Application of reagent for detecting m6A modification level of FKBP4 gene in preparation of neuroblastoma diagnostic product

Abstract

The invention belongs to the field of biomedicine, and particularly relates to application of a reagent for detecting the m6A modification level of an FKBP4 gene in preparation of a neuroblastoma diagnostic product. The invention provides a primer pair for detecting the m6A modification level of a partial region of the FKBP4 gene. High-risk neuroblastoma and medium-low-risk neuroblastoma are distinguished by detecting the m6A modification level of the FKBP4 gene, wherein the m6A modification level of the FKBP4 gene in the high-risk neuroblastoma is remarkably lower than that in the medium-low-risk neuroblastoma. Through MeRIP-seq and RNA-seq combined analysis and in combination with GEO database screening, it is determined that the m6A modification level of the FKBP4 gene is closely related to the risk degree of neuroblastoma. By detecting the m6A modification level of the FKBP4 gene, high-risk neuroblastoma and medium-low-risk neuroblastoma can be accurately distinguished.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to application of a reagent for detecting m6A modification level of FKBP4 gene in preparation of a neuroblastoma diagnosis product. BACKGROUND

[0002] Neuroblastoma (NB) originates from the sympathetic nervous system and is one of the common and fatal solid tumors in children. The incidence of neuroblastoma in children is about 10.5 cases per million children, accounting for 8-10% of all childhood tumors, and the mortality rate is 2 cases per million children, accounting for about 15% of childhood cancer-related mortality. The International Neuroblastoma Risk Group (INRG) classification system classifies NB into low-risk, intermediate-risk and high-risk types according to the age of onset, histological classification, tumor differentiation, MYCN amplification and chromosomal aberration. Patients with low-risk and intermediate-risk types have better prognosis. The treatment strategy for NB is mainly based on risk stratification, combined with surgery, chemotherapy, radiotherapy, autologous stem cell transplantation and immunotherapy. The treatment of high-risk neuroblastoma children in China is divided into induction period (chemotherapy and surgery), consolidation period (sequential autologous hematopoietic stem cell transplantation and radiotherapy for primary tumor) and maintenance treatment after consolidation period (immunotherapy and 13-cis retinoic acid treatment).

[0003] Overall, although significant progress has been made in the treatment of high-risk neuroblastoma children, the long-term survival rate is still less than 50%. The pathological mechanism of NB has not been fully elucidated, although new immunotherapy drugs have significantly improved the survival of some sensitive cases, but the mortality rate of high-risk patients remains high.

[0004] At present, there is a lack of effective molecular markers in clinical practice to accurately distinguish the risk of neuroblastoma, which leads to a certain blindness in the selection of treatment options, affecting the treatment effect and patient prognosis. Therefore, it is of great significance to find a molecular marker that can accurately identify the risk of neuroblastoma for guiding clinical treatment decisions and improving treatment effectiveness. SUMMARY

[0005] Based on this, an embodiment of the present application provides application of a reagent for detecting m6A modification level of FKBP4 gene in preparation of a neuroblastoma diagnosis product.

[0006] The present application provides a reagent for detecting m6A modification level of FKBP4 gene in preparation of a neuroblastoma diagnosis product.

[0007] In some embodiments, the reagent for detecting the m6A modification level of the FKBP4 gene comprises a detection primer pair; the detection primer pair comprises one or more pairs of primers for detecting the target site.

[0008] The target site is selected from one or more of Chr12:2795949-2796358, Chr12:2796427-2796836, Chr12:2796468-2796878 and Chr12:2796719-2797129, with the reference genome being GRCh38.p14.

[0009] In some embodiments, the detection primer pair is selected from one or more pairs of primers with nucleotide sequences as shown in SEQ ID NO. 9-SEQ ID NO. 12.

[0010] In some embodiments, the detection primer pair further comprises a qRCR detection primer pair with nucleotide sequences as shown in SEQ ID NO. 13-SEQ ID NO. 14.

[0011] Another aspect of the present application provides a detection primer pair, which comprises one or more pairs of primers for detecting the target site.

[0012] The target site is selected from one or more of Chr12:2795949-2796358, Chr12:2796427-2796836, Chr12:2796468-2796878 and Chr12:2796719-2797129, with the reference genome being GRCh38.p14.

[0013] In some embodiments, the detection primer pair is selected from one or more pairs of primers with nucleotide sequences as shown in SEQ ID NO. 9-SEQ ID NO. 12.

[0014] In some embodiments, the detection primer pair further comprises a qRCR detection primer pair with nucleotide sequences as shown in SEQ ID NO. 13-SEQ ID NO. 14.

[0015] Another aspect of the present application provides a detection kit, which comprises the detection primer pair for detecting the m6A modification level of the FKBP4 gene.

[0016] In some embodiments, the detection kit further comprises an RNA methylation co-immunoprecipitation reagent.

[0017] In some embodiments, the detection kit further comprises one or more of an RNA extraction reagent, a reverse transcription reagent.

[0018] Another aspect of the present application provides a method for detecting the m6A modification level of FKBP4 gene, comprising detecting the m6A modification level of FKBP4 gene in the sample to be tested by using the detection method, the detection primer pair or the detection kit.

[0019] In some embodiments, the detection method comprises: treating the RNA of the sample to be tested by using RNA methylation immunoprecipitation method, performing SELECT PCR by using the primer pair with the nucleotide sequence shown in SEQ ID NO. 9-SEQ ID NO. 12; and performing qPCR by using the primer pair with the nucleotide sequence shown in SEQ ID NO. 13-SEQ ID NO. 14.

