Application of plasma exosome protein CHD1 as a biomarker for clinical diagnosis of high-risk neuroblastoma

Abstract

The application belongs to the field of biological medicine, and particularly relates to a biomarker for high-risk neuroblastoma clinical diagnosis and application thereof. A product for high-risk neuroblastoma clinical diagnosis, wherein the product comprises a reagent for detecting the expression level of a biomarker, and the biomarker is a plasma exosome protein CHD1. By detecting the expression level of CHD1, the existing clinical indicators can be supplemented, precise stratification and precise treatment of neuroblastoma are realized, and the biomarker has important guiding significance for the prognosis judgment of tumor children.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of plasma exosome protein CHD1 as a biomarker for the clinical diagnosis of neuroblastoma. Background Technology

[0002] Neuroblastoma (NB) is an embryonic tumor originating from the sympathetic nervous system. NB is the most common extracranial solid tumor in infants and young children, and its clinical manifestations are complex and diverse, ranging from spontaneous tumor regression to metastasis to the bone marrow, bones, and distant organs.

[0003] NB (nephropathy of prematurity) is characterized by its insidious onset and high malignancy, with a 5-year survival rate of less than 50% for high-risk children. Currently, the main clinical challenge lies in the relatively slow early screening of suspected cases. Furthermore, due to the unique characteristics of pediatric patients, invasive procedures such as biopsy are difficult to perform. Therefore, exploring more sensitive and effective molecular markers for early screening of high-risk NB, achievable through non-invasive or minimally invasive methods, will significantly improve the diagnosis and treatment of NB in ​​children.

[0004] Exosomes are extracellular matrix microvesicles ranging in size from 30 to 150 nm (average 100 nm). Exosomes can be secreted by various cells and contain many components of their originating cell (such as DNA, RNA, proteins, lipids, and metabolites). Secreted exosomes can enter bodily fluids such as blood, saliva, urine, and breast milk, reaching other cells or tissues through fluid circulation and participating in the regulation of intercellular communication. This ability to regulate cellular pathways makes exosomes potentially useful in the treatment of many diseases. In some diseases, including neurodegenerative diseases and cancer, exosomes have been engineered as effective carriers for delivering chemotherapy drugs, immunosuppressants, etc., possessing the ability to target therapeutic drugs to desired objectives. Furthermore, because exosomes carry biological materials from their originating cells and are easily obtained through liquid biopsy, they have great potential to aid in disease diagnosis and prognosis prediction in diseases such as cancer. Summary of the Invention

[0005] This invention provides the application of plasma exosomal protein CHD1 as a biomarker for high-risk neuroblastoma.

[0006] CHD1, or Chromodomain Helicase DNA Binding Protein 1, is highly conserved in eukaryotic cells and plays a role in assembling nucleosomes, remodeling chromatin structure, regulating histone metabolism, and regulating gene transcription. CHD1 is mutated or deleted only in a few cancers (such as prostate cancer, uterine cancer, and melanoma), and its role in the progression of different tumors varies, remaining highly controversial. The relationship between CHD1 and neonatal helicobacter pylori (NB) has not yet been reported.

[0007] This invention utilizes proteomics technology to identify differentially expressed proteins in the plasma and exosomes of children with neuroblastoma (NB). CHD1, a secreted protein present in plasma exosomes, can be used to differentiate and diagnose high-risk neuroblastoma by detecting the expression level of CHD1 in plasma exosomes. CHD1 is a key regulatory molecule in the development and progression of NB tumors and can serve as a novel molecular marker for clinical diagnosis, possessing significant theoretical importance and potential application value.

[0008] This invention provides the application of plasma exosomal protein CHD1 as a biomarker in the preparation of diagnostic agents for high-risk neuroblastoma, and the expression level of plasma exosomal protein CHD1 is significantly increased in high-risk neuroblastoma.

[0009] The diagnostic reagent is used to: detect the expression of CHD1 in the plasma exosomes of a subject; compare the detected value with a reference value, and if the CHD1 level is significantly higher than the reference value, it suggests that the subject has high-risk neuroblastoma. The reference value level is the expression level of CHD1 in the plasma exosomes of normal children or children with low-risk neuroblastoma. The subject is known or suspected of having tumor cells.

