CN107164532B - Application of DNA binding site CTCF-94 of multifunctional transcription regulatory factor CTCF - Google Patents
Application of DNA binding site CTCF-94 of multifunctional transcription regulatory factor CTCF Download PDFInfo
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Abstract
The invention discloses a new application of a DNA binding site CTCF-94 of a multifunctional transcription regulatory factor CTCF, namely the application of the DNA binding site CTCF-94 in preparing a kit for early diagnosis of colorectal tumors, wherein the nucleotide sequence of the binding site is shown as SEQ ID NO. 1; the accuracy of the CTCF binding site provided by the invention for detecting colorectal adenoma and colorectal carcinoma is obviously higher than that of the reported DNA methylation molecular marker; moreover, the DNA binding site of CTCF can be used as a biomarker for monitoring the treatment effect of tumors, evaluating the prognosis effect and typing tumor molecules (such as CIMP typing). In a word, the invention provides a new idea for searching more effective molecular markers for early diagnosis of tumors, monitoring of treatment effects, evaluation of prognosis effects and molecular typing, and has important significance for prevention and treatment of tumors.
Description
Technical Field
The invention discloses an application of a DNA binding site CTCF-94 of a multifunctional transcription regulatory factor CTCF.
Background
Colorectal cancer is one of the most prevalent cancers with morbidity and mortality in the world, caused by genetic and epigenetic changes that accumulate in colonic epithelial cells; approximately 1235108 people are diagnosed as colorectal cancer each year, and approximately 609051 people die each year; the world health organization estimates that by 2030, the true diagnosis disease case of the colorectal cancer will be increased by 77 percent, the death case will be increased by 80 percent, the morbidity and the death number of the colorectal cancer in China are increasing year by year along with the rapid development of economy, and the prevention and treatment situation of the colorectal cancer is very severe.
Most colorectal cancers are currently developed from adenomas, and the time to transition from adenomas to malignancies is approximately 10-15 years, i.e., we have 10-15 years of time to discover and resect adenomas to prevent their malignancy before transitioning to malignancy. Currently, the most effective method for reducing the incidence and mortality of colorectal cancer is the early diagnosis of colorectal cancer; because patients do not have obvious clinical symptoms in early colorectal cancer, many patients are diagnosed and are in the late stage of colorectal cancer, but the treatment of the patients is most effective in the early stage of the colorectal cancer; colorectal cancer can be detected before canceration occurs or in early canceration through early diagnosis, and then effective and systematic treatment is carried out on patients as early as possible, so that the morbidity and mortality of colorectal cancer are effectively reduced; early diagnosis is mainly aimed at a large number of asymptomatic people, so an ideal early diagnosis technology should be a detection method which is simple to operate, low in cost, non-invasive to patients and high in specificity and sensitivity. The most accurate early diagnosis method of colorectal cancer in clinical application at present is colonoscopy, which has the disadvantages of higher examination cost, more complex operation procedure and more rejection of patients, and is not suitable for large-scale popularization and application in the general population. Another clinically common method is fecal occult blood test, which is relatively inexpensive and simple to operate, but has the greatest disadvantage of low sensitivity and specificity.
Epigenetic analysis shows that all colorectal cancers have abnormal DNA methylation of hundreds to thousands of genes, wherein the abnormal methylation of a part of genes is closely related to the onset of the colorectal cancers; at present, the most effective method for early diagnosis of colorectal cancer based on blood is to use DNA methylation of SEPT9 gene promoter region as biomarker, and by using the method for early diagnosis of colorectal cancer, 90% sensitivity and 88% specificity can be obtained, but the method has low sensitivity and specificity to adenoma; in addition, early colorectal cancer screening studies in asymptomatic populations over 50 years of age (7941) showed only 35%, 63%, 46% and 77.4% sensitivity to I-IV colorectal cancer, respectively, although specificity could reach 91.5%. Sensitivity to adenomas is more than 11.2%; the most successful early colorectal cancer diagnosis method based on feces is to take DNA methylation of promoter regions of 4 genes (BMP3, NDRG4, vimentin and TFPI2) as a biomarker and simultaneously detect KRAS gene mutation in feces DNA and hemoglobin content in feces, wherein the method has the sensitivity of 85 percent on colorectal cancer and 54 percent on adenoma larger than 1cm on the premise that the specificity is 90 percent; no particularly ideal diagnosis technology is suitable for wide clinical popularization and application no matter a blood-based method or a feces-based method.