[0020] The present application provides a detection primer pair for detecting the m6A modification level of a partial region of FKBP4 gene, which is used to distinguish high-risk neuroblastoma from low-risk neuroblastoma by detecting the m6A modification level of FKBP4 gene, wherein the m6A modification level of FKBP4 gene in high-risk neuroblastoma is significantly lower than that in low-risk neuroblastoma. The present application determines that the m6A modification level of FKBP4 gene is closely related to the risk of neuroblastoma by joint analysis of MeRIP-seq and RNA-seq and screening of GEO database. By detecting the m6A modification level of FKBP4 gene, high-risk neuroblastoma can be accurately distinguished from low-risk neuroblastoma, and the AUC value reaches 1, which has high diagnostic value. Moreover, the detection method is simple and reliable, and provides a new molecular marker for risk classification of neuroblastoma, which is helpful for guiding clinical treatment decision and improving treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed in the embodiment description will be briefly introduced. 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 on the basis of these drawings.

[0022] Figure 1 m6A modification characteristics of neuroblastoma tissues with different risks;

[0023] Figure 2 m6A modification abundance difference gene volcano plot of neuroblastoma with different risks;

[0024] Figure 3 Distribution of differential m6A level sites;

[0025] Figure 4Schematic diagram of common m6A motif

[0026] Figure 5 Differentially expressed genes in different risk neuroblastoma

[0027] Figure 6 Differential m6A and expression genes in different risk NB by MeRIP-seq and RNA-seq combined analysis

[0028] Figure 7 Differentially expressed genes in different risk neuroblastoma by GEO database analysis

[0029] Figure 8 Four-quadrant diagram of differential genes screened by MeRIP-seq combined with RNA-seq and GEO database

[0030] Figure 9 Expression level of FKBP4 in different stage neuroblastoma

[0031] Figure 10 Schematic diagram of FKBP4 m6A differential region in different risk stratification neuroblastoma patients

[0032] Figure 11 Schematic diagram of FKBP4 m6A modification differential site

[0033] Figure 12 m6A level of FKBP4 in neuroblastoma cells

[0034] Figure 13 FKBP4 m6A modification level in peripheral blood of children with different risk neuroblastoma

[0035] Figure 14 Accuracy of FKBP4 m6A detection in differentiating different risk neuroblastoma DETAILED DESCRIPTION

[0036] The application will be described in further detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are only used to explain the present application and not used to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be realized in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained by the modifications or changes also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0038] The term

[0039] Unless otherwise indicated or unless contradicted by context, the terms or phrases used herein have the following meanings:

[0040] The term "and / or", "or / and", "and / or" used herein is a selective range including any one of two or more related listed items, and also including any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, D, i.e. includes the combination of any two or any three of A, B, C, D, and also includes the four-item combination of A, B, C, D (i.e. the technical solution connected by "logical and").

[0041] In this application, "multiple", "multiple", "multiple", "multiple" and the like are used without special limitation, which means greater than or equal to two in quantity. For example, "one or more" means one or more than two.

[0042] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more listed items.

[0043] In this application, "suitable combination", "suitable manner", "any suitable manner" and the like are described as "suitable" to implement the technical solutions of this application, solve the technical problems of this application, and achieve the intended technical effects of this application.

[0044] In this application, "further", "further", "special" and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the scope of protection of this application.

[0045] In the present application, "optionally", "optional" or "option" means that it can be present or absent, i.e. it means that it is selected from either of the two parallel schemes "present" or "absent". If there are multiple "optionally" in a technical scheme, and there is no contradictory or mutually restrictive relationship, and no special instructions, each "optionally" is independent.

[0046] In the present application, the technical features described in an open manner include both the closed technical scheme consisting of the listed features and the open technical scheme of the listed features.

[0047] In the present application, when referring to a numerical interval (i.e. a numerical range), if no special instructions are given, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no special instructions are given, when the numerical interval only points to the integers within the numerical interval, the two end point integers of the numerical range and every integer between the two end points are equivalent to directly listing every integer in this article, for example, t is an integer selected from 1 to 10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all sub-ranges therein.

[0048] In the present application, the temperature parameters, if not specifically limited, allow both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0049] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) means volume percentage, and % (w / v) means mass volume percentage.

[0050] All documents mentioned in the present application are cited by reference in the present application as if each document was individually cited by reference. Unless it conflicts with the purpose and / or technical scheme of the present application, the cited documents are cited in their entirety and for all purposes. When referring to the cited documents in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When referring to the cited documents in the present application, the examples and preferred ways of the cited relevant technical features can also be incorporated by reference into the present application, but are limited to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified according to the description in the present application.

[0051] The present application adopts MeRIP-seq to detect different risk levels of neuroblastoma tissue binding to the GEO online database analysis and screening to find that FK506 binding protein (FK506 bingding protein 4, FKBP4) is significantly higher in high-risk neuroblastoma than in low-risk neuroblastoma, and the m6A level in high-risk neuroblastoma is significantly reduced. However, the clinical detection significance of its m6A level in neuroblastoma is still unclear.

[0052] In one aspect, the application provides the use of a reagent for detecting the m6A modification level of the FKBP4 gene in the preparation of a neuroblastoma diagnosis product.

[0053] In some embodiments, the reagent for detecting the m6A modification level of the FKBP4 gene comprises a detection primer pair; the detection primer pair comprises one or more pairs of primers in the following target sites.

