Biomarker cxcl8 and uses thereof

Abstract

The present application provides a composition or kit for screening, diagnosing and / or monitoring of coronary heart disease in an individual, comprising a reagent for detecting a CXCL8-specific sequence in a sample from the individual, including a primer or a probe, and related uses. The present application also provides a use of a compound interfering with the expression of a CXCL8-specific sequence in the manufacture of a potential medicament for treating coronary heart disease in an individual.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular the field of disease screening, diagnosis, monitoring and / or treatment; specifically, the present application provides biomarkers and related products and uses for the screening, diagnosis, monitoring and / or treatment of coronary heart disease.

[0002] Funding Information

[0003] This patent application is funded by the Ministry of Science and Technology of China, involving the National Key Research and Development Program: Application Evaluation and Quality Improvement of Newborn / Child Critical Illness Extracorporeal Life Support (Approval No: 2021YFC2701700; 2021YFC2701703). BACKGROUND

[0004] Coronary heart disease has a high mortality and disability rate, and has become a major global public health problem. Coronary heart disease is becoming younger, seriously threatening the health of young people. Early-onset coronary heart disease is considered to be acute myocardial infarction before the age of 45 or more than 70% of coronary artery stenosis observed in coronary angiography. Traditional coronary heart disease risk factors such as metabolic diseases, smoking and unhealthy lifestyle play a key role in the onset of coronary heart disease. However, the classic traditional coronary heart disease risk factors as indicators of disease prediction and risk stratification have obvious shortcomings. Epidemiological studies have found that about 40% of coronary heart disease patients have genetic susceptibility characteristics, and individuals with LDL-C≧190mg / dL are more likely to carry familial hypercholesterolemia genes, and their probability of developing coronary heart disease is 3 times that of individuals without these mutations. Genetic susceptibility is closely related to the pathogenesis of coronary heart disease. A variety of genetic variations can directly or indirectly act through a variety of biological pathways to play a pathogenic role, including blood pressure, blood lipid and glucose metabolism, vascular homeostasis, and anti-inflammatory and pro-inflammatory imbalance. The early-onset coronary heart disease population without clear traditional coronary heart disease risk factors may be more susceptible to genetic risk factors. Therefore, in the early-onset coronary heart disease population with fewer traditional coronary heart disease risk factors, genetic susceptibility is considered an important risk factor that cannot be ignored.

[0005] Therefore, elucidating the genetic susceptibility characteristics of early-onset coronary heart disease and identifying new biomarkers are crucial for early warning, diagnosis, treatment and / or improving the clinical prognosis of early-onset coronary heart disease. SUMMARY

[0006] In a first aspect, the present application provides a reagent for detecting a CXCL8 specific nucleic acid sequence or a composition comprising the reagent, wherein the reagent comprises a primer or a probe, preferably the specific nucleic acid sequence is at least about 30 nucleotides in length, more preferably 45-300 nucleotides, most preferably 57-123 nucleotides.

[0007] In a second aspect, the present application provides a kit for screening, diagnosing and / or monitoring coronary heart disease, comprising the reagent or composition of the first aspect described above, or a reagent for detecting a polypeptide encoded by the specific nucleic acid sequence of the first aspect described above.

[0008] In a third aspect, the present application provides use of a compound interfering with the expression of the specific nucleic acid sequence of the first aspect described above in the manufacture of a potential drug for treating coronary heart disease, wherein the compound inhibits or reduces the level of the nucleic acid sequence or the level of a polypeptide expressed by the nucleic acid sequence.

[0009] In another aspect, the present application provides use of a reagent for detecting the expression level of a CXCL8 specific nucleic acid sequence in a sample from an individual in the manufacture of a kit or a drug for screening, diagnosing and / or monitoring coronary heart disease in the individual, wherein if the expression level of the specific nucleic acid sequence is higher than that in a control sample, the individual has or is likely to have coronary heart disease.

[0010] In specific embodiments, the coronary heart disease described above is premature coronary heart disease.

[0011] In some embodiments, the specific nucleic acid sequence comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 1-6 and 25.

[0012] In some embodiments, the primer comprises a sequence as set forth in any one of SEQ ID NOs: 13-24.

[0013] In some embodiments, the polypeptide comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 7-12 and 26.

[0014] In some embodiments, the reagent for measuring the amount of a protein expressed by the CXCL8 gene comprises an antibody or an aptamer that specifically binds to the protein expressed by the CXCL8 gene.

[0015] In some embodiments, the amount of CXCL8 mRNA is measured by at least one method selected from the group consisting of in situ hybridization, polymerase chain reaction (PCR), reverse transcription (RT)-PCR, real-time PCR, RNAse protection assay (RPA), northern blotting, microarray, and high-throughput sequencing, etc.

