CN106319038A - Gene marker for screening early gastric cancer and application thereof - Google Patents
Gene marker for screening early gastric cancer and application thereof Download PDFInfo
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Abstract
The invention discloses a gene marker for screening early gastric cancer. The gene marker comprises a peripheral blood gastric cancer characteristic gene sequence shown as SEQ ID NO.1-SEQ ID NO.6. The invention also discloses application of the gene marker in preparation of products for screening early gastric cancer. The gene marker disclosed by the invention is used for screening early gastric cancer and is high in sensitivity and specificity; and moreover, because the peripheral blood which can be easily collected in clinical application is taken as a detection sample, the detection is convenient, the gene marker is applicable to screening and early diagnosis of a large population for gastric cancer, and the application prospects are wide.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly relate to a kind of gene marker for early gastric caacer examination and answer
With.
Background technology
Gastric cancer is the malignant tumor that a class originates from gastric epithelial, and China belongs to the High Risk For Gastric Cancer country, annual gastric cancer new cases
About 400,000 examples, how death about 350,000 example, because mortality of gastric carcinoma number occupies the 3rd in the lethal number of all malignant tumor, drop
The M & M of low China gastric cancer is great public health problem urgently to be resolved hurrily.The prognosis of gastric cancer is close for opportunity with diagnosis and treatment
Relevant, 5 years survival rates of advanced gastric carcinoma are less than 30%, and patients ' life quality is low, bring heavy bearing to family and country
Load.And major part early gastric cancer can obtain radical treatment under scope, 5 years survival rates, more than 90%, are greatly saved medical treatment
Resource.Therefore, " Cancer in China prevention and controlling planning outline (2,004 2010) " that Ministry of Public Health is issued explicitly point out,
The early discovery of cancer, early diagnosis and early treatment are to reduce mortality rate and improve the main policies of survival rate.High-risk in gastric cancer
Crowd carries out examination, promotes that the morning of gastric cancer examines and early control, be the important channel changing China's gastric cancer diagnosis and treatment severe situation.
At present, the diagnosis and treatment rate of China's early gastric cancer is less than 10%, well below Japan (70%) and Korea S's (50%).Cause me
The low reason of state's gastric cancer diagnosis and treatment rate is a lot, and one of them important reason is a lack of can be used for the simplicity, effectively of entire population's generaI investigation
Gastric cancer screening method.Under scope and scope, biopsy is the goldstandard of current diagnosis gastric cancer, but endoscopy relies on and sets
Standby and scope doctor's resource, and endoscopy expense is of a relatively high, needs to consume substantial amounts of human and material resources for Gastric Cancer Mass Screening.
The more important thing is, the diagnostic method such as endoscopy is as invasive test mode, and checking process is more painful, patient compliance
Poor, the patient of Risk of Gastric Cancer the most asymptomatic, low is difficult to accept especially.Therefore, send out even for Japan etc.
For reaching country, scope is the most not yet used to carry out extensive gastric cancer screening.
The essence that occurs of tumor is organism normal cell under the common effect of the Various Complex factors such as h and E, generation cancer base
Because activating and antioncogene inactivation, this differentiation is a kind of process relating to polygenes, multi-step, ultimately results in cell and loses
Original Growth and Differentiation regulates and paraplasm result occurs.Tumor tissues is as a kind of abnormal structure form, shape in vivo
Become and during propagation, inevitably by identification and the intervention of body immune system.Meanwhile, tumor cell is in immunity
Under editor (immunoediting) effect, autogene is expressed also can change to escape immunologic cytotoxicity, and tumor tissues
Around also can produce the microenvironment (micro environment) of a suppression immunocyte.Therefore, tumor cell is in body
Generation, dynamic process that evolution is also an immune system to interact with tumor cell.
