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

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a biomarker for clinical diagnosis of high-risk neuroblastoma and application of the biomarker. A product capable of being used for clinical diagnosis of high-risk neuroblastoma comprises a reagent for detecting the expression level of a biomarker, and the biomarker is plasma exosome protein CHD1. By detecting the CHD1 expression level, existing clinical indexes can be supplemented, precise layering and precise treatment of neuroblastoma are achieved, and meanwhile the kit has important guiding significance for prognosis judgment of tumor children.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of plasma exosome protein CHD1 as a biomarker for clinical diagnosis of neuroblastoma. BACKGROUND

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

[0003] NB has the characteristics of insidious onset and high malignancy, and the 5-year survival rate of high-risk children is less than 50%. The main problem currently faced by the clinic is that the early screening of suspected cases is relatively lagging. And due to the particularity of pediatric patients, invasive methods such as puncture biopsy are difficult to implement. Therefore, exploring more sensitive and effective high-risk NB early screening molecular markers, and realizing through non-invasive or minimally invasive methods, will effectively improve the diagnosis and treatment level of NB children.

[0004] Exosomes refer to a class of extracellular matrix microcapsules with a size of 30-150 nm (average 100 nm). Exosomes can be secreted by various cells, and contain many components (such as DNA, RNA, protein, lipid, metabolite, etc.) of the originating cells. The exosomes secreted can enter body fluids such as blood, saliva, urine and milk, circulate to other cells or tissues through body fluids, participate in the regulation of cell-to-cell communication, and have application potential in the treatment of many diseases. The characteristics of regulating cell pathways make exosomes have application potential in the treatment of many diseases. In some diseases, including neurodegenerative diseases and cancers, exosomes are designed as effective carriers for delivering chemotherapy drugs, immunosuppressants, etc., and have the ability to deliver therapeutic drugs to the desired target. In addition, since exosomes carry biological materials of the originating cells, and are easy to obtain through liquid biopsy, exosomes have great potential in helping disease diagnosis and prognosis prediction in diseases such as cancer. SUMMARY

[0005] The application provides application of plasma exosome protein CHD1 as a biomarker for high-risk neuroblastoma.

[0006] CHD1 is Chromodomain Helicase DNA Binding Protein 1, which is highly conserved in eukaryotic cells, has the functions of assembling nucleosomes, remodeling chromatin structure, regulating histone metabolism, regulating gene transcription, etc. CHD1 exists in mutations or deletions in only a few cancers (such as prostate cancer, uterine cancer, melanoma, etc.), and the role in different tumor progression is not the same, and there is still a great controversy, and the relationship between CHD1 and NB has not been reported.

[0007] The present application uses proteomics technology to mine the differential proteins in the plasma and plasma exosomes of NB children, CHD1 exists as a secretory protein in plasma exosomes, and can be used to diagnose high-risk neuroblastoma by detecting the expression level of CHD1 in plasma exosomes, is a key regulatory molecule of NB tumor occurrence and development, and can be used as a new molecular marker for clinical diagnosis, which has important theoretical significance and potential application value.

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

[0009] The use of the diagnostic preparation includes: detecting the expression of CHD1 in the plasma exosomes of the subject; comparing the detection value with the reference value, and CHD1 is significantly higher than the reference value, which indicates 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 low-risk neuroblastoma children. The subject is known or suspected to contain tumor cells.

[0010] In the present application, the term CHD1 is Chromodomain Helicase DNA Binding Protein 1, which includes CHD1 gene and the protein encoded thereby and its homologues, mutations and isoforms. The term covers full-length, unprocessed CHD1 and any form of CHD1 derived from processing in cells. Preferably, the CHD1 is a human protein, and the UniProt ID is O14646.

[0011] In the present application, the term "exosome" refers to a class of extracellular matrix microvesicles with a size of 30-150 nm (average 100 nm). Exosomes can be secreted by various cells, and contain many components (such as DNA, RNA, protein, lipid, metabolite, etc.) of the originating cells. The secreted exosomes can enter the blood, saliva, urine, and milk, etc. body fluids, circulate to other cells or tissues through body fluids, and participate in the regulation of intercellular communication. Many molecules have been confirmed to be involved in the occurrence of important diseases such as tumor, and can be used as early diagnostic markers of tumors.