[0016] In some embodiments, the amount of CXCL8 protein is measured by at least one method selected from the group consisting of Western blotting, radioimmunoassay (RIA), radial immunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, Ouchterlony double immunodiffusion, complement fixation assay, and protein chip, etc.

[0017] In some embodiments, the above-mentioned sample is a peripheral blood, whole blood, serum, or plasma sample. In a preferred embodiment, the sample is a peripheral blood sample.

[0018] In some embodiments, the above-mentioned compound interfering with the expression of CXCL8 gene can be selected from a broad-spectrum anti-inflammatory drug or a non-steroidal anti-inflammatory drug.

[0019] In the present application, a biomarker with specifically increased expression associated with coronary heart disease is identified, so that the occurrence of coronary heart disease, particularly premature coronary heart disease, can be screened, diagnosed and / or monitored by measuring the specific sequence (e.g. mRNA or protein level) of the biomarker gene. In addition, in some embodiments, the CXCL8 biomarker can also be used to develop potential drugs for treating coronary heart disease by targeting this biomarker. However, the effects of the present application are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. SUMMARY

[0020] Figure 1 Identification of differentially expressed genes (DEGs) is shown. Figure 1 A shows DEGs between coronary heart disease patient samples and healthy subject samples of GSE66360 dataset; Figure 1 B shows DEGs between premature coronary heart disease patient samples and healthy subject samples of high-throughput sequencing dataset, wherein blue dots represent down-regulated genes, gray dots represent non-significantly expressed genes, and red dots represent up-regulated genes. Figure 1 C shows DEGs between coronary heart disease patient samples and healthy subject samples of GSE66360 dataset; Figure 1 D shows DEGs between premature coronary heart disease patient samples and healthy subject samples of high-throughput sequencing dataset, wherein blue rectangles represent low expression genes, and red rectangles represent high expression genes. Figure 1 E and F show the number of up-regulated and down-regulated genes, respectively, which are shared by the two datasets.

[0021] Figure 2 Enrichment analysis of DEGs is shown. Figure 2 A and D show significantly enriched pathways of DEGs. Figure 2B and C show the significantly enriched terms of DEGs in BP and MF, respectively. Figure 2 In the bubble plot, the Y-axis represents the enriched terms, and the X-axis represents the gene proportion. In the chord plot, DEGs are shown on the left half of the plot, and significantly enriched pathways are shown on the right half of the plot.

[0022] Figure 3 The enrichment analysis of all DEGs in early-onset coronary heart disease is shown. Figure 3 A shows the significantly enriched pathways of DEGs. Figure 3 B shows the significantly enriched terms of DEGs in BP, CC, and MF, respectively.

[0023] Figure 4 The GSEA analysis of high-throughput sequencing datasets is shown.

[0024] Figure 5 The immune infiltration analysis of high-throughput sequencing datasets is shown, showing the proportion of immune cell subgroups.

[0025] Figure 6 A shows a PPI interaction network with 17 nodes and 28 edges; Figure 6 B shows the top 100 core genes selected by cytohubba algorithm after putting DEGs of all high-throughput sequencing datasets into STRING.

[0026] Figure 7 The external validation results of core genes are shown.

[0027] Figure 8 The prediction and network construction of target ncRNAs are shown, in which Figure 8 A is a Venn diagram of overlapping miRNAs from GSE31568 dataset and online miRNA database; B is a Venn diagram of overlapping circRNAs from GSE160717 dataset and online circRNAs database. Figure 8 C is the ceRNA network of CXCL8, in which red nodes represent core genes, blue nodes represent targeted miRNAs, and orange nodes represent targeted circRNAs.

[0028] Figure 9 The gene-drug interaction network diagram is shown, in which red nodes represent core genes, and blue nodes represent potential therapeutic drugs. DETAILED DESCRIPTION

[0029] Hereinafter, the present application will be described in more detail. Unless otherwise defined herein, 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. Generally, the nomenclature used herein and the experimental methods described below will be those skilled in the art.

[0030] The present application provides a composition or kit for screening, diagnosing and / or monitoring of coronary heart disease in an individual, comprising a reagent for detecting the expression level of a specific sequence of CXCL8 in a sample from the individual. The inventors identified the biomarker CXCL8 which is specifically increased in expression associated with coronary heart disease, by measuring the expression level (e.g. mRNA or protein level) of a specific sequence of the CXCL8 gene, the occurrence of coronary heart disease, particularly premature coronary heart disease, can be screened, diagnosed and / or monitored. If the expression level of the CXCL8 gene in the sample of the test individual is higher than that in the control sample, the individual has or is likely to have coronary heart disease. In addition, the CXCL8 biomarker can also be used to develop potential drugs for treating coronary heart disease by targeting this biomarker.