In the immunocyte interaction process with tumor cell, peripheral blood cells act as a key player.Blood is people
The histoorgan that body is maximum, and hemocyte is few in number, can carry out the cell of communication for information with nearly all histiocyte
Kind.When in-vivo tissue organ damage, the disease such as inflammation and tumor, hemocyte participates in the signal of systemic immune system and passes
Passing and exchange, the gene expression of self can occur respective change to carry and to transmit corresponding information.Additionally, when blood flows through tumor
When organizing the microenvironment of periphery, hemocyte also can respond the change of tumor microenvironment directly or indirectly, corresponding gene table occurs
Reach change.Hemocyte is exactly based on this regulation autogene and expresses the mode of change, participates in each systems of whole body such as immune system
Information is transmitted and is exchanged.The obvious sign change that this changes in gene expression of hemocyte far produces due to disease early than body,
Therefore, if the changes in gene expression of tumoral character can be found in the blood of tumour patient, it is possible to by closely monitoring blood
The express spectra of cytogene, captures the micromessage that the malignant disease such as in-vivo tumour occurs sensitively, for early stage of tumor detect,
Monitoring provides admissible evidence.And peripheral blood gene expression detection is as a kind of test mode easy, Non-Invasive, person under inspection is easy
In acceptance, checking that compliance is high, the early screening/diagnosis to malignant tumor has the biggest using value.
Summary of the invention
The invention solves the problems that the problem that current early gastric cancer examination technology is short of, it is provided that a kind of gene mark for early gastric caacer examination
Will thing, this mark is used for early gastric caacer examination and diagnosis, has the highest Sensitivity and Specificity, and owing to using clinic
On the peripheral blood that the most easily gathers detect, easy to use, be suitable to early gastric caacer large-scale crowd examination.
In order to solve above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, it is provided that a kind of gene marker for early gastric caacer examination, described gene marker bag
Contain: the peripheral blood stomach cancer characteristic gene order shown in SEQ ID NO.1～SEQ ID NO.6.
In another aspect of this invention, it is provided that the application in the product preparing early gastric caacer examination of a kind of said gene mark.
Preferably, the product of described examination early gastric cancer includes: with real-time quantitative PCR, chip gene expression profile detection or RNA
The product of sequence measurement examination gastric cancer.
The product of described real-time quantitative PCR examination gastric cancer comprise SLC6A6 shown in specific amplified SEQ ID NO.1～SEQ ID NO.6,
The primer of these 6 peripheral blood stomach cancer characteristic gene orders of HNRNPA3, ERBB2IP, NPEPPS, KRAS and MLL.
The product of described chip gene expression profile detection examination early gastric cancer comprises: with SEQ ID NO.1～SEQ ID NO.6 institute
Show the probe that peripheral blood stomach cancer characteristic gene order hybridizes.
In another aspect of this invention, additionally provide a kind of detection kit for early gastric caacer examination, comprise specificity for
The primer of peripheral blood stomach cancer characteristic gene order shown in SEQ ID NO.1～SEQ ID NO.6.This test kit also comprises specificity pin
Primer to GAPDH reference gene sequence.
Described test kit also comprises the SYBR Green of the hydrolysis probes specific binding with pcr amplified fragment or non-specific binding
Dyestuff.
Preferably, the sequence of described primer is as shown in SEQ ID NO.7～SEQ ID NO.20；The sequence of described hydrolysis probes is such as
Shown in SEQ ID NO.21～SEQ ID NO.27.
In another aspect of this invention, additionally provide a kind of detection kit for early gastric caacer examination, comprise specificity for
The probe of peripheral blood stomach cancer characteristic gene order shown in SEQ ID NO.1～SEQ ID NO.6.
Utilize the test kit of the present invention, early stage stomach shown in SEQ ID NO.1～SEQ ID NO.6 in person under inspection's peripheral blood can be detected
The expression of cancer characterizing gene sequence, then draws, according to the information of up-regulated or downward, the risk probability that person under inspection gets a cancer of the stomach,
Thus realize the early screening to gastric cancer and diagnosis.
The gene marker of the present invention, for early gastric caacer examination, sensitivity is high, high specificity, and owing to being with
The peripheral blood easily gathered is detection sample, easy to detect, it is adaptable to the large-scale crowd examination of gastric cancer and early diagnosis, promotes stomach
Cancer early discovery, improves gastric cancer cure rate, has broad application prospects.