[0054] With GRCh38.p14 as the reference genome, the target site is selected from one or more of the following: Chr12: 2795949-2796358, Chr12: 2796427-2796836, Chr12: 2796468-2796878, and Chr12: 2796719-2797129.

[0055] In some embodiments, the detection primer pair is selected from one or more of the following: nucleotide sequences as shown in SEQ ID NO. 9-SEQ ID NO. 12.

[0056] In some embodiments, the detection primer pair further comprises a qRCR detection primer pair with a nucleotide sequence as shown in SEQ ID NO. 13-SEQ ID NO. 14.

[0057] In another aspect, the application provides a detection primer pair for the m6A modification level of the FKBP4 gene, which comprises one or more pairs of primers in the following target sites:

[0058] With GRCh38.p14 as the reference genome, the target site is selected from one or more of the following: Chr12: 2795949-2796358, Chr12: 2796427-2796836, Chr12: 2796468-2796878, and Chr12: 2796719-2797129. The change in the m6A modification level of these sites is highly related to the risk level of neuroblastoma. By detecting the m6A modification level of these specific sites, the accuracy and specificity of the detection can be further improved.

[0059] The application distinguishes high-risk neuroblastoma from low-risk neuroblastoma by detecting the m6A modification level of FKBP4 gene, wherein the m6A modification level of FKBP4 gene is significantly lower in high-risk neuroblastoma than in low-risk neuroblastoma. The application determines that the m6A modification level of FKBP4 gene is closely related to the risk of neuroblastoma by MeRIP-seq and RNA-seq joint analysis combined with GEO database screening. By detecting the m6A modification level of FKBP4 gene, high-risk neuroblastoma can be accurately distinguished from low-risk neuroblastoma, and the AUC value reaches 1, which has extremely high diagnostic value. The detection method is simple and reliable, and provides a new molecular marker for risk classification of neuroblastoma, which helps to guide clinical treatment decision and improve treatment effect.

[0060] In some embodiments, the detection is performed by SELECT PCR;

[0061] The detection primer pair is selected from one or more of the primer pairs shown in the nucleotide sequences such as SEQ ID NO. 9-SEQ ID NO. 12.

[0062] In some embodiments, the qRCR detection primer pair is further comprised of the nucleotide sequences such as SEQ ID NO. 13-SEQ ID NO. 14.

[0063] SELECT PCR primer:

[0064] Oligo1-X-forward primer:

[0065] 5’-TAGCCAGTACCGTAGTGCGTGggtcaaagagcttgtccttg-3’ (SEQ ID NO. 9).

[0066] Oligo1-X-reverse primer:

[0067] 5’ phos-agtacccttccagtgcaaCAGAGGCTGAGTCGCTGCAT-3’ (SEQ ID NO. 10).

[0068] FKBP4 forward primer:

[0069] 5’-TAGCCAGTACCGTAGTGCGTGcgctggtcaaagagcttg-3’ (SEQ ID NO. 11).

[0070] FKBP4 reverse primer:

[0071] 5' phos-ccttgtagtacccttccagtCAGAGGCTGAGTCGCTGCAT-3' (SEQ ID NO. 12).

[0072] The product was then subjected to qPCR, with primers as follows:

[0073] forward primer: 5'-ATGCAGCGACTCAGCCTCTG-3' (SEQ ID NO. 13).

[0074] reverse primer: 5'-TAGCCAGTACCGTAGTGCGTG-3' (SEQ ID NO. 14).

[0075] Another aspect of the present application provides a detection kit, which comprises the primer pair for detecting the m6A modification level of FKBP4 gene.

[0076] In some embodiments, the kit further comprises RNA methylation co-immunoprecipitation reagents.

[0077] m6A methylated RNA immunoprecipitation (MeRIP) is a method for detecting m6A modification state and locating m6A RNA modification position in the whole transcriptome. Ribo MeRIP™ m6A Transcriptome Profiling Kit uses MeRIP method for identification and transcriptome analysis of m6A RNA modification. In the MeRIP experiment, after RNA fragmentation, immunoprecipitation is performed using monoclonal antibody against m6A.

[0078] In some embodiments, the kit further comprises RNA extraction reagents, reverse transcription reagents and qPCR reagents.

[0079] Another aspect of the present application provides a method for detecting the m6A modification level of FKBP4 gene, which comprises using the detection primer pair or the detection kit to detect the m6A modification level of FKBP4 gene in the sample to be tested.

[0080] In some embodiments, the detection method comprises: using RNA methylation co-immunoprecipitation method to process the RNA of the sample to be tested.

[0081] In some embodiments, SELECT PCR is performed using primer pairs with nucleotide sequences as shown in SEQ ID NO. 9-SEQ ID NO. 12.

[0082] In some embodiments, the qPCR is performed using a primer pair comprising nucleotide sequences as set forth in SEQ ID NO. 13-SEQ ID NO. 14.

[0083] Specifically, the method comprises treating the RNA of the sample to be tested using RNA methylation immunoprecipitation, and amplifying the treated RNA using the primer pair for detecting the m6A modification level of the FKBP4 gene.

[0084] In some embodiments, the treating the RNA of the sample to be tested using RNA methylation immunoprecipitation comprises:

[0085] The steps of RNA isolation and fragmentation of the sample to be tested, enrichment of the m6A modification region, and recovery of the enriched RNA.

[0086] In some embodiments, the m6A is enriched using magnetic beads.

[0087] In some embodiments, the method further comprises performing qPCR on the amplification product to analyze the expression level.

