CN106755464A - For the method for screening the gene marker of intestinal cancer and/or stomach cancer, the gene marker and application thereof that is screened with the method - Google Patents
For the method for screening the gene marker of intestinal cancer and/or stomach cancer, the gene marker and application thereof that is screened with the method Download PDFInfo
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- CN106755464A CN106755464A CN201710020252.4A CN201710020252A CN106755464A CN 106755464 A CN106755464 A CN 106755464A CN 201710020252 A CN201710020252 A CN 201710020252A CN 106755464 A CN106755464 A CN 106755464A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
Gene marker and application thereof the present invention relates to be used to detect intestinal cancer or stomach cancer.Method the invention further relates to be detected to intestinal cancer or stomach cancer using the gene marker.
Description
Technical field
Field the present invention relates to pass through high-flux sequence screening-gene mark.In particular it relates to pass through height
Flux is sequenced the method screened to the gene marker of intestinal cancer and/or stomach cancer, and the gene filtered out using the method
Mark and application thereof.
Background technology
With living environment and custom change, in the last few years the incidence of disease of cancer worldwide raise year by year, people
It paid close attention to be consequently increased.Stomach cancer and intestinal cancer are respectively the second largest and the third-largest cancers occurred frequently of China, belong to the incidence of disease and disease
Dead rate malignant tumour all very high, their occurrence and development are a multifactor participation and multistage to accumulate carcinogenic complexity
Process.Early treatment scheme is early found in line with existing current cancer, more early detection is more possible to early stage control and even cures cancer
Disease.How stomach cancer and intestinal cancer are early diagnosed, in time treatment, correctly judge prognosis, is gradually paid attention to by people.
Traditional screening method has gastrointestinal endoscopy and FOB Fecal Occult Blood Testing, but has itself unsurmountable shortcoming.Stomach and intestine
Mirror adds the goldstandard that pathological biopsy is considered as gastric and intestinal cancer examination and diagnosis, but because the invasive and INTESTINAL CLEANSING of microscopy
Discomfort, many patients are unwilling to receive stomach spectroscopy.Recently there are some researches show flat common to right side colon of intestines mirror
The diagnosis effect of polyp is bad, and the examination of intestines mirror can not reduce the incidence of disease of right side colon.FOB Fecal Occult Blood Testing is another
Clinically conventional screening for colorectal method, the method have the advantages that it is completely noninvasive and cheap, but it detects the accuracy of tumour
It is low, also it is only capable of detecting adenoma before the colorectal cancer of 50-60% and 30% or so cancer by immunochemical improved method.And
And, the detection method false positive rate is higher, therefore hardly results in and popularize.
Beyond upper traditional screening method, people are also constantly exploring other method, to improve early diagnostic rate, tumour
Mark is one of them.For example, finding T1151A as on mismatch repair gene hMLH_1 more than one by DHPLC analyses
State site, the candidate's index that can be screened as stomach and large bowel neoplasm, especially low age stomach and large bowel neoplasm people at highest risk (referring to
Dawn plum etc.,《Tumour》, 2005,25 (1)：62-65).Using CA19-9's (carbohydrate CA19-9) and SA (sialic acid)
Tumor markers joint-detection can reach 76.7% He respectively to the Positive rate of the knot/carcinoma of the rectum and stomach/cardia cancer
82.5% (referring to Cai Facheng etc.,《Practical medical technologies magazine》, 2005,12 (3B)：722-723).Additionally, China also developed C12
Protein chip detection system, by analyzing CA19-9, neuronspecific enolase (NSE), carcinomebryonic antigen in serum
(CEA), glycogen 242 (CA242), glycogen 125 (CA125), glycogen 153 (CA15-3), alpha-fetoprotein (AFP), ferritin, people's suede
This ten kinds of expressions of tumor markers in Chorionic Gonadotropin (HCG), human growth hormone (HGH) (HGH), to realize tumour
Early diagnosis and monitoring.However, most of tumor markerses for finding also rest on conceptual phase, clinic is not developed into and is examined
Faulted-stage section；Or have been applied in clinical diagnosis, but accuracy in detection or sensitivity is not high.For example, research finds, it is above-mentioned
C12 systems are 39.21% to the collective diagnosis rate of gastrointestinal cancer patients, and the diagnosis of I, II, III, IV phase patient is respectively
13.73%th, 33.33%, 38.30%, 58.03%, show that diagnosis of the C12 detecting systems to advanced gastrointestinal cancer has certain valency
Value, but not high to the sensitiveness of early stage (referring to Yang Xueqin etc.,《Nanjing Medical University's journal (natural science edition)》, 2008 (10)：
1285-1289)。
In the recent period, the in-vitro diagnosis method of other two kinds of intestinal cancer has been developed again on the basis of existing conventional gene label：
Cologuard technologies from Exact Sciences companies of the U.S. and the Epi proColon from Epigenomics companies
Technology.The former predominantly detect in excrement abnormal DNA change and human feces present in occult blood Lactoferrin；The latter detects blood
SEPT9 methylation markers in dissociative DNA.Larger scale clinical experiment shows that the recall rate effect of Cologuard is preferable, but
Need to operate excrement, Consumer's Experience is poor；And SEPT9 methylates and belongs to liquid biopsy, although better user experience, but specifically
Property and sensitivity are all poor (respectively 80% and 72%).
