CN107119144A - Multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 application - Google Patents

Abstract

The invention discloses a kind of multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 new application, i.e. its application being used in preparation in colorectal carcinoma early diagnosis kit, the binding site nucleotide sequence such as SEQ ID NO:Shown in 1；The CTCF binding sites that the present invention is provided either detect that the accuracy of Colon and rectum adenoma or colorectal cancer will be significantly higher than the DNA methylation molecular labeling reported；And CTCF DNA binding sites as oncotherapy effect monitoring and outcome it is also possible to assess and tumor cells parting（Such as CIMP partings）Biomarker.In a word, the present invention provides new thinking to find the molecular labeling of more efficiently early diagnosis of tumor, therapeutic effect monitoring, outcome assessment and molecule parting, and the prevention and treatment to tumour are all significant.

Description

Multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 application

Technical field

The invention discloses a kind of multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 application.

Background technology

Colorectal cancer is one of morbidity and mortality highest cancer in the world, its be in colon epithelial cell constantly The science of heredity and epigenetics of accumulation change caused；Probably there are 1235108 people to be diagnosed as colorectal cancer every year, every year Death toll is about 609051 people；The World Health Organization estimates the year two thousand thirty, and the confirmed cases of colorectal cancer will be increased newly 77%, death will increase 80% newly, with the rapid development of economy, colorectal cancer China the incidence of disease and death toll just Rising year by year, the prevention and control situation of colorectal cancer is very severe.

Current most of colorectal cancers are developed from adenoma, and the time for being changed into malignant tumour from adenoma is big It is approximately 10-15, that is to say, that before malignant tumour is changed into, we have 10-15 time to find and cut off adenoma So as to prevent its canceration.At present, the method for maximally effective reduction colorectal cancer incidence rate and the death rate is exactly the morning of colorectal cancer Phase diagnoses；Because in colorectal cancer early stage, patient does not have obvious clinical symptoms, and this results in many patients diagnosed When, late period in colorectal cancer, but just patient is carried out at the initial stage of a disease treatment be only it is maximally effective；Pass through Early diagnosis can find that human colorectal lesion before occurring canceration or find that the state of an illness early stage canceration, then to the greatest extent may be used Energy is early to be carried out effectively and the treatment of system to patient, so as to effectively reduce the morbidity and mortality of colorectal cancer；In early days Diagnosis be primarily directed to substantial amounts of Silent cerebral infarction, so a preferable early diagnosis technology should be it is simple to operate, into This is relatively low, to patient's hurtless measure and specificity and sensitiveness all higher detection methods.That is applied on Present clinical is most accurate Colorectal cancer method of early diagnosis be colonoscopy, it has the disadvantage, and inspection fee is higher, operation sequence is more complicated and disease People compares repulsion, is not appropriate for the large-scale promotion application in general population.Another method clinically commonly used is that excrement is hidden Blood examination is looked into, although this method expense is relatively low, is operated also fairly simple, and its maximum shortcoming is exactly sensitiveness and special Property is not high.

Epigenetics analysis finds that all colorectal cancers have hundreds of to thousands of genes to there occurs abnormal DNA first Base, the morbidity of the abnormal methylation and colorectal cancer of a portion gene is closely related；At present, the knot based on blood Maximally effective carcinoma of the rectum method of early diagnosis is exactly using the DNA methylation of SEPT9 gene promoter areas as biomarker, with this Method is early diagnosed to colorectal cancer, can obtain 90% susceptibility and 88% specificity, but this method is for gland The susceptibility and specificity of knurl are all very low；In addition with this method to more than 50 years old without clinical symptoms crowd（7941 people）Carry out early The phase result of study of colorectal cancer examination is shown, although specificity can reach 91.5%, but for I-IV colorectal cancer Susceptibility only has 35%, 63%, 46% and 77.4% respectively.Susceptibility for adenoma is even more only 11.2%；Knot based on excrement is straight Most successful intestinal cancer method of early diagnosis should be with 4 genes（BMP3, NDRG4, vimentin and TFPI2）Promoter region DNA methylation is as biomarker, and the mutation of KRAS genes and containing for hemoglobin in excrement in detection faeces DNA simultaneously Amount, this method is on the premise of specificity is 90%, and the susceptibility for colorectal cancer is 85%, to the quick of the adenoma more than 1cm Sensitivity is 54%；The method that either method based on blood is also based on excrement, all goes back that neither one is especially desirable to examine at present Disconnected technology is adapted to clinically wide popularization and application.

