CN107130027A - Application of the biomarker in colorectal cancer - Google Patents
Application of the biomarker in colorectal cancer Download PDFInfo
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- CN107130027A CN107130027A CN201710339698.3A CN201710339698A CN107130027A CN 107130027 A CN107130027 A CN 107130027A CN 201710339698 A CN201710339698 A CN 201710339698A CN 107130027 A CN107130027 A CN 107130027A
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
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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
The invention discloses application of the biomarker in colorectal cancer, specifically the biomarker is NHEG1, present invention firstly discovers that NHEG1 expresses in temper virtual loss type advanced colorectal cancer patient downward, the foundation for judging advanced colorectal cancer patient's parting can be turned into by pointing out detection NHEG1 expression.Simultaneously, the invention provides application of the NHEG1 genes in temper virtual loss type advanced colorectal cancer patient's therapeutic evaluation is prepared, when NHEG1 expression in temper virtual loss type advanced colorectal cancer patient and the expression no significant difference in non-temper virtual loss type advanced colorectal cancer patient, illustrate that curative effect of medication is good.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of application of biomarker in colorectal cancer, specifically should
Biomarker is NHEG1.
Background technology
Colorectal cancer already rises to the 3rd in global case fatality rate, and morbidity and mortality are in rising trend, and the whole world is every
Year number of the infected is about 900,000 people, the people of death toll about 500,000.In China, Colorectal Cancer is in obvious ascendant trend, especially
It is that situation allowed of no optimist in recent years, has been to influence the most common malignant tumour of Chinese's health at present, the incidence of disease comes the 4th
Position, the life and health of the serious threat mankind.However, the overall treatment effect of colorectal cancer is still choosing for people's sternness so far
War, 5 years survival rates are still hovered in 70% (carcinoma of the rectum) and 50% (colon cancer) left and right.II phase colon cancers, there is 60%~70%
Patient by operation can cure, even if but 15~20% patient it is postoperative receive chemotherapy, still recur.III phase colon cancers
In, operation can cure 40~50% patient, although there are about 35% postoperative can still be recurred using chemotherapy.It is to deposit for the IV phases
The patient of colon cancer is shifted a long way off, and no matter radiotherapy, its benefit of chemotherapy are lower.In recent years, the discovery of multidrug resistance more illustrated
The limitation of chemotherapeutics, therefore people begin look for drug-tolerant gene mutation site, are studied for target spot, effectively extend patient
Life span.Yet with expensive testing cost and monoclonal antibody target medical expense so that use this kind of medicine in reality
Thing crowd is few.
Therefore under the proposition of accurate medical model, Chinese medicine turns into indispensable in Multimodality Therapy of Malignant Tumors pattern
Means, due to the feature that its diagnosis and treatment means is rapid, effective, cheap, and curative effect obtain it is extensive side effect is smaller certainly, it is economical
Burden is lighter, increasingly by the accreditation of patient and favoring for scholar.
TCM Syndrome is the high level overview of body a certain stage etiology and pathogenesis in disease progression, reacts stage disease
Manage the essence of change.Since same " card " has common clinical manifestation and pathologic basis, that considers that it has common material
Basis, and this material base is probably reflected on gene level.Advanced colorectal cancer temper is lost from molecular level
The disease Essential study of asthenic symptoms provides new thinking for the treatment of colorectal cancer；Also it is the Chinese traditional treatment of colorectal cancer simultaneously
Provide fundamental basis.
The content of the invention
It is an object of the invention to provide a kind of application of biomarker in colorectal cancer.
The invention provides biomarker NHEG1 purposes, for preparing diagnosis diagnosis of colorectal carcinoma or therapeutic evaluation
Product.
Further, the colorectal cancer is temper virtual loss type advanced colorectal cancer.
Further, the product is by determining the level of biomarker and the reference water of corresponding biomarker in sample
It is flat to be determined compared to up-regulation.
Further, the biomarker includes the polynucleotides or its piece of the nucleotide sequence as shown in SEQ ID NO.1
Section, homologue, variant or derivative.
