Biomarker composition for diagnosing fetal growth restriction, comprising CCL20 and CSF3 proteins or genes encoding proteins as active ingredients

Abstract

The present invention relates to a biomarker composition for diagnosing fetal growth restriction, comprising CCL20 and CSF3 proteins or genes encoding said proteins as active ingredients, and more specifically, may provide a biomarker composition for diagnosing fetal growth restriction by identifying six core genes (CCL20, CSF3, EPO, VEGFA, IL17C, and GZMB) in which a preeclampsia mother group with fetal growth restriction is enhanced in expression in a cytokine-cytokine receptor interaction differentiated from a preeclampsia mother group without fetal growth restriction.

Description

A biomarker composition for diagnosing fetal growth restriction comprising CCL20 and CSF3 proteins or a gene encoding the proteins as an active ingredient

[0001] The present invention provides a biomarker composition for diagnosing fetal growth restriction, comprising CCL20 and CSF3 proteins or a gene encoding the proteins as an active ingredient.

[0002] Preeclampsia (PE) is a hypertensive disorder that typically develops after 20 weeks of pregnancy, with or without proteinuria. It is a leading cause of maternal and fetal death, affecting maternal organs such as the liver, heart, lungs, and kidneys, and is a major cause of fetal growth restriction (FGR) and premature birth. The incidence of PE, affecting 3–8% of all pregnancies, is increasing worldwide. Abnormal placental implantation is a known cause of PE. During placental implantation in normal pregnancy, the trophoblast of the anchoring villi penetrates deep into the inner third of the myometrium and the spiral arteries of the maternal body. These structural changes are associated with functional alterations that lower spiral arterial resistance. However, abnormal placental implantation can lead to increased maternal arterial resistance, placental ischemia, and fetal complications. This is manifested by clinical features such as maternal symptoms, hematological abnormalities, and fetal monitoring abnormalities, and is directly related to delivery. In particular, early-onset PE, which requires delivery before 34 weeks, is known to have placental dysfunction. This is associated with a worse prognosis for both mothers and fetuses compared to late-onset PE. Recent studies have shown that molecular changes play a pathogenic role in PE. However, the molecular mechanisms underlying the pathophysiology of PE remain incompletely understood. Therefore, elucidating the molecular characteristics of early-onset PE could contribute to a better understanding of the diagnosis, prevention, and treatment of PE.

[0003] Small-for-gestational age (SGA) is defined as an infant whose birth weight is below the 10th percentile for gestational age. LBW infants are broadly divided into two groups: those born in true gestational age and those with fetal growth restriction (FGR). FGR is defined as a fetus that has not reached its biologically determined growth potential, resulting from multifactorial causes such as placental development and function defects, perinatal mortality, maternal hypertension, poor maternal cardiovascular adaptation, and multiple pregnancy. Most infants with early-onset FGR are low birth weight, whereas infants with late-onset FGR have normal birth weights. Previous cohort studies have shown that low birth weight is associated with major morbidities, including bronchopulmonary dysplasia, necrotizing enterocolitis, and neurodevelopmental disorders, as well as an increased risk of neonatal death. Ultimately, more targeted studies are needed on early-onset PE, which leads to fetal growth restriction as well as preterm birth.

[0004] Proteomics has contributed to the identification of clinical biomarkers as well as therapeutic targets. In the case of PE, proteomics-based studies have identified proteins differentially expressed in serum or plasma, maternal urine, and the placenta. Previous studies have also identified protein biomarkers in amniotic fluid obtained through mid-trimester diagnostic amniocentesis. Proteomics-based studies using maternal blood or placenta collected at delivery have been published in relation to fetal growth restriction. Amniotic fluid provides information on fetal genotype and reflects maternal-fetal physiology adaptations during pregnancy. However, no studies have used amniotic fluid collected at delivery in pregnant women with PE. To investigate the molecular characteristics of preterm PE according to the presence or absence of low birth weight, we analyzed the molecular profiling of cytokines using amniotic fluid (AF) collected at cesarean section in preterm PE.

