in the population of China is greater than that of the hemorrhagic cerebral apoplexy. Clinical investigation and research show that the incidence of cerebral apoplexy is still in a continuously rising trend at present, and the cerebral apoplexy treatment agent has the characteristics of high fatality rate, high incidence rate, high disability rate and the like, so that the life quality of a patient is influenced slightly, and paralysis, aphasia, dementia and even death are caused seriously. The stroke not only seriously affects the life quality of the patient, but also brings heavy burden to families and society. Although magnetic resonance imaging (MIR) has an important reference value in stroke diagnosis, due to the practical problems of low popularization rate and high cost of MIR in china, the early diagnosis rate of stroke is still not very ideal, and timely and accurate diagnosis is an important basis for implementing therapeutic measures, so that the disability rate of stroke is reduced.

Due to the development of proteomics and genomics, the development of disease-related biomarkers has been rapidly progressed like spring shoots after rain. Biomarkers provide very important basis for rapid diagnosis, treatment guidance and prognosis evaluation of other system diseases except brain tissues, and for decades, many researchers have been dedicated to discussing the relationship between biomarkers and stroke. The search for sensitive and specific biomarkers is expected to provide a new way for the diagnosis of stroke.

Disclosure of Invention

A first object of the invention is to provide a product that can be used for diagnosing stroke.

The second object of the present invention is to provide a pharmaceutical composition for preventing or treating stroke.

It is a third object of the present invention to provide a system or apparatus for diagnosing whether a subject has stroke or predicting whether a subject is at risk for stroke.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides a product useful for diagnosing stroke, the product comprising reagents capable of detecting the expression level of biomarkers including two or three of DAAM2, ECHDC3, FOLR3 in a sample.

Further, the cerebral apoplexy comprises ischemic cerebral apoplexy and hemorrhagic cerebral apoplexy.

Furthermore, the cerebral apoplexy is ischemic cerebral apoplexy,

further, the cerebral arterial thrombosis includes cerebral arterial thrombosis caused by atherosclerosis stroke, cardiac embolism, arteriolar occlusive stroke, or ischemic stroke caused by other reasons.

Further, the ischemic stroke is cardiogenic cerebral embolism.

Further, the reagent comprises a reagent for detecting the expression level of the biomarker in the sample by a digital imaging technology, a protein immunization technology, a dye technology, a nucleic acid sequencing technology, a nucleic acid hybridization technology, a chromatographic technology and a mass spectrometry technology.

Further, the sample is blood.

Further, the reagent comprises a primer or a probe which is specifically combined with the biomarker gene; an antibody, peptide, aptamer, or compound that specifically binds to the marker protein.

Further, the product comprises a nucleic acid membrane strip, a preparation, a chip and a kit.

In another aspect, the invention provides the use of a biomarker comprising two or three of DAAM2, ECHDC3, FOLR3 in the manufacture of a product as hereinbefore described.

In another aspect, the invention provides a pharmaceutical composition comprising two or three of an inhibitor of DAAM2, an inhibitor of ECHDC3, and an inhibitor of FOLR 3.

In another aspect, the present invention provides the use of the pharmaceutical composition as described above in the preparation of a medicament for preventing or treating stroke.

Further, the cerebral apoplexy is ischemic cerebral apoplexy.

Further, the ischemic stroke is cardiogenic cerebral embolism.

In another aspect, the present invention provides a system or apparatus for diagnosing stroke in a subject or predicting the risk of stroke in a subject, the system/apparatus comprising:

an analysis unit adapted to measure the amount of a biomarker in a sample of a subject; and

an evaluation unit comprising a stored reference and a data processor having implemented an algorithm for comparing the amount of the biomarker measured by the analysis unit with the stored reference, thereby diagnosing whether the subject has a stroke or predicting whether the subject is at risk of having a stroke;

the biomarkers comprise two or three of DAAM2, ECHDC3 and FOLR 3;

Further, the cerebral apoplexy is ischemic cerebral apoplexy.