[0031] CXCL8, also known as IL-8, can induce local inflammatory response. CXCL8 is a core molecule involved in IL-17 signaling pathway, NF-κΒ signaling pathway and NOD-like receptor signaling pathway. In the present application, the inventors identified a specific nucleotide sequence of the CXCL8 gene and the corresponding amino acid sequence. In some embodiments, the specific nucleic acid sequence is at least about 30 nucleotides in length. In specific embodiments, the specific nucleic acid sequence is 45-300 nucleotides in length. In more specific embodiments, the specific nucleic acid sequence is most preferably 57-123 nucleotides in length.

[0032] Exemplary specific nucleotide sequences, corresponding amino acid sequences and primer sequences are listed in the following table. It has been verified that these sequences can be successfully used for the relevant purposes of the present application.

[0033]

[0034]

[0035] In some embodiments, the full-length sequence of CXCL8 is shown as SEQ ID NO: 25, and the encoded polypeptide is shown as SEQ ID NO: 26.

[0036] The terms "individual" or "subject" are used interchangeably herein to refer to all animals classified as mammals, and includes without limitation domestic and farm animals, primates, and humans, e.g., people, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the individual is a male or female of any age or race.

[0037] The biological sample derived from an individual can include, but is not limited to, a peripheral blood, whole blood, serum, or plasma sample, etc. In a particular embodiment, the biological sample used is a peripheral whole blood sample.

[0038] The term "diagnosis" as used herein refers to the process of attempting to determine and / or identify a possible disease in a subject, i.e., the diagnostic procedure, and the opinion reached by this process, i.e., the diagnostic opinion.

[0039] The term "screening" is understood herein as an examination or test of a group of asymptomatic individuals belonging to the general population, or of a group of individuals having one or more risk factors, i.e., subjects suspected of having or at risk of developing a disease, with the aim of distinguishing between healthy individuals and those having or suspected of having a disease. The method of screening is generally used for the early detection of a disease. Early detection refers to detection before the presence of clinical signs.

[0040] The term "monitoring" as used herein refers to determining the evolution of a disease and / or the efficacy of a therapy, for example determining whether there is a remission of the disease; or, on the contrary, whether there is a progression or a relapse of the disease.

[0041] The term "biomarker" as used herein refers to a marker of a disease, which is generally a substance that can be easily measured in a sample of an individual. The amount measured can be correlated with the underlying pathophysiology of the disease, such as the presence or absence of coronary heart disease, for example premature coronary heart disease, or with its prognosis.

[0042] In some embodiments, the reagent for detecting the expression level of the CXCL8 gene is a reagent for measuring the amount of mRNA of the CXCL8 gene. The reagent for measuring the amount of mRNA of a gene refers to a reagent capable of specifically binding to and recognizing the mRNA of the gene or amplifying the amount of mRNA of the gene. As a specific example, it can be, but is not limited to, a primer or a probe that specifically binds to the nucleotide sequence of mRNA or cDNA prepared by reverse transcription of mRNA.

[0043] The term "primer" used herein refers to a short nucleic acid sequence having a free 3'-terminal hydroxyl group, to which a complementary template strand forms a base pair, and thus serves to provide a starting point when a nucleic acid polymerase replicates and amplifies the template strand. The primer can be generally synthesized to a length of 15-30 base pairs, but can vary depending on the purpose of use, and can be modified by methylation, capping, etc. through known methods.

[0044] The term "probe" used herein refers to a nucleic acid fragment of several to several hundred bases in length, which consists of a sequence capable of specifically binding to mRNA or cDNA, and can be produced by isolation and purification by enzymatic chemistry or synthesis. The probe can be labeled with a radioisotope, an enzyme, or a phosphor to identify the presence or absence of mRNA, and can be designed and modified by known methods.

[0045] In some embodiments, the amount of mRNA of a gene can be measured using a method such as PCR, RT-PCR, competitive RT-PCR, and real-time RT-PCR using sense and antisense primers of the gene sequence, can be measured using a method such as Northern blotting and microarray using a probe having a sequence capable of specifically binding to mRNA of the gene or cDNA prepared by reverse transcription, and in addition, can be measured by a method such as RNase protection assay and sequencing, but the present application is not limited thereto, and any method known to those skilled in the art can be used to measure the mRNA expression level of a gene.

[0046] In some embodiments, the reagent for detecting the expression level of the CXCL8 gene is a reagent for measuring the amount of protein expressed from the CXCL8 gene. The reagent for measuring the amount of protein expressed from the gene refers to a reagent capable of specifically binding to and recognizing the protein. As a specific example, it can be, but is not limited to, an antibody or an aptamer that specifically binds to the protein.