[0005] The purpose of the present invention is to provide a biomarker composition for diagnosing fetal growth restriction, which comprises CCL20 (CC motif chemokine 20) and CSF3 (granulocyte colony-stimulating factor 3) proteins or a gene encoding the proteins as active ingredients.

[0006] Another object of the present invention is to provide a composition for diagnosing fetal growth restriction, which contains as an active ingredient an agent capable of measuring the expression or activity level of CCL20 and CSF3 proteins, or the expression level of a gene encoding the proteins.

[0007] Another object of the present invention is to provide a kit for diagnosing fetal growth restriction comprising the composition.

[0008] Another object of the present invention is to provide a method for providing information necessary for diagnosing fetal growth restriction, comprising the steps of (1) measuring the mRNA expression levels of CCL20 and CSF3 genes or the expression levels of CCL20 and CSF3 proteins from a sample isolated from a patient; (2) comparing the mRNA expression levels of the CCL20 and CSF3 genes or the expression levels of the CCL20 and CSF3 proteins with a control sample; and (3) determining that the fetus of the patient has fetal growth restriction if the mRNA expression levels of the CCL20 and CSF3 genes or the expression levels of the CCL20 and CSF3 proteins are higher than those of the control sample.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fetal growth restriction, comprising CCL20 and CSF3 expression or activity inhibitors as active ingredients.

[0010] To achieve the above purpose, the present invention provides a biomarker composition for diagnosing fetal growth restriction, which comprises CCL20 (CC motif chemokine 20) and CSF3 (granulocyte colony-stimulating factor 3) proteins or a gene encoding the proteins as active ingredients.

[0011] In addition, the present invention provides a composition for diagnosing fetal growth restriction, which contains as an active ingredient an agent capable of measuring the expression or activity level of CCL20 and CSF3 proteins, or the expression level of a gene encoding the proteins.

[0012] In addition, the present invention provides a kit for diagnosing fetal growth restriction comprising the composition.

[0013] In addition, the present invention provides a method for providing information necessary for diagnosing fetal growth restriction, comprising the steps of: (1) measuring the mRNA expression levels of CCL20 and CSF3 genes or the expression levels of CCL20 and CSF3 proteins from a sample isolated from a patient; (2) comparing the mRNA expression levels of the CCL20 and CSF3 genes or the expression levels of the CCL20 and CSF3 proteins with a control sample; and (3) determining that the fetus of the patient has fetal growth restriction when the mRNA expression levels of the CCL20 and CSF3 genes or the expression levels of the CCL20 and CSF3 proteins are higher than those of the control sample.

[0014] In addition, the present invention provides a pharmaceutical composition for preventing or treating fetal growth restriction, comprising CCL20 and CSF3 expression or activity inhibitors as active ingredients.

[0015] The present invention relates to a biomarker composition for diagnosing fetal growth restriction, comprising CCL20 and CSF3 proteins or a gene encoding the proteins as an active ingredient, and more specifically, to a biomarker composition for diagnosing fetal growth restriction by identifying six key genes (CCL20, CSF3, EPO, VEGFA, IL17C and GZMB) whose expression is enhanced in a cytokine-cytokine receptor interaction that differentiates a preeclamptic mother group with fetal growth restriction from a preeclamptic mother group without fetal growth restriction.

[0016] Figure 1 is a diagram illustrating the classification results after patient and sample collection (SGA: low birth weight, AGA: appropriate birth weight).

[0017] Figure 2 shows a heatmap showing differentially expressed genes between the group with fetal growth restriction and the control group in the amniotic fluid of mothers with preeclampsia.

[0018] Figure 3 shows a protein-protein interaction network consisting of 16 nodes.

[0019] Figure 4 shows the results of confirming six hub genes.

[0020] Figure 5 shows the results of receiver operating characteristic curve analysis performed on six hub genes to distinguish low birth weight from premature PE.

[0021] Hereinafter, the present invention will be described in more detail.

[0022]

[0023] The present invention provides a biomarker composition for diagnosing fetal growth restriction, comprising CCL20 (CC motif chemokine 20) and CSF3 (granulocyte colony-stimulating factor 3) proteins or a gene encoding the proteins as active ingredients.