[0012] In the present application, the term "biomarker" refers to an indicator that can be detected in a sample, such as a predictive, diagnostic, and / or prognostic indicator. A biomarker can serve as an indicator of a particular disease or condition (e.g., cancer) subtype characterized by particular 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), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate and / or glycolipid-based molecular markers.

[0013] In the present application, 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., genetic code and / or epigenetic) is translated into structures that exist and function in a cell. Thus, "expression" as used in the present application 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). Fragments of a transcribed polynucleotide, a translated polypeptide, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered as expression, whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide (e.g., by proteolysis).

[0014] According to the embodiment of the present application, a product for clinical diagnosis of high-risk neuroblastoma is provided, wherein the product comprises a reagent for detecting the expression level of a biomarker CHD1 in plasma exosome protein.

[0015] The present application provides the use of a reagent for detecting the expression level of CHD1 in plasma exosome protein in the preparation of a diagnostic product for diagnosing high-risk neuroblastoma.

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

[0017] In the present application, the term "chip" refers to a gene chip, also known as a DNA chip or a biochip, which immobilizes probe molecules on a support and hybridizes them with labeled sample molecules, and obtains the quantity and sequence information of the sample molecules by detecting the hybridization signal intensity of each probe molecule.

[0018] The diagnostic product further comprises a negative control, a positive control, a primer, a probe or an antibody.

[0019] In the present application, the term "primer" refers to a single-stranded polynucleotide that can hybridize to a nucleic acid and allow the polymerization of the complementary nucleic acid, generally by providing a free 3'-OH group.

[0020] In the present application, the term "antibody" is used in the broadest sense and encompasses various 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 application provides a kit for diagnosing high-risk neuroblastoma, which comprises the detection reagent for the expression level of the plasma exosome protein CHD1.

[0022] Preferably, the detection reagent further comprises a negative control, a positive control, a primer, a probe or an antibody.

[0023] The present application also provides the use of a reagent for inhibiting the expression of CHD1 gene in the preparation of a medicament for treating neuroblastoma, wherein the reagent for inhibiting the expression of CHD1 gene is an siRNA or shRNA specifically targeting the CHD1 gene.

[0024] Preferably, the nucleotide sequence of the siRNA is 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 the specific embodiment of the present application contains any one or more of the following:

[0028] (1) a reagent for down-regulating the expression amount of CHD1 gene in the body;

[0029] (2) a reagent for inhibiting or blocking the expression of CHD1 gene in the body.

[0030] The drug for treating neuroblastoma according to the specific embodiment of the present application comprises shRNA or siRNA specifically targeting the CHD1 gene.

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

[0032] In the present application, the inhibition or reduction of the expression of the coding gene of the active protein to be inhibited is achieved, specifically, by gene knockout or by gene silencing.

[0033] The gene knockout refers to the phenomenon of inactivating a specific target gene by homologous recombination. The gene knockout is to inactivate a specific target gene by changing the DNA sequence.

[0034] The gene silencing refers to the phenomenon of not expressing or lowly expressing a gene without damaging the original DNA. The gene silencing can occur at two levels, one is the transcriptional level of gene silencing caused by DNA methylation, heterochromatinization and position effect, and the other is the post-transcriptional gene silencing, that is, inactivating a gene by specifically inhibiting the target RNA after the gene transcription, including antisense RNA, co-suppression, gene suppression, RNA interference (RNAi) and microRNA (miRNA) mediated translation inhibition.

[0035] The beneficial effects of the present application are:

[0036] The present application uses proteomics technology to mine the differential proteins in the plasma and plasma exosomes of NB children, and uses the CHD1 protein expression level to identify high-risk NB, which has good external validity, universality and applicability, and good prediction performance.

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

[0038] Based on the above finding, the present application designs siRNA to knock down the CHD1 gene, and after the CHD1 gene is knocked down, the proliferation and clonogenicity of neuroblastoma are significantly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0040] Figure 1 Screening process of CHD1, a differentially expressed protein in plasma exosomes of children with neuroblastoma, is shown;

[0041] Figure 2 The relationship between the expression amount of CHD1 in the public database GSE62564 and the high-risk group, event-free survival and overall survival rate is shown;

[0042] A. CHD1 is highly expressed in tumor tissues of children with high-risk NB (P<0.01);

[0043] B. The event-free survival rate of children with high expression of CHD1 in tumor tissues is low (P<0.05);

[0044] C. The overall survival rate of children with high expression of CHD1 in tumor tissues is poor (P<0.0001);

[0045] Figure 3 The expression amount of CHD1 in the verification set samples is verified by ELISA experiment.