Therefore in the urgent need to high specific and high sensitivity and being available for the gene mark of the human primary gastrointestinal cancers that clinical detection uses
Will thing, allows to noninvasive or minimally invasive, efficiently method efficient diagnosis human primary gastrointestinal cancers, to improve the meaning that patient receives long term monitoring
It is willing to.
The content of the invention
Inventor by the way that normal specimens and intestinal cancer or stomach cancer samples are carried out with high-flux sequence, and on wherein each gene
5-hydroxymethyl cytosine (5-hmC) content is analyzed, it has surprisingly been found that multiple great information can be used for detect intestines
The gene marker of cancer or stomach cancer.
Therefore, the first aspect of the invention is related to the gene marker for detecting intestinal cancer, including one or more choosings
From following gene：ADAM metallopeptidases domain 20 (ADAM20), F boxes and full asphalt mixture albumen 7 (FBXL7), follistatin
(FST), TP53 apoptosis effects device (PERP), pleckstrin homology similar regions family A member 3 (PHLDA3), Runt phases
Close the mobile companion 1 (RUNX1T1) of transcription factor 1, syntrophism albumen γ 2 (SNTG2), sperm antigen 4 (SPAG4), sulfuric ester
Enzyme 1 (SULF1) and NME/NM23 nucleoside diphosphokinases (NME3).Preferably, the gene marker include at least two, extremely
Three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten are selected from following less
Gene：ADAM20, FBXL7, FST, PERP, PHLDA3, RUNX1T1, SNTG2, SPAG4, SULF1 and NME3.It is furthermore preferred that institute
Stating gene marker includes ADAM20, FBXL7, FS hole PERP, PHLDA3, RUNX1T1, SNTG2, SPAG4, SULF1 and NME3.
The second aspect of the invention is related to the gene marker for detecting stomach cancer, including one or more are selected from following
Gene：Rho gtpase activating proteins 28 (ARHGAP28), BMP combination endotheliums regulation and control person (BMPER), chromosome 9 open reading code
Frame 92 (C9orf92), calcium dependent secretory swash reviver 2 (CADPS2), cadherin 11 (CDH11), F boxes and full asphalt mixture egg
The homologous frame 2 (MEOX2) of white 7 (FBXL7), interstitial, nitric oxide synthetase 1 (NOS1), oncostatinM acceptor (OSMR), cell membrane
Modulin paralemmin2 (PALM2), phosphodiesterase 10 A (PDE10A), the single-stranded interaction protein 3 of RNA combination die bodys
(RBMS3), sulfatase 1 (SULF1), wntless Wnt parts regulation of secretion person (WLS), the action protein of Wilms knurls 1
(WTIP), zinc finger protein 51 8B (ZNF518B), zinc finger protein 714 (ZNF714) and Rad52 die bodys include 1 (RDM1).It is preferred that
, the gene marker include at least two, at least three, at least four, at least five, at least six, at least seven, extremely
Few eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16
Individual, at least 17 or 18 are selected from following gene：ARHGAP28、BMPER、C9orf92、CADPS2、CDH11、FBXL7、
MEOX2, NOS1, OSMR, PALM2, PDE10A, RBMS3, SULF1, WLS, WTIP, ANF518B, ZNF714 and RDM1.More preferably
, the gene marker include ARHGAP28, BMPER, C9orf92, CADPS2, CDH11, FBXL7, MEOX2, NOS1,
OSMR, PALM2, PDE10A, RBMS3, SULF1, WLS, WTIP, ANF518B, ZNF714 and RDM1.
The invention further relates to purposes of the said gene mark in detection intestinal cancer or stomach cancer.The invention further relates to using upper
Stating gene marker carries out the kit of intestinal cancer or stomach cancer detection, and it includes containing for determining the 5-hmC of said gene mark
The reagent and specification of amount.