It is noted that the previous studies basically used DNA methylation of specific gene promoter regions as biomarkers for early diagnosis of colorectal cancer, and few people focused on transcriptional regulatory regions other than the promoter regions. The promoter of a gene, like a "switch," determines the activity of the gene, controlling the initiation time and the extent of expression of the gene transcription. However, transcription of a gene is also affected by a transcription regulatory region other than a promoter region, and the initiation time and the degree of expression of gene transcription are also determined in many cases, and a transcription regulatory region other than a promoter region of a gene should be paid the same attention.
CTCF is a multifunctional transcription factor widely present in eukaryotes, a highly conserved multi-zinc-finger, DNA-binding nucleoprotein in evolution. CTCF can selectively recognize various DNA sequences through different combinations of zinc finger structures of the CTCF, forms different CTCF-DNA complexes, plays a role in regulating and controlling gene expression, and has various biological functions of promoter inhibition and activation, gene silencing, enhancer blockage, gene imprinting regulation, X chromosome inactivation and the like. The DNA binding site of CTCF is ubiquitous in the human genome, ranging from about 5 to 6 ten thousand, and each site recognizes about 34 nucleotides; at present, no report that the DNA binding site of the multifunctional transcription regulatory factor CTCF is used as a biomarker for early diagnosis of colorectal tumors is found.
Disclosure of Invention
The invention aims to provide a new application of a DNA binding site CTCF _94 of a multifunctional transcription regulatory factor CTCF, namely the application of the DNA binding site CTCF _94 of the multifunctional transcription regulatory factor CTCF in preparing a kit for early diagnosis, screening and disease risk prediction of colorectal tumors, wherein the nucleotide sequence of the binding site is shown as SEQ ID NO. 1.
The detection kit comprises a conventional genomic DNA extraction reagent, a bisulfite treatment reagent of methylated DNA, a reagent required for DNA methylation detection and a primer pair, wherein the primer pair can amplify a sequence shown in bisulfite converted methylated or unmethylated SEQ ID NO:1 and carry out subsequent DNA methylation detection on the amplified sequence by a DNA mass spectrometry method. This binding site CTCF 94 exhibits a hypermethylated state in colorectal tumor cells and a hypomethylated state in normal tissues.
The sequences of the primer pair are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
The application takes the DNA binding site of the multifunctional transcription regulatory factor-CTCF as a biomarker for early diagnosis of colorectal tumors for the first time. First, by analyzing the published data of DNA binding sites of CTCF in the human genome and combining the published data of DNA methylation in the human genome, we screened that there was a significant correlation between the binding pattern of CTCF at 121 sites and the DNA methylation state, i.e. the degree of DNA methylation at these sites was inversely correlated with how much CTCF binds. The 121 CTCF binding sites are specific in tumor cell lines and there is a significant correlation between their binding pattern and DNA methylation status. In the second step, the DNA methylation level of the 121 sites is detected in 33 colorectal tumor tissue samples and corresponding normal samples by a high resolution melting curve method (HRM), and 23 sites with tumor specificity in methylation patterns are screened out. Since the HRM method cannot perform accurate quantitative analysis in the application of detecting DNA methylation, the advantages and disadvantages of the 23 selected sites cannot be further compared, and the subsequent DNA methylation detection is completed by a DNA mass spectrometry; and thirdly, detecting the DNA methylation levels of 23 sites in 20 colorectal tumor tissue samples and corresponding normal samples by using a DNA mass spectrometry method, and screening 10 sites with the best performance from the samples by expanding the samples according to the performances of the 23 sites in distinguishing the tumor tissues from the normal tissues. Fourthly, detecting the DNA methylation level of 10 sites in 70 colorectal tumor tissue samples and 20 normal samples by using a DNA mass spectrometry method, and selecting 5 sites with best performance in a large sample for verification according to the performances of the 10 sites on distinguishing the tumor tissue from the normal tissue. Fifthly, detecting the DNA methylation level of 5 sites in 295 colorectal tumor tissue samples and 84 normal samples by using a DNA mass spectrometry method, and finally screening and identifying the CTCF binding site for the molecular marker for early diagnosis of colorectal tumors. The site shows a hypermethylated state in colorectal tumor cells and a hypomethylated state in normal tissues, and the fact that the DNA methylation of the CTCF binding site CTCF-94 is a molecular marker for early diagnosis of colorectal tumors is suggested.
