CN115058512A - Application of iron death related gene in identifying cerebral arterial thrombosis - Google Patents

Application of iron death related gene in identifying cerebral arterial thrombosis Download PDF

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CN115058512A
CN115058512A CN202210912577.4A CN202210912577A CN115058512A CN 115058512 A CN115058512 A CN 115058512A CN 202210912577 A CN202210912577 A CN 202210912577A CN 115058512 A CN115058512 A CN 115058512A
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iron death
marker gene
related marker
slc40a1
slc2a3
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王玉峰
蒯东
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Shanxi Cardiovascular Hospital Shanxi Institute Of Cardiovascular Disease
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Shanxi Cardiovascular Hospital Shanxi Institute Of Cardiovascular Disease
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    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
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    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event

Abstract

The invention discloses application of iron death related genes in identifying ischemic stroke, wherein the iron death related genes are SLC40A1, SLC2A3 and ACSL4, and the invention finds that the combination of SLC40A1, SLC2A3 and ACSL4 can be used in early diagnosis of ischemic stroke and has higher accuracy, sensitivity and specificity.

Description

Application of iron death related gene in identifying cerebral arterial thrombosis
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of an iron death related gene in identification of cerebral arterial thrombosis.
Background
Ischemic Stroke (CIS) is an acute cerebrovascular disease that causes cerebral blood supply dysfunction, ischemia, and hypoxia due to various causes, and further causes necrosis and softening of brain tissue, resulting in focal or complete neurological impairment. Statistically, the mortality rate of ischemic stroke is as high as the third world, the chronic disability rate is the first of various diseases (Bevan S, Markus H S. genetic profile in ischemic stroke [ J ]. Current disorders Reports,2013,15(8):342.), and is the main cause of adult death and disability, with about 10% of the deaths and 5% of the disability-adjusting life losses worldwide being caused by ischemic stroke (GBD 2016DALYs and HALE Collaborators. Global, regional, and national diagnosis-assisted life-areas DAYEARs (Sep) for diseases and infections and diseases and clinical life-adjusting mechanisms; 201333D 2016 and Escherichia and clinical life-adjusting mechanisms (HALE) for experiments 195. Study and 195. 1990. 2016. about 80. about the same kind of ischemic stroke). In recent years, the incidence of ischemic stroke is rising, becoming one of the most common high-incidence diseases in neurology, gradually presenting a trend of younger, becoming an important killer seriously threatening the quality of life and physical and mental health of people, and causing heavy economic burden for many families and even the whole society.
There are many hypotheses about the causes of ischemic stroke, including heredity, endocrine, systemic metabolism, and congenital development, but there is no hypothesis that can fully explain the pathogenesis and progression of the disease. The traditional risk factors include hypertension, coronary heart disease, diabetes, smoking and drinking history, hyperlipidemia, etc., but these cannot explain the cause of ischemic stroke patients without risk factors, and cannot explain the difference of attack when exposed to the same environmental factors. In recent years, genetic research is rapidly developed, more and more researchers try to explore the genetic basis of ischemic stroke from a molecular level, and key genes related to ischemic stroke are discovered by utilizing an advanced bioinformatics technology, so that the related researchers can explain the potential molecular mechanism in the ischemic stroke pathogenesis from a genetic perspective. Currently, the causes of ischemic stroke are identified mainly based on cranial CT scans, Magnetic Resonance Imaging (MRI) and arteriography. However, CT and MRI scanning are not completely reliable methods, because CT has only 16% sensitivity to ischemic stroke and 89% sensitivity to hemorrhagic stroke in early diagnosis. 20% of ischemic strokes cannot be detected by MRI scanning techniques. And the related equipment of the image diagnosis technology is not present in all diagnosis and treatment centers, which hinders the rapid diagnosis of stroke and the timely application of medicines. Biomarkers are effective tools in drug development processes, provide information about drug performance and disease progression, and reflect specific drug treatment effects. Diabetes, immune diseases and other diseases are clinically treated according to the guidance of biomarkers. However, biomarkers are still relatively lacking in cerebrovascular disease. Therefore, the exploration of biomarkers related to ischemic stroke is of great significance to the field.
