CN112921081B - Use of osteoarthritis-associated methylation sites - Google Patents
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Abstract
The invention discloses application of osteoarthritis-related methylation sites, wherein osteoarthritis-related differential methylation sites are screened out by a high-throughput DNA methylation sequencing analysis technology, namely a TNXB gene methylation site cg15265085 and a STOML1 gene methylation site cg03328299, and further diagnosis performance verification results show that the TNXB gene methylation site cg15265085, the STOML1 gene methylation site cg03328299 and the combination of the TNXB gene methylation site cg 65085 and the STOML1 gene methylation site cg03328299 can be used as new osteoarthritis biomarkers and have good clinical application value in osteoarthritis diagnosis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of methylation sites related to osteoarthritis, in particular to application of methylation sites cg15265085 of TNXB genes and methylation sites cg03328299 of STOML1 genes in diagnosis and treatment of osteoarthritis.
Background
Osteoarthritis (OA) is a chronic joint disease common in middle-aged and elderly people, and is also the most common cause of joint pain in middle-aged and elderly people, and is characterized by degenerative change, destruction and secondary hyperosteogeny of articular cartilage, and has the main clinical manifestations of repeated pain, swelling, gradual aggravation, joint deformity and dyskinesia of joints. DNA Methylation has been reported to have a great potential role in the development and progression of osteoarthritis (Wu Z, shou L, wang J, huang T, xu X. The Methylation Pattern for Knee and Hip Osteohritis. Front Cell Dev. 2020Nov 6), but related studies are relatively deficient, and more studies are still needed to reveal Methylation genes related to osteoarthritis and elucidate the relationship between the Methylation sites and osteoarthritis, so as to provide effective action targets for osteoarthritis diagnosis and treatment and further lay the foundation for the elucidation of the pathogenesis of osteoarthritis.
Currently, clinical diagnosis of osteoarthritis relies primarily on symptomatic signs and imaging examinations, but these diagnostic methods are insensitive to early stage patients, rendering osteoarthritis patients unable to be treated in a timely manner. With the continuous development of molecular biology, more and more researches show that various cytokines such as interleukins, transforming growth factor-beta, tumor necrosis factor-alpha, matrix metalloproteinases, insulin-like growth factors, osteopontin and the like influence the occurrence and development of osteoarthritis, but as the pathogenesis of osteoarthritis is not clear and the influencing factors are complex and changeable, the cytokines cannot be well applied to the early diagnosis, treatment and prognosis evaluation of osteoarthritis. Therefore, the research on specific biomarkers related to osteoarthritis and capable of being used for early diagnosis, treatment and prognosis evaluation of osteoarthritis has important significance for revealing the mechanism of osteoarthritis generation and development and predicting the osteoarthritis generation and development conditions. In view of the above, the differential methylation sites related to osteoarthritis are screened by a high-throughput DNA methylation sequencing analysis technology, and further diagnostic efficacy verification results show that the methylation site cg15265085 of the TNXB gene and the methylation site cg03328299 of the STOML1 gene can be used as new biomarkers of osteoarthritis.
Disclosure of Invention
Aiming at the defects existing in the prior art in the field, the differential methylation sites related to osteoarthritis are screened out by a high-throughput DNA methylation sequencing analysis technology, and further diagnosis efficiency verification results show that the methylation site cg15265085 of the TNXB gene and the methylation site cg03328299 of the STOML1 gene can be used as new biomarkers of osteoarthritis, and whether a subject suffers from osteoarthritis can be judged according to the methylation level of the methylation site cg15265085 and/or the methylation site cg03328299.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect of the invention, a kit for detecting osteoarthritis is provided.
Further, the kit comprises reagents for detecting the methylation level of at least one gene locus selected from the group consisting of: TNXB gene, STOML1 gene.
Further, the site of the TNXB gene includes cg15265085.
Further, the sites of the STOML1 gene include cg03328299.
Further, the reagents include primers and/or probes for the TNXB gene, and/or the STOML1 gene.
Further, the detection kit also comprises a reagent for detecting the reference gene;
preferably, the reagent for detecting the internal reference gene comprises a primer and/or a probe aiming at the internal reference gene.