The invention provides a kind of product of NHEG1 expressions in vitro detection sample, the product includes：By surveying
Sequence technology, nucleic acid hybridization technique, nucleic acid amplification technologies or the method for immunoassays detect the expression of NHEG1 genes.
Further, the nucleic acid amplification technologies are selected from PCR, reverse transcriptase polymerase chain reaction, transcription Jie
Amplification, ligase chain reaction, strand displacement amplification and the amplification based on nucleotide sequence led.
Further, the product includes preparation, chip or kit.
Further, the product includes the reagent of specific recognition NHEG1 genes.
Further, the reagent of the specific recognition NHEG1 genes is selected from：
The primer of specific amplification NHEG1 genes；Or
The probe of specific recognition NHEG1 genes.
The diagnosis of temper virtual loss type advanced colorectal cancer or therapeutic evaluation instrument are being prepared the invention provides the said goods
In application.
The advantages of the present invention：
, accordingly can be with present invention firstly discovers that NHEG1 genes are to the treatment of temper virtual loss type advanced colorectal cancer related
Doctor is instructed to determine use and the usage cycles of clinical medicine.
The invention provides a kind of diagnostic products, by detecting NHEG1 expression, so as to judge whether patient is spleen
Gas deficiency patient, and then carry out Coryza Treated by Syndrome Differentiation.
Brief description of the drawings
Fig. 1 is to utilize differential expression statistical chart of the genechip detection NHEG1 genes in colorectal cancer patients；
Fig. 2 is to detect differential expression statistical chart of the NHEG1 genes in colorectal cancer patients using QPCR.
Embodiment
By the present invention in that being found that NHEG1 and spleen first with four gentleman's granule therapy temper virtual loss type advanced colorectal cancers
Gas deficiency advanced colorectal cancer is related, and demonstrates the NHEG1 in blood in temper virtual loss type advanced colorectal cancer patient
Height expression.NHEG1 can as colorectal cancer independentpredictor, can also be with other biomarker use in conjunction.
Biomarker
Term " biomarker " is its expression and normal or healthy cell or tissue in tissue or cell
Expression compares any gene or albumen changed.
It would be recognized by those skilled in the art that the practicality of the present invention is not limited to the marker gene of the present invention
The gene expression of any specific variants is quantified.As nonrestrictive example, marker gene can have SEQ ID NO.1
The polynucleotide sequence specified.In some embodiments, it has same or analogous with listed sequence at least 85%
CDNA sequence, all listed sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% as described above or
At least 99% same or analogous cDNA sequence.
Term " fragment " refers to a part of of polynucleotides.For biomarker nucleotide sequence fragment it is many
Nucleotides generally comprises at least 10,15,20,50,75,100,150,200,250,300,350,400,450,500,550,600,
650th, 700,800,900,1,000,1,100,1,200,1,300 or the continuous nucleotides of Isosorbide-5-Nitrae 00, or be at most present in herein
Nucleotides number in disclosed total length biomarker polynucleotides.
" variant " is intended to indicate that essentially similar sequence.In general, the variant of particular organisms mark of the invention
By with determined by alignment programs with the biomarker at least about 40%, 45%, 50%, 55%, 60%,
65%th, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
Higher sequence identity.
" polynucleotides " in the present invention include RNA, cDNA, genomic DNA, synthesized form and the polymer of mixing, have
Adopted chain and antisense strand, and can be chemistry or biochemical modification, or non-natural or derivative nucleosides soda acid can be contained
Base, this is easily recognized by those skilled in the art.It is this modification include for example mark, methylate, with analog substitution one or
Modification such as uncharged key connects (such as methyl phosphorodithioate, phosphotriester, phosphoric acid between multiple nucleotides naturally occurred, nucleotides
Acid amides, carbamate etc.), electrically charged key connect (such as thiophosphate, phosphorodithioate etc.), overhang
(pendent moieties) (such as polypeptide), insert (such as acridine, psoralen), chelating agent, alkylating agent, and modification
Key connect (such as α anomeric nucleic acids).Also synthetic molecules are included, there are simulation polynucleotides to pass through hydrogen bond and otherization for it
Learn the ability that interaction combines specified sequence.This molecule is known in the art, including the peptide for example wherein in molecular backbone
Key replaces phosphate bond.