[0088] It can be understood that the method for detecting the m6A modification level of the FKBP4 gene of the present application comprises disease diagnosis and treatment and non-disease diagnosis and treatment related application scenarios. The non-disease diagnosis and treatment scenarios include detecting the m6A modification level of the FKBP4 gene for genetic polymorphism research, etc.

[0089] In some embodiments, the sample to be tested comprises a peripheral blood sample. Peripheral blood samples are easy to collect, cause little trauma to patients, and are convenient for clinical application, providing a non-invasive or minimally invasive detection method for neuroblastoma risk classification.

[0090] Further, the correlation between the m6A modification level of the FKBP4 gene and the risk of neuroblastoma is analyzed by ROC curve, and the AUC value is 1. The AUC value of 1 indicates that the m6A modification level of the FKBP4 gene as a marker for neuroblastoma risk classification has very high diagnostic value and can accurately distinguish between high-risk and low-risk neuroblastoma.

[0091] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods not specified in the following examples are preferred to refer to the guidelines given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturer, or by referring to the known experimental methods in the art.

[0092] In the following detailed description, the amount of raw material components are described in terms of measurement parameters, which can have slight variations within the range of weighing accuracy unless otherwise specified. In terms of temperature and time parameters, acceptable variations are allowed due to instrument testing accuracy or operating accuracy.

[0093] It should be understood that the magnitude of the serial number of the above processes does not mean the order of execution in various embodiments of the present application, and the execution order of the processes should be determined according to its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0094] Example 1

[0095] I. Screening genes with differential m6A levels in high-risk and low-to-intermediate risk NB using MeRIP-seq

[0096] The total RNA of the sample was separated and purified by TRIzol (Invitrogen, CA, USA) according to the operation scheme provided by the manufacturer. Then the amount and purity of the total RNA were controlled by NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The integrity of the RNA was detected by Bioanalyzer 2100 (Agilent, CA, USA), and verified by the scheme of agarose electrophoresis. The concentration was >50 ng / μL, the RIN value was >7.0, OD260 / 280 was >1.8, and the total RNA was >1 μg, which met the downstream experiment. The magnesium ion breaking kit (Magnesium RNA Fragmentation Module, item number E6150S, USA) was added to the total RNA for fragmentation treatment at 94℃ for 5 min. The m6A antibody (No. 202003, Synaptic Systems, Germany) was pre-mixed with the immunomagnetic beads Dynabeads Antibody Coupling Kit (Thermo Fisher, CA, USA), and the pre-mixed m6A-immunomagnetic beads were immunoprecipitated (IP) with the fragmented RNA (containing ribosomal RNA fragments), thereby obtaining the IP product.

[0097] The IP product was reverse transcribed by SMARTScribe Reverse Transcriptase (Clontech, CA, USA) to obtain the cDNA product. TM ​One strand cDNA was synthesized under the catalysis of reverse transcriptase (CloneTech, Cat No. 634414, Japan), and then a linker was added for the first round of PCR: pre-denaturation at 94°C for 1 min, denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 68°C for 30 s, and finally extension at 68°C for 2 min for a total of 5 cycles to synthesize double-stranded DNA. The amplified DNA library was purified using purification beads. R-Probes v2 probes (for mammals) and ZapR v2 enzyme were added to the DNA library to remove ribosomal RNA reverse-transcribed cDNA sequences, incubated at 72°C for 2 min, 4°C for 2 min, 37°C for 1 h, 72°C for 10 min, and finally 4°C. The final library was amplified by a second round of PCR, with the same PCR program as the first round, and the number of cycles increased to 12-16.

[0098] Finally, the Illumina Novaseq TM 6000 (LC Bio Technology CO., Ltd. Hangzhou, China) was sequenced according to the standard operation, and the sequencing mode was PE150. The RNA-seq data was processed by bioinformatics analysis process. First, the raw sequencing data was quality controlled by fastp software, and the low-quality reads were discarded to obtain high-quality data. Then, HISAT2 was used to align the quality-controlled data to the reference genome to generate an alignment file. On this basis, exomePeak2 software was used for Peak calling analysis and identification of differential Peak regions. ANNOVAR was used for functional annotation. At the same time, in order to further explore the regulation mechanism of the peak region, MEME and HOMER software were used for motif analysis to identify potential transcription factor binding sites. StringTie was used for transcript assembly and gene expression quantification to obtain FPKM values. Finally, based on the edgeR software, differential expression analysis was performed, and |log2FC|≥1 and P<0.05 were used as the standard to screen for significantly differentially expressed genes.

[0099] The results show that the differential m6A level genes in 2 cases of high-risk neuroblastoma, 4 cases of low-risk neuroblastoma and 2 cases of medium-risk neuroblastoma were analyzed by (MeRIP-Seq). It was found that the tissue samples of high-risk patients showed a significant trend of reduced m6A modification level, and the modification peak was significantly lower than that of low-risk and medium-risk groups (see Figure 1 ). Screening found that there were 841 genes with increased m6A levels and 656 genes with decreased m6A abundance (see Figure 2). The differential m6A level sites were mainly located in 3'UTR region (46.14%) and exon region (32.74%), and also in 5'UTR (16.70%), intron (4.12%) and intergenic region (0.30%) (see Figure 3 ) In high-risk neuroblastoma tissues, the specific motif of m6A modification, in which the occurrence frequency of three motifs of GGACU, CAUACUGGAG and CCGAAGA was significantly higher than that of other sequences (see Figure 4 ).