It is worth noting that, research before this be substantially the DNA methylation of specific gene promoter region is straight as knot The sight of concern is placed on the transcription regulating region beyond promoter region by the biomarker of intestinal cancer early diagnosis, few people.Base The promoter of cause determines the activity of gene just as " switch ", controls the initial time of genetic transcription and the degree of expression.But open Transcription regulating region beyond mover area equally can also influence the transcription of gene, and rising for genetic transcription can be also determined in many cases Transcription regulating region beyond time beginning and the degree of expression, gene promoter area also should be by same concern.

CTCF is a kind of multi-functional transcription factor being widely present in eucaryote, for upper highly conserved many zinc of evolving Finger, DNA combination nucleoprotein.CTCF, can be with a variety of DNA sequence dnas of Selective recognition by the various combination of its zinc fingers, and shape Into different CTCF-DNA complexs, play and the expression regulation of gene is acted on, suppress with promoter and activate, gene sinks The various biological functions such as silent, enhancer blocking, gene imprinting regulation and control, x chromosome inactivation.CTCF DNA binding sites are in people Genoid group is middle generally existing, probably there is 5-6 ten thousand, and each site recognizes about 34 nucleotides；Yet there are no will The report for the biomarker that multi-functional transcription regulatory factor CTCF DNA binding sites are early diagnosed as colorectal carcinoma.

The content of the invention

Present invention aims at provide a kind of the new of multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 Purposes, i.e., multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 is used for colorectal carcinoma in preparation and examined in early days Application in disconnected, examination and risk prediction kit, the nucleotide sequence such as SEQ ID NO of the binding site:Shown in 1.

Detection kit of the present invention includes conventional gene group DNA extracts reagents, at the bisulfites of methylate DNA Manage reagent, carry out the required reagent of DNA methylation detection and primer pair, the primer pair can be expanded through bisulfite conversion That crosses methylates or unmethylated SEQ ID NO:Sequence shown in 1 is simultaneously carried out subsequently to the sequence expanded with DNA mass spectrographies DNA methylation detection.Binding site CTCF_55 shows hyper-methylation state in colorectal carcinoma cell, and just Often hypomethylation is in tissue.

The primer pair sequence such as SEQ ID NO:2 and SEQ ID NO:Shown in 3.

The application is examined multi-functional transcription regulatory factor-CTCF DNA binding sites as colorectal carcinoma early stage first Disconnected biomarker.First, by analyzing DNA binding site data of the CTCF delivered in human genome, and combine The human genome DNA delivered methylates data, and we have filtered out the CTCF binding patterns and DNA first in 121 sites altogether There is significant correlation in base state, i.e. the number that the height of these site DNA methylation degree is combined with CTCF has negative Close.This 121 CTCF binding sites just belong to specific in tumor cell line, and its binding pattern and DNA methylation There is significant correlation in state.Second step, passes through high-resolution solubility curve method（HRM）In 33 colorectal tumor tissue samples And its DNA methylation level in this 121 sites is have detected in corresponding normal sample, therefrom filter out 23 moulds that methylate Formula has the site of tumour-specific.Because HRM methods can not carry out accurate quantitative analysis in detection DNA methylation application, So also cannot further compare 23 filtered out site qualities, so follow-up DNA methylation detection is all to use DNA What mass spectrography was completed；3rd step, by DNA mass spectrographies in 20 colorectal tumor tissue samples and its corresponding normal sample The DNA methylation level in 23 sites is have detected, according to performance of this 23 sites on tumor tissues and normal structure is distinguished, 10 sites behaved oneself best therefrom are filtered out and further screening are made by enlarged sample.4th step, is existed by DNA mass spectrographies The DNA methylation level in 10 sites is have detected in 70 colorectal tumor tissue samples and 20 normal samples, according to this 10 Performance of the individual site on tumor tissues and normal structure is distinguished, have selected 5 sites behaved oneself best and is carried out in large sample Checking.5th step, 5 positions are have detected by DNA mass spectrographies in 295 colorectal tumor tissue samples and 84 normal samples The DNA methylation level of point, finally screening and identifying the application is used for colorectal carcinoma early diagnosis molecular labeling CTCF binding sites.The site shows hyper-methylation state in colorectal carcinoma cell, and in the normal tissue in low Methylation state, the DNA methylation for pointing out CTCF binding sites CTCF_55 is the molecular labeling of colorectal carcinoma early diagnosis.