Further, the ischemic stroke is cardiogenic cerebral embolism.

The present invention also provides a computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements the system/apparatus as described above.

Drawings

FIG. 1 is a boxplot of the differential expression of DAAM 2;

FIG. 2 is a box line plot of differential expression of ECHDC 3;

FIG. 3 is a box line plot of FOLR3 differential expression;

FIG. 4 is a ROC plot of DAAM2 diagnosis of cardiogenic cerebral embolism;

FIG. 5 is a ROC plot of ECHDC3 diagnosis of cardiogenic cerebral embolism;

FIG. 6 is a ROC plot of FOLR3 diagnosis of cardiogenic cerebral embolism;

FIG. 7 is a ROC plot of DAAM2 and ECHDC3 in combination to diagnose cardiogenic cerebral embolism;

FIG. 8 is a ROC plot of ECHDC3 and FOLR3 in combination to diagnose cardiogenic cerebral embolism;

FIG. 9 is a ROC plot of the combined diagnosis of cardiogenic cerebral embolism by DAAM2 and FOLR 3;

FIG. 10 is a ROC plot of DAAM2+ ECHDC3+ FOLR3 in combination for diagnosis of cardiogenic cerebral embolism.

Detailed Description

Biomarkers

The invention provides a product for diagnosing cerebral apoplexy, which comprises reagents capable of detecting biomarkers, wherein the biomarkers comprise two or three of DAAM2, ECHDC3 and FOLR 3.

The term "biomarker" refers to a biological molecule present in an individual at varying concentrations that can be used to predict the disease state of the individual. Biomarkers can include, but are not limited to, nucleic acids, proteins, and variants and fragments thereof. A biomarker may be DNA comprising all or part of a nucleic acid sequence encoding the biomarker, or the complement of such a sequence. Biomarker nucleic acids useful in the present invention are considered to include DNA and RNA comprising all or part of any nucleic acid sequence of interest.

The DAAM2 refers to a Gene with Gene ID 23500.

The ECHDC3 refers to a Gene with the Gene ID of 79746.

The FOLR3 refers to Gene with Gene ID 2352.

The term "primer" or "probe" encompasses an oligonucleotide having a specific sequence or an oligonucleotide having a specific sequence. In other embodiments, the nucleic acid is detected by an indirect detection method. For example, biotinylated probes can be combined with streptavidin-conjugated dyes to detect bound nucleic acids. The streptavidin molecules bind the biotin labels on the amplified biomarkers, and the bound biomarkers are detected by detecting dye molecules attached to the streptavidin molecules. In one embodiment, the streptavidin-conjugated dye molecule comprises PHYCOLINK. Streptavidin R-phycoerythrin (PROzyme). Other conjugated dye molecules are known to those skilled in the art.

Markers include, but are not limited to: luminescent, light scattering, and light absorbing compounds that produce or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal. In some embodiments, a dual-labeled fluorescent probe comprising a reporter fluorophore and a quencher fluorophore is used. It will be appreciated that pairs of fluorophores with different emission spectra are selected so that they can be readily distinguished. In certain embodiments, the label is a hybridization stabilizing moiety that is used to enhance, stabilize or affect hybridization of the duplex, e.g., an intercalator and an intercalating dye.

The term "antibody" refers to an immunoglobulin that specifically recognizes an epitope on a target antigen as determined by the binding properties of the immunoglobulin variable regions of the heavy and light chains (VHS and VLS), and more specifically, the Complementarity Determining Regions (CDRs). Many potential antibody formats are known in the art and may include, but are not limited to: a plurality of intact monoclonal antibodies or polyclonal mixtures comprising intact monoclonal antibodies, antibody fragments (such as Fab, Fab', and Fv fragments, multispecific antibodies and linear single chain antibodies comprising antibody fragments), single chain variable fragments (scFvS), multispecific antibodies, chimeric antibodies, humanized antibodies, and fusion proteins comprising domains necessary to recognize a given epitope on a target antigen. Preferably, antibodies cited in the context of the present invention refer to monoclonal antibodies. The antibody may also be coupled to various detectable labels to enable detection, including, but not limited to, radionuclides, fluorescent probes, dyes, or enzymes, such as horseradish peroxidase and alkaline phosphatase, among others.