Disclosure of Invention
The invention aims to provide application of an iron death related gene in identifying ischemic stroke so as to make up for the defects in the prior art.
The above object of the present invention is achieved by the following technical solutions:
the first aspect of the invention provides application of a reagent for detecting the expression level of an iron death-related marker gene in preparing a product for early diagnosis of ischemic stroke.
Further, the iron death-related marker gene is any one or more of SLC40A1, SLC2A3 and ACSL 4;
preferably, the iron death-related marker gene is a combination of SLC40a1, SLC2A3, ACSL 4.
In a specific embodiment of the invention, the iron-death-related marker gene further comprises any two combinations of SLC40a1, SLC2A3, ACSL4, i.e. combination of both SLC40a1 and SLC2A3, combination of both SLC40a1 and ACSL4, and combination of both SLC2A3 and ACSL.
The invention discovers and verifies that the combination of SLC40A1 and SLC2A3, the combination of SLC40A1 and ACSL4, the combination of SLC2A3 and ACSL, and the combination of SLC40A1, SLC2A3 and ACSL4 have higher diagnostic efficacy on ischemic stroke for the first time, wherein the combined effect of the SLC40A1, the SLC2A3 and the ACSL4 is optimal.
Further, the reagent comprises a reagent for detecting the expression level of the iron death related marker gene in the sample by adopting a nucleic acid hybridization technology, a nucleic acid sequencing technology, a digital imaging technology, a protein immunity technology, a dye technology, a chromatography technology and a mass spectrometry technology.
Further, the reagent comprises a primer that specifically binds to a sequence of the iron death-related marker gene, a probe that is specifically complementary to a sequence of the iron death-related marker gene, an antibody specific for an epitope of the iron death-related marker gene, and/or a dye specific for the iron death-related marker gene.
Further, the sample is a peripheral blood sample from a subject.
Further, the subject is preferably a human.
In a second aspect of the invention, a product for early diagnosis of ischemic stroke is provided.
Further, the product comprises a reagent for detecting the expression level of the iron death-related marker gene in the sample;
the iron death-related marker gene is the iron death-related marker gene described in the first aspect of the present invention.
Furthermore, the product comprises a detection kit, a chip and test paper.
Further, the detection kit comprises a primer and/or a probe for detecting the iron death-related marker gene;
the detection kit also comprises a reagent for detecting the reference gene;
the reagent for detecting the internal reference gene comprises a primer and/or a probe aiming at the internal reference gene;
the detection kit also comprises dNTPs and Mg 2+ Ions, DNA polymerase or DNA polymerase containing dNTPs, Mg 2+ Ionic, DNA polymerase PCR systems;
the detection kit also comprises bisulfite or hydrazine salt.
Further, the detection kit comprises a qPCR detection kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry detection kit, an immunohistochemical detection kit, an ELISA detection kit and an electrochemiluminescence detection kit.
Further, the chip comprises a gene chip and a protein chip, wherein the gene chip comprises an oligonucleotide probe aiming at the iron death-related marker gene and used for detecting the transcription level of the iron death-related marker gene, and the protein chip comprises a specific binding agent of the protein coded by the iron death-related marker gene.
Further, the test paper comprises a test paper carrier and oligonucleotide fixed on the test paper carrier, wherein the oligonucleotide can detect the expression level of the iron death-related marker gene or the functional fragment thereof.
A third aspect of the invention provides a device/system for early diagnosis of ischemic stroke.
Further, the apparatus/system comprises a detection module and an evaluation module;
the detection module is used for detecting the expression level of an iron death-related marker gene in a sample of a subject, wherein the iron death-related marker gene is the iron death-related marker gene in the first aspect of the invention;
the evaluation module is used for comparing the detected value of the subject obtained by the detection module with the detected value of the normal sample or the normal reference value, and judging that the subject is the patient with the ischemic stroke when the expression level of one or more of SLC40A1, SLC2A3 and ACSL4 is significantly higher than the detected value of the normal sample or the normal reference value.
Further, the sample is a peripheral blood sample from a subject.