The third aspect of the invention is related to the method for detecting stomach cancer or intestinal cancer, comprises the following steps：
A () determines the content of the 5-hmC of gene marker of the present invention in normal specimens and Samples subjects；
B () uses the 5-hmC contents of gene marker described in normal specimens as reference, will be corresponding in Samples subjects
The 5-hmC content standards of gene marker；
C () carries out mathematical to the 5-hmC contents of the normalised gene marker, and scored；With
D () obtains testing result according to the scoring.
In one embodiment, the sample be subject or normal person's body fluid middle reaches from DNA fragmentation, or derive from
Complete genome group DNA in organelle, cell and tissue.Wherein, body fluid is blood, urine, sweat, sputum, excrement, brain ridge
Liquid, ascites, hydrothorax, bile, pancreas liquid etc..
In one embodiment, the 5-hmC contents of gene marker of the present invention can be by people in the art
The known any method of member is measured, for example, include but is not limited to, glucosylation method, restriction enzyme enzyme process, chemical labeling
Method and the real-time PCR sequencing PCR of the precipitation method, unimolecule (SMRT), oxidation bisulfite PCR sequencing PCR associated with high-flux sequence method
(OxBS-Seq) etc..The principle of glucosylation method is using T4 bacteriophages β-glucosyl transferase (β-GT), in grape saccharide donor
In the presence of substrate UDP-glucose (UDP-Glu), by suction pressure to hydroxy position, so as to generate β-glucose
Base -5-hydroxymethyl cytosine (5-ghmC).Can be quantified using isotope labelled substrates simultaneously.On the basis of glucosylation method
Further develop restriction enzyme enzyme process and chemical labeling method.The principle of restriction enzyme enzyme process is：Glucosylation reaction changes
The digestion characteristic of some restriction enzymes is become.Methylate dependence restriction endonuclease MspI and HpaII it is recognizable same
Sequence (CCGG), but their sensitiveness to methylation state are different：MspI is recognized and is cut 5-methylcytosine (5-
MC) and 5-hmC, but 5-ghmC can not be cut；HpaII only cuts completely unmodified site, any modification (5- on cytimidine
MC, 5-hmC, 5-ghmC) hinder cutting.If CpG contains 5-hmC in site, then can detect bar after glycosylation, enzymolysis
Band, not glycosylating in control reaction does not have band；Quantitative analysis can be carried out using qPCR simultaneously.In addition, other restriction enzymes
Similarly there is the situation for hindering 5-ghmC digestions in enzyme, can be applied to 5-hmC detections (such as：GmrSD, MspJI, PvuRts1I,
TaqI etc.).The principle of chemical labeling method is：Glucose on enzyme reaction substrate is chemically modified and is transformed into UDP-6-N3-
Glucose, hydroxymethyl position is transferred to by 6-N3-glucose, generates N3-5ghmC.Then, by click chemistry method every
Molecular biosciences element is added on individual 5-hmC, with reference to high flux DNA sequencing technology of future generation or single-molecule sequencing technology, can be analyzed
Distribution situations of the 5-hmC in genomic DNA.The precipitation method be by 5-hmC with particular form modify after again by its specifically from
Captured in genomic DNA, and carry out sequencing analysis.Oxidation bisulfite PCR sequencing PCR is first with single base resolution ratio pair
5-hmC is carried out KRuO4 oxidation processes by the method that 5-hmC carries out quantitative sequencing first, generates 5- formyls cytimidine (5fC), so
It is sequenced using bisulfite afterwards.In the process, 5-hmC initial oxidations are 5fC, and then deamination forms U.Generally, at the same using
Various detection methods carry out quantitative determination to 5-hmC.