[0046] Figure 4 The ROC curve of dividing NB high-risk or low-risk by the expression level of CHD1 protein in the verification set is shown.

[0047] Figure 5 The qPCR verifies the CHD1 knockdown effect.

[0048] Figure 6 The Western blot experiment verifies the CHD1 knockdown effect.

[0049] Figure 7 The real-time label-free cell analysis experiment detects the proliferation of neuroblastoma cell line SH-SY5Y cells after CHD1 gene knockdown.

[0050] Figure 8 The change of the clonality of neuroblastoma cell line SH-SY5Y cells after CHD1 gene knockdown is shown. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0052] In the present application, high-risk NB children: according to the International NB Risk Cooperative Group (INRG), the risk of patients before treatment is stratified based on factors such as diagnosis age, INRGSS stage, histological type and MYCN gene. High-risk children have poor prognosis, and the 5-year event-free survival rate is less than 50%.

[0053] Low-risk NB children: according to the International NB Risk Cooperative Group (INRG) stratification of the pre-treatment risk of NB, low-risk children have better prognosis, and the 5-year event-free survival rate is greater than 75%.

[0054] Event-free survival: Event Free Survival, EFS refers to the time from randomization (or the start of treatment in a single-arm trial) to the first occurrence of any of the following events: disease progression that cannot be treated surgically, local or distant recurrence, death from any cause, etc.

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

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

[0057] Non-M stage: According to the International NB Risk Cooperative Group (INRG) staging, the staging of M stage NB is excluded, including L1 stage, L2 stage and Ms stage.

[0058] The neuroblastoma cell line SH-SY5Y used in the present application is purchased from The Global Bioresource Center, ATCC.

[0059] The other reagents used in the present application are commercially available.

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

[0061] 1.1 Extraction of exosomes in plasma

[0062] The previous application includes 30 cases of high-risk NB children with clear NB diagnosis and risk classification, and 30 cases of low-risk NB children without surgical resection of tumor and chemotherapy. Collect plasma samples from children, and collect plasma samples from children with no potential impact on plasma components (such as: short lingual frenulum, strabismus, phimosis, scoliosis, etc.) during the same period as healthy control group. All samples use Invitrogen Total Exosome Isolation (from plasma) kit (item number: 4484450) for plasma exosome extraction. The extraction method is as follows:

[0063] (1) Take 1 mL of fresh whole blood, centrifuge at 2,000 g for 10 min at room temperature, and aspirate the upper plasma. Store in a-80℃ refrigerator for standby;

[0064] (2) Take out the sample, melt in a water bath at room temperature, and place on ice;

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

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

[0067] (5) Add PBS (V2 = V1 x 0.5), vortex well, at least 30 s;

[0068] (6) Add Exosome Precipitation Reagent: V3 = 0.2 x (V1 + V2), vortex well, and the liquid is cloudy;

[0069] (7) Room temperature for 10 min;

[0070] (8) 10,000 g, 5 min, room temperature centrifugation, carefully discard the supernatant, and leave the tube bottom precipitate;

[0071] (9) 10,000 g, 1 min, room temperature centrifugation, carefully discard the supernatant, and leave the tube bottom precipitate;

[0072] (10) Resuspend the precipitate with PBS, mix well, avoid air bubbles, and V4 = 0.3 x V1;

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

[0074] (12) Add 2x zwitter lysis buffer (cocktail added in advance) to the remaining exosome volume 1:1, and ultrasonic lysis on ice for 1 h;

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

[0076] (14) BCA method for protein quantification, 562nm to measure absorbance. Take 100μg protein for subsequent mass spectrometry.

[0077] 1.2 Plasma exosome proteomics pretreatment

[0078] The FASP method was used for plasma exosome proteomics pretreatment, and the steps were as follows:

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

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

[0081] (3) Equilibrium membrane: 30K membrane was pre-washed twice with 200μL Tris (4°C x 14,000g x 5min), centrifuged until no liquid in the sleeve, and the liquid was discarded.