[0010] In this invention, the term CHD1 refers to Chromodomain Helicase DNA Binding Protein 1, including the CHD1 gene and its encoded protein, as well as its homologs, mutations, and isotypes. This term encompasses full-length, unprocessed CHD1, as well as any form of CHD1 derived from and processed within cells. Preferably, the CHD1 is a human protein with UniProt ID O14646.

[0011] In this invention, the term "exosome" refers to a class of extracellular matrix microvesicles with a size ranging from 30-150 nm (average 100 nm). Exosomes can be secreted by various cells and contain many components of their originating cells (such as DNA, RNA, proteins, lipids, metabolites, etc.). Secreted exosomes can enter bodily fluids such as blood, saliva, urine, and breast milk, reaching other cells or tissues through fluid circulation and participating in the regulation of intercellular communication. Many molecules have been shown to participate in important disease processes such as tumorigenesis and can serve as early diagnostic markers for tumors.

[0012] In this invention, the term "biomarker" refers to an indicator that can be detected in a sample, such as a predictive, diagnostic, and / or prognostic indicator. Biomarkers can serve as indicators of a specific disease or condition (e.g., cancer) subtype characterized by specific molecular, pathological, histological, and / or clinical features. In some embodiments, a biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number alterations (e.g., DNA copy number), peptides, peptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate and / or glycolipid-based molecular markers.

[0013] In this invention, the term "expression level" generally refers to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., gene coding and / or epigenetics) is transformed into structures present and functioning in the cell. Therefore, "expression" as used in this invention can refer to transcription into a polynucleotide, translation into a polypeptide, or polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Transcribed polynucleotide fragments, translated polypeptide fragments, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide) should also be considered expressed, regardless of whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides (e.g., through proteolysis).

[0014] According to a specific embodiment of the present invention, a product that can be used for the clinical diagnosis of high-risk neuroblastoma includes a reagent for detecting the expression level of a biomarker, wherein the biomarker is CHD1 in plasma exosome protein.

[0015] This invention provides the application of a reagent for detecting the expression level of plasma exosome protein CHD1 in the preparation of diagnostic products for high-risk neuroblastoma.

[0016] Preferably, the diagnostic product is a chip or a reagent kit.

[0017] In this invention, the term "chip" refers to a gene chip, also known as a DNA chip or biochip, which involves immobilizing probe molecules on a support and then hybridizing them with labeled sample molecules. The quantity and sequence information of the sample molecules are obtained by detecting the hybridization signal intensity of each probe molecule.

[0018] The diagnostic products also include negative controls, positive controls, primers, probes, or antibodies.

[0019] In this invention, the term "primer" refers to a single-stranded polynucleotide capable of hybridizing with nucleic acids and allowing the polymerization of complementary nucleic acids (generally by providing a free 3'-OH group).

[0020] In this invention, the term "antibody" is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0021] Meanwhile, the present invention provides a kit for diagnosing high-risk neuroblastoma, the kit comprising the aforementioned reagent for detecting the expression level of plasma exosome protein CHD1.

[0022] Preferably, the detection reagent further includes a negative control, a positive control, primers, probes, or antibodies.

[0023] The present invention also provides the use of an agent that inhibits CHD1 gene expression in the preparation of a drug for treating neuroblastoma, wherein the agent that inhibits CHD1 gene expression is a siRNA or shRNA that specifically targets the CHD1 gene.

[0024] Preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0025] SEQ ID NO.1: 5'-GAAGCACACCGATTAAAGA-3';

[0026] SEQ ID NO. 2: 5'-GGACTATTCCTCGGGAGAA-3'.

[0027] The medicament for treating neuroblastoma according to a specific embodiment of the present invention contains one or more of the following:

[0028] (1) Contains reagents that downregulate the expression level of the CHD1 gene in vivo;

[0029] (2) Reagents that inhibit or block the expression of the CHD1 gene in the body.

[0030] The medicament for treating neuroblastoma according to a specific embodiment of the present invention includes shRNA or siRNA that specifically targets the CHD1 gene.

[0031] Preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0032] In this invention, the expression of the gene encoding the active protein to be inhibited is reduced or suppressed. Specifically, this can be achieved through gene knockout or gene silencing.