Further, the reference genes include (but are not limited to): beta-actin, GAPDH.
Furthermore, the detection kit also comprises dNTPs and Mg 2+ Ions, DNA polymerase or DNA polymerase containing dNTPs, mg 2+ Ionic, DNA polymerase PCR system.
Further, the detection kit also comprises bisulfite or hydrazine salt.
Further, the detection kit further comprises (but is not limited to): quality control, negative control and/or kit instructions.
Further, the kit instructions describe how to use the test kit to detect the methylation level of a gene site in the TNXB gene, and/or the STOML1 gene;
preferably, the site of the TNXB gene comprises cg15265085;
preferably, the sites of the STOML1 gene include cg03328299.
The osteoarthritis detection kit can be used for detecting the methylation level of at least one gene site in TNXB gene and STOML1 gene in a biological sample of a subject, so that the osteoarthritis detection kit is applied to auxiliary diagnosis of osteoarthritis and medication guidance.
Further, the above-mentioned detection methods include (but are not limited to): methylation chips, methylation specific PCR, bisulfite sequencing, restriction enzyme analysis with sodium bisulfite, fluorometry, high throughput sequencing, and other methods that can be used to detect the level of methylation at the sites.
Furthermore, the methylation chip is a methylation specific oligonucleotide chip, the basic principle is that the DNA is treated by bisulfite to convert cytosine C which is not methylated into uracil U, then PCR amplification is carried out, the product is brought with fluorescence by fluorescence labeling, U and A are paired to generate T, and methylated C is not modified by sulfite, so that methylation information contained in the DNA is converted into DNA sequences with differences, the PCR product is hybridized with the oligonucleotide on the chip, and different gene locus methylation conditions can be measured by detecting the intensity of fluorescence signals. The first world DNA Methylation chip Human Methylation27Beadchip was introduced in 2009, and 2.7 ten thousand Methylation sites of a Human genome can be detected, and then 27K chip upgrade versions are introduced in sequence. The chip-based methylation map analysis currently includes an Agilent platform, an Illumina platform, a Roche NimbleGen platform, and the like. The most common Methylation chips in current research are Illumina Human Methylation 450K bead chip and Infinium Methylation epic bead chip (850K chip for short).
Furthermore, methylation specific PCR (MS-PCR/MSP) is a specific site Methylation detection technique. The basic principle is that a target DNA fragment is treated by bisulfite, and then two corresponding primers are designed according to the methylation and non-methylation conditions of a target site, so that the target DNA can be amplified in corresponding PCR groups according to the methylation state of the target DNA, and the result is displayed by a gel electrophoresis image. The methylation state of the site can be judged according to the PCR group with the amplified band, if the site is methylated, the band can be amplified by using a methylated primer, and if the site is not methylated, the band can be amplified by using an unmethylated primer. The method has high sensitivity, does not need special instruments, is economical and practical, and is the most widely applied detection method.
Further, bisulfite Sequencing (BS) is a method for further studying methylation of CpG islands at various sites based on MSP, and its basic principle is that Bisulfite deaminates unmethylated cytosine in DNA to uracil, while methylated cytosine remains unchanged, and PCR amplification (design of primers avoids CpG as much as possible to avoid methylation) of the desired fragment results in complete conversion of uracil to thymine. Finally, the PCR product is sequenced and compared to the untreated sequence to determine whether methylation of the CpG site has occurred. The method has high reliability and accuracy, and can be used for determining the methylation state of each CpG site in a target fragment. There are advantages over other approaches in finding key CpG sites of interest. The sequencing method takes a section of sequence without CpG points at two sides of the CpG island as a primer pairing region, so that methylated and unmethylated target sequences can be amplified simultaneously.