[0082] (4) Sample loading: Transfer the protein sample to the 30K membrane, centrifuge at 4°C x 14,000g x 30min until no liquid in the sleeve, and the membrane shows a small crescent.

[0083] (5) Washing: Add 200μL 20mM Tris, shake well, and centrifuge at 4°C x 14,000g x 30min until no liquid in the sleeve, repeat 3 times.

[0084] (6) Clean the sleeve: Use 20mM Tris to clean the tube twice.

[0085] (7) Enzymatic digestion: Add enzyme according to the mass ratio of sample: enzyme = 50:1, add Tris to 30μL, shake well, spin the liquid onto the membrane, place the tube on the beaker containing tap water, microwave high fire 1min, 2 times, change tap water in between, 37°C water bath overnight.

[0086] (8) Collect the enzyme solution: centrifuge at 14,000g until the membrane is clean and no liquid, the liquid below is the enzyme solution.

[0087] (9) BCA method to determine the concentration of peptides.

[0088] 1.3 Independent Data Acquisition (DIA) mass spectrometry analysis

[0089] Each sample took 2 pg of peptide segments, and mixed with iRT standard peptide segments at the volume ratio of 1:20. Each sample was tested by DIA mass spectrometry once.

[0090] Chromatographic separation was performed on a 1.5 pL / min flow rate UPLC system UltiMate 3000. Buffer: A liquid was 0.1% formic acid aqueous solution, B liquid was 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 80%). Peptide segment samples were directly introduced into the chromatographic analysis column (length: 50 cm, inner diameter: 75 pm) for mass spectrometry detection.

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

[0092] The samples after liquid chromatography separation were detected by Orbitrap Exploris 480 high resolution mass spectrometer (Thermo Scientific).

[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 collection settings 60 collection 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 processing was performed using Spectronaut Pulsar (18.0) software, and the database used was SwissProthuman database (20,386 sequences, 2022.6 released). The search method was direct search. Qualitative and quantitative parameter settings: Precursor Q value: 0.01, Protein Q value: 0.01, MS1 level quantification based on peak area, correction method: Cross-Run Normalization, quantitative method: MaxLFQ, Inference Algorithm: IDPicker.

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

[0097]

[0098]

[0099] As shown in the above table, among the numerous proteins, CHD1, HSP90AB1, BASP1, TAGLN2, and C17orf100 are proteins with increased expression common to the three groups. Through further screening, CHD1 was determined as a candidate up-regulated differential protein for investigation.

[0100] Example 2

[0101] The public database GSE62564 was used to investigate the relationship between CHD1 expression and NB patient grouping and survival.

[0102] The public database GSE62564 used RNA-seq (RNA sequencing, transcriptome sequencing technology) technology to sequence and analyze NB samples, forming a gene expression matrix, the numerical value of which represents the expression abundance of the corresponding gene of the corresponding sample, reflecting the expression amount of the gene.

[0103] In the public database GSE62564, there were 176 high-risk children, with a median CHD1 expression of 5.601, and 322 non-high-risk children, with a median expression of 5.425. The t-test gave a P value <0.01, indicating that high-risk NB children had high expression of CHD1 in tumor tissues, as shown in Figure 2 -A;

[0104] Taking the survival outcome of the patient, death or not, as a binary classification basis, ROC analysis was performed on the CHD1 gene expression, and the point with the largest Youden index (sensitivity + specificity - 1) was selected as the cutoff value, dividing the patient data into two groups: high expression (151) and low expression (347).

[0105] AsFigure 2 C, the 5-year overall survival rate of NB children with high expression of CHD1 was 66.07%, while the 5-year overall survival rate of NB children with low expression of CHD1 was 84.36%, and the overall survival rate of NB children with high expression of CHD1 was worse (P<0.0001).

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

[0107] Example 3 verifies the expression amount of CHD1 in the verification set samples by ELISA experiment

[0108] The present application further collects plasma samples as a verification set of proteomics results, including 15 healthy controls, 15 low-risk NB children and 15 high-risk NB children. After extracting the plasma exosome protein, ELISA experiment is performed for verification.

[0109] The ELISA experiment process is as follows:

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

[0111] (2) Set standard wells, 0-value wells, blank wells and sample wells, add 50 μL of different concentrations of standard to each standard well, add 50 μL of sample diluent to the 0-value well, do not add to the blank well, and add 50 μL of sample to be tested to the sample well.