The binding site shows very high accuracy.On the premise of specificity is 95%, it is in detection adenoma, I phase Susceptibility in colorectal cancer, II phase colorectal cancer, III phase colorectal cancer and all colorectal carcinomas is respectively 73.52%, 95.13%th, 88.87%, 80.77% and 84.66%.That is, CTCF_55 detects the accuracy highest of I phase colorectal carcinoma（It is special The opposite sex is that susceptibility is 95.13% on the premise of 95%）.Moreover, on the premise of specificity is 95%, it is detecting I phase Colon and rectum Susceptibility in cancer, II phase colorectal cancer, III phase colorectal cancer and all colorectal carcinomas is both greater than 80%, although detection adenoma Susceptibility it is minimum, but also reached 73.52%.Illustrate that this site all has for the early diagnosis of all colorectal carcinomas There is very high accuracy.

In addition, the DNA binding sites CTCF_55 and other 4 of the multi-functional transcription regulatory factor CTCF when the application offer Individual CTCF DNA binding sites, which constitute one group, is used for the molecular labeling that colorectal carcinoma is early diagnosed, and with any in 5 sites 2 or more than 2 for positive criterion as tumer positive when, optimal specificity and susceptibility can be obtained（Respectively For 94.05% and 93.54%）, 94.05% and 91.67% are respectively reached especially for the specific and susceptibility that adenoma is detected.

I.e. the present invention is another object is that by multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 and following 4 Any one in individual CTCF DNA binding sites or several apply are used in colorectal carcinoma early diagnosis kit in preparation；

（1）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 4；

（2）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 5；

（3）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 6；

（4）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 7.

Although BMP3 and NDRG4 be reported colorectal carcinoma early diagnosis molecular labeling in behave oneself best, It is that we have detected BMP3 and NDRG4 by DNA mass spectrographies in 62 colorectal tumor tissue samples and 58 normal samples And CTCF_55 DNA methylation level, as a result show, on the premise of specificity is 90%, CTCF_55 susceptibility is 81.02%, and BMP3 and NDRG4 susceptibility is respectively 48.56% and 69.56%.That is, the Colon and rectum that the application is provided The molecular labeling that the molecular labeling CTCF_55 of early diagnosis of tumor detection accuracy will have been reported significantly larger than.

Advantages of the present invention and technique effect are as follows:

The CTCF binding sites that the present invention is provided either detect that the accuracy of Colon and rectum adenoma or colorectal cancer will be notable Higher than the DNA methylation molecular labeling reported；It is early that the application not only provides a kind of more efficiently colorectal carcinoma Phase diagnostic techniques, also has reference, and CTCF DNA binding sites for setting up the early diagnosis technology of other tumours It is also possible to being assessed and tumor cells parting as oncotherapy effect monitoring and outcome（Such as CIMP partings）Biology Mark.In a word, this research is monitored to find more efficiently early diagnosis of tumor, therapeutic effect, outcome is assessed and divided The molecular labeling of sub- parting provides new thinking, and the prevention and treatment to tumour are all significant.

Brief description of the drawings

Fig. 1 is that the 23 DNA mass spectral results for having the CTCF binding sites of tumour-specific in 40 samples layerings are poly- Class figure；What row were represented in figure is sample, and what row was represented is CTCF DNA binding sites.

Embodiment

The present invention is described in further detail below by drawings and examples, but the scope of the present invention is not limited to Method is conventional method unless otherwise specified in the content, embodiment, and the reagent used is routine unless otherwise specified Commercial reagent or the reagent prepared according to a conventional method.