[0033] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0034] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing can occur at two levels: one is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects; the other is post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA, including antisense RNA, co-inhibition, gene repression, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0035] The beneficial effects of this invention are:

[0036] This invention utilizes proteomics technology to identify differentially expressed proteins in the plasma and exosomes of children with neonatal NB (NB). The expression level of CHD1 protein is used to identify high-risk NB. This method has good external validity, universality, applicability, and good predictive performance.

[0037] This invention found that CHD1 is highly expressed in the exosomes of high-risk NB and is positively correlated with clinical progression; in the plasma exosomes of newly diagnosed NB children, the high expression level of CHD1 is positively correlated with M-stage NB, high-risk NB, and high serum lactate dehydrogenase (LDH) levels.

[0038] Based on the above findings, this invention designs siRNA to knock down the CHD1 gene. After knocking down the CHD1 gene, the proliferation and clonogenic ability of neuroblastoma are significantly reduced. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This document outlines the screening process for the differentially expressed protein CHD1 in plasma exosomes from children with neuroblastoma.

[0041] Figure 2 The relationship between CHD1 expression levels in the public database GSE62564 and high-risk grouping, event-free survival, and overall survival is shown.

[0042] A. High expression of CHD1 in tumor tissues of high-risk NB children (P < 0.01);

[0043] B. Children with high expression of CHD1 in tumor tissue had low event-free survival (P < 0.05);

[0044] C. Children with high expression of CHD1 in tumor tissue had poor overall survival (P < 0.0001);

[0045] Figure 3 This demonstrates the use of ELISA to verify the expression level of CHD1 in the validation set samples;

[0046] Figure 4 The ROC curves in the validation set are shown to divide NB into high-risk and low-risk categories based on CHD1 protein expression levels.

[0047] Figure 5 The qPCR results demonstrate the effectiveness of CHD1 knockdown.

[0048] Figure 6 The Western blot assay showed the effect of CHD1 knockdown.

[0049] Figure 7 This experiment demonstrates real-time label-free cell analysis to detect the proliferation of the neuroblastoma cell line SH-SY5Y after CHD1 gene knockout.

[0050] Figure 8 This shows the changes in the cloning ability of the neuroblastoma cell line SH-SY5Y after CHD1 gene knockdown. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0052] In this invention, high-risk NB patients are grouped according to the International NB Risk Group (INRG) based on factors such as age at diagnosis, INRGSS stage, histological type, and MYCN gene. High-risk patients have a poor prognosis, with a 5-year event-free survival rate of less than 50%.

[0053] Low-risk NB patients: According to the International NB Risk Group (INRG) for pre-treatment risk stratification of NB, low-risk patients have a better prognosis, with a 5-year event-free survival rate of more than 75%.

[0054] Event-free survival (EFS) refers to the time from the start of randomization (or the start of treatment in a single-arm trial) to the first occurrence of any of the following events: disease progression that makes surgical treatment impossible, local or distant recurrence, death from any cause, etc.

[0055] Overall survival (OS) refers to the time from the start of randomization (or the start of treatment in a single-arm trial) to death from any cause.

[0056] M stage: According to the International New Balance Risk Group (INRG) staging, any primary tumor with distant lymph node, bone marrow, liver, skin and / or other organ dissemination (excluding Ms stage).

[0057] Non-M stage: According to the International New Balance Risk Group (INRG) staging system, the staging of New Balance NB excluding the M stage includes L1, L2 and Ms stages.

[0058] The neuroblastoma cell line SH-SY5Y used in this invention was purchased from The Global Bioresource Center, ATCC.

[0059] All other reagents used in this invention are commercially available.

[0060] Example 1: Screening for differentially expressed protein CHD1 in plasma exosomes of children with neuroblastoma.

[0061] 1.1 Extraction of exosomes from plasma

[0062] This invention initially included 30 high-risk and 30 low-risk infants with confirmed nephrotic syndrome (NB) diagnosis and risk classification who had not undergone surgical resection of the lesion or chemotherapy. Plasma samples were collected from these infants. Simultaneously, plasma samples were collected from 30 other infants whose conditions did not potentially affect plasma composition (e.g., short frenulum, strabismus, phimosis, scoliosis, etc.) who presented during the same period, serving as a healthy control group. All samples underwent plasma exosome extraction using Invitrogen's Total Exosome Isolation (from plasma) kit (catalog number: 4484450). The extraction method is as follows:

[0063] (1) Take 1 mL of fresh whole blood, 2,000 g, centrifuge at room temperature for 10 min, collect the upper plasma layer, and store it in a -80℃ refrigerator for later use.