Further, the subject is preferably a human.
Further, the evaluation module includes a memory and a data processor.
Further, the memory is for storing the detected value of one or more of SLC40a1, SLC2A3, ACSL4 or the reference value of one or more of normal SLC40a1, SLC2A3, ACSL4 in the normal sample.
Further, the data processor is configured to compare the detection value of the subject obtained by the analysis detection module with the normal sample detection value or the normal reference value stored in the memory, thereby diagnosing whether the subject has ischemic stroke.
The fourth aspect of the invention provides application of an agent for detecting the expression level of an iron death-related marker gene in preparing a device/system for early diagnosis of ischemic stroke.
Further, the iron death-related marker gene is the iron death-related marker gene described in the first aspect of the present invention.
In a fifth aspect of the present invention, an in vitro screening method for a candidate drug for treating ischemic stroke is provided.
Further, the method comprises the steps of:
(1) adding the drug to be tested into a system expressing or containing one or more of SLC40A1, SLC2A3 and ACSL 4;
(2) detecting the expression level of one or more of SLC40A1, SLC2A3 and ACSL4 in the system;
(3) selecting as the drug candidate a drug that significantly reduces the expression level of one or more of SLC40a1, SLC2A3, ACSL 4.
Further, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
Further, the drugs to be tested include, but are not limited to: interfering molecules, nucleic acid inhibitors, small molecule compounds and the like designed against one or more of the SLC40A1, SLC2A3, ACSL4 genes or upstream or downstream genes thereof.
In addition, the invention also provides application of the reagent for detecting the expression level of one or more of SLC40A1, SLC2A3 and ACSL4 in-vitro screening of drugs for treating ischemic stroke.
Further, the screening method is the method according to the fifth aspect of the present invention.
Further, the screening method judges whether the drug to be tested has a treatment effect on ischemic stroke by detecting the influence of the drug to be tested on the expression level of one or more of SLC40A1, SLC2A3 and ACSL4 in the system.
In addition, the invention also provides application of an agent for inhibiting the expression level of one or more of SLC40A1, SLC2A3 and ACSL4 in preparing a medicament for treating ischemic stroke.
Further, the medicament comprises an agent for inhibiting the expression level of one or more of SLC40A1, SLC2A3 and ACSL4, and a pharmaceutically acceptable carrier and/or auxiliary material.
Compared with the prior art, the invention has the following advantages and beneficial effects:
based on the iron death-related marker genes SLC40A1, SLC2A3 and ACSL4 provided by the invention, patients with ischemic stroke and non-ischemic stroke can be effectively diagnosed and distinguished; the invention is based on that the AUC value of the SLC40A1, the SLC2A3 and the ACSL4 which are combined and predicted in a training set for diagnosing the ischemic stroke is up to 0.971, the sensitivity and the specificity are respectively up to 1.000 and 0.897, the AUC value of the SLC40A1, the SLC2A3 and the ACSL4 which are combined and predicted in a verification set for diagnosing the ischemic stroke is up to 0.963, the sensitivity and the specificity are respectively up to 0.957 and 0.870, and the effect is obviously better than that of the single use of the SLC40A1, the SLC2A3 or the ACSL 4. In addition, the method for early diagnosis of ischemic stroke provided by the invention is simple, convenient and objective, is less influenced by human subjectivity, has higher accuracy, sensitivity and specificity compared with the traditional diagnosis method, and can be used for early accurate and effective screening and diagnosis of ischemic stroke.