The binding site exhibits high accuracy. It has sensitivity in detecting adenoma, stage i colorectal cancer, stage ii colorectal cancer, stage iii colorectal cancer and all colorectal tumors of 79.44%, 83.59%, 74.46%, 74.04% and 77.41%, respectively, with a specificity of 95%. That is, CTCF 94 detected stage i colorectal tumors with the highest accuracy (sensitivity 83.59% with a specificity of 95%). Moreover, it has a sensitivity of more than 74% in the detection of adenoma, colorectal cancer stage I, colorectal cancer stage II, colorectal cancer stage III and all colorectal tumors with a specificity of 95%, indicating that this site has a high accuracy for the early diagnosis of all colorectal tumors.
In addition, when the DNA binding site CTCF-94 of the multifunctional transcription regulatory factor CTCF provided by the application and the DNA binding sites of the other 4 CTCF form a group of molecular markers for early diagnosis of colorectal tumors, and any 2 or more than 2 of the 5 sites are positive as the judgment standard of tumor positivity, the optimal specificity and sensitivity (94.05% and 93.54% respectively) can be obtained, and particularly, the specificity and sensitivity for detecting adenomas reach 94.05% and 91.67% respectively.
The invention also aims to provide the application of the DNA binding site CTCF-94 of the multifunctional transcription regulatory factor CTCF and any one or more of the DNA binding sites of the following 4 CTCF in the preparation of a kit for early diagnosis of colorectal tumors;
(1) CTCF DNA binding site with the nucleotide sequence shown as SEQ ID NO. 4;
(2) CTCF DNA binding site with the nucleotide sequence shown as SEQ ID NO. 5;
(3) CTCF DNA binding site with the nucleotide sequence shown as SEQ ID NO. 6;
(4) CTCF DNA binding site with nucleotide sequence shown in SEQ ID NO. 7.
Although BMP3 and NDRG4 performed best in the reported molecular markers for early diagnosis of colorectal tumours, we examined the DNA methylation levels of BMP3 and NDRG4 and CTCF _94 in 62 colorectal tumour tissue samples and 58 normal samples by DNA mass spectrometry, and the results showed that the sensitivity of CTCF _94 was 73.83% and the sensitivity of BMP3 and NDRG4 was 48.56% and 69.56%, respectively, with a specificity of 90%. That is to say, the detection accuracy of the molecular marker CTCF-94 for the early diagnosis of colorectal tumors provided by the application is far higher than that of the reported molecular marker.
The invention has the following advantages and technical effects:
the accuracy of the CTCF binding site provided by the invention for detecting colorectal adenoma and colorectal carcinoma is obviously higher than that of the reported DNA methylation molecular marker; the application not only provides a more effective colorectal tumor early diagnosis technology, but also has reference significance for establishing other tumor early diagnosis technologies, and the DNA binding site of CTCF can also become a biomarker for monitoring tumor treatment effect, evaluating prognosis effect and typing tumor molecules (such as CIMP typing). In a word, the research provides a new idea for finding more effective molecular markers for early diagnosis of tumors, monitoring of treatment effects, evaluation of prognosis effects and molecular typing, and has important significance for prevention and treatment of tumors.
Drawings
FIG. 1 is a DNA mass spectrometry results hierarchical clustering plot of 23 tumor-specific CTCF binding sites in 40 samples; in the figure, columns represent samples and rows represent the DNA binding sites of CTCF.
Detailed Description
The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited to the above description, and the examples are conventional methods unless otherwise specified, and reagents used are conventional commercially available reagents or reagents formulated according to conventional methods unless otherwise specified.