The term "aptamer" refers to an oligonucleotide molecule or polypeptide molecule that can specifically bind to a target molecule. Oligonucleotide aptamers can be nucleotides (RNA) or Deoxynucleotides (DNA), typically consisting of short-chain oligonucleotides. Polypeptide aptamers typically consist of short peptide domains, which can be attached to one or both ends of a protein scaffold.

The term "specific binding", in the context of an antibody-epitope interaction, refers to an interaction in which the binding of an antibody to an epitope is more frequent or faster, or longer in duration or with greater affinity, or a combination of any of the above, than if either of the antibody or epitope were replaced by a surrogate, such as a non-related protein. Typically, but not necessarily, reference to binding refers to specific recognition. Furthermore, it is understood that one antibody may specifically recognize more than one antigen. Techniques known in the art for determining specific binding of a target or lack thereof by monoclonal antibodies include, but are not limited to: FACS analysis, immunocytochemical staining, immunohistochemistry, western/dot blotting, ELISA and affinity chromatography. By way of example and not limitation, specific binding or lack thereof can be determined by comparative analysis with controls comprising antibodies known in the art to specifically recognize the target, and/or controls that do not specifically recognize or minimally specifically recognize the target (e.g., the controls comprise the use of non-specific antibodies). The comparative analysis may be a qualitative or quantitative analysis. However, it will be appreciated that an antibody or binding portion specifically recognizing a given target is reported to have a higher specificity for that target than an antibody, e.g., an antibody that specifically recognizes both the target and a homologous protein.

Reagent kit

The present invention provides kits for diagnosing stroke for determining levels of biomarkers (wherein the sequence optionally comprises uracil in place of one, more than one, or all of the disclosed thymines), and combinations thereof. The kit may comprise materials and reagents suitable for selectively detecting the presence of a biomarker or a set of biomarkers for diagnosing stroke in a sample derived from a subject. For example, in one embodiment, the kit can include reagents that specifically hybridize to the biomarkers. Such reagents may be nucleic acid molecules in a form suitable for detecting a biomarker, e.g., probes or primers. The kit may include reagents for performing an assay to detect one or more biomarkers, e.g., reagents that may be used to detect one or more biomarkers in a qPCR reaction. The kit may also include a microarray for detecting one or more biomarkers.

In further embodiments, the kit may contain instructions for appropriate operating parameters in the form of labels or product inserts. For example, the instructions may include information or guidance on how to collect the sample, how to determine the level of one or more biomarkers in the sample, or how to correlate the level of one or more biomarkers in the sample with the stroke status of the subject.

In another embodiment, the kit may contain one or more containers with a biomarker sample to be used as a reference standard, a suitable control, or for calibration of an assay to detect a biomarker in a test sample.

Pharmaceutical composition

The invention provides a pharmaceutical composition which comprises two or three of an inhibitor of DAAM2, an inhibitor of ECHDC3 and an inhibitor of FOLR 3.

The inhibitor comprises a nucleic acid molecule, a carbohydrate, a small molecule compound or an interfering lentivirus; the nucleic acid molecule is selected from: antisense oligonucleotides, double-stranded RNA, small interfering RNA or short hairpin RNA.