[0112] (3) Except for the blank well, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard well, 0-value well and sample well.

[0113] (4) Cover the reaction plate with a sealing film, and incubate in a 37℃ water bath or incubator for 60 min in the dark.

[0114] (5) Remove the sealing film, discard the liquid, and dry with a paper towel. Add enough washing solution to each well, stand for 20 seconds, shake off the washing solution, and dry with a paper towel. Repeat this process 5 times.

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

[0116] (7) Add 50 μL of stop solution to all wells, and read the absorbance (OD value) of each well on a 450 nm wavelength enzyme-labeled instrument.

[0117] (8) Result calculation: taking the standard concentration as the horizontal coordinate, and the corresponding absorbance (OD value) as the vertical coordinate, using computer software, a four-parameter Logistic curve fitting (4-pl) was used to create a standard curve equation. Through the absorbance (OD value) of the sample, the concentration value of the sample was calculated by the equation.

[0118] As shown in Figure 3 , the median of CHD1 expression in the control group, low-risk group and high-risk group was 0.533, 0.537 and 0.829, respectively. The t-test result P value was <0.05 when comparing the high-risk group with the control group and the low-risk group, indicating that the CHD1 expression in the high-risk group was significantly increased.

[0119] Therefore, it is proved that the CHD1 expression level is significantly increased in high-risk NB children.

[0120] Example 4: Draw ROC curve in verification set to identify high-risk or low-risk NB with CHD1 protein expression level

[0121] Based on the CHD1 protein expression level, the ROC curve was drawn to identify high-risk NB or low-risk NB, and the results are shown in Figure 4 , the obtained ROC curve has an AUC value of 0.7467 and P = 0.0213.

[0122] It can be seen that through external verification, the CHD1 protein expression level has good external validity for identifying high-risk NB, and has universality and applicability, and good prediction performance.

[0123] Example 5: Investigation of the correlation between CHD1 expression and clinical progression

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

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

[0126]

[0127]

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

[0129] As shown in Table 2, among the children with high expression of CHD1 in plasma exosomes, the proportion of M-phase children was significantly higher than that of non-M-phase children (73.3% vs. 26.7%, P=0.011), and the proportion of high-risk children was significantly higher than that of non-high-risk children (73.3% vs. 26.7%, P=0.011). At the same time, the proportion of children with elevated serum LDH levels was significantly higher than that of children with normal values (14.3% vs. 42.9% vs. 28.6% vs. 14.3%, P=0.028). The above all prove that the high expression of CHD1 in NB plasma exosomes is positively correlated with the high malignant degree and clinical progression of NB.

[0130] In the plasma exosomes of newly diagnosed NB children, high expression of CHD1 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] In this example, siRNA was designed for the CHD1 all-transcript homologous sequence region:

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

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

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

[0137] (1) One day before transfection, well-grown transfection neuroblastoma cell line SH-SY5Y was washed, digested, resuspended, and then counted. The cells were seeded in a 6-well plate at a concentration of 1-2x10 5 cells / well, shaken, and then placed in a constant temperature incubator for 20-24 hours. When the cell density reached 50%-60% and the cell distribution in each well was even, transfection was performed.

[0138] (2) One hour before transfection, the 6-well plate was taken out of the incubator, the old culture medium was discarded, and 1 mL of fresh DMEM complete culture medium was replaced. It was placed in a constant temperature incubator for standby.

[0139] (3) Transfection using Lipofectamine RNAiMAX transfection reagent, according to the reagent instructions to system configuration. For example, a hole of a 6-well plate (operated in a biological safety cabinet). 100 μL of Opti-MEM medium was used to dilute 4 μL of 20 μM siRNA solution, and the siRNA diluent was gently mixed. 100 μL of Opti-MEM medium was used to dilute 5 μL of Lipofectamine RNAiMAX, and the RNAiMAX diluent was gently mixed.

[0140] (4) Mix the siRNA diluent and the RNAiMAX diluent, and blow and mix well, and incubate at room temperature for 20 min.

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

[0142] (6) After 6-8 hours of transfection, change the cell solution, replace each hole with 2 mL of complete culture medium, and continue to culture.

[0143] (7) After 72 hours of transfection, collect the cells for subsequent experiments.