[0100] II. Screening of differentially expressed genes in high-risk and low-risk NB by transcriptome sequencing (RNA-seq)

[0101] The mRNA was enriched and purified by oligo(dT) magnetic beads, and the mRNA was fragmented by breaking reagent to obtain mRNA fragments of appropriate length. In the cDNA synthesis stage, the first and second strand cDNA were synthesized by two-step method, and the cDNA ends were repaired and A-tailed to prepare for subsequent adapter ligation. Then, adapter ligation and PCR amplification were performed to construct a complete library. The PCR amplification product was converted into single-stranded state by heat denaturation, and then intramolecular circularization reaction was performed.

[0102] DNA nanoballs (DNB) were formed by the replication process of single-stranded circular DNA molecules, and the formed DNA nanoballs were accurately loaded into the micropore structure of the chip using high-density DNA nanochip technology. Finally, the combined probe anchor polymerization technology (cPAS) mode was selected.

[0103] The initial sequencing results were subjected to multiple quality control processes using SOA Pnuke (v1.5.2) tool. The clean data after quality control was accurately aligned with the reference genome using HISAT and Bowtie2. After obtaining the alignment results, the gene expression level was quantitatively calculated using RESM software. DESeq was used for intragroup differential gene analysis with the conditions of FoldChange≥2 and Pvalue<0.05. PossionDis was used for intergroup differential analysis with the conditions of Fold Change≥2 and Pvalue<0.05.

[0104] The results showed that: by performing transcriptome sequencing (RNA-Seq) on 8 neuroblastoma samples, 2690 differentially expressed genes were screened, of which 1388 were down-regulated and 1302 were up-regulated (see Figure 5 ).

[0105] III. Intersect analysis of differentially m6A modified genes and differentially expressed genes screened by MeRIPseq and RNAseq to screen target genes

[0106] The differentially expressed genes screened in the above two parts were analyzed by R (ggplot2) package.

[0107] The results showed that: the intersection of MeRIP-seq and RNAseq data found 72 common significantly changed genes. These intersection genes showed different expression patterns in the four quadrant distribution map: 16 genes with increased transcription level and decreased m6A modification, 30 genes with increased transcription level and m6A modification, 6 genes with decreased transcription level and m6A modification, and 20 genes with decreased transcription level but increased m6A modification (see Figure 6 ).

[0108] Four, analysis of differentially expressed genes in NB tissues in GEO database by bioinformatics method, further screening of m6A abundance and expression difference genes in NB tissues

[0109] The NB tissue RNAseq sequencing data set in GEO database was screened, and the samples were divided into low-risk and high-risk groups according to the risk degree. The R language DESeq2 package was used for differential gene analysis, and the screening standard was set as |log2FC|>2 and P<0.05, so as to define the significant differential genes between the two groups. Then the expression difference genes were analyzed by intersection analysis with the genes screened in 5.1.3 to determine the target genes.

[0110] The two data sets GSE45547 and GSE62419 were integrated and analyzed. The R language platform was used for statistical processing of the combined data, and finally 635 genes with significant expression difference were screened.

[0111] The results showed that: the expression level of 547 genes was increased, while the expression level of 88 genes was decreased (see Figure 7 ). By intersecting the differentially expressed genes screened and the m6A modification and expression difference genes screened by MeRIP-seq combined RNA-seq, 22 significantly different genes were finally determined, including FKBP4 (see Figure 8 ).

[0112] Five, analysis of expression difference of target genes in different stages of NB tissues in GEO data set

[0113] The GSE45547 data set in GEO database was screened, and the samples were divided into 1, 2 stage and 3, 4 stage, two groups according to the clinical stage. The R language DESeq2 package was used for differential analysis of FKBP4 expression difference in the two groups, and the screening standard was set as |log2FC|>2 and P<0.05. The expression of FKBP4 in clinical 1, 2 stage NB was significantly lower than that in clinical 3, 4 stage NB (seeFigure 9 ).

[0114] VI. Determining the site and abundance of m6A modification of the target gene in cultured neuroblastoma cells by MeRIP-qPCR

[0115] (1) Cell culture

[0116] Culture SK-N-SH, SH-SY5Y, SK-N-AS, BE-(2), SK-N-DZ and other neuroblastoma cell lines, SK-N-SH cells are cultured in EMEM medium, SH-SY5Y and BE-(2) cells are cultured in DMEM / F12 medium, SK-N-AS cells are cultured in RPMI 1640 medium, and SK-N-DZ cells are cultured in DMEM medium, with a serum concentration of 10%. All cells are cultured under standard culture conditions (37°C, 5% CO2).

[0117] (2) Total RNA extraction

[0118] Total RNA extraction: 0.25% trypsin (Thermo Fisher, C25200072) was used to digest 5 kinds of cells, then phosphate buffered saline (PBS) was used for washing, and the cells were separated by centrifugation. Then, 1 milliliter of Trizol Reagent (Invitrogen, 15596018) was used to lyse the cells, and 200 μL of chloroform was added for layering, 4°C, 12000g, 15min centrifugation. The supernatant was transferred to a fresh EP tube and mixed with an equal amount of isopropanol, shaken gently and then left to stand for 5 min. After centrifugation at 4°C, 12000 x g for 10 min, the isopropanol was discarded, 500 μL of 75% ethanol was added to wash the precipitate, and the impurities were removed by centrifugation at 4°C, 12000 x g for 5 min. Then the precipitate was placed in a clean environment to dry. Finally, the purified RNA was dissolved in an appropriate amount of enzyme-free water, placed on ice and repeatedly blown with a pipette tip until the precipitate was completely dissolved. The RNA concentration was measured using a microplate reader, and the RNA concentration was recorded on the tube wall. Continue to the next step or store in a -80°C refrigerator.