[0024] The above fetal growth restriction may be caused by preeclampsia.

[0025] The above biomarker may further include, as an active ingredient, one or more proteins selected from the group consisting of EPO (erythropoietin), VEGFA (vascular endothelial growth factor A), IL-17C (Interleukin-17C), and GZMB (Granzyme B), or a gene encoding the protein, but is not limited thereto.

[0026] The "biomarker" of the present invention is a substance that can diagnose tissues or cells of a subject with fetal growth restriction by distinguishing them from tissues or cells of a normal control group, and includes organic biomolecules such as proteins, nucleic acids, lipids, glycolipids, and glycoproteins that show an increase or decrease in tissues or cells of a subject with a disease compared to the normal control group.

[0027] “Diagnosis” of the present invention includes determining the susceptibility of an individual to a particular disease or condition, determining whether an individual currently has a particular disease or condition, determining the prognosis of an individual with a particular disease or condition, or therametrics (e.g., monitoring the condition of an individual to provide information about the efficacy of a treatment).

[0028]

[0029] In addition, the present invention provides a composition for diagnosing fetal growth restriction, which contains as an active ingredient an agent capable of measuring the expression or activity level of CCL20 and CSF3 proteins, or the expression level of a gene encoding the proteins.

[0030] The above formulation is a primer or probe that specifically binds to the CCL20 and CSF3 genes, and may be, but is not limited to, an antibody, peptide, aptamer or compound that specifically binds to the CCL20 and CSF3 proteins.

[0031] The term "primer" used in the present invention refers to a short genetic sequence that serves as the starting point for DNA synthesis, and is an oligonucleotide synthesized for purposes such as diagnosis and DNA sequencing. The primers are typically synthesized and used in lengths of 15 to 30 base pairs, but this may vary depending on the intended use, and may be modified by methylation, capping, etc. using known methods.

[0032] The term "probe" used in the present invention refers to a nucleic acid capable of specifically binding to mRNA, ranging from a few bases to several hundred bases in length, produced through enzymatic, chemical, or synthetic separation and purification processes. The presence or absence of mRNA can be confirmed by labeling with a radioisotope or enzyme, and the probe can be designed and modified using known methods.

[0033] The term "antibody" used in the present invention is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to CCL20 or CSF3 of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes special antibodies such as humanized antibodies.

[0034] The term "peptide" used in the present invention has the advantage of high binding affinity to target substances and is resistant to denaturation even during heat and chemical treatments. Furthermore, due to its small molecular size, it can be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymer protein chains, making it suitable for use as a diagnostic kit and drug delivery material.

[0035] The term "aptamer" used in the present invention refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being able to bind to a target molecule with high affinity and specificity. As described above, an aptamer can specifically bind to an antigenic substance in the same way as an antibody, but is composed of a polynucleotide that is more stable than a protein, has a simpler structure, and is easy to synthesize, and therefore can be used as a substitute for an antibody.

[0036]

[0037] In addition, the present invention provides a kit for diagnosing fetal growth restriction comprising the composition.

[0038] The “kit” of the present invention may include an antibody that specifically binds to a biomarker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with a substrate, a chromogenic substrate solution that reacts with the label, a washing solution, an enzyme reaction stop solution, and the like, and may be manufactured into a plurality of separate packagings or compartments containing reagent components to be used.

[0039]

[0040] In addition, the present invention provides a method for providing information necessary for diagnosing fetal growth restriction, comprising the steps of: (1) measuring the mRNA expression levels of CCL20 and CSF3 genes or the expression levels of CCL20 and CSF3 proteins from a sample isolated from a patient; (2) comparing the mRNA expression levels of the CCL20 and CSF3 genes or the expression levels of the CCL20 and CSF3 proteins with a control sample; and (3) determining that the fetus of the patient has fetal growth restriction when the mRNA expression levels of the CCL20 and CSF3 genes or the expression levels of the CCL20 and CSF3 proteins are higher than those of the control sample.

[0041]

[0042] In addition, the present invention provides a pharmaceutical composition for preventing or treating fetal growth restriction, comprising CCL20 and CSF3 expression or activity inhibitors as active ingredients.