Example 1: screening of DNA binding site of multifunctional transcription regulatory factor CTCF
1. Sample collection
187 fresh tumor tissues and 84 corresponding normal tissues (more than 6cm away from the tumor edge) of colorectal cancer patients from 4 months to 2016 months 8 of first subsidiary hospital 2014 of Kunming medical university are collected; 108 cases of fresh adenoma tissue were collected in the enteroscope. 379 samples were counted, including 84 normal tissues and 295 tumor tissues; the clinical data of the above samples are shown in table 1:
TABLE 1 sample information Table
2. Genomic DNA extraction and bisulfite conversion
The genomic DNA was extracted using QIAamp DNA Mini Kit (cat # 51304) from QIAGEN, all according to the instructions of the Kit. The Bisulfite conversion was carried out using the Kit EpiTect Fast DNA bisufite Kit (cat # 59826) from QIAGEN, all according to the instructions of the Kit.
3. Screening for candidate CTCF binding sites
Wang et al, by analyzing ChIP-seq data for CTCF in 19 human cell lines (including 7 tumor cell lines and 12 normal cell lines), found that the binding pattern of 1236 sites was tumor cell line specific, and these specific CTCF binding sites distinguished 7 tumor cell lines from 12 normal cell lines. In combination with published DNA methylation data of CTCF binding sites in 13 cell lines (including 6 tumor cell lines and 7 normal cell lines), we found that there was a significant correlation between the CTCF binding pattern of 121 sites in the 1236 tumor cell line-specific CTCF binding sites and the DNA methylation status, i.e., the higher the DNA methylation degree of these sites, the less CTCF binding. These 121 CTCF binding sites belong to the CTCF binding sites that are specific in tumor cell lines and whose binding pattern has a significant correlation with DNA methylation status. We downloaded the reference sequence of these 121 CTCF binding sites from UCSC database according to their genomic coordinates, and then designed PCR primers for DNA methylation detection by HRM.
4. High resolution melting Curve method (HRM) for DNA methylation analysis
In this example, the DNA methylation status of 121 CTCF binding sites in 66 samples (33 tumor tissue samples and their corresponding normal tissue samples, wherein 33 tumor tissues include 6 adenomas, 9 stage I tumors, 10 stage II tumors and 8 stage III tumors) was first analyzed by HRM method in order to find out CTCF binding sites whose DNA methylation status is specific in tumor tissues, i.e., those sites whose DNA methylation status can distinguish normal tissues from tumor tissues. First, we downloaded the genomic sequences of 121 selected CTCF binding sites (each binding site is 135bp), predicted the most likely binding site of CTCF on each sequence from the online CTCF binding site database CTCFBSDB 2.0(http:// ins. mu. latordb. uthsc. edu.), and then designed HRM primers for each CTCF binding site based on the predicted binding site of CTCF, the amplified region of HRM primers must contain the binding site of CTCF. The instrument used for PCR amplification and HRM analysis was the ABI StepOne Plus real-time PCR system (Applied Biosystems, USA). The reagent used was MeltDoctor from ABITMHRM Master Mix (Applied Biosystems, USA); all operations were performed according to the manufacturer's instructions; the HRM PCR reaction system and amplification procedure are shown in tables 2 and 3:
TABLE 2 HRM PCR amplification reaction System
TABLE 3 HRM PCR amplification and analysis procedure
In each experiment, a standard curve is made by using a standard substance (EpiTect Control DNA, QIAGEN company) with a known methylation ratio to judge the methylation levels of a tumor sample and a normal sample; the HRM data obtained was analyzed using high-resolution formatting software (Applied Biosystems, USA) software from ABI.
Of the 121 CTCF binding sites examined, 23 sites were found to have better tumor specificity for DNA methylation status (see table 4, where N indicates no tumor specificity and blank boxes indicate tumor specificity), with 16 sites with methylation patterns significantly higher in tumor tissue than in normal tissue (table 4, T > N), 7 sites with methylation patterns significantly lower in tumor tissue than in normal tissue (table 4, T < N), and 23 sites with 64% -94% tumor specificity in 33 tumor patients.