[0119] (3) RNA methylation immunoprecipitation (MeRIP)

[0120] The experiment was performed according to the instructions of riboMeRIP m6A Transcriptome Profiling Kit (C11051-1, Ribobio, Shanghai), as follows:

[0121] 1) RNA isolation and fragmentation

[0122] A. After removing the culture medium of the cultured cells, wash 3 times with PBS, and use the Trisol method to separate total RNA. Dilute the obtained RNA with RNase-free water to a concentration of 1 μg / μL. Take 18 μL (about 18 μg) of total RNA and transfer it to a 200 μL PCR tube, add 2 μL of 10×RNA fragmentation buffer, and shake to mix. A total of 5 tubes are prepared, and the total amount of RNA for each batch of reaction is 90 μg.

[0123] B. Preheat the PCR instrument to 70°C, place the prepared fragmentation reaction system therein, and react for 7 minutes to fragment the RNA into fragments of about 200 nt. After the reaction is completed, quickly transfer the PCR tube to ice, add 2 μL of 0.5M EDTA to terminate the reaction, and shake to mix. If the number of reaction tubes exceeds 5, perform batch processing, 5 tubes per batch, to ensure the fragmentation efficiency.

[0124] C. Add water to the reaction products of the same batch to a total volume of 180 μL, and transfer them to a 1.5 mL EP tube. Add 18 μL of 3M sodium acetate, 1 μL of glycogen, mix, wherein the concentration of glycogen is 20 mg / mL, add 600 μL of pre-cooled anhydrous ethanol, and stand in a -20°C refrigerator for 12-15 hours.

[0125] D. Centrifuge the precipitate at 12000×g at 4°C for 30 minutes, then remove the supernatant, add 800 μL of -20°C pre-cooled 75% ethanol, centrifuge again at 12000×g at 4°C for 5 minutes, remove the supernatant, and repeat the above steps once.

[0126] E. After the RNA is briefly dried, dissolve it with 50 μL of nuclease-free water, and detect the fragment size by 1.5% concentration gel electrophoresis. Take 1 / 10 of the fragmented RNA as input RNA, and store it at -80°C for use.

[0127] 2) Preparation of m6A magnetic beads

[0128] A. Prepare 1×IP Buffer: Mix 5×IP Buffer with nuclease-free water at a ratio of 1:4, invert thoroughly to mix, and place on ice for standby.

[0129] B. Place the magnetic beads A / G in a small centrifuge and centrifuge for 5 seconds, then gently blow them with a pipette to ensure that the magnetic beads are completely resuspended.

[0130] C. For each reaction, 250 μL of 1×IP Buffer and 25 μL of magnetic beads A / G are required. After gentle blowing and mixing, place the mixture on a magnetic column for 1 minute, discard the supernatant with a pipette, and wash a total of twice.

[0131] D. Each reaction system requires 100 μL of 1x immunoprecipitation buffer, and 5 μg of anti-m6A antibody, placed at room temperature, and mixed well with a rotating mixer for 30 minutes; remove and place in a small centrifuge for instantaneous separation, and then use a magnetic column to absorb the liquid to clarify, and remove the supernatant.

[0132] E. After removing the centrifuge tube from the magnetic column, 500 μL of 1x immunoprecipitation buffer is added, and the mixture is mixed well several times with a pipette to ensure thorough mixing. The mixture is then transferred to the magnetic column again, and the supernatant is removed after observing the liquid clarification. A total of 3 washes are performed, and as much residual liquid as possible is removed during each wash. The centrifuge tube is removed from the magnetic column, covered with a cap, and placed on ice for standby, and then subsequent experimental steps are continued.

[0133] 3) Immunoprecipitation

[0134] A. Fragmented RNA is added to a total volume of 395 μL with enzyme-free water, and MeRIP reaction solution is prepared according to Table 1 below

[0135] Table 1

[0136] Fragmented RNA in Nuclease free water 395 μL RNase Inhibitor 5 μL 5x IP Buffer 100 μL Total 500 μL

[0137] B. 500 μL of MeRIP reaction buffer is slowly added to the pre-treated anti-m6A magnetic bead suspension, and the mixture is gently blown and sucked several times to ensure that the magnetic beads are fully dispersed.

[0138] C. The mixture is continuously stirred at 4°C for 2 hours in a rotating mixer to ensure complete reaction.

[0139] D. The liquid is precipitated to the bottom of the tube by brief centrifugation, and then the centrifuge tube is placed on the magnetic column for adsorption for 1 minute. The supernatant is carefully removed to avoid disturbing the magnetic beads.

[0140] E. The centrifuge tube is removed from the magnetic column, 500 μL of 1x IP buffer is added, and the magnetic beads are fully resuspended by gentle blowing and sucking. The mixture is placed on the magnetic column again for adsorption for 1 minute, and the supernatant is removed.

[0141] F. The above washing operation is repeated twice, a total of 3 washes are completed, and finally the sample is stored on ice for standby.

[0142] 4) Elution and RNA recovery

[0143] A. Elution buffer is prepared according to Table 2 below

[0144] Table 2

[0145] 5x IP Buffer 45 μL m6A (20 mM) 75 μL RNase Inhibitor 3.5 μL Nuclease free water 101.5 μL Total 225 μL

[0146] B. 100 μL of elution buffer is added to the sample that has completed the IP reaction, and the mixture is mixed well and resuspended by gentle blowing.