[0047] The term "antibody" used herein refers to an immunoglobulin molecule that immunologically specifically binds to a protein epitope and has reactivity, including but not limited to a monoclonal antibody, a polyclonal antibody, an antibody having a full-length chain structure, an antibody having a functional fragment of at least an antigen-binding function, and a recombinant antibody. The term "aptamer" refers to a single-stranded nucleic acid molecule having a stable three-dimensional structure having the property of being able to target and specifically bind to a protein, and an aptamer specific to a protein can be synthesized using a system evolution of ligands by the exponential enrichment (SELEX) technique or the like.

[0048] In some embodiments, the amount of protein expressed by a gene can be measured by at least one method such as Western blotting, protein microarray (protein chip), enzyme-linked immunosorbent assay (ELISA), two-dimensional electrophoresis, immunohistochemistry (IHC), immunofluorescence, flow cytometry, co-immunoprecipitation assay, fluorescence-activated cell sorter (FACS), radioimmunoassay (RIA), radial immunodiffusion, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), Ouchterlony double immunodiffusion, complement fixation assay, etc., but the present application is not limited thereto, and any method known to those skilled in the art can be used to measure the expression level of a protein.

[0049] The wide application of high-throughput sequencing, microarray analysis and clinical bioinformatics technology methods has made breakthrough progress in exploring differentially expressed genes (DEGs) involved in various diseases. These DEGs are closely related to various biological functions of coronary heart disease, so these technologies will provide a deep understanding of the pathogenesis of coronary heart disease from the whole genome dimension. The competing endogenous RNA (ceRNA) network constructed based on DEGs can elucidate the transcriptional regulation mechanism during disease progression and recovery. In addition, DEGs in the blood circulation have become promising candidate biomarkers due to their relatively stable, easy to detect and disease-specific properties. In specific embodiments, the inventors identified potential targets that can be used for early warning, risk stratification and / or precision intervention, such as the CXCL8 gene, by comparing the whole blood transcriptome differences between early-onset coronary heart disease patients and young healthy individuals through high-throughput sequencing and bioinformatics analysis methods.

[0050] In some embodiments, high-throughput sequencing directly obtains the sequence and number of all captured fragments through next-generation sequencing technology. However, microarray sequencing requires the prior synthesis of gene sequences of interest and the determination of whether these genes are expressed through the fluorescence signal introduced by nucleic acid hybridization. The directness of high-throughput sequencing necessarily makes its overall accuracy and sequencing depth superior to microarray sequencing.

[0051] In some embodiments, the biological characteristics of all DEGs are summarized into three levels of molecular function (MF), biological process (BP) and cell components (CC) by using the method of gene ontology (GO) enrichment analysis. In addition, the molecular pathways in which each DEG is involved can be determined, for example, by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis, and the interactions between genes can be comprehensively understood.

[0052] In some embodiments, potential therapeutic drugs related to the core genes can be screened by using the Drug Gene Interaction Database (DGIdb; http: / / www.dgidb.org).

[0053] In specific embodiments, the compositions or kits disclosed herein are used to detect the expression level of the CXCL8 gene in a sample from an individual, and if the expression level of the CXCL8 gene is significantly higher than that in a control sample, it indicates that the individual has or is at risk of developing coronary heart disease.

[0054] The control sample can be a sample from a healthy individual, a sample from an individual who does not have coronary heart disease, a mixed sample from multiple individuals who do not have coronary heart disease, or a control or baseline expression level known to be from an individual who does not have coronary heart disease.

[0055] In certain embodiments, the methods and kits of the present application can also be used to monitor the effectiveness of chemotherapy treatment. In alternative embodiments, if a treatment regimen is effective in an individual, the level of the CXCL8 biomarker can decrease over time; if the treatment regimen is ineffective, the level of the biomarker will not change or can increase over time.

[0056] In certain embodiments, the methods and kits of the present application can also be used to screen potential drugs for treating coronary heart disease. For example, if the expression of the CXCL8 gene is inhibited or decreased after a certain compound is administered to an individual, the compound can be further studied or developed as a candidate for treating coronary heart disease.

[0057] In some embodiments, the inventors identified compounds that interfere with the expression of CXCL8 include broad-spectrum anti-inflammatory drugs and non-steroidal anti-inflammatory drugs, etc. The broad-spectrum anti-inflammatory drugs can be selected from leflunomide, colchicine, paclitaxel, cetuximab, bevacizumab, etc. The non-steroidal anti-inflammatory drugs can be selected from acetaminophen, aspirin, ibuprofen, etc.

[0058] In this specification and in the claims, the words "comprise", "contain", and "include" and the like are not meant to be limiting, and are intended to mean "including but not limited to".

[0059] It should be understood that features, aspects, components or steps described in a particular aspect, embodiment or example of the application can be applicable to any other aspect, embodiment or example, unless incompatible therewith, or with the context.