Further, a Combined restriction enzyme analysis (COBRA) using sodium bisulfite is a method for quantitative study of methylation level without prior knowledge of CpG sites and sample sequences, and the basic principle is to treat sample DNA with sodium bisulfite, PCR amplify a target fragment, and then digest it with a restriction enzyme that requires a CG sequence, such as BstUI (CGCG), in its recognition sequence. If C in the recognition sequence is completely methylated (5 mCG5 mCG), the C is reserved as CGCG after PCR amplification, and BstU I can recognize and cut; if C in the sequence to be detected is not methylated, the C is converted into TGTG after PCR, bstU I recognition sites are lost, and the cleavage cannot be carried out. Thus, the proportion of methylation in the original sample can be obtained after electrophoretic separation, probe hybridization and scanning quantification of the enzyme digestion product. Brena et al combined COBRA and Agilent 2100Bioanalyzer directly analyzed the enzyme digestion product, making the quantification of COBRA faster, more accurate and free of radioactive contamination.
Further, such other methods that can be used to detect the methylation level at the above-mentioned sites include (but are not limited to): pyrosequencing quantification, southern blotting, restriction landmark genome scanning, single nucleotide primer extension, cpG island microarray, single nucleotide primer extension SNUPE, mass spectrometry.
A second aspect of the invention provides a method for non-diagnostically detecting the level of methylation of a genomic locus in the TNXB gene, and/or the STOML1 gene in vitro.
Further, the method comprises the steps of:
(1) Carrying out bisulfite treatment or hydrazine treatment on a sample to be detected to obtain a treated sample to be detected;
(2) Detecting the sample to be detected processed in the step (1) by using the detection kit of the first aspect of the invention to obtain the methylation level of the gene locus in the TNXB gene and/or the STOML1 gene;
preferably, the site in the TNXB gene comprises cg15265085;
preferably, the site in the STOML1 gene includes cg03328299.
Further, the sample to be tested in step (1) includes (but is not limited to): tissue samples, body fluid samples;
further, the tissue sample comprises a tissue sample of a lesion site;
preferably, the tissue sample of the lesion site includes (but is not limited to): cartilage tissue, bone tissue, synovial tissue;
further, the bodily fluid sample includes (but is not limited to): blood, saliva, urine, serum, plasma, synovial fluid;
preferably, the sample to be tested is a tissue sample;
more preferably, the tissue is cartilage tissue;
most preferably, the tissue is knee joint cartilage tissue.
Further, the methylation level in step (2) is an average methylation value M-value.
In a specific embodiment of the present invention, the methylation site cg15265085 of the TNXB gene is selected to have an increased methylation level in osteoarthritis, and the methylation site cg03328299 of the selected STOML1 gene is selected to have a decreased methylation level in osteoarthritis.
A third aspect of the invention provides a system for detecting osteoarthritis.
Further, the detection system comprises the following modules:
(1) A detection module for detecting the methylation level of a gene locus in the TNXB gene and/or the STOML1 gene in a sample of a subject by using the method of the second aspect of the invention;
(2) The evaluation module is used for comparing the detected value of the tested subject obtained by the module (1) with the detected value of the normal sample or the normal reference value to obtain the detection result of osteoarthritis;
preferably, the site in the TNXB gene comprises cg15265085;
preferably, the sites in the STOML1 gene include cg03328299;
preferably, in module (2), when the methylation level of the cg15265085 site in the TNXB gene is increased and/or the methylation level of the cg03328299 site in the STOML1 gene is decreased, the osteoarthritis patient is determined or the risk of osteoarthritis in the subject is determined to be high.
The osteoarthritis detection system is simple and convenient to operate, the result is visual, only specific methylation sites in the TNXB gene and/or the STOML1 gene need to be directionally detected, the osteoarthritis detection system not only reduces the difficulty of osteoarthritis diagnosis, but also is simple and easy to operate, and the diagnosis cost is reduced.
The fourth aspect of the invention provides the use of a reagent for detecting the methylation level of a gene locus in a TNXB gene and/or a STOML1 gene in a biological sample for the preparation of an osteoarthritis detection kit, an osteoarthritis detection system, an osteoarthritis diagnostic reagent or diagnostic equipment;
preferably, the site in the TNXB gene comprises cg15265085;
preferably, the sites in the STOML1 gene include cg03328299;
preferably, the test kit is a test kit according to the first aspect of the invention;
preferably, the detection system is as described in the third aspect of the invention.
Further, the reagents include those used in the methods employed to detect methylation levels.
Further, the method includes (but is not limited to): methylation chips, methylation specific PCR, bisulfite sequencing, restriction enzyme analysis with sodium bisulfite, fluorometry, high throughput sequencing, and other methods that can be used to detect the level of methylation at the sites.