In one embodiment of the invention, base of the invention is determined using chemical labeling method combination high-flux sequence
Because of the 5-hmC contents of mark.In the specific embodiment, the 5-hmC contents of gene marker of the invention are determined
Method is comprised the following steps：By the DNA fragmentation of the sample from intestinal cancer or patients with gastric cancer and normal person；By the fragmentation
Repair and blunt end DNA ends；The DNA of blunt end is connected with sequence measuring joints, connection product is obtained；Reacted by marking
5-hydroxymethyl cytosine in connection product is marked；The DNA fragmentation containing 5-hydroxymethyl cytosine mark is enriched with, is obtained
Enriched product；Enter performing PCR amplification to enriched product, obtain sequencing library；High-flux sequence is carried out to sequencing library, is sequenced
As a result；Content of the 5-hydroxymethyl cytosine on gene is determined according to sequencing result.Wherein, mark reaction includes：I) using sugar
Based transferase will be covalently attached on the methylol of 5-hydroxymethyl cytosine with the sugar of modification group, and ii) will directly or
The click chemistry substrate and the 5-hydroxymethyl cytosine with modification group for having biotin in succession react.Wherein, step i) and step
Rapid ii) can carry out in order, it is also possible to carried out simultaneously in being reacted at one.This labeling method reduces the sample needed for being sequenced
Biotin label in this amount, and 5-hydroxymethyl cytosine makes it that dynamic signal higher is shown in sequencing, improves
The accuracy of nucleotides identification.In this embodiment, the glycosyl transferase is included but is not limited to：T4 bacteriophages β-glucityl
It is transferase (β-GT), T4 bacteriophage alpha-glucosyls transferase (α-GT) and its derivative with same or similar activity, similar
Thing or recombinase；The sugar with modification group is included but is not limited to：Carbohydrate (such as 6-N3- grapes with nitrine modification
Sugar) or with other chemical modification (such as carbonyl, sulfydryl, hydroxyl, carboxyl, carbon-to-carbon double bond, carbon-to-carbon triple bond, disulfide bond, amine
Base, amide groups, diene etc.) carbohydrate, wherein it is preferred that with nitrine modification carbohydrate；It is described for being indirectly connected with biotin and point
The chemical group for hitting chemical substrate is included but is not limited to：Carbonyl, sulfydryl, hydroxyl, carboxyl, carbon-to-carbon double bond, carbon-to-carbon triple bond, two sulphur
Key, amido, amide groups, diene.In this embodiment it is preferred to be enriched with the DNA pieces containing 5-hmC marks by solid phase material
Section.Specifically, can by solid phase compatible reaction or other specifically bind reaction and will be marked containing 5-hydroxymethyl cytosine
DNA fragmentation is combined on solid phase material, then removes uncombined DNA fragmentation by repeatedly washing.Solid phase material include but not
It is limited to silicon chip or other chips with surface modification, such as artificial macromolecule bead (preferably a diameter of 1nm-100um), magnetic
(preferably a diameter of 1nm-100um) such as bead (preferably a diameter of 1nm-100um), agarose beads.It is used in solid phase enrichment
Cleaning solution is buffer solution well known to those skilled in the art, including but not limited to：Contain Tris-HCl, MOPS, HEPES (pH=
6.0-10.0, concentration is between 1mM to 1M), the buffering of NaCl (0-2M) or surfactant such as Tween20 (0.01%-5%)
Liquid.In this embodiment it is preferred to directly in the enterprising performing PCR amplification of solid phase so as to prepare sequencing library.If it is desired, in solid phase
After the amplification of enterprising performing PCR, can reclaim carry out the second wheel PCR amplifications after amplified production and prepare sequencing library.Second wheel
PCR amplifications can be carried out with conventional method well known by persons skilled in the art.Optionally, can enter during sequencing library is prepared
One step includes one or more purification steps.Any purification kit that those skilled in the art know or commercially available can use
In the present invention.Purification process is included but is not limited to：Gel electrophoresis gel extraction, pellosil centrifugal column method, paramagnetic particle method, ethanol or different
The propyl alcohol precipitation method or its combination.Optionally, before high-flux sequence, quality examination is carried out to sequencing library.For example, to library
Carry out clip size analysis and absolute quantitation is carried out to the concentration in library using qPCR methods.By the sequencing library of quality examination
Can be used for high-flux sequence.Then the library of certain amount (1-96) containing different barcode is mixed simultaneously by same concentrations
Machine sequencing in machine method, obtains sequencing result in standard according to two generation sequenators.Various two generations sequencings known in the art are flat
The reagent of platform and its correlation can be used for the present invention.
In one embodiment of the invention, preferably sequencing result is compared with standard human's genome reference sequences
It is right, pick out the sequence wherein compared on gene marker of the present invention, that is, select than loci and gene expression characteristicses (such as histone
Decorating site, Binding site for transcription factor, gene extron include subregion and gene promoter etc.) read of overlapping region
Quantity, to represent modification levels of the 5-hmC on the gene, so as to determine contents of the 5-hmC on the gene marker.It is preferred that
Before comparing, sequencing result is removed into low quality sequencing site first, wherein the factor for weighing sequencing site quality includes
But it is not limited to：Base quality, reads mass, G/C content, repetitive sequence and Overrepresented sequence quantity etc..The step
In the various comparison softwares that are related to and analysis method be known in the art.