[0147] C. Place the sample in a thermomixer at 4°C for 1 hour.

[0148] D. Perform a brief centrifugation to collect the liquid at the bottom of the tube, then use the magnetic column to adsorb for 1 minute.

[0149] E. Carefully transfer 100 μL of the supernatant to a new 1.5 mL centrifuge tube, avoiding the absorption of magnetic beads.

[0150] F. Remove the centrifuge tube from the magnetic column, repeat steps 2-4, and combine the two eluents, with a final volume of 200 μL.

[0151] 5) Micro RNA extraction

[0152] A. Add an equal volume of phenol:chloroform:isopropanol (125:24:1) to the combined eluents, mix well, and incubate on ice for 5 minutes.

[0153] B. Centrifuge at 14800 rpm, 4°C for 15 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0154] C. Add 30 μL of sodium acetate (pH 5.5) to each tube, and mix well.

[0155] D. Add 10 μg of glycogen, mix, and add 3 volumes of anhydrous ethanol, mix well, and incubate at -80°C overnight for precipitation.

[0156] E. Centrifuge at 15000 x g, 4°C for 30 minutes, and discard the supernatant.

[0157] F. Wash the precipitate with 1 mL of 75% anhydrous ethanol, centrifuge at 15000 x g, 4°C for 15 minutes, and discard the supernatant.

[0158] G. Dissolve the precipitate with an appropriate amount of enzyme-free water, and determine the RNA concentration.

[0159] 6) Reverse transcription and real-time fluorescent quantitative PCR (qRT-PCR)

[0160] Use the PrimeScript™ RT kit from TAKARA for reverse transcription.

[0161] A. Prepare the reagents according to the following Table 3

[0162] Table 3

[0163] gDNA Eraser 1 μL 5x gDNA Eraser Buffer 2 μL Total RNA 1 μg RNase Free ddH2O Up to 10.0 μL

[0164] Reaction conditions: 42°C, 2 min.

[0165] B. Add the reagents prepared according to the following Table 4 to the tube from Step 1.

[0166] Table 4

[0167] Prime Script RT Enzyme Mix I 1 μL RT Primer Mix 1 μL 5x Prime Script Buffer 4 μL RNase Free ddH2O 4 μL

[0168] Reaction conditions: 37°C, 15 min; 85°C, 5 s; 4°C, 5 min.

[0169] C. Real-time fluorescence quantification

[0170] After reagents were prepared according to Table 5 below, 95°C, 30 s; then 95°C, 5 s, 60°C, 30 s, 40 cycles were performed.

[0171] Table 5

[0172]

[0173]

[0174] According to the MeRIP-seq results, the positions of FKBP4 m6A abundance change on chromosome 12 are 2795949-2796358, 2796427-2796836, 2796468-2796878 and 2796719-2797129 (see Figure 10 ).

[0175] According to the m6A abundance change site, the MeRIP-qPCR primer was designed (see Figure 11 ), as follows:

[0176] MeRIP-qPCR primer sequence:

[0177] m6AF1 5'-GAAGAGGAAGATGGCGGAATC-3' (SEQ ID NO. 1).

[0178] m6AR1 5'-AGTACCCTTCCAGTGCAACCT-3' (SEQ ID NO. 2).

[0179] m6AF2 5'-GCTATCGTGGAGGTTGCACTG-3' (SEQ ID NO. 3).

[0180] m6AR2 5'-CTGCCAAAAGCATAGCTGGGC-3' (SEQ ID NO. 4)

[0181] m6AF3 5'-GTGTACCTCAAGCCCAGCTAT-3' (SEQ ID NO. 5)

[0182] m6AR3 5'-AAGACTCCTTGGCCTTTTCAA-3' (SEQ ID NO. 6)

[0183] m6AF4 5'-TCCAATGAGGAAGCACAGAAA-3' (SEQ ID NO. 7)

[0184] m6AR4 5'-TGTCCAGTTCTAGGGCCTTGT-3' (SEQ ID NO. 8)

[0185] The m6A modification levels of four regions in neuroblastoma cell lines SH-SY-5Y, SK-N-SH, BE-(2), SK-N-DZ, and SK-N-AS were detected by MeRIP-qPCR. With SH-SY-5Y as the reference, SK-N-SH had higher m6A modification levels in the four regions, BE-(2) almost could not detect m6A modification, SK-N-DZ had higher m6A modification than the control cells. While SK-N-AS had no significant difference with SH-SY-5Y (see Figure 10 ). Figure 12

[0186] Seven, the level of m6A modification of the target gene in the peripheral blood of neuroblastoma children was detected by Select PCR

[0187] The FKBP4 m6A levels of 20 high-risk neuroblastoma patients and 30 low-risk neuroblastoma patients were detected by SELECT PCR. It was found that the FKBP4 m6A level of high-risk neuroblastoma patients was significantly lower than that of low-risk neuroblastoma patients (P<0.001) (see Figure 13 ).

[0188] The SELECT PCR primers are shown in Table 6 as follows:

[0189] Table 6

[0190]

[0191]

[0192] The product was then subjected to qPCR, and the primers were as follows:

[0193] forward primer: 5'-ATGCAGCGACTCAGCCTCTG-3' (SEQ ID NO. 13).

[0194] reverse primer: 5'-TAGCCAGTACCGTAGTGCGTG-3' (SEQ ID NO. 14).​

[0195] (1)Peripheral blood RNA extraction

[0196] A, Collect high-risk, low-risk neuroblastoma patients peripheral blood, after receiving the specimen, centrifugation, 3500 rpm, 10 min, 4℃.