[0043] The above expression inhibitor may be any one selected from the group consisting of antisense nucleotide small interfering RNA (siRNA) and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the CCL20 and CSF3 genes, but is not limited thereto.

[0044] The above activity inhibitor may be any one selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies and natural products that specifically bind to CCL20 and CSF3 proteins, but is not limited thereto.

[0045] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0046] Specifically, carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like, into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0047] According to one embodiment of the present invention, the pharmaceutical composition can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes.

[0048] The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and degree of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, and more preferably 0.1 mg / kg to 100 mg / kg. Administration may be once a day or divided into several times, and the scope of the present invention is not limited thereby.

[0049] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0050]

[0051] <Experimental Example 1> Patient and Sample Collection

[0052] From January 2017 to May 2022, 46 women with preeclampsia who delivered at less than 34 weeks of gestation due to uncontrolled hypertension were enrolled in the study at Keimyung University Dongsan Hospital in Daegu.

[0053] Amniotic fluid was collected at the time of delivery, and amniotic fluid extracted during cesarean sections in high-risk pregnant women has been stored at the Keimyung University Human Resources Bank since 2017. Before participating in the study, all patients were educated on the purpose of the study and obtained individual informed consent. The study protocol was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (IRB Approval No. 2022-04-041-002).

[0054] Amniotic fluid and maternal hospital registration numbers were provided by the Keimyung University Biobank and analyzed. The clinical characteristics of pregnant women and infants were retrospectively reviewed. As shown in Figure 1, 110 of the 343 cases studied developed PE. Thirteen cases of polychorionic pregnancies, which are known to be the main cause of fetal growth restriction through mechanisms different from fetal growth, were excluded. Twelve cases with exposure to premature premature rupture of membranes or histologic chorioamnionitis were excluded due to the bacterial inflammatory response of the amniotic fluid. Other risk factors, including placental abruption (n=10) and congenital malformations (n=2), were also excluded. Finally, 73 cases with early-onset PE were investigated in this study (Figure 1).

[0055] Of the 46 enrolled cases, 21 were low birth weight (i.e., birth weight below the 10th percentile for gestational age). Twenty-five cases of appropriate-for-gestational-age (AGA) infants with birth weights between the 25th and 75th percentiles for gestational age were randomly selected as controls.

[0056] We retrospectively reviewed clinical characteristics including maternal age, maternal body mass index (BMI), gestational age at delivery, birth weight, skin / color, pulse, reflexes, muscle tone / motor activity, activity, and respiration (APGAR) scores, as well as nulliparity, history of assisted reproductive technology and pre-pregnancy hypertension, gestational diabetes mellitus (GDM), hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome.

[0057]

[0058] <Experimental Example 2> Proteome Analysis

[0059] Amniotic fluid was collected from 46 pregnant women at the time of delivery for proteomic analysis and stored at -80°C. Samples were sent to Olink (Watertown, MA, USA) and analyzed using the Olink Explore 384 Inflammation Panel. This panel was selected because it contains a variety of inflammatory cytokines. Samples were processed using the Olink Explore platform, which consists of next-generation sequencing (NGS) and proximity extension assays (PEA). Potential protein biomarkers were evaluated using PEA as described on the manufacturer's website (https: / / www.olink.com). The manufacturer's website (https: / / www.olink.com) provides an extensive list of potential protein biomarkers, consisting of 368 items. The website also provides comprehensive assay validation data, including details on the limit of detection (LOD), lower and upper limits of quantification, and coefficients of variation of intra- and inter-run precision. The final protein concentration output from the analysis was expressed as a Normalized Protein Expression (NPX) value. NPX is a logarithmic scale based on 2, with higher NPX values ​​indicating higher protein concentration. Protein expression data were obtained from 46 patients. Based on an adjusted p-value of less than 0.05, 35 potential protein biomarkers were identified. Among the three proteins, those with an absolute value greater than 1 were considered differentially expressed proteins (DEPs). As a result, 16 DEPs were selected and utilized for further analysis.