[0060] The foregoing disclosure generally describes the present application, which is further illustrated by the following examples. The description of these examples is merely meant to be illustrative of the present application, and is not meant to limit the scope of the application. Although specific terms and values are used herein, they are also understood to be exemplary and not limiting to the present application. Unless otherwise specified, the experimental methods and techniques in the specification are those that are well known and commonly used in the art.

[0061] Examples

[0062] The following examples are provided for illustration of some embodiments of the present application, and are not intended to be limiting.

[0063] The methods used in the following examples are routine unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0064] Materials and Methods

[0065] Inclusion of subjects and sample collection

[0066] This study was approved by the Ethics Committee of General Hospital of People's Liberation Army. Forty-five patients with premature coronary heart disease were enrolled in this study. Inclusion criteria: ① age ≤ 45 years; ② definite clinical manifestations of chest tightness or chest pain; ③ complete admission or laboratory tests and examinations; ④ confirmed diagnosis of coronary heart disease by coronary angiography. Exclusion criteria: ① patients who have received coronary artery bypass grafting or heart transplantation; ② patients without coronary angiography results; ③ patients with definite coagulation abnormalities or active bleeding; ④ patients with large artery inflammation, rheumatic diseases or cancer; ⑤ patients with suspected aortic dissection, pulmonary embolism, heart valve disease and other chest pain; ⑥ patients with current infection or autoimmune diseases. At the same time, 8 healthy individuals under the age of 45 were enrolled in the control group. All participants were fully informed of the study and gave their informed consent. Peripheral blood samples were collected after admission for high-throughput sequencing.

[0067] Peripheral blood RNA extraction, sequencing and data processing

[0068] According to the product instructions, the total RNA was isolated from whole blood samples using RNA was isolated from whole blood samples using the Trizol LS Reagent (Wuhan ServiceBio Technology, Wuhan, China). The quality of the RNA was assessed by 1% agarose gel electrophoresis to evaluate whether the RNA was degraded or contaminated. The purity and concentration of the RNA were calculated by a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA). The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to accurately assess the integrity of the RNA. Then, the cDNA library required for RNA sequencing was constructed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB, Ispawich, USA). The quality of the library was then tested by the Agilent 2100 Bioanalyzer, quantified by qPCR (Kapa Biosystems, Woburn, MA, USA), and sequenced on the Illumina HiSeq TMSequencing was performed on the HiSeq® 2000 Sequencing Platform (Illumina, San Diego, CA, USA). We filtered the raw sequencing reads by the following three criteria to ensure the quality of information analysis: (1) if the percentage of unknown bases in single-end reads was more than 10%, the paired reads were discarded; (2) the paired reads with sequencing adapters or adapters were discarded; (3) if the percentage of low-quality bases in single-end reads was more than 50%, the paired reads were discarded. In addition, Q20, Q30 and GC base content of all sequencing reads were also detected, and high-quality, clean reads were selected for subsequent analysis.

[0069] Genome reference files and gene model annotations were downloaded from the genome website. The reference genome index was generated by Bowtie2 software, and then the paired-end clean reads were aligned to the reference genome based on HISA T2 software. The Cufflinks2.0 program was applied to assemble the transcriptome of each sample, respectively. We used Cuffmerge to combine all the transcriptomes to produce the final transcriptome, and the abundance of all transcripts can be quantified and presented in the form of Transcripts per million (TPM) after standardization by Cuffdiff software.

[0070] Microarray data acquisition and screening

[0071] The coronary heart disease related microarray data used in this study were downloaded from the Gene Expression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / geo), which is an online available genomic database containing abundant gene expression profiles and related clinical information. We used the following search strategy: (1) the search key is ('coronary disease'[MeSH Terms] OR 'CHD'[All Fields]) AND ('Expression profiling by array'[Study type] AND 'Homo sapiens'[Top Organisms]); (2) all blood samples were from human; (3) each dataset should contain more than six individuals. After strict screening, we selected a GPL570 dataset GSE66360, which was composed of 21 coronary heart disease patient blood samples and 22 healthy human blood samples, together with high-throughput sequencing data as the data test set of this study. In addition, GPL570 dataset GSE19339, GPL9040 dataset GSE31568 and GPL21825 dataset GSE160717 were also included, which contained 26 coronary heart disease patient blood samples and 26 healthy human blood samples, as the data validation set to validate the core genes, miRNAs and cicrRNAs involved in the pathogenesis of coronary heart disease, respectively.