Embodiment 1：The screening of multi-functional transcription regulatory factor CTCF DNA binding sites

1st, sample is collected

First Affiliated Hospital of Kunming Medical University's in April, 2014 is collected to the fresh group of the colorectal cancer patients of in August, 2016 Knit 187 and its corresponding normal structure（Apart from more than borderline tumor 6cm）84；Fresh adenoma tissue is have collected in colonoscopy room 108.All samples amount to 379, wherein normal structure 84, tumor tissues 295；Above-mentioned sample clinical data situation is such as Shown in table 1：

The sample information table of table 1

。

2nd, extracting genome DNA and bisulfite conversion

The extraction of genomic DNA uses the QIAamp DNA Mini Kit of QIAGEN companies（Article No. 51304）Kit, All operations are completed according to the specification of kit.Bisulfite conversion uses QIAGEN companies EpiTect Fast DNA Bisulfite Kit（Article No. 59826）Kit, all operations are all the explanations according to kit Book is completed.

3rd, the screening of candidate CTCF binding sites

Wang et al. is by analyzing 19 kinds of Human cell lines（Including 7 kinds of tumor cell lines and 12 kinds of normal cell systems）Middle CTCF's ChIP-seq data, the binding pattern that 1236 sites are found that altogether is that tumor cell line is special, these specific CTCF Binding site can distinguish 7 kinds of tumor cell lines with 12 kinds of normal cell systems.With reference to the 13 kinds of cell lines delivered（Including 6 kinds of tumor cell lines and 7 kinds of normal cell systems）The DNA methylation data of middle CTCF binding sites, we are in this 1236 tumours The CTCF binding patterns and methylation state of DNA for finding 121 sites in the special CTCF binding sites of cell line altogether exist aobvious The DNA methylation degree for writing correlation, i.e. these sites is higher, and CTCF combination is fewer.This 121 CTCF binding sites are just Belong to specific in tumor cell line, and there is the CTCF of significant correlation with methylation state of DNA in its binding pattern Binding site.We have downloaded this 121 positions according to the genomic coordinates of this 121 CTCF binding sites from UCSC databases The reference sequences of point, then design carries out the PCR primer of DNA methylation detection with HRM methods.

4th, high-resolution solubility curve method（HRM）Carry out DNA methylation analysis

The present embodiment is first with HRM methods in 66 samples（33 tumor tissues samples and its corresponding normal structure sample, wherein 33 tumor tissues include 6 adenomas, 9 I phase tumours, 10 II phases tumours and 8 III phase tumours）In 121 CTCF are tied The methylation state of DNA for closing site is analyzed, to find out methylation state of DNA in tumor tissues with specific CTCF binding sites, that is, the site that those methylation state of DNA can distinguish normal structure and tumor tissues.It is first First, we have downloaded the genome sequence of the 121 CTCF binding sites filtered out（Each binding site is 135bp）, together When according to the online (http of CTCF binding site databases CTCFBSDB 2.0://ins μ latordb.uthsc.edu/) it is pre- The most probable binding sites of CTCF in every sequence are surveyed, each CTCF knots are then designed according to the CTCF binding sites predicted The HRM primer in site is closed, the amplification region of HRM primer must include CTCF binding site.PCR is expanded and HRM analyses are made Instrument is ABI StepOne Plus real-time PCR systems (Applied Biosystems, USA).Used Reagent be ABI companies MeltDoctor HRM Master Mix (Applied Biosystems, USA)；All behaviour All it is to be completed according to the operation instruction of manufacturer；HRM PCR reaction systems and amplification program are as shown in table 2 and table 3：

The HRM pcr amplification reaction systems of table 2

;

The HRM PCR of table 3 are expanded and analysis program

;

Experiment can all use the standard items of the known ratio that methylates every time（EpiTect Control DNA, QIAGEN companies）Do one Individual standard curve judges the methylation level of tumor sample and normal sample；Resulting HRM data use ABI companies High-resolution melting software (Applied Biosystems, USA) software is analyzed.