[0060]

[0061] <Experimental Example 3> Gene Ontology (GO) Analysis of DEP

[0062] The biological significance of DEPs was assessed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) 2021, a web-based tool (DAVID, http: / / david.ncifcrf.gov / , accessed April 10, 2023). Gene Ontology (GO) terms were classified into three groups: biological process (BP), cellular component (CC), and molecular function (MF). A p-value <0.05 was considered significant.

[0063]

[0064]

[0065] <Experimental Example 4> Protein-Protein Interaction (PPI) Network Analysis

[0066] To analyze and visually represent the functional PPI network of the identified DEPs, Cytoscape software (version 3.9.1) and the Search Tool for the Retrieval of Interacting Genes, version 11.5 (STRING, http: / / string-db.org, accessed April 10, 2023) were used. The criteria for selecting interactions were that the maximum number of interactors was 0 and the confidence score was ≥0.9.

[0067] We used the Cytoscape plugin Molecular Complex Detection (MCODE) to identify clustered modules within the PPI network using the following parameters: degree cutoff = 2, node score cutoff = 0.2, k117 score = 2, maximum depth = 100.

[0068]

[0069] <Experimental Example 5> Enrichment Analysis of Hub Genes

[0070] Enrichment analysis of hub genes was performed using the MetaScape database (https: / / metascape.org, accessed April 11, 2023). The MetaScape database integrates various authoritative data resources, including GO, Kyoto Encyclopedia of Genes and Genomes (KEGG), UniProt, and DrugBank. This integration significantly improved the enrichment and annotation of biological processes, providing comprehensive and reliable insights into the functional significance of genes and proteins.

[0071]

[0072] <Experimental Example 6> Statistical Analysis

[0073] Data analysis was performed using SPSS version 26.0 for Windows, developed by SPSS, Inc., Chicago, IL, USA. Differences in clinical information between groups were analyzed using the chi-square test for categorical variables. Analysis of variance (Student's t-test) was used for continuous variables. Statistical significance was defined as a p value of less than 0.05. Receiver operating characteristic (ROC) curve analysis was performed using SPSS. Results were considered statistically significant at p < 0.05, and an area under the curve (AUC) greater than 0.7 was considered to indicate a reliable prognostic model.

[0074]

[0075] <Example 1> Patient and demographic characteristics

[0076] Among 46 pregnant women, 21 were in the SGA group and 25 were in the AGA group. According to Table 1, there were no differences in maternal age, maternal body mass index (BMI), nulliparity, assisted reproductive technology, pre-eclampsia, GDM, or HELLP syndrome between the SGA and AGA groups. The mean birth weight was significantly lower in the SGA group than in the AGA group (945.2 ± 302.3 vs. 1590.0 ± 393.2, p < 0.001), but there was no difference in the mean gestational age at birth (p > 0.05). The 1-minute APGAR score, an indicator of neonatal status at birth, was significantly lower in the SGA group than in the AGA group (5.8 ± 1.3 vs. 6.7 ± 1.3, respectively, p 0.019), but there was no significant difference in the 5-minute APGAR score between the two groups.

[0077] SGA groupAGA groupp value(n = 21)(n = 25)Maternal factorsAge (year)32.8 ± 3.433.9 ± 3.80.284BMI28.2 ± 7.330.4 ± 5.00.228Nulliparity (n) (%)7 (33.3)9 (36.0)0.850ART, n (%)3 (14.3)2 (8.0)0.495Pregnancy hypertension, n (%)1 (4.8)1 (4.0)0.900GDM, n (%)1 (4.8)2 (8.0)0.658HELLP Syndrome, n (%)5 (23.8)5 (20)0.516Fetal factorsGestational age at delivery (week)30.3 ± 2.631.4 ± 1.90.112Birth weight (g)945.2 ± 302.31590.0 ± 393.2< 0.0011 min APGAR score5.8 ± 1.36.7 ± 1.30.0195 min APGAR score7.9 ± 0.88.2 ± 0.70.135

[0078]

[0079] <Example 2> DEP Identification

[0080] Sixteen DEPs were identified. All 16 genes were upregulated in the SGA group compared to the AGA group. Gene symbols, descriptions, log2 fold changes, and p values ​​are listed in Table 2, and a heatmap showing the identified DEPs is shown in Figure 2.