TABLE 4 HRM screening statistics
5. DNA methylation analysis by DNA Mass Spectrometry
In order to further verify the specificity of 23 CTCF binding sites selected by the HRM method in tumor tissues and accurately quantify the methylation ratio of different CpG sites in each CTCF binding site and the specific difference of DNA methylation level of each CpG site in tumor tissues and normal tissues so as to find the CTCF binding site most suitable for the early diagnosis of colorectal tumors, we firstly detected the DNA methylation level of 23 CTCF binding sites selected by the HRM method in tumor tissues by the DNA mass spectrometry method of the Sequenom MassARRAY EpiTYPER platform in 40 samples (20 tumor tissue samples and corresponding normal tissue samples, wherein 20 tumor tissues comprise 7 adenomas, 6 stage I tumors and 7 stage II tumors). The primers used were designed using the Sequenom in-line primer design software SequenomEpiDesigner, and each reverse primer contained the promoter sequence of T7RNA polymerase. Mass spectrometric DNA methylation detection uses a combination of base-specific cleavage and mass spectrometry to detect the methylation level of DNA fragments containing one or more CpG sites.
The specific operation steps are briefly described as follows:
step 1, converting all non-methylated C in sample DNA into U (corresponding to T in DNA) by using bisulfite;
step 2, amplifying the DNA sample converted by the bisulfite by using a pair of specially designed primers to obtain an amplification product with a T7RNA polymerase promoter sequence;
step 3, in an in vitro transcription system, transcription of the amplified product into RNA fragments by using T7RNA polymerase;
step 4, in the system of the step 3, utilizing the characteristic that RNase A can specifically recognize and cut the U3' end in RNA, cutting the RNA fragment into small fragments (CpG units) carrying CpG sites;
In order to ensure the reliability of the data, all the data are subjected to strict quality control selection. Briefly, if more than 30% of samples from a CpG analysis unit are not successfully detected, the data from that CpG analysis unit will be discarded. If more than 30% of CpG sites in a CTCF binding site in a sample are not successfully detected, the data for that sample at the CTCF binding site will also be excluded.
The detection result of the DNA mass spectrum shows that in 23 CTCF binding sites, the methylation pattern of 16 sites is that the methylation level in the tumor tissue is obviously higher than that in the normal tissue, and the methylation pattern of 7 sites is that the methylation level in the tumor tissue is obviously lower than that in the normal tissue, and the result is consistent with the result of the HRM detection, and further verifies the reliability of the DNA methylation detection by the HRM method.
To determine whether these 23 tumor-specific CTCF binding sites can distinguish tumor tissue from normal tissue, the study performed hierarchical clustering analysis using the percentage methylation of these 23 sites in 40 samples (fig. 1). As can be seen from the figure, 40 samples are clustered into two branches by hierarchical clustering: n and T. The N shoots contained 22 samples, 20 of which were normal tissue (91%), and 2 of which were tumor tissue (9%); t shoots contained 18 samples, all tumor tissue (100%); that is, tumor tissue can be well distinguished from normal tissue based on the methylation status of the 23 CTCF binding sites.
To differentiate the behavior of these 23 CTCF binding sites in tumor and normal tissues, we further screened the sites that are most suitable as molecular markers for early diagnosis of colorectal tumors and analyzed the contribution of each site in differentiating tumor and normal tissues (table 5). As can be seen from the table below, the AUC >0.8 for 19 of the 23 sites indicates that these sites perform well in differentiating between tumor tissue and normal tissue. Considering the AUC values of these 23 sites together with the magnitude of the difference between their percentage of methylation in tumor tissue and normal tissue, we selected 10 sites from them for further screening in subsequent experiments (sites in bold in the table below).