As used herein, a "small interfering RNA" is a double-stranded small RNA molecule consisting of a first and second strand that are perfectly complementary, processed by Dicer (an enzyme of the RNAase III family that is specific for double-stranded RNA). The first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is identical to the target sequence in the biomarker gene described above, or a sequence that hybridizes to the target sequence under high stringency conditions. The length of the first strand and the second strand of the double-stranded RNA are both 15-27 nucleotides; preferably, each 19-23 nucleotides in length; more preferably, each 19, 20 or 21 nucleotides in length. siRNA is a major member of siRISC, triggering silencing of the target gene to which it is complementary. RNA interference (RNAi) refers to the phenomenon of specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsrna), resulting in silencing of expression of a target gene and the corresponding loss of a functional phenotype.

As used herein, the shRNA, i.e., a small hairpin RNA or short hairpin RNA (shRNA), is a segment of RNA sequence with a tight hairpin loop (light hairpin turn) comprising a sense strand segment, an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, commonly used for expression of RNA interference silencing target genes. Wherein the sequences of the sense and antisense strands are complementary and the sequence of the sense strand fragment is identical to 15-27 contiguous nucleotide sequences in the previously described biomarker genes, preferably the sense strand fragment is identical to 19-23 contiguous nucleotide sequences in the previously described biomarker genes; more preferably, the sense strand fragment is identical to a nucleotide sequence of 19, 20 or 21 consecutive nucleotides of the biomarker gene described above, or a sequence that hybridizes to each of the above sequences under conditions of high stringency. The hairpin structure of the shRNA can be cleaved into siRNA by cellular machinery, and the siRNA is then bound to an RNA-induced silencing complex (RISC) which is capable of binding to and degrading the gene of interest.

The small interfering RNA of the invention can be a chemically synthesized double-stranded RNA; it may also be a double-stranded RNA expressed by a vector or expression framework in which small interfering RNA expression in mammalian cells is regulated using, for example, an RNA polymerase III promoter including human or murine U6 promoter and human H1 promoter, and an RNA polymerase III terminator.

The small interfering RNA of the invention can be composed of a single small interfering RNA acting on a target sequence, or can be composed of a plurality of small interfering RNAs acting on a plurality of target sequences of a gene or target sequences on a plurality of genes; the target sequence may be the genomic sequence of the biomarker gene as described above, or the cDNA sequence of the biomarker gene as described above.

The sirnas of the invention can be screened by the methods disclosed in the examples herein or by methods known in the art.

The first strand in the siRNA of the present invention and the sense strand in the shRNA have the same sequence as the target sequence, and have at least 10 (preferably at least 15, and more preferably at least 18) consecutive identical nucleotide sequences as compared to the siRNA target sequence, or a sequence that hybridizes to the target sequence under high stringency conditions.

In the present invention, the nucleic acid construct is meant to include a replication system and sequences capable of transcription within a given target cell. It can be obtained by cloning a fragment encoding the shRNA described herein into a known vector. Further, the nucleic acid construct is a lentiviral vector. After the lentiviral vector is packaged into infectious viral particles by virus, the cells are infected, the shRNA is further transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the biomarker genes.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term carrier includes any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like suitable for use in preparing the particular dosage form desired. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol and phosphate buffer, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

In preparing the pharmaceutical compositions of the present invention, the active ingredient is typically mixed with, or diluted with, excipients or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The preparation may further comprise a humectant, an emulsifier, a preservative (such as methyl and propyl hydroxybenzoate), a sweetener, etc.

The application of the pharmaceutical composition provides a method for treating cerebral apoplexy, in particular to a method for preventing or treating cerebral apoplexy of a subject, which comprises the step of administering an effective dose of the pharmaceutical composition to the subject.

The pharmaceutical composition is used for preventing or treating cerebral apoplexy of a subject, and an effective dose of the pharmaceutical composition is required to be administered to the subject. Typical dosage forms for topical administration include creams, ointments, sprays, lotions and plasters. However, the pharmaceutical composition may be formulated for any type of administration, for example, intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intranasal injection, intracerebral injection, intratracheal injection, intraarterial injection, intraperitoneal injection, intravesical injection, intrapleural injection, intracoronary injection, or intratumoral injection using a syringe or other device. Dosage forms for administration by inhalation (e.g., aerosol) or oral, rectal or vaginal administration are also contemplated.