[0064] (2) Remove the sample, thaw it in a water bath at room temperature, and place it on ice;

[0065] (3) Centrifuge at 2,000g for 20 min at room temperature to remove cells and debris, and transfer the supernatant to a new EP tube;

[0066] (4) Centrifuge at 10,000g for 20 min at room temperature, transfer the supernatant to a new EP tube and place it on ice (record plasma volume V1);

[0067] (5) Add PBS (V2 = V1 × 0.5), vortex to mix, and let stand for at least 30 seconds;

[0068] (6) Add Exosome Precipitation Reagent: V3 = 0.2 × (V1 + V2), vortex mix, the liquid will be cloud-like;

[0069] (7) Let stand at room temperature for 10 minutes;

[0070] (8) Centrifuge at 10,000g for 5 minutes at room temperature, carefully discard the supernatant and keep the precipitate at the bottom of the tube;

[0071] (9) Centrifuge at 10,000g for 1 min at room temperature, carefully discard the supernatant and keep the precipitate at the bottom of the tube;

[0072] (10) Resuspend the precipitate in PBS, mix well, and avoid bubbles. V4 = 0.3 × V1;

[0073] (11) Take 1 / 3 of the exosome sample and freeze it directly for exosome electron microscopy and particle number analysis;

[0074] (12) Add 2×Zwitter lysis buffer (pre-added cocktail) at a 1:1 ratio to the remaining exosome volume, and sonicate on ice for 1 h;

[0075] (13) 12,000g, 20min, 4℃, take 40μL, add 5*loading 10μL, 98℃, 5min.

[0076] (14) Protein quantification was performed using the BCA method, and absorbance was measured at 562 nm. 100 μg of protein was then subjected to subsequent mass spectrometry.

[0077] 1.2 Preprocessing of plasma exosomal proteins for proteomics

[0078] Plasma exosomal protein proteomics pretreatment was performed using the filter-assisted sample preparation (FASP) method, with the following steps:

[0079] (1) Reduction: Add DDT to a final concentration of 20 mM to 100 μL of plasma exosome protein lysis buffer (containing 100 μg of exosome protein), vortex, briefly ionize, and heat at 95 °C for five minutes. Equilibrate to room temperature.

[0080] (2) Alkylation: Add IAM with a final concentration of 50mM, vortex, detach and react in the dark for 45min.

[0081] (3) Equilibrium membrane: The 30K membrane was washed twice with 200 μL Tris (4℃×14,000g×5min) before centrifugation until there was no liquid in the tube and the liquid was discarded.

[0082] (4) Sample loading: Transfer the protein sample to a 30K membrane and centrifuge at 4℃ for 14,000g for 30min until there is no liquid in the tube and the membrane shows a small crescent shape.

[0083] (5) Cleaning: Add 200 μL of 20 mM Tris, shake to mix, 4℃×14,000g×30min until there is no liquid in the cannula, repeat 3 times.

[0084] (6) Cleaning the sleeve: Clean the lower tube twice with 20mM Tris.

[0085] (7) Enzymatic hydrolysis: Add enzyme according to the mass ratio of sample:enzyme = 50:1, add Tris to 30μL, shake to mix, swirl the liquid onto the membrane, place the test tube on the float in a beaker containing tap water, microwave on high for 1min, twice, changing the tap water in between, and incubate overnight in a 37℃ water bath.

[0086] (8) Collect the enzyme hydrolysate: Centrifuge at 14,000g until the membrane is clean and free of liquid. The liquid below is the enzyme hydrolysate.

[0087] (9) BCA method for determining peptide concentration.

[0088] 1.3 Independent Data Acquisition (DIA) Mass Spectrometry Analysis

[0089] Take 2 μg of peptide from each sample and incorporate iRT standard peptide at a volume ratio of 1:20. Perform DIA mass spectrometry test on each sample once.

[0090] Chromatographic separation was performed using a UltiMate 3000 UPLC system with a flow rate of 1.5 μL / min. Buffer solutions: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile content 80%). Peptide samples were directly introduced into the chromatographic column (length: 50 cm, inner diameter: 75 μm) for mass spectrometry detection.