In the 121 CTCF binding sites detected, find that the methylation state of DNA in 23 sites has altogether and preferably swell Knurl specificity（It is shown in Table N in 4, table and represents and do not have tumour-specific, space, which is represented, has tumour-specific）, wherein 16 sites Methylation patterns be tumor tissues in methylation level be significantly higher than normal structure（Table 4, T>N）, 7 sites methylate Pattern is that the methylation level in tumor tissues is substantially less than normal structure（Table 4, T<N）, 23 sites are in 33 tumor patients In tumour-specific be 64%-94%.

Table 4HRM the selection result statistical forms

5th, DNA mass spectrums carry out DNA methylation analysis

In order to further verify specificity of the 23 CTCF binding sites screened by HRM methods in tumor tissues, and In each CTCF binding sites of accurate quantitative analysis methylate ratio and each CpG sites in difference CpG sites in tumor tissues and The specific difference of DNA methylation level in normal structure, so as to find out the CTCF early diagnosed most suitable as colorectal carcinoma Binding site, we are first in 40 samples（20 tumor tissues samples and its corresponding normal structure sample, wherein 20 Tumor tissues include 7 adenomas, 6 I phase tumours and 7 II phase tumours）It is flat with Sequenom MassARRAY EpiTYPER The DNA mass spectrometry methods of platform have detected 23 filtered out by HRM methods has specific CTCF combinations in tumor tissues The DNA methylation level in site.Primer used is the online primer-design software Sequenom with Sequenom companies Designed by EpiDesigner, every reverse primer all includes the promoter sequence of T7 RNA polymerases.Mass spectrum DNA methylation Detection uses base shearing specifically and mass spectral analysis is combined to detect the DNA for including one or more CpG site The methylation level of fragment.

Concrete operation step is summarized as follows：

Step 1, using bisulfites, the C not methylated in sample DNA is completely converted into U（Equivalent in DNA T）；

Step 2, the DNA sample crossed using the pair of primers amplification of particular design through bisulfite conversion, obtain carrying T7 The amplified production of rna polymerase promoter sequence；

In step 3, in vitro transcription system, using T7 RNA polymerases, amplified production is transcribed into RNA segments；

Step 4, in the system of step 3, specific recognition and the characteristic that U 3 ' in RNA is held can be cut using RNase A, will RNA segments cut into the small pieces for carrying CpG sites（CpG units）；

Step 5, using Sequenom MassARRAY EpiTYPER platforms flight mass spectrum analysis system detecting step 4 production Thing；Due in same segment, between only CpG and CpA in 16Da molecular weight difference, i.e. mass spectrogram both peaks gap；Institute The obtained mass spectrometric data that methylates is analyzed using the softwares of Typer 4.0 of Sequenom companies, so as to obtain target dna piece The percentage that methylates of each CpG units in section.CTCF binding sites for including multiple CpG units, we unite selection Methylate percentage of the percentage as the site that methylates of AUC highest CpG units in meter analysis.

In order to ensure the reliability of data, all data all have passed through strict Quality Control selection.Briefly, if one CpG analytic units have the sample more than 30% not to be successfully detected, and the data of this CpG analytic unit will be excluded.If There are the CpG sites more than 30% not to be successfully detected in one CTCF binding site of one sample, this sample at this The data of CTCF binding sites will be also excluded.

The mass spectrographic testing results of DNA are shown, in 23 CTCF binding sites, wherein the methylation patterns in 16 sites are Methylation level in tumor tissues is significantly higher than normal structure, and the methylation patterns in 7 sites are the methyl in tumor tissues Change level is substantially less than normal structure, and this result is consistent with HRM testing results, further demonstrates with HRM methods detection DNA first The reliability of base.

In order to judge that can the CTCF binding sites of this 23 tumour-specifics distinguish tumor tissues and normal structure, This research has done hierarchical cluster analysis with this percentage that methylates of 23 sites in 40 samples（Fig. 1）.Can be with from figure Find out, 40 samples are poly- for two by hierarchical cluster：N and T.N include 22 samples, wherein 20 are normal structure （91%）, 2 are tumor tissues（9%）；T include 18 samples, entirely tumor tissues（100%）；That is according to this The methylation state of 23 CTCF binding sites can be very good to distinguish tumor tissues and normal structure.