[0197] B, transfer the upper plasma, then add the bottom red blood cells to the 15 mL centrifuge tube containing 8 mL red cell lysis solution (ACK), mix well, centrifugation 800 rpm, 10 min.

[0198] C, remove supernatant, add 3 mL ACK, mix well.

[0199] D, centrifugation 800 rpm, 5 min, remove supernatant.

[0200] E, add 3 mL PBS, mix well, centrifugation, 1500 rpm, 5 min.

[0201] F, remove supernatant, add 1 mL Trisol.

[0202] G, the rest of the steps are the same as total RNA extraction

[0203] (2)SELECT PCR

[0204] The reaction system is shown in Table 7:

[0205] Table 7

[0206] RNA 1500 ng up primer 40 nm down primer 40 nm dNTP 5 μm 1x custmat buffer Total volume 17 μL

[0207] Reaction conditions: 90℃, 1 min, 80℃, 1 min, 70℃, 1 min, 60℃, 1 min, 50℃, 1 min, 40℃, 6 min.

[0208] Prepare 0.01 U Bst 2.0 DNA polymerase, 0.5 U SpinR ligase, 10 nM ATP, and add water to 3 μL.

[0209] Reaction conditions: 40℃, 20 min, 80℃, 20 min.

[0210] Real-time fluorescence quantification was performed. With Oliogo X as Input, the target gene as IP, and the ΔCT value obtained by subtracting Input from IP, 2 -ΔΔCT , which is the relative level of m6A.

[0211] Eight, calculate the AUC area to determine the diagnostic value of the m6A modification level of the target gene in neuroblastoma

[0212] ROC curve drawing and result interpretation: the receiver operating characteristic (ROC) curve was drawn by using the pROC package (version 1.18.5) in R language, and the AUC was calculated to evaluate the diagnostic ability of all candidate indicators.

[0213] ROC curve interpretation: the closer the curve is to the upper left corner, the better the diagnostic performance; the diagonal line represents random guessing with no diagnostic value. AUC value interpretation: 0.5-0.7: low diagnostic value; 0.7-0.9: moderate diagnostic value; 0.9: high diagnostic value.

[0214] The receiver operating characteristic (ROC) curve of FKBP4 m6A levels in neuroblastoma patients with different risk stratifications was drawn by the R language pROC package (version 1.18.5), and the AUC was analyzed.

[0215] The results suggest that the AUC value is 1, and the FKBP4 gene m6A modification level has high diagnostic value in neuroblastoma (see Figure 14 ).

[0216] The above-described examples only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the scope of patent protection. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of protection of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained are also within the scope of protection of the present application. It should also be understood that those skilled in the art can obtain technical solutions on the basis of the technical solutions provided by the present application through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the patent of the present application should be based on the contents of the appended claims, and the specification can be used to explain the contents of the claims.

Claims

1. Use of a reagent for detecting m6A modification level of FKBP4 gene in the preparation of a neuroblastoma diagnosis product.

2. Use according to claim 1, characterized in that, The reagent for detecting m6A modification level of FKBP4 gene comprises a detection primer pair; the detection primer pair comprises one or more pairs of primer pairs for detecting the following target sites: The target sites are selected from one or more of Chr12: 2795949-2796358, Chr12: 2796427-2796836, Chr12: 2796468-2796878 and Chr12: 2796719-2797129, with GRCh38.p14 as the reference genome.

3. Use according to claim 2, characterized in that, The detection primer pair is selected from one or more of the primer pairs with nucleotide sequences as shown in SEQ ID NO. 9-SEQ ID NO.

12.

4. Use according to claim 3, characterized in that, The detection primer pair further comprises a qRCR detection primer pair with nucleotide sequences as shown in SEQ ID NO. 13-SEQ ID NO.

14.

5. A detection primer pair, characterized in that, The detection primer pair comprises one or more pairs of primer pairs for detecting the following target sites: The target sites are selected from one or more of Chr12: 2795949-2796358, Chr12: 2796427-2796836, Chr12: 2796468-2796878 and Chr12: 2796719-2797129, with GRCh38.p14 as the reference genome.

6. The detection primer pair of claim 5, wherein, The detection primer pair is selected from one or more of the primer pairs with nucleotide sequences as shown in SEQ ID NO. 9-SEQ ID NO.

12. Optionally, the detection primer pair further comprises a qRCR detection primer pair with nucleotide sequences as shown in SEQ ID NO. 13-SEQ ID NO.

14.

7. A test kit characterized in that, The detection kit comprises the detection primer pair of any one of claims 5-6.

8. The test kit according to claim 7, characterized in that The detection kit further comprises an RNA methylation immunoprecipitation reagent; Optionally, the detection kit further comprises one or more of an RNA extraction reagent, a reverse transcription reagent.

9. A method for detecting the m6A modification level of FKBP4 gene, characterized in that, The detection method comprises detecting the m6A modification level of FKBP4 gene in the sample to be tested using the detection primer pair of 5 or 6 or the detection kit of claim 7 or 8. 10.The method for detecting the m6A modification level of FKBP4 gene according to claim 9, characterized in that, The detection method comprises: treating the RNA of the sample to be tested using an RNA methylation immunoprecipitation method, performing SELECT PCR using the primer pair with nucleotide sequences as shown in SEQ ID NO. 9-SEQ ID NO. 12; and performing qPCR using the primer pair with nucleotide sequences as shown in SEQ ID NO. 13-SEQ ID NO. 14.

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