[0091] The liquid phase separation gradients are as follows: 0 min - 50 min, linear gradient of liquid B from 1% to 20%; 50 min - 55 min, linear gradient of liquid B from 20% to 30%; 55 min - 56 min, linear gradient of liquid B from 30% to 50%; 55 min - 56 min, linear gradient of liquid B from 30% to 50%; 56 min - 60 min, linear gradient of liquid B from 50% to 90%.

[0092] The samples separated by liquid chromatography were analyzed by mass spectrometry using an Orbitrap Exploris 480 high-resolution mass spectrometer (ThermoScientific).

[0093] Detection mode: positive ion; Primary mass spectrometry scan range: 350-1200 m / z; Mass spectrometry resolution: 120,000; AGC target: Custom; Maximum IT: 50 ms; Data type: Profile. MS2 acquisition settings: 60 acquisition windows; Secondary resolution: 30,000; AGC target: Custom; Maximum IT: 50 ms; Data type: Centroid. HCD collision energy: 30%.

[0094] 1.4 Mass Spectrometry Data Processing

[0095] Mass spectrometry data were processed using Spectronaut Pulsar (18.0) software and the SwissProthuman database (20,386 sequences, released in June 2022). Direct search was used for the search. Qualitative and quantitative parameters were set as follows: Precursor Qvalue: 0.01, Protein Qvalue: 0.01, quantification based on peak area at MS1 level, correction method: Cross-Run Normalization, quantification method: MaxLFQ, Inference Algorithm: IDPicker.

[0096] Table 1. Screening results of proteins with increased expression levels between the two groups.

[0097]

[0098]

[0099] As shown in the table above, among the numerous proteins, CHD1, HSP90AB1, BASP1, TAGLN2, and C17orf100 are three groups of proteins with increased expression levels. Through further screening, CHD1 was identified as a candidate differentially expressed protein for further investigation.

[0100] Example 2

[0101] The relationship between CHD1 expression and NB patient grouping and survival was investigated using the public database GSE62564.

[0102] The public database GSE62564 uses RNA-seq (RNA sequencing, transcriptome sequencing technology) to perform sequencing analysis on NB samples, forming a gene expression matrix. The values ​​represent the expression abundance of the corresponding genes in the sample, reflecting the gene expression level.

[0103] In the public database GSE62564, 176 high-risk infants had a median CHD1 expression level of 5.601, while 322 non-high-risk infants had a median expression level of 5.425. A t-test showed a p-value <0.01, indicating high CHD1 expression in the tumor tissues of high-risk non-high-risk infants (NB). Figure 2 -A;

[0104] Based on the patient's survival outcome of death, ROC analysis was performed on the expression level of CHD1 gene. The cutoff value was selected as the point with the largest Youden index (sensitivity + specificity - 1), and the data of the children were divided into two groups: high expression (151 patients) and low expression (347 patients).

[0105] like Figure 2 -B, the 5-year event-free survival rate of NB children with high CHD1 expression was 55.83%, while that of NB children with low CHD1 expression was 65.55%. The event-free survival rate was even lower in children with high CHD1 expression in tumor tissue (P<0.05).

[0106] like Figure 2 -C, the 5-year overall survival rate of NB children with high CHD1 expression was 66.07%, while the 5-year overall survival rate of NB children with low CHD1 expression was 84.36%, and the overall survival rate of NB children with high CHD1 expression was worse (P<0.0001).

[0107] Example 3 uses ELISA to verify the expression level of CHD1 in the validation set samples.

[0108] This invention also collects plasma samples as a validation set for proteomics results, including 15 healthy controls, 15 low-risk NB infants, and 15 high-risk NB infants. After extracting plasma exosome proteins, ELISA experiments were performed for validation.

[0109] The ELISA experimental procedure is as follows:

[0110] (1) Prepare reagents: dilute concentrated washing solution with distilled water at a ratio of 1:20; mix substrate solutions A and B at a volume ratio of 1:1 and use within 15 minutes of mixing; take out the pre-coated enzyme-labeled strips for later use.

[0111] (2) Set up standard wells, zero value wells, blank wells and sample wells. Add 50 μL of standard of different concentration to each standard well, add 50 μL of sample diluent to the zero value well, do not add to the blank well, and add 50 μL of the sample to be tested to the sample well.