[0081] Gene descriptionLog2FCAdjustedpvalueEPOErythropoietin1.980.037WFIKKN2Wap, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 21.670.006CLSTN2Calsyntenin-21.460.017CSF3Granulocyte colony-stimulating factor1.440.017COL9A1Collagen alpha-1(IX) chain1.330.031SCG3Secretogranin-31.300.028CCL23C-C motif chemokine 231.260.038SKAP2Src kinase-associated phosphoprotein 21.250.037CCL20C-C motif chemokine 201.150.006GZMBGranzyme B1.140.047TIMP3Metalloproteinase inhibitor 31.140.029FIS1Mitochondrial fission 1 protein1.060.028IL17CInterleukin-17C1.030.017PON3Serum paraoxonase / lactonase 31.020.006VEGFAVascular endothelial growth factor A1.020.006CXCL8Interleukin-81.020.017

[0082]

[0083] <Example 3> DEP Enhancement Analysis

[0084] To analyze the functional profile of DEPs, GO term analysis was performed using DAVID software. In biological processes, DEPs were mainly involved in positive regulation of the ERK1 and ERK2 cascades, chemokine-mediated signaling pathways, neutrophil chemotaxis, cellular response to interleukin-1, inflammatory response, chemotaxis, cellular response to tumor necrosis factor, immune response, positive chemotaxis induction, cell-cell signaling, signal transduction, calcium-mediated signaling using intracellular calcium sources, lymphocyte chemotaxis, monocyte chemotaxis, negative regulation of neuronal apoptosis, and negative regulation of cell proliferation.

[0085] In cellular components, these genes were mainly distributed in the extracellular region, space, and matrix.

[0086] In molecular function analysis, DEP was mainly involved in cytokine activity, chemokine activity, heparin binding, metalloendopeptidase inhibitor activity, and CCR chemokine receptor binding (Tables 3 and 4).

[0087] 카테고리용어유전자 이름유전자 개수p값GO_BPGO:0070374 Positive regulation of ERK1 and ERK2 cascadeCCL20,CCL23, EPO,VEGFA4< 0.05GO:0070098 Chemokine-mediated signaling pathwayCCL20,CCL23, CXCL83< 0.05GO:0030593 Neutrophil chemotaxisCCL20,CCL23, CXCL83< 0.05GO:0071347 Cellular response to interleukin-1CCL20,CCL23, CXCL83< 0.05GO:0006954 Inflammatory responseCCL20,CCL23, CXCL8,IL17C4< 0.05GO:006935 ChemotaxisCCL20,CCL23, CXCL83< 0.05GO:0071356 Cellular response to tumor necrosis factorCCL20,CCL23, CXCL83< 0.05GO:0006955 Immune responseCCL20,CCL23, CXCL8,CSF34< 0.05GO:0050930 Induction of positive chemotaxisCXCL8,VEGFA2< 0.05GO:0007267 Cell-cell signalingCCL20,CCL23, IL17C3< 0.05GO:0007165 Signal transductionCCL20,CCL23, CXCL8, EPO, SKAP25< 0.05GO:0035584 Calcium-mediated signaling using intracellular calcium sourceCCL20,FIS12< 0.05GO:0048247 Lymphocyte chemotaxisCCL20,CCL232< 0.05GO:0002548 Monocyte chemotaxisCCL20,CCL232< 0.05GO:1901215 Negative regulation of neuron deathCSF3, EPO2< 0.05

[0088] Category TermsGene NameGene Numberp ValueGO_BPGO:0008285 Negative regulation of cell proliferationCCL23,CXCL8, SKAP23< 0.05GO_CCGO:0005576 Extracellular regionCCL20,CCL23, CXCL8,TIMP3, COL9A1,CSF3, EPO,GZMB, IL17C,PON3, SCG3,VEGFA12< 0.05GO:0005615 Extracellular spaceCCL20,CCL23, CXCL8,TIMP3, WFIKKN2,COL9A1, CSF3, EPO, IL17C,PON3, VEGFA11< 0.05GO:0031012 Extracellular matrixTIMP3,COL9A1, VEGFA3< 0.05GO_MFGO:0005125 Cytokine activityCSF3, EPO, IL17C,VEGFA4< 0.05GO:0008009 Chemokine activityCCL20,CCL23, CXCL83< 0.05GO:0008201 Heparin bindingCCL23,CXCL8, VEGFA3< 0.05GO:0008191 Metalloendopeptidase inhibitor activityTIMP3,WFIKKN22< 0.05GO:0048020 CCR chemokine receptor bindingCCL20,CCL232< 0.05