Systems or arrangements

The present invention relates to a system/apparatus for diagnosing whether a subject has stroke or predicting whether a subject is at risk for stroke, comprising:

an evaluation unit comprising a stored reference and a data processor having implemented an algorithm for comparing the amount of the biomarker measured by the analysis unit with the stored reference, thereby diagnosing whether the subject has a stroke or predicting whether the subject is at risk of having a stroke.

A device as applied herein shall at least comprise the above-mentioned units. The units of the device are operatively connected to each other. How the units are operatively linked will depend on the type of unit contained in the device. For example, in case a tool for automatic quantitative measurement of biomarkers is applied in the analysis unit, the data obtained by said automatic operation unit may be processed by the evaluation unit, e.g. by a computer program running on a computer as data processor, in order to facilitate the diagnosis. In one embodiment, the data processor performs a comparison of the amount of the biomarker to a reference.

Further, in this case, the unit is constituted by a single device. However, the analysis unit and the evaluation unit may also be physically separate. In this case, operational connection (operational connection) may be realized via wired and wireless connection between units allowing data transmission. The wireless connection may use a wireless lan (wlan) or the internet. The wired connection may be achieved by optical and non-optical cable connections between the units. The cable for wired connection is further suitable for high-throughput data transmission.

The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present application, are given by way of illustration and explanation only, and are not intended to limit the present application.

Example 1 high throughput sequencing analysis

1. Sample collection

Blood samples were collected from 4 stroke patients and 4 healthy controls.

1.1 stroke patients inclusion criteria:

(1) diagnosing stroke;

(2) the treatment for the cerebral apoplexy by a medicament mode or an operation and the like is not received;

(3) no infectious, autoimmune or severe chronic disease.

1.2 inclusion criteria for healthy controls:

(1) male or female volunteers between the ages of 20 and 90 years;

(2) healthy, without any chronic, infectious or genetic disease;

(3) no conventional prescription is taken recently;

(4) no blood donation, pregnancy, no vaccination within 3 months prior to study initiation;

(5) non-allergic constitution;

(6) the physical health was confirmed by conventional physical examination.

2. Experimental methods

2.1 extraction of total RNA from blood

Total RNA from peripheral Blood was extracted using a PAXgene Blood RNA extraction Kit (PAXgene Blood RNA Kit).

2.2 sample detection

Total RNA concentration, RIN value, 28S/18S and fragment size were measured using an Agilent 2100Bioanalyzer (Agilent RNA 6000Nano Kit).

2.3 construction of the library and transcriptome sequencing

1) DNase digestion to remove DNA: DNA fragments existing in Total RNA samples are digested by DNase I, and reaction products are recovered by magnetic bead purification and finally dissolved in DEPC water.

2) Removing rRNA: taking a digested Total RNA sample, removing rRNA by using a kit, carrying out Agilent 2100 detection after the rRNA is removed, and verifying the rRNA removal effect;

3) RNA disruption: taking the sample in the previous step, adding a breaking Buffer, and placing the sample in a PCR instrument for thermal breaking to 130-;

4) reverse transcription one-strand synthesis: adding a proper amount of primers into the broken sample, fully and uniformly mixing, reacting for a certain time at a proper temperature of a Thermomixer to open a secondary structure and combine with the primers, adding a one-chain synthesis reaction system Mix prepared in advance, and synthesizing one-chain cDNA on a PCR instrument according to a corresponding procedure;

5) synthesis of reverse transcription duplex: preparing a double-chain synthesis reaction system, reacting on a Thermomixer at a proper temperature for a certain time to synthesize double-chain cDNA, and purifying and recovering reaction products by using magnetic beads. Purifying and recovering the product by using magnetic beads;