Gene expression detection technology
The present invention can determine gene expression using any method known in the art.Those skilled in the art should manage
Solution, the means for determining gene expression are not the importances of the present invention.The table of biomarker can be detected on transcriptional level
Up to level.
Generally, PCR is followed using denaturation, primer pair and the annealing of opposite strand and the multiple of primer extend
Ring, exponentially increases the copy number of target nucleic acid sequence；Reverse transcriptase (RT) is then used for by reverse transcriptase polymerase chain reaction
Complementary DNA (cDNA) is prepared from mRNA, then expands cDNA by PCR to produce DNA multiple copies；Transcriptive intermediate
Amplification autocatalytically synthesizes the multiple of target nucleic acid sequence under conditions of the temperature, ionic strength and pH of substantial constant and copied
Multiple RNA copies of shellfish, wherein target sequence autocatalytically generate other copy, and the amplification of transcriptive intermediate optionally includes making
With blocking, part, dwell section and other modified parts, with the sensitivity and the degree of accuracy of the amplification procedure for improving transcriptive intermediate；
Ligase chain reaction uses two groups of complementary DNA oligonucleotides that the adjacent area with target nucleic acid hybridizes.DNA oligonucleotides is in warm
It is covalently attached in the multiple circulations of repetition of denaturation, hybridization and connection by DNA ligase, it is few to produce detectable double-strand connection
Oligonucleotide product；Strand displacement amplification uses multiple circulations of following steps：The opposite strand of primer sequence pair and target sequence is moved back
Fire, primer extend is carried out in the case where there is dNTP α S to produce (hemiphosphorothioated) of the thiophosphorylation of double-strand half
Primer extension product, the nicking for the endonuclease mediation that semi-modified restriction enzyme enzyme recognition site is carried out, and from cutting
The polymerase-mediated thing drawn that mouth 3' ends are carried out extends to be put with replacing existing chain and producing for next round primer annealing, nicking and chain
The chain changed, so as to cause the geometry of product to expand.
The selection of nucleic acid hybridization formats is not crucial.Multiple nucleic acids hybrid versions include but is not limited to sandwich assay and competing
Strive or substitute measure.The detection of hybridization complex can need to produce pair of Signaling complex and target and probe polynucleotide or nucleic acid
The combination of spiral.Generally, this combination is interacted by part and anti-ligand and occurred, the probe and idol of such as ligand coupling
It is associated with the interaction between the anti-ligand of signal.By the way that exposed to ultrasonic energy, the combination of signal generation compound is also easy to
To acceleration.
Preparation, chip or kit
Term " chip " is also referred to as " array ", refers to the solid support of the nucleic acid comprising connection or peptide probes.Array is usual
Include a variety of different nucleic acid or peptide probes that substrate surface is connected to according to different known locations.These arrays, also referred to as
" microarray ".
" microarray " is that hybridised arrays original paper is ordered in matrix, and the hybridised arrays original paper such as polynucleotide is visited
Pin (such as oligonucleotides) or bonding agent (such as antibody).The matrix can be solid matrix, for example, glass or silica
Slide, pearl, fibre optics binding agent or semi-solid matrix, such as nitrocellulose filter.Nucleotide sequence can be DNA, RNA or
Any arrangement therein.
Term " probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.Unless another
Point out, term " probe " is often referred to can be by complementary base pairing and another polynucleotides (often referred to as " target polynucleotide ")
With reference to polynucleotide probes.Lack sufficient sequence complementarity according to the preciseness of hybridization conditions, probe energy and with the probe
Target polynucleotide is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, including, but do not limit
In：Solution, solid phase, mixed phase or in situ hybridization determination method.