[0072] Identification of differentially expressed genes

[0073] Considering the different methods and depths of different types of data sequencing, the following threshold criteria were used to screen DEGs: (1) high-throughput sequencing data: log2 [fold change (FC)] > 4 or <-4, adjusted P value (Q value) <0.01; (2) microarray data: log2 (FC) > 0.5 or <-0.5 and adjusted P value (Q value) <0.05. The volcano plot and heat map of DEGs from each dataset were made by using the limma package and pheatmap package of R language software. In addition, the system / organ specificity of all DEGs was identified by the online tool BioGPS (http: / / biogps.org / ), which can better indicate the distribution of each DEG in the tissue. Finally, the online tool Venn (http: / / www.bioinformatics.com.cn / static / others / jvenn / ) was used to display the DEGs common to each dataset.

[0074] Functional and pathway enrichment analysis

[0075] In our study, the results of GO and KEGG pathway enrichment analysis were visualized by R language software. The screening criteria was adjusted to P value < 0.05, which was statistically significant.

[0076] Gene set enrichment analysis (GSEA) software can be used to evaluate the distribution trend of genes in the gene list sorted by the degree of correlation with the phenotype, so as to determine the contribution of genes to the phenotype. The screening criteria for gene sets with significant differences are as follows: P value < 5%, false discovery rate (FDR) < 25%.

[0077] Immune infiltration analysis

[0078] The standardized expression profile of DEGs obtained can be imported into CIBERSORT ( / / cibersort.stanford.edu / ) to evaluate the relative content of various immune cells in the sequencing data, so as to determine the immunological characteristics of the data set.

[0079] Protein-protein interaction network construction

[0080] Protein-protein interaction (PPI) network can predict and display the interaction of genes or proteins. We constructed the PPI network of DEGs in this study by searching the online tool Search Tool for the Retrieval of Interacting Genes / Proteins database (STRING; http: / / www.string-db.org / ). Cytoscape software was used to optimize the visual display form of the PPI network. At the same time, the software can find important interaction gene clusters through the Minimal Common Oncology Data Elements (MCODE) module, and identify core genes through the Cytohubba plug-in.

[0081] ceRNAs network construction

[0082] The obtained core genes were input into three online miRNA databases, miRDB, miRWalk and targetScan databases, to predict the miRNAs targeted by the core genes, and the miRNAs commonly existing in the three databases were selected for subsequent analysis. StarBase database (http: / / starbase.sysu.edu.cn / contact.php) was used to identify the circRNAs targeted by the above-mentioned screened miRNAs. Finally, the miRNAs and circRNAs screened from the online databases were collated and intersected with the coronary heart disease related miRNAs and circRNAs obtained from the GEO database, so as to obtain the miRNAs and circRNAs related to both the core genes and the pathogenesis of coronary heart disease, and the Cytoscape software was used for visual display of the results.

[0083] Screening of potential coronary heart disease treatment drugs

[0084] The potential therapeutic drugs related to the core genes were screened by using the Drug Gene Interaction Database (DGIdb; http: / / www.dgidb.org).

[0085] Statistical analysis

[0086] All statistical data processing and analysis were performed by R language software and SPSS Statistics 26.0. The t test was used for comparison of quantitative data between groups. Pearson correlation analysis was used to show the correlation between genes. P<0.05 was considered statistically significant.

[0087] Results

[0088] Identification of DEGs

[0089] According to the pre-set log2(FC) value and Q value, 1692 and 885 DEGs were identified from the high-throughput sequencing data set and the GSE66360 data set, respectively, and the volcano plot and heat map were used for visualization (A-D). Among them, the high-throughput sequencing data set contained 235 up-regulated genes and 1457 down-regulated genes, and the GSE66360 data set contained 666 up-regulated genes and 219 down-regulated genes. The Venn diagram showed that the two data sets had 35 overlapping DEGs, including 31 up-regulated genes and 4 down-regulated genes (E-F). Figure 1 Figure 1

[0090] Enrichment analysis results ​​

[0091] KEGG pathway analysis was first performed on the 35 overlapping DEGs common to both datasets, which were mainly enriched in vigorous immune responses, including IL-17 signaling pathway, Nuclear factor kappa beta (NF-κβ) signaling pathway, Tumor necrosis factor (TNF) signaling pathway, nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway (A and D). In the GO enrichment analysis of these 35 DEGs, the BP level was significantly enriched in the positive regulation of response to external stimulus, hematopoietic or neutrophil activation, and the positive regulation of inflammatory response, and the MF level contained cytokine activity and Toll-like receptor (TLR) binding (B and C). Figure 2 Figure 2