Further, such other methods that can be used to detect the methylation level at the above-mentioned sites include (but are not limited to): pyrosequencing quantification, southern blotting, restriction landmark genome scanning, single nucleotide primer extension, cpG island microarray, single nucleotide primer extension SNUPE, mass spectrometry.
A fifth aspect of the present invention provides a method for drug screening for prevention and/or treatment of osteoarthritis.
Further, the method comprises the step of detecting the level of methylation of a gene site in the TNXB gene, and/or the STOML1 gene of the subject;
preferably, the site in the TNXB gene comprises cg15265085;
preferably, the sites in the STOML1 gene include cg03328299.
Further, a decrease in the methylation level of the methylation site cg15265085 of the TNXB gene and/or an increase in the methylation level of the methylation site cg03328299 of the STOML1 gene, as compared to before the use of the screening drug, is indicative that the screening drug is effective in the subject.
Unless defined otherwise, 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 invention belongs. The terms used in the specification of the present invention are only for describing specific embodiments and are not intended to limit the present invention, and furthermore, some terms are explained as follows.
The term "methylation" as used herein refers to a natural modification of DNA, which mainly refers to the process of converting a methyl group to 5-methylcytosine (5-methylcytosine) by linking a carbon atom at the 5' position of cytosine in CpG dinucleotides (5-methylcytosine) under the action of methylated CpG-binding domain (MBD) and DNA methyltransferase (DNMT) in eukaryotes. DNA methylation is a more deep form of epigenetics research, has multiple biological meanings, is closely related to normal development of embryos, gene expression regulation, inactivation of X chromosomes of female individuals, inhibition of parasitic DNA sequences, stability of imprinted genes and genome structures and the like, and has remarkable progress in the aspects of research on DNA methylation mechanisms, DNA methyltransferases, methylation transcription inhibition mechanisms, relations between methylation and tumors and diseases, detection methods and the like, and the research on DNA methylation is becoming an important frontier field in disease and tumor research.
The term "and/or," as used herein, is intended to include any and all combinations of one or more of the associated listed items.
The term "test sample" as used herein refers to a nucleic acid sample to be tested, which contains a nucleic acid or nucleic acids, in which it is desired to know whether or not a target nucleic acid is present. In the present invention, the sample to be tested includes a tissue sample including cartilage tissue, bone tissue, and synovial tissue, and a body fluid sample including blood, saliva, urine, serum, plasma, and joint fluid.
The term "probe" as used herein refers to a single-stranded nucleic acid having a known nucleotide sequence, which has a nucleotide sequence structure substantially complementary to a target nucleic acid and can form a double strand with the target nucleic acid. The probe may carry a label. For example, the label can be attached to the 5 'end or the 3' end of the probe.
The term "biomarker," as used herein, refers to a molecular indicator with a specific biological property, biochemical characteristic, or other aspect, that can be used to determine the presence or absence and/or severity of a particular disease or condition.
The invention has the advantages and beneficial effects that:
from the clinical practical angle, the invention aims to solve the clinical practical problems, differential methylation sites related to osteoarthritis are screened out by a high-throughput DNA methylation sequencing analysis technology, namely a TNXB gene methylation site cg15265085 and a STOML1 gene methylation site cg03328299, the relationship between the sites and osteoarthritis is not reported at present, and further diagnosis efficiency verification results show that the TNXB gene methylation site cg15265085 and the STOML1 gene methylation site cg03328299 can be used as new biomarkers of osteoarthritis.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows a graph of the results of differential methylation site cg15265085 methylation levels in osteoarthritis patients;
FIG. 2 is a graph showing the results of differential methylation site cg03328299 methylation levels in osteoarthritis patients;
FIG. 3 shows a ROC plot of the differential methylation site cg15265085;
FIG. 4 shows a ROC plot of the differential methylation site cg03328299;
FIG. 5 shows a ROC plot of the combination of differential methylation sites cg15265085 and cg03328299.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example 1 screening for differentially methylated sites in osteoarthritis
1. GEO data retrieval
In the GEO database, the search was performed using "osteoparthritis" AND "Homo sapiens" AND "gse" as a keyword, AND the search results are shown in table 1.