In one embodiment of the invention, the 5-hmC contents for determining gene marker refer to determine the genetic marker
5-hmC contents in thing total length determine 5-hmC contents or its combination of a certain fragment on the gene marker.
According to the present invention, on each gene marker is determined after 5-hmC contents, with genetic marker described in normal specimens
The 5-hmC contents of thing as reference, by the 5-hmC content standards of corresponding gene marker in Samples subjects.Citing and
Speech, the 5-hmC contents of same gene mark are respectively X and Y in normal specimens and Samples subjects, then should in Samples subjects
The standardization 5-hmC contents of gene marker are Y/X.
According to the present invention, after data normalization, the standardization 5-hmC contents to each gene marker carry out mathematical
To be scored, so as to obtain testing result according to the scoring.As used herein, " mathematical " refers to by from biological sample
Any computational methods or machine learning side that the 5-hmC contents of the gene marker of product are associated with intestinal cancer or diagnosing gastric cancer result
Method.Those of ordinary skill in the art understand that optional different computational methods or instrument are used to provide mathematical of the invention,
For example elastomeric network regularization, decision tree, generalized linear model, logistic regression, highest score are to, neutral net, linear and two
Secondary discriminant analysis (LQA and QDA), naive Bayesian, random forest and SVMs.
In one embodiment of the invention, the standardization 5-hmC contents to each gene marker carry out mathematical
And obtain comprising the following steps that for scoring：The standardization 5-hmC contents of each gene marker are multiplied by weight coefficient, the base is obtained
Because of the predictive factor t of mark；The predictive factor t of each gene marker is added, total predictive factor T is obtained；To always predict because
Sub- T obtains scoring P by Logistic conversions；If P ＞ 0.5, the Samples subjects suffer from intestinal cancer or stomach cancer；If P ＜ 0.5,
Then the Samples subjects are normal.Weight coefficient as herein described refer to consider may influence 5-hmC contents factor (for example
Subject region, the age, sex, less than, smoking history, history of drinking history, family history etc.) in the case of, by various advanced statisticals point
The coefficient that analysis method is obtained.
Compared with prior art, for detecting the method for intestinal cancer and/or stomach cancer it is based on gene marker in the present invention
5-hmC contents, therefore can be originated using more extensive DNA sample.Therefore, the present invention in for detect intestinal cancer and/or
The method of stomach cancer has following advantage：(1) safety is noninvasive, even if Silent cerebral infarction is also high to the detection acceptance；(2)
DNA wide material sources, in the absence of the check frequency in iconography；(3) accuracy is high, there is sensitivity and specificity higher；(4) grasp
Facilitate, Consumer's Experience is good, easily carry out disease dynamic monitoring.
Brief description of the drawings
Fig. 1：The result of first sample is distinguished with intestinal oncofetal gene mark of the invention.
Fig. 2：The result of second batch sample is distinguished with intestinal oncofetal gene mark of the invention.
Fig. 3：The 3rd batch of result of sample is distinguished with stomach cancer gene marker of the invention.
Fig. 4：The 4th batch of result of sample is distinguished with stomach cancer gene marker of the invention.
Specific embodiment
The present invention is described in detail below with reference to the accompanying drawings and in conjunction with the embodiments, so that those skilled in the art can be more
Good understanding is of the invention and can be practiced.It should be noted that it should be appreciated by those skilled in the art accompanying drawing of the invention
And the purpose that embodiment is merely to illustrate that, any limitation can not be constituted to the present invention.In the case of reconcilable, this
The feature in embodiment and embodiment in application can be mutually combined.
The screening of the intestinal oncofetal gene mark of embodiment 1.
(1) plasma dna is extracted：
10ng plasma dnas are extracted respectively from from 15 patients with bowel cancer and 18 samples of normal person.Using ability
Any method and reagent suitable for extracting plasma dna known to field technique personnel carries out this step.
(2) plasma dna carried out into blunt end, hang A and be connected with sequence measuring joints：
Prepared according to Kapa Hyper PerpKit specifications and contain 50uL plasma dnas, 7uLEnd Repair＆A-
(cumulative volume is the reaction mixture of Tailing Buffer and 3uL End Repair＆A-Tailing Enzyme mix
60uL), in 20 DEG C of warm bath 30 minutes, then in 65 DEG C of warm bath 30 minutes.Connection is anti-below configuration in the low absorption EP pipes of 1.5mL
Answer mixture：5uL Nuclease free water, 30uL Ligation Buffer and 10uL DNA Ligase.To
The sequence measuring joints of 5uL are added in 45uL coupled reaction mixtures, mixing is heated 20 minutes in 20 DEG C, is then held in 4 DEG C.Make
Product is purified with AmpureXP beads, Tris-HCl (10mM, pH=8.0) and EDTA is contained with 20uL
The buffer solution of (0.1mM) carries out wash-out and obtains final DNA connection samples.