Drawings
FIG. 1 is a graph of the results of the differential expression of the iron death-related genes SLC40A1, SLC2A3, ACSL4 between patients with ischemic stroke and healthy controls in the training set, wherein, A is a graph: SLC40a1, panel B: SLC2a3, panel C: ACSL 4;
FIG. 2 is a graph showing the results of differential expression of the iron death-related genes SLC40A1, SLC2A3 and ACSL4 between patients with ischemic stroke and healthy controls in the validation set, wherein, A is a graph: SLC40a1, panel B: SLC2a3, panel C: ACSL 4;
fig. 3 is a graph showing the results of the diagnostic efficacy of the iron death-related genes SLC40a1, SLC2A3, ACSL4 in differentiating ischemic stroke patients from healthy controls in the training set, wherein, a is a graph: SLC40a1, panel B: SLC2a3, panel C: ACSL 4;
FIG. 4 is a graph showing the results of the diagnostic efficacy of the iron death-related genes SLC40A1, SLC2A3 and ACSL4 in differentiating patients with ischemic stroke from healthy controls in the validation set, wherein, A is a graph: SLC40a1, panel B: SLC2a3, panel C: ACSL 4;
figure 5 is a graph of the results of the diagnostic efficacy of any two of the iron death-related genes SLC40a1, SLC2A3, ACSL4 in combination in differentiating ischemic stroke patients from healthy controls in a training set, wherein, a graph: SLC40A1+ SLC2A3, Panel B: SLC40a1+ ACSL4, panel C: SLC2a3+ ACSL 4;
fig. 6 is a graph showing the results of the combined diagnosis effect of any two of the iron death-related genes SLC40a1, SLC2A3, ACSL4 in differentiating ischemic stroke patients from healthy controls in the validation set, wherein, a graph: SLC40A1+ SLC2A3, Panel B: SLC40a1+ ACSL4, panel C: SLC2a3+ ACSL 4;
FIG. 7 is a graph showing the results of the combined diagnostic efficacy of the iron death-related genes SLC40A1, SLC2A3 and ACSL4 in the training set in differentiating patients with ischemic stroke from healthy controls;
FIG. 8 is a graph showing the results of the combined effect of the SLC40A1, SLC2A3 and ACSL4 genes on the diagnosis of ischemic stroke patients and healthy controls in the validation set.
Detailed Description
The invention will now be further illustrated with reference to specific examples, which are provided for illustration only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.
According to the invention, through extensive and intensive research, gene expression data of 155 ischemic stroke patients and healthy contrast persons are comprehensively analyzed based on key genes related to iron death, the key genes related to early diagnosis of ischemic stroke are screened out and used as diagnostic markers of ischemic stroke, and the method for early diagnosis of ischemic stroke is constructed based on the markers, so that the method has higher accuracy, sensitivity and specificity.
As used in this disclosure, "and/or" should be viewed as specifically disclosing each of the two specified features or components, with or without the other. For example, "a and/or B" will be considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed in the present invention.
Marker substance
As used herein, "marker", as well as "marker gene" and "biomarker", refer to a biological molecule that is present in an individual at varying concentrations that can be used to predict the disease state of the individual. Such markers include, but are not limited to, nucleic acids, proteins, and variants and fragments thereof. The marker may be a DNA comprising all or part of a nucleic acid sequence encoding the marker or the complement of such a sequence. Marker 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.
Further, markers exist with a level difference of statistical significance (i.e. p-value less than 0.05 and/or q-value less than 0.10, as determined using Welch's T-Test or Wilcoxon rank-sum Test).
In a particular embodiment of the invention, the marker comprises the iron death-related gene SLC40a1, SLC2A3 and/or ACSL 4. In another embodiment, the marker is a combination of any two of SLC40a1, SLC2A3 and/or ACSL4, and in a preferred embodiment, the marker is a combination of SLC40a1, SLC2A3 and ACSL 4.
Iron death-related gene SLC40a 1: the typical homo sapiens mRNA and protein sequences can be found in the NCBI database as gene ID 30061 by the source carrier family 40member 1.
Iron death-related gene SLC2a 3: the typical homo sapiens mRNA and protein sequences can be found in the NCBI database under the name of gene ID:2182 by the source carrier family 2member 3.
Iron death-related gene ACSL 4: acyl-CoA synthesizing chain protein family member 4, gene ID:6515 in NCBI database can find typical homo sapiens mRNA and protein sequences.
As used herein, the terms "marker", "marker gene", "biomarker" are used interchangeably and refer to a molecule that is differentially present in a sample taken from an ischemic stroke subject compared to a comparable sample taken from a control subject, e.g., a healthy subject. Thus, the markers of the invention provide information about the likely course of ischemic stroke and correlate with early diagnosis of ischemic stroke.