In order to distinguish performance of this 23 CTCF binding sites in tumor tissues and normal structure is distinguished, further screening Go out to early diagnose the site of molecular labeling most suitable as colorectal carcinoma, we analyze each site and are distinguishing tumor tissues With the contribution in normal structure（Table 5）.In 23 sites it can be seen from following table, there is the AUC in 19 sites>0.8, illustrate this Performance of a little sites in tumor tissues and normal structure is distinguished is all fine.Consider the AUC in this 23 sites, and it Difference size of the percentage between tumor tissues and normal structure that methylate, we therefrom have selected 10 sites rear Make further screening in continuous experiment（The site marked in following table with runic）.

The detection accuracy of 5 23 CTCF binding sites of table compares

。

Embodiment 2：Enlarged sample amount does further screening to 10 sites

In order to improve specificity and susceptibility of the filtered out molecular labeling in detection Colon and rectum adenoma, we add again 10 CTCF binding sites that 50 Colon and rectum adenoma samples are filtered out to embodiment 1 do further screening；Plus embodiment 1 In 40 samples mass spectrometric data, the screening of this step altogether comprising 90 samples, wherein 20 be normal control, 57 be gland Knurl, 6 be I phase tumour and 7 are II phase tumour.Table 6 shows that this 10 CTCF binding sites are distinguishing tumor tissues and just The often performance in tissue.As can be seen from Table 6, this 10 CTCF binding sites all have very strong differentiation tumor tissues and normal The ability of tissue（AUC≥ 0.85）.Specificity be 95% on the premise of, their susceptibility 44.64% -88.89% it Between.Our target is to set up a kind of Colon and rectum that molecular labeling as few as possible is included on the premise of detection accuracy is ensured Early diagnosis of tumor technology, because only that so just can guarantee that the economy and accuracy of detection.Therefore, according to this 10 sites Performance in tumor tissues and normal structure is distinguished（AUC in table 6）, it is most strong that we have selected 5 separating capacities CTCF binding sites are used as optimal molecular labeling（The site that runic is represented in table 6）, next step we will be tested in large sample Demonstrate,prove this accuracy of 5 molecular labelings in being early diagnosed to colorectal carcinoma.In this 5 sites, only 1 site （CTCF_33）Methylation patterns be tumor tissues in methylation level be substantially less than normal structure.Each site has The very strong ability for distinguishing tumor tissues and normal structure（AUC ≥ 0.916）, on the premise of specificity is 95%, they Susceptibility is between 73.08% -88.89%.Wherein, CTCF_55 is the application binding site to be protected, its nucleotide sequence Such as SEQ ID NO：Shown in 1.

The detection accuracy of 6 10 CTCF binding sites of table compares

。

Embodiment 3：The CTCF binding sites CTCF_55 of the application detection accuracy is verified in large sample

In order to verify the application binding site CTCF_55 to be protected detection accuracy, we have chosen 379 Colon and rectum samples This（Table 1）, wherein normal sample 84, tumor sample 295（Adenoma 108, I 39, phase tumour, II 101, phase tumour and III 47, phase tumour）, continue to have detected DNA methylation water of the binding site in 379 Colon and rectum samples with DNA mass spectrographies Flat, " DNA mass spectrums carry out DNA methylation analysis " content is identical in detection method be the same as Example 1, using such as SEQ ID NO:2 Hes SEQ ID NO:Primer pair shown in 3.