Detection accuracy of 523 CTCF binding sites in Table was compared
Example 2: further screening 10 sites with expanded sample size
In order to improve the specificity and sensitivity of the screened molecular marker in detecting the colorectal adenoma, 50 colorectal adenoma samples are additionally added for further screening 10 CTCF binding sites screened in the example 1; this step of screening included a total of 90 samples, 20 of which were normal controls, 57 were adenomas, 6 were stage I tumors, and 7 were stage II tumors, in addition to the mass spectral data of 40 samples from example 1. Table 6 shows the performance of these 10 CTCF binding sites in differentiating between tumor and normal tissues. As can be seen from Table 6, all 10 CTCF binding sites have strong ability to distinguish tumor tissue from normal tissue (AUC ≧ 0.85). Their sensitivity is between 44.64% and 88.89% with a specificity of 95%. Our goal is to establish an early diagnosis technique for colorectal tumours that contains as few molecular markers as possible, while ensuring the accuracy of the detection, since only this ensures the economy and accuracy of the detection. Therefore, based on the 10 sites' performance in differentiating tumor tissue from normal tissue (AUC values in table 6), we selected 5 most discriminating CTCF binding sites as the optimal molecular markers (sites indicated in bold in table 6), and we next verified the accuracy of these 5 molecular markers in early diagnosis of colorectal tumors in large samples. Of these 5 sites, only 1 site (CTCF 33) had a methylation pattern with significantly lower levels of methylation in tumor tissue than in normal tissue. Each site has strong ability to distinguish tumor tissue from normal tissue (AUC ≧ 0.916), and their sensitivity is between 73.08% -88.89% with 95% specificity. Wherein CTCF-94 is a binding site to be protected in the application, and the nucleotide sequence thereof is shown as SEQ ID NO. 1.
TABLE 610 detection accuracy comparison of CTCF binding sites
Example 3: verification of the detection accuracy of the CTCF binding site CTCF _94 of the present application in Large samples
To verify the accuracy of detection of the binding site CTCF 94 claimed in the present application, 379 colorectal samples (Table 1) were selected, wherein 84 normal samples, 295 tumor samples (108 adenomas, 39 stage I tumors, 101 stage II tumors and 47 stage III tumors) were selected, DNA methylation level of the binding site in 379 colorectal samples was continuously detected by DNA mass spectrometry, and the detection method was the same as that of "DNA methylation analysis by DNA mass spectrometry" in example 1, and the primer pairs shown in SEQ ID NO:2 and SEQ ID NO:3 were used.
Based on the results of DNA mass spectrometry, we statistically analyzed the sensitivity of CTCF-94 in detecting the AUC value and specificity of 95% of adenoma, stage I tumor, stage II tumor, stage III tumor and all tumors. The results show that CTCF 94 has a strong ability to differentiate tumor tissue from normal tissue (AUC values of 0.92, 0.96, 0.9, 0.88 and 0.91, respectively) regardless of whether adenoma, stage i, stage ii, stage iii or all tumors were detected, and that all analyses had significant statistical significance (p < 0.0001). The sensitivity of CTCF 94 to detect adenomas, stage i, ii, iii and all tumors was 79.44%, 83.59%, 74.46%, 74.04% and 77.41% with a specificity of 95%. That is, CTCF 94 detected stage i colorectal tumors with the highest accuracy (sensitivity 83.59% with a specificity of 95%). Moreover, it has a sensitivity of more than 74% in the detection of adenoma, colorectal cancer stage I, colorectal cancer stage II, colorectal cancer stage III and all colorectal tumors with a specificity of 95%, indicating that this site has a high accuracy for the early diagnosis of all colorectal tumors.
Example 4: detection accuracy for the CTCF binding site CTCF-94 of the present application in combination with other CTCF binding sites
To determine whether the combination of the 5 optimal CTCF binding sites selected by example 1 and example 2 can improve detection accuracy, we simultaneously detected the DNA methylation levels of CTCF _13, CTCF _33, CTCF _55, and CTCF _113 in large samples using DNA mass spectrometry. The samples and the detection method used were the same as in example 3.
We respectively take any 1 or more than 1, any 2 or more than 2, any 3 or more than 3, any 4 or more than 4 or all 5 sites in the 5 sites as positive judgment criteria of tumor positivity, and respectively analyze the specificity and the sensitivity of the detection of adenoma, stage I tumor, stage II tumor, stage III tumor and all tumors, and finally find that when any 2 or more than 2 sites in the 5 sites are positive judgment criteria of tumor positivity, the detection accuracy can be remarkably improved (Table 7). The sensitivity for detection of adenomas, stage I, II, III and all tumors was 91.67%, 97.44%, 94.06%, 93.62% and 93.54% at a specificity of 94.05%.