TABLE 1 GEO database search results
2. Data pre-processing
383335 loci are obtained after the intersection of GSE73626 and GSE63695, and the loci are asexual chromosome loci, so that 383335 loci are still obtained after the sex chromosome loci are removed. The difference between the different datasets was large, and the quantile normalization was performed using the normaize.quantiles function of the preprocessorcore package, preserving sites with non-NA greater than 80%, again 383335.
3. Differential methylation analysis
R package COHCAP was used. The criteria for the screening were: i delta beta is >0.2, FDR is less than 0.05.
4. Functional enrichment of differentially methylated genes GO and KEGG
The differential methylation sites correspond to 69 genes, and the 69 differential methylation genes are subjected to GO functional enrichment and KEGG functional enrichment analysis by using Metascape (http:// Metascape. Org/gp/index. Html #/main/step 1). The criteria for the screening were: pvalue <0.05.
5. Feature selection with LASSO
Using the tool, R-3.3.3, R package glmnet (https:// cran.r-project. Org/web/packages/glmnet /).
6. Screening for optimal biomarkers using machine learning
Tool: random forest: r packet randomForest (https:// cran.r-project.org/web/packages/randomForest /); support Vector Machine (SVM): r package e1071 (https:// cran.r-project.org/web/packages/e 1071/index.html); decision tree: r package rpart (https:// cran.r-project.org/web/packages/rpart/index. Html).
And (3) constructing a classification model by using a random forest algorithm, and sequencing the importance of the DNA methylation sites from large to small according to the MeanDecreaAccurary value.
7. Results of the experiment
The results of the analysis showed that there were 305 differentially methylated sites in the GEO database, including 213 hypermethylated sites and 92 hypomethylated sites. 9 important sites are screened from the 305 differential expression sites obtained in the prior art by using a LASSO algorithm, a classification model is constructed by using a random forest algorithm, the 9 DNA methylation sites are subjected to importance sequencing according to the MeaneDesequeneaAccurary value from large to small, and an optimal biomarker is screened out, wherein the result graphs of the methylation levels of the differential methylation sites cg15265085 and cg03328299 in osteoarthritis patients are respectively shown in a figure 1 and a figure 2, the specific site information is shown in a figure 2, and the difference has a statistical significance (P < 0.05).
TABLE 2 detailed information on the differentially methylated sites cg15265085, cg03328299
Example 2 diagnostic Performance validation
1. Experimental methods
And (3) drawing a receiver working curve (ROC curve) by adopting R package 'pROC' (version 1.15.0) for the screened differential methylation sites, analyzing sensitivity, specificity and AUC values of the differential methylation sites to judge the diagnostic efficacy of the differential methylation sites, and verifying the methylation levels of the methylation sites cg15265085 of the TNXB gene and cg03328299 of the methylation sites cg1 of the STOML1 gene.
2. Results of the experiment
The results are shown in FIGS. 3-5 and Table 3, and indicate that the methylation site cg15265085 of TNXB gene, the methylation site cg03328299 of STOML1 gene and the combination of the two have higher diagnostic efficacy.
The above results suggest that the methylation site cg15265085 of the TNXB gene, the methylation site cg03328299 of the STOML1 gene and a combination of the two can be used in the early diagnosis and treatment of osteoarthritis.
TABLE 3 diagnostic potency of the combination of differentially methylated sites cg15265085, cg03328299, cg15265085 and cg03328299
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Claims (2)
1. The application of a reagent for detecting the methylation level of gene loci in TNXB gene and STOML1 gene in a biological sample in the preparation of an osteoarthritis detection kit;
the gene locus in the TNXB gene is cg15265085;
the gene site in the STOML1 gene is cg03328299.
2. The application of the reagent for detecting the methylation level of gene loci in TNXB gene and STOML1 gene in a biological sample in the preparation of osteoarthritis diagnostic reagents;
the gene locus in the TNXB gene is cg15265085;
the gene site in the STOML1 gene is cg03328299.
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| "cg15265085""cg03328299";CRMs;《MethBank》;20200930;第1-2页 * |
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