Probe is generally directly marked, such as with isotope, chromophore, illuminophore, chromogen, or by indirect labelling, example
Biotin is such as used, Streptavidin compound can then be combined with biotin.Therefore, it is used during the present invention is determined to can detect
Mark can be that (wherein the mark includes the element that can directly detect or can produce directly detectable element primary marker
Element) or two grades of marks (wherein detectable mark is combined with primary marker, for example, conventional in immune labeled).Generally, mark
The signal nucleic acid of note is used to detect and hybridized.Can by be usually used in detect hybrid polynucleotide exist several methods in any
Plant mark complementary nucleic acid or signal nucleic acid.The most frequently used detection method is to utilize use3H、125I、35S、14C or32The spy of P marks
The autoradiography of pin etc..Other marks include, for example, can the part of binding marker antibody, fluorogen, chemiluminescence agent,
Enzyme and can as the specific binding pair members of tagged ligand antibody.
The polynucleotides used as probe be preferably sized to 18 or more nucleotides, more preferably 20 or
More nucleotides, and transcript regions total length or less.As primer in use, the polynucleotides are preferably sized to 18
Or more nucleotides, and 50 or more Oligonucleotide.
Above-mentioned probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ",
As long as hybridization, can not be complete complementary.These polynucleotides, which are commonly angled relative to the specific base sequence, to be had
More than 80%, preferably more than 90%, more preferably more than 95%, particularly preferred 100% homology.These probes can be DNA,
Can also be RNA, furthermore it is possible to be artificial by PNA, LNA, ENA, GNA, TNA etc. in one part or whole nucleotides
The polynucleotides that replacement nucleic acid is obtained.
In the present invention, genetic chip can be used for multiple genes of the detection including NHEG1 genes (for example, straight with knot
The related multiple genes of intestinal cancer) expression.Protein-chip can be used for multiple albumen of the detection including NHEG1 albumen
The expression of matter (such as multiple protein related to colorectal cancer).
Term " kit " includes the reagent of the detection NHEG1 genes of detection effective dose, the one or more being selected from the group
Material：Container, operation instructions, positive control, negative control thing, buffer, auxiliary agent or solvent.For example for being suspended or admittedly
Determine the solution of cell, detectable label or tag makes nucleic acid be easy to the solution of hybridization, for the solution of cell lysis, or uses
In the solution of nucleic acid purification.
Can also have the operation instructions of kit in the kit of the present invention, how be entered wherein describing using kit
Row detection, and how tumor development to be judged using testing result, therapeutic scheme is selected.
Using the kit of the present invention, NHEG1 can be detected by the various methods (including but is not limited to) being selected from the group：It is real
When Quantitative Reverse Transcription PCR, biochip test method, southern blotting technique method or RNA blottings or hybridization in situ.The common skill in this area
Art personnel can be according to physical condition and needing to be adjusted detection mode and change.
In the context of the present invention, " diagnosis colorectal cancer " both includes judging whether subject suffers from Colon and rectum
Cancer, also include judge subject with the presence or absence of suffer from colorectal cancer risk.
Term " sample " includes but is not limited to, can be blood, tissue, urine, serum, blood plasma, amniotic fluid, cerebrospinal fluid,
Placenta cells or tissue, endothelial cell, leucocyte or monocyte.Sample can derive from patient or object is directly used, or
Person is pre-processed, and is such as handled by filtering, distilling, extract, concentrate, centrifuge, inactivating interference component, addition reagent mode,
The characteristic of sample is modified in some modes as described herein or known in the art.In a particular embodiment of the present invention, institute
" sample " is stated for blood.
When the biomarker of the present invention is used to diagnose, when the expression water of the NHEG1 genes in late period colorectal cancer patients
During flat substantially less than non-temper virtual loss type colorectal cancer, then it represents that the colorectal cancer patients are temper virtual loss type colorectal cancer.
When the biomarker of the present invention is used to evaluate curative effect, when giving the medicine of temper virtual loss type colorectal cancer patients,
Lost when the expression for detecting the NHEG1 genes in temper virtual loss type advanced colorectal cancer patient is significantly higher than temper before treatment
Deficiency advanced colorectal cancer patient or with the expression no significant difference in non-temper virtual loss type advanced colorectal cancer patient
When, then show that the therapeutic effect of the medicine is preferable.When detecting the NHEG1 genes in temper virtual loss type advanced colorectal cancer patient
Expression when being substantially less than the expression in non-temper virtual loss type advanced colorectal cancer patient, then show that patient also needs to
Continue medication treatment.