TABLE 7 detection accuracy of CTCF 94 in combination with the other 4 CTCF binding sites
Example 5: detection accuracy of the CTCF binding site CTCF-94 of the present application versus reported molecular markers
In order to compare the detection accuracy of the CTCF binding site CTCF _94 of the present application with the reported optimal molecular markers BMP3 and NDRG4, the present example examined the DNA methylation level of the promoter regions of CTCF _94 and BMP3 and NDRG4 in 120 samples. These 120 samples included 58 normal tissues, 46 adenomas, 4 stage I tumors, 8 stage II tumors, and 4 stage III tumors. From the results of the tests, we calculated AUC of CTCF _94, BMP3 and NDRG4 and sensitivity at a specificity of 90%, respectively (table 8). As can be seen from table 8, the sensitivity of the CTCF binding site CTCF _94 of the present application at 90% specificity was 73.83%, whereas the sensitivity of BMP3 and NDRG4 at 90% specificity was 48.56% and 69.56%, respectively. That is, the accuracy of the CTCF binding site CTCF _94 of the application in detecting colorectal tumors of Chinese population is significantly higher than that of the reported optimal molecular markers BMP3 and NDRG 4;
TABLE 8 comparison of the accuracy of CTCF binding sites CTCF-94 with the reported optimal molecular markers
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<213> human
<400>7
gcccagagga gaaaggaacc tctgcctcga atttccccac tgcgccgggc gctgcggaga 60
gcggcgaggg tgggcgcgag gcggagaacg cgatgaatga gttctcccct cgcctcggag 120
ttgtctgagt tggcggcgct gcgcccaggc ttccggctct cagcgcccca cgcgcgcgtg 180
gctccccggg ctgccaccca cgcccgcggc cggggccgag ccagccacgc agggcagccg 240
aggctccgga gctcctgtcc cggccccagt ccgggtaaaa ggagggttgt ccccag 296
Claims (3)
1. The application of the primer for detecting the DNA binding site CTCF-94 of the multifunctional transcription regulatory factor CTCF in preparing the kit for early diagnosis of colorectal tumors is disclosed, and the nucleotide sequence of the binding site is shown as SEQ ID NO. 1.
2. Use according to claim 1, characterized in that: the primer sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
3. The application of a primer for detecting the DNA binding site CTCF-94 of the multifunctional transcription regulatory factor CTCF and a primer for detecting any one or more of the DNA binding sites of the following 2 CTCF in preparing a kit for early diagnosis of colorectal tumors, wherein the nucleotide sequence of the DNA binding site CTCF-94 of the multifunctional transcription regulatory factor CTCF is shown as SEQ ID NO. 1;
(1) CTCF DNA binding site with the nucleotide sequence shown as SEQ ID NO. 4;
(2) CTCF DNA binding site with nucleotide sequence shown in SEQ ID NO. 5.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164516A (en) * | 2014-09-05 | 2014-11-26 | 中国科学院上海微系统与信息技术研究所 | Primer and kit based on human colorectal cancer specific methylation detection |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050014165A1 (en) * | 2003-07-18 | 2005-01-20 | California Pacific Medical Center | Biomarker panel for colorectal cancer |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164516A (en) * | 2014-09-05 | 2014-11-26 | 中国科学院上海微系统与信息技术研究所 | Primer and kit based on human colorectal cancer specific methylation detection |
Non-Patent Citations (3)
| Title |
|---|
| 《CTCF/cohesin-binding sites are frequently mutated in cancer》;Riku Katainen等;《Nature genetics》;20150608;第47卷(第7期);第818-823页 * |
| IGF2基因组印迹介导结直肠癌的靶向治疗作用的实验研究;聂珍琳;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20140515(第5期);第1536-1540页 * |
| Loss of imprinting of IGF2 correlates with hypomethylation of the H19 differentially methylated region in the tumor tissue of colorectal cancer patients;Tian Fuliang等;《Molecular medicine reports》;20120630;第5卷(第6期);E072-280 * |
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| CN107164532A (en) | 2017-09-15 |
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