(3) 5-hydroxymethyl cytosine is marked：
Prepare the mark reaction mixture that cumulative volume is 26uL：UDp-glc (the i.e. UDP-N3- of nitrine modification
Glu, final concentration of 50uM), β-GT (final concentration of 1uM), Mg2+(final concentration of 25mM), HEPES (pH=8.0, it is final concentration of
20uL DNA 50mM) and from above-mentioned steps.By mixed liquor in 37 DEG C of warm bath 1 hour.Mixed liquor is taken out, AmpureXP is used
Beads is purified, and obtains the 20uL DNA of purifying.
Then 1uL is added to be connected with the diphenyl cyclooctyne (DBCO- of biotin in the 20uL DNA of above-mentioned purifying
Biotin), reacted 2 hours in 37 DEG C, then purified with AmpureXP beads, obtain the marked product of purifying.
(4) DNA fragmentation of the solid phase enrichment containing markd 5-hydroxymethyl cytosine：
First, magnetic bead is prepared according to the following steps：Take out 0.5uL C1streptadvin beads (life
Technology) and 100uL buffer solutions (5mM Tris, pH=7.5,1M NaCl, 0.02%Tween20), vortex mixed are added
30 seconds, then wash magnetic bead 3 times with 100uL cleaning solutions (5mM Tris, pH=7.5,1M NaCl, 0.02%Tween20), most
25uL combination buffers (10mM Tris, pH=7.5,2M NaCl, 0.04%Tween20 or other surfaces activity are added afterwards
Agent), and be well mixed.
Then, the marked product of the purifying of above-mentioned steps acquisition is added in magnetic bead mixed liquor, and in impeller
Mixing 15min makes it fully combine.
Finally, magnetic bead 3 is washed with 100uL cleaning solutions (5mM Tris, pH=7.5,1M NaCl, 0.02%Tween20)
Secondary, supernatant is removed in centrifugation, adds water of the 23.75uL without nuclease.
(5) PCR amplifications：
To being added in the final system of above-mentioned steps, 2X PCR master mix and the 1.25uL PCR primer of 25uL are (total
Volume is 50uL), expanded according to the temperature and condition of following PCR reaction cycles：
Amplified production AmpureXP beads are purified, final sequencing library is obtained.
(6) high-flux sequence is carried out after carrying out quality inspection to sequencing library：
The sequencing library of acquisition is carried out into concentration mensuration by qPCR, and it is big to DNA fragmentation in library with Agilent2100
Small content is determined.To be mixed with same concentrations by the sequencing library of quality inspection, surveyed with Illumina Hiseq 4000
Sequence.
(7) the 5-hmC contents and weight coefficient of each gene marker are determined
The sequencing result of acquisition is carried out into preliminary Quality Control assessment, after removing low quality sequencing site, sequencing quality is up to
The read of standard is compared using Bowtie2 instruments with human standard genome reference sequences.Then utilize
FeatureCounts and HtSeq-Count instruments are come the 5-hmC contents that count read quantity to determine each gene marker.Together
Shi Liyong high-flux sequence results, it would be possible to the factor of 5-hmC contents is influenceed as covariate, by logistic regression and elastic network(s)
Network regularization obtains the weight coefficient of each gene marker.Result is as shown in table 1.
Table 1：The Average normalized 5-hmC contents and weight coefficient of intestinal oncofetal gene mark of the invention
As described above, Average normalized 5-hmC contents refer to the average 5-hmC contents of the gene marker in intestinal cancer sample
The ratio between with the average 5-hmC contents of same gene mark in normal specimens.As it can be seen from table 1 intestinal oncofetal gene of the invention
There is significant difference in normal specimens and in intestinal cancer sample in the 5-hmC contents of mark, and in addition to NME3, remaining gene
The 5-hmC contents of mark relative to normally dramatically increasing per capita.
The validity of the intestinal oncofetal gene mark of embodiment 2.
The present embodiment verifies that intestinal oncofetal gene mark of the invention is used to detect the validity of intestinal cancer.
Method according to embodiment 1 determines of the present invention in first 59 sample (24 intestinal cancer and 35 controls)
10 5-hmC contents of intestinal oncofetal gene mark, and determine the weight coefficient of each gene marker.