[0092] To comprehensively evaluate the genetic properties of early-onset coronary heart disease and its possible pathogenesis, we performed separate enrichment analysis on the high-throughput sequencing dataset. KEGG pathway enrichment analysis showed that all DEGs were mainly enriched in focal adhesion, tight junction, and extracellular matrix (ECM) receptor interaction (A). GO enrichment analysis of the BP level contained humoral immune response, phagocytosis, complement activation, immunoglobulin-mediated immune response, and B cell-mediated immune response; the CC level mainly contained immunoglobulin complex and the outer side of the plasma membrane; the MF level mainly contained channel activity and passive transmembrane transporter activity (B). GSEA analysis found that the significantly enriched gene sets contained cardiac muscle contraction, innate immune response activation signal transduction, IL-1-mediated signaling pathway, and response to IL-12 (C). Figure 3 Figure 3 Figure 4

[0093] Immune infiltration characteristics of high-throughput sequencing data sets

[0094] ​​​​​To clarify the immune infiltrate environment of early-onset coronary heart disease, through CIBERSORT algorithm analysis, it can be known that the content of resting memory CD4+ T lymphocytes, resting mast cells, eosinophils and neutrophils in the body of early-onset coronary heart disease patients is relatively rich, while the content of resting natural killer cells (Natural killer cell, NK), M2 (Macrophages2) macrophages, memory B lymphocytes, CD8 T+ lymphocytes and naive CD4 T+ lymphocytes is relatively less. The proportion of neutrophils is negatively correlated with the proportion of CD8 T+ lymphocytes and regulatory T (Regulatory T, Treg) lymphocytes. See Figure 5 .

[0095] PPI network analysis results

[0096] The 35 co-expressed DEGs were input into the online tool STRING, and after deleting the scattered nodes, a PPI interaction network (A) with 17 nodes and 28 edges was constructed. Figure 6 A) 10 relatively important core genes (marked with red and yellow) were identified by cytohubba algorithm, including CXCL8 (such as SEQ ID NO: 25), JUN, BCL2A1, CXCL2, NFKBIA, CD83, NFKBIZ, FOSB, NR4A2 and S100A9. In addition, the DEGs of all high-throughput sequencing data sets were put into STRING, and the top 100 core genes were screened using the cytohubba algorithm (B). Figure 6 B).

[0097] External validation of core genes

[0098] In the external GSE19339 data set, the expression of 10 core genes was verified. The results showed that the levels of CD83, CXCL2, CXCL8 (such as SEQ ID NO: 25), JUN and NR4A2 were still significantly increased (P<0.05) Figure 7 ).

[0099] ceRNA network construction

[0100] Based on 3 miRNA databases, 201 miRNAs related to core genes were found, and 397 miRNAs related to coronary heart disease were screened in the GSE31568 data set by differential gene expression analysis. Finally, 19 target miRNAs were determined by Venn diagram overlap intersection, and the interaction network diagram was visualized (A). The same method as above was used to predict the target circRNA (B). Figure 8 A). Figure 8B). We searched StarBase database, analyzed GSE160717 dataset, and took the intersection as targeted circRNA. Finally, according to the interaction relationship, the ceRNA network of CXCL8 was constructed, which might be the potential pathogenesis of premature coronary heart disease Figure 8 C).

[0101] Screening of potential treatment drugs for early-onset coronary heart disease

[0102] The potential therapeutic drugs that can reverse the harmful effects of DEGs in the pathogenesis of premature coronary heart disease were predicted using the DGIdb database. The results showed that CXCL8 can be affected by multiple drugs, including broad-spectrum anti-inflammatory drugs (leflunomide, colchicine, paclitaxel, cetuximab, bevacizumab, etc.), non-steroidal anti-inflammatory drugs (acetaminophen, aspirin, ibuprofen, etc.). See Figure 9 .

[0103] DISCUSSION

[0104] In this study, 45 patients with premature coronary heart disease were taken as the main research object, and peripheral blood samples were extracted for high-throughput sequencing. After data processing and differential gene expression analysis, the inventors found that there were clear differential gene expressions in patients with premature coronary heart disease compared with healthy individuals, suggesting that premature coronary heart disease is clearly related to gene expression. The inventors determined 35 overlapping DEGs from the high-throughput sequencing gene set and the public database coronary heart disease dataset. GO and KEGG pathway enrichment analysis was performed. It was found that compared with healthy people, the immune response of neutrophil activation, IL-17 signaling pathway, NF-κβ signaling pathway, TNF signaling pathway and NOD-like receptor signaling pathway in patients with coronary heart disease was more significant.