6) and (3) repairing the tail end: preparing a terminal repair reaction system, reacting in a Thermomixer at a proper temperature for a certain time, and repairing the cohesive terminal of the cDNA double-chain obtained by reverse transcription under the action of enzyme. Purifying and recovering the end repairing product by using magnetic beads, and finally dissolving a sample in EB Solution;

7) the cDNA ends were added with "A": preparing an A reaction system, reacting in a Thermomixer at a proper temperature for a certain time, and adding A basic groups to the 3' end of a product cDNA with repaired end under the action of enzyme;

8) ligation of cDNA adapter: preparing a joint connection reaction system, reacting in a Thermomixer at a proper temperature for a certain time, connecting a joint with the A base under the action of enzyme, and purifying and recovering a product by using magnetic beads.

9) PCR reaction and product recovery: preparing a PCR reaction system, selecting a proper PCR reaction program, and amplifying the product obtained in the previous step. And (5) carrying out magnetic bead purification and recovery on the PCR product. The recovered product was dissolved in EB solution. Labeling and library preparation are completed.

10) And (3) detecting the quality of the library: the size and concentration of fragments of the library were determined using an Agilent 2100Bioanalyzer (Agilent DNA 1000 Reagents).

11) Cyclization of PCR products: and (3) after the PCR product is denatured into a single chain, preparing a cyclization reaction system, fully mixing uniformly, reacting at a proper temperature for a certain time to obtain a single-chain cyclic product, and digesting the linear DNA molecules which are not cyclized to obtain the final library.

12) And (3) machine sequencing: the single-stranded circular DNA molecule replicates through rolling circles to form a DNA Nanosphere (DNB) containing more than 200 copies. The obtained DNBs are added into the mesh pores on the chip by adopting a high-density DNA nano chip technology. The sequencing read length of 50bp/100bp is obtained by a sequencing-by-synthesis method.

2.4 sequencing data quality control

Filtering the raw sequencing data to obtain high-quality sequencing data (clean data), comprising the following steps: removing the adapter sequence in reads; removing bases containing non-AGCT at the 5' end before shearing; pruning ends of reads with lower sequencing quality (sequencing quality value less than Q20); removing reads with the N content of 10%; discarding small fragments with length less than 25bp after removing the adapter and mass pruning.

2.5 alignment with reference genome

Sequencing data were aligned to the reference genome using hisat2 analysis software. The reference genome was from the Ensembl database.

2.6 Gene expression level analysis

The expression level of the gene was calculated by aligning the number of sequences (clean reads) to the reference genomic region. The FPKM value of each gene/transcript in the sample was calculated using Stringtie according to the alignment of Hisat2 software, and this value was used as the expression level of the gene/transcript in the sample.

2.7 differential mRNA expression analysis

The expression difference of mRNA of the control group and the disease group is compared by using DESeq2, and the difference analysis steps are as follows: firstly, standardizing (normalization) the original read count, mainly correcting the sequencing depth; carrying out hypothesis test probability (P-value) calculation through a statistical model, carrying out multiple hypothesis test correction (BH) to obtain a padj value (false discovery rate), wherein the screening standard of the differential expression genes is as follows: pvalue <0.05 and | log2foldchange | > 1.

3. Results and analysis

Compared with a control group, the biomarkers DAAM2, ECHDC3 and FOLR3 involved in the invention are significantly up-regulated in the stroke patients, and the difference is statistically significant.

Example 2 biomarker expression

Differential analysis was performed on gene expression data using blood from subjects with a history of cardiovascular disease (n 23; 11 females, 12 males, and 11 females) and asymptomatic vascular disease (n 23; 11 females, 12 males) derived from a database as a sample (the data set was GSE 58294). The probes corresponding to the multiple genes were removed, and then only one probe with the largest average expression level was retained for the gene corresponding to the multiple probes. The data set scale is normalized. The tool used for the differential analysis was R-4.0.5, and the method used for p-value combination in meta analysis was invert normal method using meta MA, limma package analysis. MetaMA package description of P, FDR value calculation (scales differential expression p-values and effect sizes from data from structural or modified t-tests (Limma, SMVar) for test and combinations of the p-values by the inverse normal method, FDR calculation: Benjamini Hochberg threshold. by default, the False Discovery Rate is controlled at 5%).