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.
Embodiment 1 screens the biomarker related to colorectal cancer
1st, case is collected
Collect III~IV phase colorectal cancer patients that hospital's oncology is clarified a diagnosis through pathology；Temper virtual loss is selected to demonstrate,prove 5
As experimental group, non-temper virtual loss demonstrate,proves 5 as a control group；Only receive pure Chinese traditional treatment.
The diagnosis of 1.1 colorectal cancers and staging scale
Write in October, 2010 with reference to Department of Medical Administration of Ministry of Health of the People's Republic of China《Colorectal cancer diagnosis and treatment specification》,
2011NCCN clinical tumors practice guideline (Chinese version) first edition TNM stage standard.
The diagnostic criteria of 1.2 TCM syndromes
Temper virtual loss is demonstrate,proved：
Main symptom：Belly secret anguish, though inhibited defecation or there is the difficulty of awareness of defecation solution, loose stool；
Secondary card：Make swollen after spiritlessness and weakness, poor appetite, food, sallow complexion, pale tongue is fat, tongue is white, deep thready pulse.Possesses main symptom 1
, secondary card 3 is diagnosable.
1.3 include, exclusion standard
Inclusive criteria：(1) Han nationality III~IV phase colorectal cancer patients, only receive pure Chinese traditional treatment；(2) 18~75 years old；
(3) Cattell scoring (KPS) >=60 point；(4) estimated 3 months life cycles ＞；(5) compliance is good, signed informed consent form.
Exclusion standard：(1) cardiovascular and cerebrovascular, liver, kidney and disease of hematopoietic system serious or that need treatment are associated with；(2)
Pregnant woman, nursing period and mental patient；(3) there is obvious compound card patient.
2nd, treatment method
Patient in group is extracted after peripheral blood, with four gentleman's particles (Jian-Yisheng Pharmaceutical Co., Ltd., Jilin's production)
Intervened, each 15g, mixing in water for oral taking, 3 times/d, treatment time is 21d,.During treatment, patient do not receive other doctors trained in Western medicine and in
Medical treatment, rejects case lost to follow-up, terminates and obvious adverse reaction occurs and because other reasons trigger the case of adverse events.21d
Extract peripheral blood again afterwards, carry out genechip detection, and with the chip before intervention
3rd, total mRNA is extracted
The RNA in blood is extracted using the Trizol kits of sigma companies of the U.S., is comprised the following steps that：
Early morning gathers patient empty stomach peripheric venous blood 5mL, and leucocyte is isolated with erythrocyte cracked liquid, adds 20 times of cell
The Trizol of volume.Pipettor is lashed cell until not seeing pockets of cell block repeatedly, is allowed to be completely dissolved in
Formed in limpid not sticky liquid, -80 DEG C of refrigerators and preserved in Trizol.Trizol one-step method extracted total RNAs.Quantitative analysis:
Determine absorbance respectively at 260nm and 280nm with spectrophotometer, A260/A280 ratios are purer 1.8~2.0
RNA。
4th, Agilent chip of expression spectrum hybridizes
After RNA quality inspections are qualified, the hybridization and elution of the mark, chip of sample are with reference to chip standard flow.First, always
CRNA of the RNA reverse transcriptions into double-strand cDNA, further synthesis Cyanine-3-CTp (Cy3) marks.The cRNA that has marked and
Chip hybridization, original graph is obtained after elution using Agilent Scanner G2505C (Agilent Technologies) scannings
Picture.
5th, data processing
Handled using Feature Extraction softwares (version10.7.1.1, Agilent Technologies)
Original image extracts initial data.Followed by Genespring softwares (version 12.5：Agilent
Technologies quantile standardization and subsequent treatment) are carried out.Data after standardization are filtered, for comparing
At least one group 100% probe labeled as Detected leaves carry out subsequent analysis in every group of sample.Examine what is obtained using t
Significance of difference P values and the fold differences value of normalized signal value carry out differential gene screening, the standard of screening for up-regulation or
Lower fold change value >=2.0 and P<0.05.