[0112] (3) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard wells, zero value wells and sample wells.

[0113] (4) Cover the reaction plate with a sealing film and incubate in a 37°C water bath or constant temperature incubator for 60 minutes in the dark.

[0114] (5) Remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 20 seconds, shake off the washing liquid, pat dry on absorbent paper, and repeat this process 5 times.

[0115] (6) Mix substrates A and B thoroughly at a 1:1 volume ratio, and add 100 μL of the substrate mixture to all wells. Cover the reaction plate with a sealing film and incubate in a water bath or incubator at 37°C in the dark for 15 min.

[0116] (7) Add 50 μL of stop solution to all wells and read the absorbance (OD value) of each well on a microplate reader at a wavelength of 450 nm.

[0117] (8) Result calculation: Using the concentration of the standard as the abscissa and the corresponding absorbance (OD value) as the ordinate, a standard curve equation is created by using computer software and a four-parameter Logistic curve fitting (4-pl). The concentration value of the sample is calculated by using the equation based on the absorbance (OD value) of the sample.

[0118] like Figure 3 As shown, the median CHD1 expression in the control group, low-risk group, and high-risk group were 0.533, 0.537, and 0.829, respectively. Compared with the control group and low-risk group, the t-test results showed that the high-risk group had a significantly increased CHD1 expression.

[0119] This demonstrates that CHD1 expression levels are significantly elevated in high-risk NB children.

[0120] Example 4: Plotting ROC curves in the validation set to identify high-risk or low-risk NBs based on CHD1 protein expression levels.

[0121] ROC curves were plotted based on CHD1 protein expression levels to identify high-risk or low-risk neonatal piglets (NBs). The results are as follows: Figure 4 As shown, the obtained ROC curve has an AUC value of 0.7467 and P = 0.0213.

[0122] It is evident that, through external validation, the expression level of CHD1 protein has good external validity for identifying high-risk NB, demonstrating universality, applicability, and good predictive performance.

[0123] Example 5 examines the correlation between CHD1 expression levels and clinical progression.

[0124] Using the cutoff value of 0.779 obtained from the ROC curve of CHD1 in Example 4 as the distinguishing criterion, patients were divided into a high CHD1 expression group and a low CHD1 expression group. Chi-square test was used for analysis.

[0125] Table 2. Analysis of CHD1 expression and clinical characteristics in NB children.

[0126]

[0127]

[0128] LDH: Lactate dehydrogenase; NSE: Neuron-specific enolase. * P<0.05, chi-square test.

[0129] As shown in Table 2, among children with high CHD1 expression in plasma exosomal tissues, the proportion of children in the M stage was significantly higher than that in non-M stage children (73.3% vs. 26.7%, P = 0.011), and the proportion of children in the high-risk group was significantly higher than that in the non-high-risk group (73.3% vs. 26.7%, P = 0.011). Simultaneously, the proportion of children with elevated serum LDH levels was significantly higher than that of children with normal levels (14.3% vs. 42.9% vs. 28.6% vs. 14.3%, P = 0.028). All of the above demonstrates that high expression of CHD1 in NB plasma exosomal tissues is positively correlated with the high malignancy and clinical progression of NB.

[0130] In newly diagnosed NB patients, high expression of CHD1 in plasma exosomes was positively correlated with M-phase NB, high-risk NB, and high serum lactate dehydrogenase (LDH) levels.

[0131] Example 6

[0132] 1. CHD1 gene knockdown experiment

[0133] This embodiment designs siRNA targeting homologous sequence regions of all CHD1 transcripts:

[0134] siCHD1-1# 5'-GAAGCACACCGATTAAAGA-3';

[0135] siCHD1-2# 5'-GGACTATTCCTCGGGAGAA-3';

[0136] siNC 5'-GGCTCTAGAAAAGCCTATGC-3'.

[0137] (1) One day before transfection, the well-grown neuroblastoma cell line SH-SY5Y was washed, digested, and resuspended, and cell counts were performed at a rate of 1-2 × 10⁻⁶ cells / year. 5 Seed cells per well in 6-well plates, shake well, and incubate in a constant temperature incubator for 20-24 hours. Transfection is performed when the cell density reaches 50%-60% and the cells are evenly distributed in each well.