[0105] To better reveal the genetic characteristics of early-onset coronary heart disease, the inventors further performed the above two enrichment analyses on the DEGs in all high-throughput sequencing data sets related to early-onset coronary heart disease. The results also showed that these DEGs were mostly enriched in excessive immune activation pathways and rich signal transduction pathways. GSEA analysis found that most of the genes of early-onset coronary heart disease patients were mainly expressed in myocardial contraction, innate immune response activation signal transduction, IL-12 response, and IL-1 mediated signal pathways, suggesting that immune dysfunction is an important pathogenic mechanism of early-onset coronary heart disease. Through CIBERSORT algorithm to calculate the immune infiltration of high-throughput sequencing data set, the results found that a higher proportion of neutrophils and eosinophils were associated with early-onset coronary heart disease, which was consistent with the results of GO and KEGG enrichment analysis in this study, and M2 macrophages, CD8 T+lymphocytes and Treg lymphocytes were relatively less. This study determined the important inflammatory pathways and immune cell subsets in the pathogenesis of early-onset coronary heart disease patients at the transcriptome and cellular levels through comprehensive bioinformatics methods, providing a detailed theoretical basis for a more in-depth understanding of the pathogenesis of the disease.

[0106] The inventors found that CXCL8, JUN, BCL2A1, CXCL2, NFKBIA, CD83, NFKBIZ, FOSB, NR4A2, and S100A9 were the 10 core genes that played an important role in the network by constructing a PPI network and analyzing the network using Cytoscape. In order to reduce the bias and error of the experiment, this study selected another set of coronary heart disease-related data sets for external verification of the expression levels of core genes, and the results showed that only five genes, CD83, CXCL2, JUN, CXCL8 (such as SEQ ID NO: 25), and NR4A2, showed significant differences between coronary heart disease and healthy populations. miRNA can bind to target genes, down-regulate gene expression, and even silence it, while the upstream circRNA can interfere with the miRNA response element to regulate gene expression. In order to more clearly understand the molecular regulation mechanism of core genes, this study constructed a ceRNA network of CXCL8 through GEO, miRNA-related databases, and StarBase databases.

[0107] The present study shows that the up-regulated CXCL8 gene is closely related to the high incidence of premature coronary heart disease. The research results also show that CXCL8 can be interfered by a variety of compounds, including leflunomide, colchicine, paclitaxel, cetuximab, bevacizumab and other broad-spectrum anti-inflammatory drugs, and acetaminophen, aspirin, ibuprofen and other non-steroidal anti-inflammatory drugs, suggesting that these predictive drugs can be used in future individualized treatment of PCHD. The CXCL8 gene identified in the present study and its therapeutic drugs will play an important role in individualized diagnosis and precision treatment of premature coronary heart disease, and optimize the current coronary heart disease management program.

[0108] In summary, the present study determines that the CXCL8 gene is closely related to the occurrence and development of premature coronary heart disease, and can be used as a biomarker for early warning, precision treatment and / or prognosis evaluation of premature coronary heart disease.

[0109] It can be understood that although the present application is described in a certain form, the present application is not limited to what is shown and described in the present specification. It is obvious to those skilled in the art that various changes can be made without departing from the scope of the present application. These changes are within the scope of the present application.

Claims

1. Use of an agent or a composition comprising said agent for the manufacture of a kit or a medicament for screening for premature coronary heart disease in an individual aged < 45 years, wherein said agent comprises a primer or a probe for detecting a CXCL8 specific nucleic acid sequence.

2. Use according to claim 1, wherein said CXCL8 specific nucleic acid sequence has a length of at least 30 nucleotides.

3. Use according to claim 2, wherein said CXCL8 specific nucleic acid sequence has a length of 45-300 nucleotides.

4. Use according to claim 3, wherein said CXCL8 specific nucleic acid sequence has a length of 57-123 nucleotides.

5. Use according to any one of claims 1-4, wherein said CXCL8 specific nucleic acid sequence comprises or consists of a sequence as set forth in any one of SEQ ID NO: 1-6 and 25.

6. Use according to any one of claims 1-4, wherein said primer comprises a sequence as set forth in any one of SEQ ID NO: 13-24.

7. Use according to any one of claims 1-4, wherein the amount of said CXCL8 specific nucleic acid sequence is measured by at least one method selected from the group consisting of in situ hybridization, polymerase chain reaction (PCR), RNase protection assay (RPA), northern blotting, microarray and high-throughput sequencing.

8. Use according to claim 7, wherein the amount of said CXCL8 specific nucleic acid sequence is measured by at least one method selected from the group consisting of reverse transcription (RT)-PCR and real-time PCR.

9. Use of an agent for detecting a polypeptide encoded by a CXCL8 specific nucleic acid sequence for the manufacture of a kit for screening for premature coronary heart disease in an individual aged < 45 years.

10. Use according to claim 9, wherein said polypeptide comprises or consists of a sequence as set forth in any one of SEQ ID NO: 7-12 and 26.

11. Use according to claim 9, wherein the amount of said polypeptide is measured by at least one method selected from the group consisting of western blotting, radioimmunoassay (RIA), radial immunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, Ouchterlony double immunodiffusion, complement fixation assay and protein chip.

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