6th, result
As a result show, compared with non-temper virtual loss type patient, temper virtual loss type advanced colorectal cancer patient variation expression base
Because of 126, wherein 51 raised, 55 of downward.
Temper virtual loss type patient has found 15 differential gene sites altogether using four gentleman's particles in patients before and after intervention, wherein 12
Up-regulation, 3 downwards.
Wherein, as shown in figure 1, difference expression gene NHEG1 expression is compared to significantly up-regulation before intervening.
The differential expression of embodiment 2QPCR sequence verification NHEG1 genes
1st, NHEG1 genes are selected to carry out large sample QPCR checkings according to the testing result of high-flux sequence.According to embodiment 1
In sample collection mode selection temper virtual loss type advanced colorectal cancer patient and non-temper virtual loss type colorectal cancer patients each 50
Example.
2nd, treatment method step be the same as Example 1
3rd, RNA extraction steps be the same as Example 1.
4th, reverse transcription：Operated using the reverse transcription reagent box of TIANGEN companies, kit article No. is KR106.Specifically
Step is as follows：
Genomic DNA reaction is removed, 5 × DNA Buffer 2.0,1 μ g of μ l, RNA are added in test tube, RNase is removed in addition
ddH2O makes cumulative volume to 10 μ l, 42 DEG C of heating 3min in water-bath, then by 10 × Fast RT Buffer 2.0 μ L, RT
The μ L of 1.0 μ L, FQ-RT Primer Mix of Enzyme Mix 2.0, remove RNase ddH2The μ L of O 5.0, add above-mentioned test tube after mixing
In be mixed together, 42 DEG C of heating 15min, 95 DEG C of heating 3min in water-bath, when the cDNA of synthesis needs long-term preserve, please in-
20 DEG C or lower temperature preservation.
5th, QPCR is expanded
(1) design of primers
According to NHEG1 genes in Genebank and the sequences Design QPCR amplimers of GAPDH genes, by Bo Maide companies
Synthesis.Specific primer sequence is as follows：
NHEG1 genes：
Forward primer is 5 '-GCAACAAGCAAGGCAGAG-3 ' (SEQ ID NO.2)；
Reverse primer is 5 '-GAAGTTCAGAGATTGGCAGTC-3 ' (SEQ ID NO.3).
GAPDH genes：
Forward primer is 5 '-GAAGGTGAAGGTCGGAGT-3 ' (SEQ ID NO.4)；
Reverse primer is 5 '-CATGGGTGGAATCATATTGGAA-3 ' (SEQ ID NO.5).
(2) PCR reaction systems are prepared according to table 1：
Wherein, SYBR Green PCRs system is purchased from TIANGEN companies (article No. FP205).
Table 1PCR reaction systems
| Reagent | Volume |
| Forward primer | 0.6μl |
| Reverse primer | 0.6μl |
| 2×SuperReal PreMix Plus | 10μl |
| DNA profiling | 2μl |
| 50×Reference Dye△ | 2μl |
| Distilled water | Complement to 20 μ l |
(3) PCR reaction conditions：95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations.With SYBR
Green is as fluorescent marker, in the enterprising performing PCR reaction of the type quantitative real time PCR Instruments of ABI 7300, by melt curve analysis analysis and
Electrophoresis determines purpose band, and Δ Δ CT methods carry out relative quantification.
6th, statistical method
Experiment is tested using 3 repetitions, and result data is represented in the way of mean+SD, is used
SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that work as P<There is system when 0.05
Meter learns meaning.