Sample(s)
As used herein, "sample" may refer to a biological sample, typically a clinical sample, and includes, for example, blood and other bodily fluids, including but not limited to peripheral blood, serum, plasma, urine, and saliva; and solid tissue samples, such as biopsy specimens. In certain embodiments, peripheral blood samples are the preferred type of sample to be used in the present invention. Typically, the process of obtaining a sample to be analyzed from a subject is not part of the early diagnostic method of the present invention. The sample in a particular embodiment of the invention is a peripheral blood sample.
Expression level
The "expression level" as used herein, together with the "expression level of a marker gene" refers to the level of mRNA expression of the marker gene of the present invention in a sample from a subject and/or the level of expression of a polypeptide and/or protein encoded by the marker gene of the present invention in the sample.
The present invention can detect the expression levels of the marker genes SLC40a1, SLC2A3 and ACSL4 using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, including but not limited to: protein immunization technology, dye technology, nucleic acid sequencing technology, nucleic acid hybridization technology, chromatographic technology and mass spectrum technology. The following are listed in detail herein.
The protein immunization methods of the invention include sandwich immunoassays, such as sandwich ELISA, in which the detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.
Illustrative, non-limiting examples of the nucleic acid sequencing methods of the present invention include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.
Another illustrative, non-limiting example of a nucleic acid sequencing method of the present invention includes next generation sequencing (deep sequencing/high throughput sequencing), a high throughput sequencing technique that is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.
Methods of nucleic acid hybridization in the present invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization using a labeled complementary DNA or RNA strand as a probe to locate a tissue portion or section (in situ) or a specific DNA or RNA sequence in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNAISH can be used to determine the structure of chromosomes. Rnash is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.
Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.
In the present invention, "chip", also referred to as "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.
In the present invention, "primer" refers to an oligonucleotide that hybridizes to a sequence in a target nucleic acid ("primer binding site") and is capable of serving as a point at which synthesis is initiated along the complementary strand of the nucleic acid under conditions suitable for such synthesis.
In the present invention, a "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled. Hybridization formats, including but not limited to solution phase, solid phase, mixed phase or in situ hybridization assays.
In the present invention, "binding agent for a protein" refers to, for example, a receptor for a protein, a lectin that binds to a protein, an antibody against a protein, a peptide antibody (peptidebody) against a protein, a bispecific dual binding agent, or a bispecific antibody format.
In the present invention, "subject" refers to an animal subject, in particular a vertebrate subject, more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present invention include, but are not limited to, any member of the subphylum chordata, including primates, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cows), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, ducks, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards, etc.), and fish. Preferred subjects are primates (e.g., humans, apes, monkeys, chimpanzees). The subject as preferred according to the invention is a human.
Example 1 identification and screening of key genes (iron death-related genes) in ischemic stroke 1, sample data Collection and Pre-treatment
GEO is a common functional genomic data repository in which gene expression profiles are selected for current studies. The invention uses keywords 'click' and 'Homo sapiens' to filter gene expression profile data. The corresponding data sets were then screened using the following criteria. The inclusion criteria were: not less than 5 samples are obtained; ② there is normal contrast in data set. Exclusion criteria were: research on cell lines or animal level; research of single sample; (iii) repeated or overlapping studies. Finally GSE16561, GSE58294 were included in the study of the present invention. GSE16561 peripheral blood samples containing 39 CIS patients and 24 healthy controls were submitted by Barr TL et al. GSE58294 was a peripheral blood sample submitted by Stamova BB et al containing 69 CIS patients and 23 healthy controls, with GSE16561 as the training set and GSE58294 as the validation set, detailed in table 1, for both data sets, from which the invention downloaded their corresponding gene expression matrix files for analysis. And (3) annotating the gene expression profile by using a GPL platform annotation file, and converting the gene probes into gene symbols, wherein a plurality of probes correspond to the average value of the same gene. The combath function in the R-package "sva" was used to remove the batch effect.