According to DNA Mass Spectrometer Method results, we distinguish statistical analysis CTCF_55 in detection adenoma, I phase tumour, II phase Susceptibility when tumour, the AUC of III phase tumour and all tumours and specificity are 95%.As a result show, either detect gland Knurl, I phase tumour, II phase tumour, III phase tumour or all tumours, CTCF_55 is with very strong differentiation tumor tissues and just The ability often organized（AUC is respectively 0.93,0.98,0.97,0.94 and 0.94）, and all analyses all have significantly system Meter learns meaning（p < 0.0001）.Specificity be 95% on the premise of, CTCF_55 detection adenoma, I phase tumour, II phase tumour, The susceptibility of III phase tumour and all tumours is respectively 73.52%, 95.13%, 88.87%, 80.77% and 84.66%.That is, CTCF_55 detects the accuracy highest of I phase colorectal carcinoma（Specificity is that susceptibility is 95.13% on the premise of 95%）.Moreover, On the premise of specificity is 95%, it is detecting that I phase colorectal cancer, II phase colorectal cancer, III phase colorectal cancer and all knots are straight Susceptibility in intestinal tumor is both greater than 80%, although the susceptibility of detection adenoma is minimum, but has also reached 73.52%.Illustrate this Individual site all has very high accuracy for the early diagnosis of all colorectal carcinomas.

Embodiment 4：The CTCF binding sites CTCF_55 of the application and the detection that other CTCF binding sites are used in combination are accurate True property

Whether can to determine that the 5 optimal CTCF binding sites filtered out by embodiment 1 and embodiment 2 are used in combination Improve detection accuracy, we be have detected simultaneously in large sample with DNA mass spectrographies CTCF_13, CTCF_33, CTCF_94 and CTCF_113 DNA methylation level.Sample used and detection method be the same as Example 3.

We respectively by any 1 in 5 sites or more than 1, any 2 or more than 2, any 3 or 3 with Above, any 4 or more than 4 or all 5 sites are the positive criterion as tumer positive, and analyze it respectively Detect the specificity and susceptibility of adenoma, I phase tumour, II phase tumour, III phase tumour and all tumours, finally found that when with 5 In individual site any 2 or more than 2 for positive criterion as tumer positive when, detection accuracy can be significantly improved （Table 7）.When specificity is 94.05%, the susceptibility of adenoma, I phase tumour, II phase tumour, III phase tumour and all tumours is detected Respectively 91.67%, 97.44%, 94.06%, 93.62% and 93.54%.

The detection accuracy that the CTCF_55 of table 7 is used in combination with other 4 CTCF binding sites

。

Embodiment 5：The CTCF binding sites CTCF_55 of the application is compared with the detection accuracy for the molecular labeling reported

In order to compare the CTCF binding sites CTCF_55 of the application and the optimal molecular labeling BMP3 and NDRG4 reported inspection Accuracy is surveyed, the present embodiment have detected CTCF_55 and BMP3 and NDRG4 promoter region DNA methylation in 120 samples Level.It is swollen that this 120 samples include 58 normal structures, 46 adenomas, 4 I phases tumours, 8 II phases tumours and 4 III phases Knurl.According to testing result, the sensitivity when AUC and specificity that we calculate CTCF_55, BMP3 and NDRG4 respectively are 90% Degree（Table 8）.As can be seen from Table 8, susceptibilitys of the CTCF binding sites CTCF_55 of the application when specificity is 90% is 81.02%, and susceptibilitys of the BMP3 and NDRG4 when specificity is 90% is respectively 48.56% and 69.56%.That is, this Shen Accuracys of the CTCF binding sites CTCF_55 please in the detection on state crowd colorectal carcinoma, which will be significantly higher than, have been reported most Excellent molecular labeling BMP3 and NDRG4；

The CTCF binding sites CTCF_55 of table 8 is with having reported that the accuracy of optimal molecular labeling is compared

。

Sequence table

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Claims (4)

1. multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 is being prepared for colorectal carcinoma early diagnosis Application in kit, the binding site nucleotide sequence such as SEQ ID NO:Shown in 1.

2. kit according to claim 1, it includes primer pair, and the primer pair can be expanded to be turned through bisulfites That changed methylates or unmethylated SEQ ID NO:Sequence shown in 1.

3. kit according to claim 2, it is characterised in that：Primer pair sequence such as SEQ ID NO:2 and SEQ ID NO:Shown in 3.

4. in multi-functional transcription regulatory factor CTCF DNA binding sites CTCF_55 and following 4 CTCF DNA binding sites Any one or it is several prepare be used for colorectal carcinoma early diagnosis kit in application；

（1）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 4；

（2）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 5；

（3）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 6；

（4）Nucleotide sequence such as SEQ ID NO:CTCF DNA binding sites shown in 7.