[0144] 2. qPCR verification of CHD1 knockdown effect

[0145] (1) siRNA (siCHD1-1# and siCHD1-#2) was transfected into neuroblastoma cell line SH-SY5Y to knock down CHD1 gene, and after 72 h, the cells were lysed, total RNA was extracted and cDNA was prepared by reverse transcription. The reverse transcription reagent is from TaKaRa company, and the operation of preparing cDNA by reverse transcription is carried out according to the instructions of PrimeScriptTM RT Master Mix (Perfect Real Time) cDNA reverse transcription kit.

[0146] (2) The reagents required for real-time fluorescent quantitative PCR (qPCR) are from Bio-Rad company, and the operation is carried out according to the instructions of SYBR Green Supermix reagent, and a 20 μL reaction system is constructed.

[0147] The reaction system includes: SYBR Green (2x) 10.0 μL; cDNA 1.0 μL; upstream primer (10 μmol / L) 0.5 μL; downstream primer (10 μmol / L) 0.5 μL; sterilized water 8.0 μL. The primer sequence is as follows:

[0148] The solution was mixed well according to the above system, and after the instant separation, it was put into a real-time quantitative PCR instrument (ViiA7, Singapore ABI / VIIA7). The amplification program was set as follows: denaturation at 95°C for 10 min; 95°C for 15 sec, 60°C for 35 sec, 70°C for 30 sec, for a total of 40 cycles.

[0149] (3) The analysis of the melting curve was carried out according to the system default conditions, and the cycle number (Ct value) corresponding to the inflection point of the fluorescence signal from the background into the exponential growth period during amplification was used as an indirect indicator of the initial concentration of the template. The results were corrected according to the Ct value of the internal reference gene. The experimental results were calculated by formula (2-ΔΔCt method):

[0150]

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

[0152] ΔCt (experimental) = Ct Mean (experimental CHD1) - Ct Mean (experimental GAPDH);

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

[0154] The results are shown in Figure 5 .

[0155] 3. Western Blot verification of CHD1 knockdown effect

[0156] siRNA (siCHD1-1# and siCHD1-#2) was transfected into neuroblastoma cell line SH-SY5Y to knock down CHD1 gene. After 72 h, the cells were lysed and Western Blot was performed for detection.

[0157] CHD1 antibody (cat. sc-271626, 1:500, Santa Cruz Biotechnology), and reference protein GAPDH antibody (cat. KM9002, 1:10000, Sungene Biotech) were used to detect the expression level of CHD1 protein. A chemiluminescence kit was used, and a multicolor fluorescence and chemiluminescence imaging system (ChemiDoc MP, Singapore Bio-Rad) was used for exposure and development.

[0158] The results are shown in 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. Use of plasma exosomal protein CHD1 as a biomarker in the preparation of diagnostic preparations for high-risk neuroblastoma, characterized in that, The expression level of plasma exosome protein CHD1 is significantly increased in high-risk neuroblastoma.

2. The use of a reagent for detecting the expression level of plasma exosome protein CHD1 in the preparation of a diagnostic product for diagnosing high-risk neuroblastoma.

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

4. Use according to claim 3, characterized in that, The diagnostic product further comprises a negative control, a positive control, primers, probes or antibodies.

5. A kit for diagnosing high-risk neuroblastoma, characterized by, The kit comprises a detection reagent for detecting the expression level of plasma exosome protein CHD1 according to claim 2.

6. The kit of claim 5, wherein The detection reagent further comprises a negative control, a positive control, primers, probes or antibodies.

7. Use of an agent that inhibits expression of a CHD1 gene in the manufacture of a medicament for treating high-risk neuroblastoma, characterized in that, The reagent for inhibiting the expression of CHD1 gene is siRNA or shRNA specifically targeting CHD1 gene.

8. Use according to claim 7, characterized in that, The nucleotide sequence of siRNA is SEQ ID NO. 1 or SEQ ID NO.

2.

9. A medicament for treating neuroblastoma, characterized by comprising the compound of claim 1. It contains any one or more of the following: (1) a reagent containing CHD1 gene expression down-regulation in the body; (2) a reagent for inhibiting or blocking the expression of CHD1 gene in the body.

10. The medicament for treating neuroblastoma according to claim 9, wherein It comprises shRNA or siRNA specifically targeting CHD1 gene.

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