TABLE 1 data set-related information
Figure BDA0003774359190000111
2. Collection of iron death-related genes
Iron death-related genes (FRGs) were from FerrDb and related literature (PMID 32760210). Html is the first database in the world for iron death, providing a more up-to-date database of modulators and markers associated with disease for iron death. The present invention incorporates 259 iron death-related genes in the FerrDb database and is supplemented by the iron death gene in the literature (PMID 32760210). Finally, the present invention obtained 267 FRGs. Of these, 216 FRGs exist in the GEO data set collected above.
3. Differential expression gene analysis
After the above pre-processing of the two data sets, differential expression analysis is performed using the "limma" package, thereby obtaining the DEGs of the CIS. The screening criteria for the DEGs was set to adj<0.05，|log 2 FC|>0.2, and visualized using a volcanic image. FRGs were screened for differences and the results were shown in a heatmap.
4. Results of the experiment
The invention reads processed data into R, and uses 'limma' packet as adj<0.05，|log 2 FC|>The standard of 0.2 was used for differential expression analysis. In the CIS patient group, 27 iron death-related differentially expressed genes were identified compared to the control group, and the results are shown in tables 2 and 3. The differential expression result graphs of the iron death-related genes SLC40A1, SLC2A3 and ACSL4 in the training set and the verification set are shown in fig. 1 and fig. 2, the results show that SLC40A1, SLC2A3 and ACSL4 have significant differential expression between peripheral blood samples of patients with ischemic stroke and healthy controls, and the expression trends of SLC40A1, SLC2A3 and ACSL4 in the peripheral blood samples of patients with ischemic stroke are significantly up-regulated. The result indicates that the iron death related genes SLC40A1, SLC2A3 and ACSL4 are expected to be biomarkers for early diagnosis of ischemic stroke.
TABLE 2 identification of 29 iron death-related differentially expressed genes in CIS patient groups (GSE16561)
Figure BDA0003774359190000121
Figure BDA0003774359190000131
TABLE 3 29 iron death-related differentially expressed genes identified in CIS patient group (GSE58294)
Figure BDA0003774359190000132
Figure BDA0003774359190000141
Example 2 application of the marker genes SLC40A1, SLC2A3 and ACSL4 related to iron death in early diagnosis of cerebral ischemic stroke
1. Experimental method
In order to further evaluate the accuracy of the iron death-related marker genes SLC40a1, SLC2A3 and ACSL4 obtained by screening in example 1 in distinguishing CIS patients from healthy patients, this example performed a diagnostic efficacy analysis, wherein the area under the ROC curve (AUC), which is also called AUC value, is closer to 1, and the authenticity of the diagnostic test is better when any one or more of the iron death-related genes SLC40a1, SLC2A3 and ACSL4 are used in combination for diagnosing ischemic stroke. The AUC is the possibility of correctly distinguishing between patients and non-patients in a diagnostic test, and if the AUC is less than 0.7, it means that the diagnostic accuracy is low, if the AUC is between 0.7 and 0.9, it means that the diagnostic accuracy is medium, and if the AUC is greater than 0.9, it means that the diagnostic accuracy is high, and it is able to very well and accurately distinguish between patients and non-patients. In addition, the present example evaluated the classification effect by calculating sensitivity and specificity, where sensitivity is true positive/(true positive + false negative), and specificity is true negative/(true negative + false positive).
2. Results of the experiment
The results show that the AUC values of the selected iron death-related marker genes SLC40a1, SLC2A3 and ACSL4 in example 1 of the present invention are greater than 0.850 in both training set and validation set, and show higher sensitivity and specificity, as shown in fig. 3, fig. 4, table 4 and table 5. Furthermore, the combination of SLC40a1, SLC2A3 and ACSL4 has AUC values up to 0.971 in the training set, sensitivity and specificity up to 1.000 and 0.897, respectively, the combination of SLC40a1, SLC2A3 and ACSL4 has AUC values up to 0.963 in the validation set, sensitivity and specificity up to 0.957 and 0.870, respectively, the effect is significantly better than that of SLC40a1, SLC2A3 or ACSL4 used alone, and significantly better than that of any two of SLC40a1, SLC2A3 and ACSL4 used in combination, as shown in fig. 5-8, table 4 and table 5. In addition, compared with the traditional diagnosis method, the combination of the SLC40A1, the SLC2A3 and the ACSL4 has higher accuracy, sensitivity and specificity for the diagnosis of the ischemic stroke, and can be used for the early accurate and effective screening and diagnosis of the ischemic stroke.