[0138] (2) One hour before transfection, remove the 6-well plate from the incubator, discard the old culture medium, replace it with 1 mL of fresh DMEM complete culture medium, and place it in a constant temperature incubator for later use.

[0139] (3) Transfection was performed using Lipofectamine RNAiMAX transfection reagent. The system was prepared according to the reagent instructions. For example, one well of a 6-well plate (operated in a biosafety cabinet). Dilute 4 μL of 20 μM siRNA solution with 100 μL of Opti-MEM medium and mix gently to obtain the siRNA dilution. Dilute 5 μL of Lipofectamine RNAiMAX with 100 μL of Opti-MEM medium and mix gently to obtain the RNAiMAX dilution.

[0140] (4) Mix the siRNA dilution buffer and RNAiMAX dilution buffer, mix well by pipetting, and incubate at room temperature for 20 min.

[0141] (5) Take out the spare 6-well plate from the incubator, add all the siRNA-transfection reagent mixture to the culture medium, shake gently and then put it into the incubator.

[0142] (6) Change the medium 6-8 hours after transfection, replacing 2 mL of complete culture medium in each well and continue culturing.

[0143] (7) 72 hours after transfection, cells were collected for subsequent experiments.

[0144] 2. qPCR verification of CHD1 knockdown effect

[0145] (1) siRNA (siCHD1-1# and siCHD1-#2) were transfected into the neuroblastoma cell line SH-SY5Y to knock down the CHD1 gene. After 72 hours, the cells were lysed, and total RNA was extracted and reverse transcribed to prepare cDNA. The reverse transcription reagent was obtained from TaKaRa, and the reverse transcription to prepare cDNA was performed according to the instructions of the PrimeScript™ RT Master Mix (Perfect Real Time) cDNA Reverse Transcription Kit.

[0146] (2) The reagents required for real-time quantitative PCR (qPCR) were obtained from Bio-Rad. The SYBR Green Supermix reagent instructions were followed to construct a 20 μL reaction system.

[0147] The reaction system consisted of: 10.0 μL SYBR Green (2×); 1.0 μL cDNA; 0.5 μL upstream primer (10 μmol / L); 0.5 μL downstream primer (10 μmol / L); and 8.0 μL sterile water. The primer sequences are as follows:

[0148] The solutions were thoroughly mixed according to the above system, and after brief infiltration, they were placed in a real-time quantitative PCR instrument (ViiA7, Singapore ABI / VIIA7). The amplification program was set as follows: 95℃ denaturation for 10 min; 95℃ denaturation for 15 sec, 60℃ annealing for 35 sec, 70℃ extension for 30 sec, for a total of 40 cycles.

[0149] (3) The melting curve analysis was performed under the system default conditions. The cycle number (Ct value) corresponding to the inflection point of the fluorescence signal entering the exponential growth phase during amplification was used as an indirect indicator of the initial template concentration. The results were corrected based on the Ct value of the internal reference gene. The experimental results were calculated using the formula (2-ΔΔCt method):

[0150]

[0151] ΔΔCt=ΔCt(experimental)-ΔCt(control);

[0152] ΔCt(experimental)=Ct Mean(experimental CHD1)–Ct Mean(experimentalGAPDH);

[0153] ΔCt(control)=Ct Mean(control CHD1)–Ct Mean(control GAPDH).

[0154] The results are as follows Figure 5 As shown.

[0155] 3. Western blotting to verify the CHD1 knockdown effect.

[0156] siRNAs (siCHD1-1# and siCHD1-#2) were transfected into the neuroblastoma cell line SH-SY5Y to knock down the CHD1 gene. Cells were lysed after 72 hours and Western blot analysis was performed.

[0157] The expression level of CHD1 protein was detected using CHD1 antibody (cat.sc-271626, 1:500, Santa Cruz Biotechnology) and internal reference protein GAPDH antibody (cat.KM9002, 1:10000, Sungene Biotech). Exposure and development were performed using a chemiluminescence kit and a multicolor fluorescence and chemiluminescence imaging system (ChemiDoc MP, Singapore Bio-Rad).

[0158] The results are as follows Figure 6As shown, after knocking down the CHD1 gene with siCHD1-1# and siCHD1-#2, the expression level of CHD1 gene protein decreased significantly, indicating that the synthesized siRNA can effectively reduce the protein expression of CHD1.