7th, result
As a result as shown in Fig. 2 compared with non-temper virtual loss type patient, NHEG1 genes are expressed in temper virtual loss type patient
Lower, after four gentleman's particles are intervened, NHEG1 up-regulated expressions in temper virtual loss type patient, expression quantity is suffered from non-temper virtual loss type
There was no significant difference for the expression of person, consistent with RNA-sep results.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Hospital of the Production and Construction Corps of Xinjiang of Urumqi City Hospital of Traditional Chinese Medicine of affiliated hospital of Xinjiang Medicine University the 4th（Stone
The sub- affiliated hospital of University Medical College second in river）
<120>Application of the biomarker in colorectal cancer
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 1360
<212> DNA
<213>People source
<400> 1
taagaagtgt ctttcgcctc ctgccatgat tctgaggtct ccccagccat gtggaactgg 60
gaagaatgga aggcaacgcc tccgaattaa attttgggac ccccagtaaa atttcaggaa 120
agcccagaac tgatctgaga atgcacaaac ttggcatgtc tttgacagag aaggctctgg 180
gatttcagcg cgttcattgc cctccactaa ggagctgctc agcaaagggg cttccctctg 240
ctctgctctc gggttcggaa tccggagcga gtttccagtg agcggcgccc gctgagcgag 300
tgggaaagca tcgcgcgtgc ctttccccgc gcgcgtctgc ggatcagcgc aggcgaggtg 360
ggtgggaatg cagatgtgag gctggaggca gtagagccat ctcgcaatca tgcagcaaca 420
agcaaggcag agctggcatg ttgttgactg ataacatcga gaagctgcta tattaatcct 480
ggactgccaa tctctgaact tcttggtata ttgggaacat aaactctttt ggttaggcac 540
tgcgtcagat ttctactaca tgcagccaaa tgcaatctag attgaaatac ccctttacct 600
aagttagctt tagctgttct cacaacttgc tgtttgtgct ccacccacag catatgagat 660
cccttttgcc cacagcaccc tccctcagtg tgatgtcgtc ccccaaccag tatttgcagg 720
ttttttgttt ttttttttta atcacaaaac tgctctcagt ctgccaaatc tgttggccct 780
agagccagaa gtgctatctc ctgggaattt tttttttttt tgagacagga tcttgctgtc 840
atccaggctg gagggcagtg gcacacactc ctgggctcaa gcgatcctcc tgcttcagcc 900
ttccgagtgg ttgggactat aggtgagtgc caccactccc agctaattgt ttttattttt 960
tgtagagatg gggatgggca gggatcttac tttttgccca ggctggtctt gaactcctgg 1020
actcaagcca tcctcctgcc ttgatctctc aaagtgctgg gattacaggc atgagccacc 1080
atgcccggcc tgggatctat tcttaaccct tgccctagtc agggtggtct tcctgcagga 1140
ttaagagtta gactctgaga ctctgatatc acaccctggc ttggccttcc cttccctgtc 1200
ctacctcccc accctgctac cagtttcttc tggagatatt tcctgataga ctacagtagt 1260
cctcccttac ctgaggttta actttctgca gtttcagtta ttggcagtca acttaggtct 1320
gaaaatacta aataaaatat ccagaaataa aaaatttata 1360
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
gcaacaagca aggcagag 18
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
gaagttcaga gattggcagt c 21
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
gaaggtgaag gtcggagt 18
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
catgggtgga atcatattgg aa 22
Claims (10)
1. biomarker NHEG1 purposes, it is characterised in that the product for preparing diagnosis of colorectal carcinoma or therapeutic evaluation.
2. purposes according to claim 1, it is characterised in that the colorectal cancer is temper virtual loss type late period Colon and rectum
Cancer.
3. purposes according to claim 2, it is characterised in that the product is by determining the water of biomarker in sample
Put down and raise and determine compared with the reference levels of corresponding biomarker.
4. purposes according to claim 3, it is characterised in that the biomarker is included as shown in SEQ ID NO.1
The polynucleotides of nucleotide sequence or its fragment, homologue, variant or derivative.
5. the product of NHEG1 expressions in a kind of vitro detection sample, it is characterised in that the product includes：Pass through sequencing
Technology, nucleic acid hybridization technique, nucleic acid amplification technologies or the method for immunoassays detect the expression of NHEG1 genes.
6. product according to claim 5, it is characterised in that the nucleic acid amplification technologies be selected from PCR,
Reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement amplification and based on nucleotide sequence
Amplification.