TABLE 4 statistical results of diagnostic efficacy of the iron death-related genes SLC40A1, SLC2A3 or ACSL4 on ischemic stroke alone or in combination (GSE16561)
Figure BDA0003774359190000151
TABLE 5 statistical results of the diagnosis efficacy of iron-death-related genes SLC40A1, SLC2A3 or ACSL4 (GSE58294) alone or in combination
Figure BDA0003774359190000152
Figure BDA0003774359190000161
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims (10)

1. The application of the reagent for detecting the expression level of the iron death-related marker gene in preparing products for early diagnosing ischemic stroke is characterized in that the iron death-related marker gene is any one or more of SLC40A1, SLC2A3 and ACSL 4;
preferably, the ferrodeath-related marker gene is a combination of SLC40a1, SLC2A3, ACSL 4.
2. The use of claim 1, wherein the reagents comprise reagents for detecting the expression level of the iron death-related marker gene in the sample using nucleic acid hybridization techniques, nucleic acid sequencing techniques, digital imaging techniques, protein immunization techniques, dye techniques, chromatography techniques, mass spectrometry techniques.
3. The use according to claim 2, wherein the agent comprises a primer that specifically binds to a sequence of the iron death-related marker gene, a probe that is specifically complementary to a sequence of the iron death-related marker gene, an antibody specific for an epitope of the iron death-related marker gene, and/or a dye specific for the iron death-related marker gene.
4. The use of claim 2, wherein the sample is a peripheral blood sample from a subject.
5. A product for early diagnosis of ischemic stroke, comprising an agent for detecting the expression level of an iron death-related marker gene in a sample;
the iron death-related marker gene is the iron death-related marker gene according to claim 1.
6. The product of claim 5, wherein the product comprises a test kit, chip, strip.
7. The product according to claim 6, wherein the detection kit comprises primers and/or probes for detecting the iron death-related marker gene;
the detection kit also comprises a reagent for detecting the reference gene;
the reagent for detecting the reference gene comprises a primer and/or a probe aiming at the reference gene;
the detection kit also comprises dNTPs and Mg 2+ Ions, DNA polymerase or DNA polymerase containing dNTPs, Mg 2+ Ionic, DNA polymerase PCR systems;
the detection kit also comprises bisulfite or hydrazine salt.
8. An apparatus/system for early diagnosis of ischemic stroke, characterized in that the apparatus/system comprises a detection module and an evaluation module;
the detection module is used for detecting the expression level of an iron death-related marker gene in a sample of a subject, wherein the iron death-related marker gene is the iron death-related marker gene in claim 1;
the evaluation module is used for comparing the detected value of the subject obtained by the detection module with the detected value of the normal sample or the normal reference value, and when the expression level of one or more of SLC40A1, SLC2A3 and ACSL4 is significantly higher than the detected value of the normal sample or the normal reference value, the subject is judged to be the patient with ischemic stroke.
9. Use of an agent for detecting the expression level of an iron death-related marker gene in the preparation of a device/system for early diagnosis of ischemic stroke, wherein the iron death-related marker gene is the iron death-related marker gene according to claim 1.
10. An in vitro screening method for a drug candidate for treating ischemic stroke, comprising the steps of:
(1) adding the drug to be tested into a system expressing or containing one or more of SLC40A1, SLC2A3 and ACSL 4;
(2) detecting the expression level of one or more of SLC40A1, SLC2A3 and ACSL4 in the system;
(3) selecting as the drug candidate a drug that significantly reduces the expression level of one or more of SLC40a1, SLC2A3, ACSL 4.
CN202210912577.4A 2022-07-30 2022-07-30 Application of iron death related gene in identifying cerebral arterial thrombosis Pending CN115058512A (en)

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