The standardization 5-hmC contents of each gene marker are multiplied by corresponding weight coefficient, the genetic marker is obtained
After the predictive factor t of thing, the predictive factor t of each gene marker is added, obtains total predictive factor T, then will always predict because
Sub- T obtains scoring P according to below equation by Logistic conversions：
If P ＞ 0.5, the Samples subjects suffer from intestinal cancer；If P ＜ 0.5, the Samples subjects are normal.
Fig. 1 shows that the method according to the invention distinguishes this batch of result of sample.As shown in figure 1, method of the present invention energy
Enough reach 83% sensitivity and 94% specificity.Inventor further distinguishes 69 samples of second batch with the method for the present invention
(32 intestinal cancer and 37 controls), its result shows that the method can reach 88% sensitivity and 89% specificity (Fig. 2).
These results indicate that compared with prior art, the method according to the invention can be with sensitivity higher and special
Property detection intestinal cancer.
The screening of the stomach cancer gene marker of embodiment 3.
Method according to embodiment 1 screens the gene marker of stomach cancer, and unique difference is that specimen in use is from 7
Position patients with gastric cancer and 18 plasma DNAs of normal person.The stomach cancer marker for screening is as shown in table 2.
Table 2：The Average normalized 5-hmC contents and weight coefficient of stomach cancer gene marker of the invention
As described above, Average normalized 5-hmC contents refer to the average 5-hmC contents of the gene marker in stomach cancer samples
The ratio between with the average 5-hmC contents of same gene mark in normal specimens.From table 2 it can be seen that stomach oncogene of the invention
There is significant difference in normal specimens and in intestinal cancer sample in the 5-hmC contents of mark, wherein in stomach cancer samples, 3 kinds of bases
Because of mark display 5-hmC content reductions：RDM1, ZNF714 and ZNF518B, 15 kinds of gene markers show 5-hmC content liters
It is high：ARHGAP28、BMPER、C9orf92、CADPS2、CDH11、FBXL7、MEOX2、NOS1、OSMR、PALM2、PDE10A、
RBMS3, SULF1, WLS and WTIPo
The validity of the stomach cancer gene marker of embodiment 4. is determined
The present embodiment verifies that stomach cancer gene marker of the invention is used to detect the validity of stomach cancer.
Method according to embodiment 1 determines of the present invention in the 3rd batch of 60 samples (25 stomach cancers and 35 control)
18 5-hmC contents of stomach cancer gene marker, and determine the weight coefficient of each gene marker.
The standardization 5-hmC contents of each gene marker are multiplied by corresponding weight coefficient, the genetic marker is obtained
After the predictive factor t of thing, the predictive factor t of each gene marker is added, obtains total predictive factor T, then will always predict because
Sub- T obtains scoring P according to below equation by Logistic conversions：
If P ＞ 0.5, the Samples subjects suffer from stomach cancer；If P ＜ 0.5, the Samples subjects are normal.
Fig. 3 shows that the method according to the invention distinguishes this batch of result of sample.As shown in figure 3, method of the present invention energy
Enough reach 92% sensitivity and 91% specificity.Inventor further distinguishes the 4th batch of 63 samples with the method for the present invention
(29 stomach cancers and 37 controls), its result shows that the method can reach 90% sensitivity and 97% specificity (Fig. 4).
These results indicate that compared with prior art, the method according to the invention can be with sensitivity higher and special
Property detection intestinal cancer.
Claims (13)
1. it is used to detect the gene marker of intestinal cancer, including one or more are selected from following gene：ADAM metallopeptidases domain 20
(ADAM20), F boxes and full asphalt mixture albumen 7 (FBXL7), follistatin (FST), TP53 apoptosis effects device (PERP), general
The homologous similar regions family A member 3 (PHLDA3) of lek substrate protein, the mobile companion 1 (RUNX1T1) of Runt associated transcription factors 1,
Syntrophism albumen γ 2 (SNTG2), sperm antigen 4 (SPAG4), sulfatase 1 (SULF1) and NME/NM23 nucleoside diphosphates
Kinases (NME3).
2. the gene marker described in claim 1, including ADAM20, FBXL7, FST, PERP, PHLDA3, RUNX1T1,
SNTG2, SPAG4, SULF1 and NME3.
3. purposes of the gene marker described in claim 1 or 2 in for the method for detecting intestinal cancer.