7. product according to claim 5, it is characterised in that the product includes preparation, chip or kit.
8. product according to claim 7, it is characterised in that the product includes the examination of specific recognition NHEG1 genes
Agent.
9. product according to claim 8, it is characterised in that the reagent of the specific recognition NHEG1 genes is selected from：
The primer of specific amplification NHEG1 genes；Or
The probe of specific recognition NHEG1 genes.
10. the product described in claim any one of 5-9 is commented in the diagnosis or curative effect for preparing temper virtual loss type advanced colorectal cancer
Application in valency instrument.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710339698.3A CN107130027B (en) | 2017-05-15 | 2017-05-15 | Application of the biomarker in colorectal cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710339698.3A CN107130027B (en) | 2017-05-15 | 2017-05-15 | Application of the biomarker in colorectal cancer |
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| CN107130027A true CN107130027A (en) | 2017-09-05 |
| CN107130027B CN107130027B (en) | 2018-02-16 |
Family
ID=59733034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710339698.3A Active CN107130027B (en) | 2017-05-15 | 2017-05-15 | Application of the biomarker in colorectal cancer |
Country Status (1)
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| CN (1) | CN107130027B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107447033A (en) * | 2017-09-15 | 2017-12-08 | 东南大学 | A kind of diagnosis of colorectal carcinoma biomarker and its application |
| CN110387422A (en) * | 2019-08-29 | 2019-10-29 | 浙江省中医药研究院 | The diagnostic assays of colorectal cancer |
| CN111321220A (en) * | 2018-12-14 | 2020-06-23 | 中国医学科学院肿瘤医院 | Composition, microarray and computer system for detecting sensitivity of radiotherapy and chemotherapy of rectal cancer |
| CN112921083A (en) * | 2021-03-31 | 2021-06-08 | 青岛泱深生物医药有限公司 | Genetic markers in the assessment of intestinal polyps and colorectal cancer |
| CN113122628A (en) * | 2019-12-30 | 2021-07-16 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of CLEC5A gene as marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer |
| CN113862353A (en) * | 2021-09-09 | 2021-12-31 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | M of total RNA of peripheral blood immune cells6Application of detection reagent for A modification level in preparation of colorectal cancer diagnosis product |
-
2017
- 2017-05-15 CN CN201710339698.3A patent/CN107130027B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| NCBI: "NR_027994.1", 《GENBANK》 * |
| 杨德华: "神经母细胞瘤高表达基因1（NHEG1）的生物学功能及其机制研究", 《华中科技大学硕士学位论文》 * |
| 谢新梅等: "晚期结直肠癌脾气亏虚证患者差异表达基因的临床研究", 《河南中医》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107447033A (en) * | 2017-09-15 | 2017-12-08 | 东南大学 | A kind of diagnosis of colorectal carcinoma biomarker and its application |
| CN111321220A (en) * | 2018-12-14 | 2020-06-23 | 中国医学科学院肿瘤医院 | Composition, microarray and computer system for detecting sensitivity of radiotherapy and chemotherapy of rectal cancer |
| CN110387422A (en) * | 2019-08-29 | 2019-10-29 | 浙江省中医药研究院 | The diagnostic assays of colorectal cancer |
| CN113122628A (en) * | 2019-12-30 | 2021-07-16 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of CLEC5A gene as marker in diagnosis and treatment of gastric cancer, colorectal cancer, endometrial cancer and ovarian cancer |
| CN113122628B (en) * | 2019-12-30 | 2023-08-11 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of CLEC5A gene as marker in diagnosis and treatment of endometrial cancer |
| CN112921083A (en) * | 2021-03-31 | 2021-06-08 | 青岛泱深生物医药有限公司 | Genetic markers in the assessment of intestinal polyps and colorectal cancer |
| CN113862353A (en) * | 2021-09-09 | 2021-12-31 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | M of total RNA of peripheral blood immune cells6Application of detection reagent for A modification level in preparation of colorectal cancer diagnosis product |
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|---|---|
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