[0089]

[0090] <Example 4> PPI network and hub gene analysis

[0091] To identify highly interacting genes, hub genes, that play a significant role in early-onset PE with SGA, we obtained a PPI network consisting of 16 nodes and 19 edges, with an average node degree of 2.38 (p = 2.36E-10) (Fig. 3). Further analysis was performed using the MCODE plugin, and six hub genes were identified: CC motif chemokine 20 (CCL20), granulocyte colony-stimulating factor 3 (CSF3), erythropoietin (EPO), vascular endothelial growth factor A (VEGFA), interleukin-17C (IL-17C), and granzyme B (GZMB) (Fig. 4).

[0092]

[0093] <Example 5> Enrichment analysis of hub genes

[0094] CCL20, CSF3, EPO, VEGFA, IL17C, and GZMB were further enriched using the Metascape database, which combines GO function and KEGG pathway analysis. These genes were primarily enriched in processes related to positive regulation of peptidyl-tyrosine phosphorylation, cytokine-mediated signaling pathways, and mitogen-activated protein kinase cascades. KEGG pathway enrichment analysis revealed that hub genes were significantly enriched in the cytokine-cytokine receptor pathway (Figure 5).

[0095]

[0096] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0097] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A biomarker composition for diagnosing fetal growth restriction, comprising CCL20 (CC motif chemokine 20) and CSF3 (granulocyte colony-stimulating factor 3) proteins or a gene encoding the proteins as active ingredients.

2. In claim 1, A biomarker composition for diagnosing fetal growth restriction, characterized in that the biomarker further comprises as an active ingredient at least one protein selected from the group consisting of EPO (erythropoietin), VEGFA (vascular endothelial growth factor A), IL-17C (Interleukin-17C), and GZMB (Granzyme B) or a gene encoding the protein.

3. A composition for diagnosing fetal growth restriction, comprising as an active ingredient an agent capable of measuring the expression or activity level of CCL20 and CSF3 proteins, or the expression level of a gene encoding the proteins.

4. In claim 3, A composition for diagnosing fetal growth restriction, characterized in that the above formulation is a primer or probe that specifically binds to the CCL20 and CSF3 genes, and an antibody, peptide, aptamer or compound that specifically binds to the CCL20 and CSF3 proteins.

5. A kit for diagnosing fetal growth restriction comprising the composition of claim 3 or claim 4. 6.(1) A step of measuring the mRNA expression level of CCL20 and CSF3 genes or the expression level of CCL20 and CSF3 proteins from a sample isolated from a patient; (2) a step of comparing the mRNA expression levels of the CCL20 and CSF3 genes or the expression levels of CCL20 and CSF3 proteins with a control sample; and (3) A method for providing information necessary for diagnosing fetal growth restriction, including a step of determining that a fetus of a patient with preeclampsia has fetal growth restriction if the mRNA expression level of the CCL20 and CSF3 genes or the expression level of the CCL20 and CSF3 proteins is higher than that of a control group sample.

7. A pharmaceutical composition for preventing or treating fetal growth restriction, comprising an inhibitor of CCL20 and CSF3 expression or activity as an active ingredient.

8. In claim 7, A pharmaceutical composition for preventing or treating fetal growth restriction, characterized in that the expression inhibitor is any one selected from the group consisting of antisense nucleotide small interfering RNA (siRNA) and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the CCL20 and CSF3 genes.

9. In claim 7, A pharmaceutical composition for preventing or treating fetal growth restriction, characterized in that the above activity inhibitor is any one selected from the group consisting of low-molecular-weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to CCL20 and CSF3 proteins.

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