[0159] 4. CHD1 gene knockout slows down neuroblastoma cell proliferation.

[0160] xCELLigence real-time cell analysis dual-purpose (RTCA DP) assay was used to detect the proliferation of the neuroblastoma cell line SH-SY5Y after CHD1 gene knockout.

[0161] The experimental steps are as follows:

[0162] (1) Neuroblastoma cell line SH-SY5Y was transfected with siRNA to knock down CHD1. After treatment for 72 hours, the cells were washed with PBS, digested with trypsin, and resuspended in DMEM complete medium. After gentle pipetting and mixing, cell counts were performed to determine the original cell concentration in each group.

[0163] (2) Calculate the volume of cell stock solution to be added for each group according to 5000 cells per well, a final volume of 100 μL, and 3 replicates per group (calculate the system according to 4 replicates). Then add DMEM complete culture medium to the final volume and mix thoroughly.

[0164] (3) Add 50 μL of complete culture medium to the wells of E-Plate 16 (use reverse aspiration to avoid air bubble formation), place it on the xCELLigence RTCA DP analyzer, and measure the baseline after ensuring good machine contact. The baseline calibration is considered successful if the ordinate of all wells is less than 0.063.

[0165] (4) Take out the E-Plate 16 plate and add 100 μL of the cell suspension prepared above evenly to each well (note that the sample must be mixed evenly). Let it stand at room temperature for 30 minutes in a clean bench, then perform the detection (in a constant temperature incubator), counting once every 15 minutes, and continuously timing for 100 hours.

[0166] like Figure 7 As shown, the proliferation of neuroblastoma cell line SH-SY5Y cells was slowed after CHD1 gene knockdown.

[0167] 5. Colony Formation Experiment

[0168] The experimental steps are as follows:

[0169] (1) Neuroblastoma cell line SH-SY5Y was transfected with siRNA to knock down CHD1. After treatment for 72 hours, the cells were washed with PBS, digested with trypsin, and resuspended in DMEM complete medium. After gentle pipetting and mixing, cell counts were performed to determine the original cell concentration in each group.

[0170] (2) Calculate the volume of cell stock solution to be added for each group based on 800 cells per well, a final volume of 1 mL, and 3 replicates per group (calculate the system based on 4 replicates). Then, add DMEM complete culture medium to the final volume.

[0171] (3) After thoroughly mixing, add 1 mL of cell dilution solution to each well of the 6-well plate, and then add 1 mL of complete culture medium to each well to a final volume of 2 mL. Shake the 6-well plate well using the cross-hatching method and place it in an incubator for culture. Gently replenish the culture medium periodically (do not move it during the first week).

[0172] (4) Once visible clones are observed, terminate the culture (approximately 14 days). Remove all 6-well plates, discard the culture medium, and gently wash once with PBS. Add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 15 minutes. Discard the fixative, add 1 mL of crystal violet staining solution to each well, and stain at room temperature in the dark for 15 minutes. Wash several more times with PBS until the washing buffer becomes clear. Aspirate the washing buffer from the 6-well plates, allow them to air dry at room temperature, and count the clones.

[0173] like Figure 8 As shown, the cloning ability of the neuroblastoma cell line SH-SY5Y was significantly reduced after CHD1 gene knockdown, and knockdown of CHD1 could inhibit NB cell cloning.

[0174] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of plasma exosome protein CHD1 as a biomarker in the preparation of diagnostic agents for high-risk neuroblastoma, characterized in that, The expression level of plasma exosomal protein CHD1 is significantly increased in high-risk neuroblastomas.

2. Application of reagents for detecting the expression level of plasma exosomal protein CHD1 in the preparation of diagnostic products for high-risk neuroblastoma.

3. The application according to claim 2, characterized in that, The diagnostic product is a chip or a reagent kit.

4. The application according to claim 3, characterized in that, The diagnostic products also include negative controls, positive controls, or antibodies.

5. The application of a reagent that inhibits CHD1 gene expression in the preparation of drugs for treating high-risk neuroblastoma, characterized in that, The reagent used to inhibit CHD1 gene expression is a siRNA or shRNA that specifically targets the CHD1 gene.

6. The application according to claim 5, characterized in that, The nucleotide sequence of siRNA is as shown in SEQ ID NO.1 or SEQ ID NO.2.

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