Screening criteria for differentially expressed genes: val <0.05& | logFC | >1, obtaining 208 differentially expressed genes, wherein the expression of the biomarkers related to the invention in the cardiac cerebral embolism subjects is shown in the table 1 and the figures 1 to 3, the biomarkers are up-regulated in the disease groups, and the differences have statistical significance.

TABLE 1 expression of biomarkers in blood samples of cardiogenic cerebral embolism patients

Example 3 diagnostic efficacy

The diagnostic performance of each diagnostic index can be visually identified by a receiver operating characteristic curve (ROC), and The closer The ROC curve is to The upper left corner, The larger The area under The curve (AUC), and The higher The diagnostic value (refer to The means and use of The area under a Receiver Operating Characteristic (ROC) curve [ J ]. Radiology, 1982). The invention uses R language pROC to carry out ROC diagnosis analysis on the gene so as to screen biomarkers with diagnostic value.

As a result:

when AUC > 0.5, the closer the AUC value is to 1, the better the diagnostic effect of the diagnostic marker is. Based on the GEO data set, the ROC curve of the gene related to the invention is drawn by using the R language. The results are shown in fig. 4-6, the AUC values of the genes of the present invention are substantially in the range of 0.70-0.85, wherein DAAM2(AUC is 0.733), ECHDC3(AUC is 0.829), FOLR3(AUC is 0.826), and the diagnostic efficacy of the biomarker combinations are further analyzed, as shown in fig. 7-10, table 2.

TABLE 2 biomarker combination diagnostic potency

Biomarker combinations	AUC
		DAAM2+ECHDC3	0.825
ECHDC3+FOLR3	0.909
		DAAM2+FOLR3	0.872
DAAM2+ECHDC3+FOLR3	0.922

The three biomarker combination has better diagnostic efficacy compared to either a single biomarker or two biomarkers. The above results demonstrate that the biomarkers of the invention can be used for diagnosing cardiogenic cerebral embolism.

The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application. It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application. In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.

Claims

1. A product useful for diagnosing stroke, comprising reagents for detecting the level of expression of biomarkers in a sample, said biomarkers comprising two or three of DAAM2, ECHDC3, FOLR 3.

2. The product of claim 1, wherein the reagents comprise reagents for detecting the level of expression of a biomarker in a sample by digital imaging techniques, protein immunization techniques, dye techniques, nucleic acid sequencing techniques, nucleic acid hybridization techniques, chromatography techniques, and mass spectrometry techniques.

3. The product of claim 1, wherein the sample is blood.

4. The product of claim 1, wherein the reagents comprise primers or probes that specifically bind to the biomarker genes; an antibody, peptide, aptamer, or compound that specifically binds to the marker protein.

5. The product of claim 1, wherein the product comprises a nucleic acid membrane strip, a formulation, a chip, a kit.

6. Use of a biomarker for the manufacture of a product according to any of claims 1 to 5, wherein the biomarker comprises two or three of DAAM2, ECHDC3, FOLR 3.

7. A pharmaceutical composition comprising two or three of an inhibitor of DAAM2, an inhibitor of ECHDC3, and an inhibitor of FOLR 3.

8. Use of the pharmaceutical composition of claim 7 for the preparation of a medicament for the prevention or treatment of stroke.

9. A system or apparatus for diagnosing whether a subject has a stroke or predicting whether a subject is at risk for developing a stroke, the system/apparatus comprising:

the biomarkers comprise two or three of DAAM2, ECHDC3 and FOLR 3.

10. A computer-readable storage medium, having stored thereon a computer program which, when executed by a processor, implements the system/apparatus of claim 9.