4. it is used to detect the gene marker of stomach cancer, including one or more are selected from following gene：Rho gtpase activating proteins
28 (ARHGAP28), BMP combination endotheliums regulation and control person (BMPER), the opening code-reading frame 92 (C9orf92) of chromosome 9, calcium dependent secretory
Swash reviver 2 (CADPS2), cadherin 11 (CDH11), F boxes and the homologous frame 2 of full asphalt mixture albumen 7 (FBXL7), interstitial
(MEOX2), nitric oxide synthetase 1 (NOS1), oncostatinM acceptor (OSMR), cell membrane modulin paralemmin2
(PALM2), the single-stranded interaction protein 3 (RBMS3) of phosphodiesterase 10 A (PDE10A), RNA combination die bodys, sulfatase 1
(SULF1), wntless Wnt parts regulation of secretion person (WLS), the action protein of Wilms knurls 1 (WTIP), zinc finger protein 51 8B
(ZNF518B), zinc finger protein 714 (ZNF714) and RadS2 die bodys include 1 (RDM1).
5. the gene marker described in claim 4, including ARHGAP28, BMPER, C9orf92, CADPS2, CDH11,
FBXL7, MEOX2, NOS1, OSMR, PALM2, PDE10A, RBMS3, SULF1, WLS, WTIP, ANF518B, ZNF714 and RDM1.
6. purposes of the gene marker described in claim 4 or 5 in for the method for detecting stomach cancer.
7. a kind of method for detecting intestinal cancer, comprises the following steps：
The 5-hydroxymethyl cytosine of the gene marker in (a) measure normal specimens and Samples subjects described in claim 1 or 2
The content of (5-hmC)；
B () uses the 5-hmC contents of gene marker described in normal specimens as reference, by corresponding gene in Samples subjects
The 5-hmC content standards of mark；
C () carries out mathematical to the 5-hmC contents of the normalised gene marker in step (b), and scored；
With
D () obtains testing result according to the scoring.
8. a kind of method for detecting stomach cancer, comprises the following steps：
The 5-hmC contents of the gene marker in (a) measure normal specimens and Samples subjects described in claim 4 or 5；
(b) with the 5-hmC contents of gene marker described in normal product as reference, by corresponding gene mark in Samples subjects
The 5-hmC content standards of will thing；
C () carries out mathematical to the 5-hmC contents of the normalised gene marker, and scored；With
D () obtains testing result according to the scoring.
9. the method described in claim 7 or 8, wherein step (a) are determined in the gene marker total length or its fragment
The content of 5-hmC.
10. the method described in claim 7 or 8, wherein the sample be from normal person or subject's body fluid middle reaches from DNA
Fragment, or the complete genome group DNA in organelle, cell and tissue.
Method described in 11. claims 10, wherein the body fluid is blood, urine, sweat, sputum, excrement, cerebrospinal fluid, abdomen
Water, hydrothorax, bile or pancreas liquid.
A kind of 12. kits for detecting intestinal cancer, including：
A () is used for the reagent of the 5-hmC contents for determining the gene marker described in claim 1 or 2；With
(b) specification.
A kind of 13. kits for detecting stomach cancer, including：
A () is used for the reagent of the 5-hmC contents for determining the gene marker described in claim 4 or 5；With
(b) specification.
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| CN201710598499.4A CN108300783A (en) | 2017-01-11 | 2017-01-11 | The method of gene marker for screening intestinal cancer and/or gastric cancer, the gene marker and application thereof screened with this method |
| CN201710020252.4A CN106755464A (en) | 2017-01-11 | 2017-01-11 | For the method for screening the gene marker of intestinal cancer and/or stomach cancer, the gene marker and application thereof that is screened with the method |
| PCT/CN2017/108423 WO2018129989A1 (en) | 2017-01-11 | 2017-10-30 | Method for screening for gene marker of intestinal cancer and/or stomach cancer, gene marker for screening via method, and application of gene marker |
| TW106146503A TWI647312B (en) | 2017-01-11 | 2017-12-29 | Method for screening gene markers of intestinal cancer, gene markers screened by the method, and uses thereof |
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| CN107385050A (en) * | 2017-08-04 | 2017-11-24 | 上海易毕恩生物技术有限公司 | For detecting the gene marker, kit and cancer of pancreas detection method of cancer of pancreas |
| WO2018129989A1 (en) * | 2017-01-11 | 2018-07-19 | 上海易毕恩基因科技有限公司 | Method for screening for gene marker of intestinal cancer and/or stomach cancer, gene marker for screening via method, and application of gene marker |
| WO2018129988A1 (en) * | 2017-01-11 | 2018-07-19 | 上海易毕恩基因科技有限公司 | Method for detecting intestinal cancer by gene markers, gene markers screened using method and use thereof |
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2018129989A1 (en) | 2018-07-19 |
| TWI647312B (en) | 2019-01-11 |
| TW201829781A (en) | 2018-08-16 |
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