Accompanying drawing explanation
The present invention is further detailed explanation with detailed description of the invention below in conjunction with the accompanying drawings.
Fig. 1 is that the present invention utilizes the characterizing gene filtered out to screen the box traction substation of gastric cancer；
Fig. 2 is that the present invention utilizes the characterizing gene filtered out to scheme the ROC AUC of diagnosing gastric cancer.
Detailed description of the invention
On the basis of the present invention is built upon mankind's full genome express spectra quantitative analysis, by the gene of quantitative analysis peripheral blood total serum IgE
Expression situation, compares other 5 kinds of malignant tumor patients (hepatocarcinoma, pulmonary carcinoma, noses beyond Patients with Gastric Cancer, Healthy People and gastric cancer
Pharyngeal cancer, breast carcinoma, carcinoma of prostate) etc. the differential expression of peripheral blood gene in three groups of samples, then screened by logistic regression analysis
Go out 6 gene signals that differential expression (gene expression is raised or lowered) occurs in peripheral blood from patients with gastric cancer in early days, i.e. periphery
The expression signal of stomach cancer characteristic gene (biomarker) in blood.Use logistic regression analysis method, based on 6 filtered out
Individual peripheral blood of gastric cancer characterizing gene sets up the risk forecast model of gastric cancer.Finally, fluorescence quantitative RT-RCR, gene expression profile are used
The relative expression quantity of these 6 early gastric cancer gene markers in chip or RNA sequencing technologies detection by quantitative person under inspection's peripheral blood, profit
Differentiate whether person under inspection gets a cancer of the stomach and corresponding risk probability with the risk forecast model of the gastric cancer set up.
The screening of embodiment 1 stomach cancer characteristic gene marker
The screening of stomach cancer characteristic gene comprises the following steps:
1) PAXgene is usedTMBlood RNA Tube blood taking tube is collected 20 examples and is diagnosed as the patient of gastric cancer, 20 examples through pathologic finding
Healthy People and 40 examples are the peripheral blood sample of other 5 kinds of malignant tumor patients in addition to gastric cancer, each sample collection 2-3mL
Peripheral blood.
2) PAXgene Blood RNA Kit extraction agent box is used to extract the total serum IgE of above-mentioned peripheral blood sample.Use Agilent
The complete fragment degree (RIN) of Bioanalyzer 2100 biological analyser detection RNA sample, divides by Nano1000 trace ultraviolet
The purity of light photometer detection RNA sample.All RNA samples have to comply with following Quality Control condition: RNA productivity is more than 2 micrograms,
The ratio at 28S/18S peak is more than 1, and RIN value is more than 7, and the absorbance ratio of 260nm/280nm is more than 1.8.
3) Affymetrix Gene Profiling Array cGMP U133P2 chip (mankind's full genome chip of expression spectrum) is used
Detect the gene expression signal of above-mentioned RNA sample.
4) gene expression to each sample of the MAS5 statistical method in AffymetrixExpression Console software is utilized
Spectrum chip testing result is normalized (Normalization), only retains and expresses signal value in the range of 100-10000
And the gene all expressed in all samples carries out subsequent analysis as candidate gene.
5) analyze the dependency of above-mentioned candidate gene and gastric cancer, be ranked up by the height of gene with the relative coefficient of gastric cancer, will be with
Disease associated high candidate gene and matching two-by-two with disease associated low candidate gene, forms the combination of a series of candidate gene.
6) assess the every kind of candidate gene combination diagnostic effect to gastric cancer with logistic regression statistical analysis technique, calculate every kind of candidate gene
Receiver operating curve's (receiver operator characteristic curve, ROC curve) of combination and song
Under line, area value (Area Under Curve, AUC), filters out the assortment of genes to gastric cancer with optimal discrimination capabilities.
7) utilize real time fluorescence quantifying PCR method that the assortment of genes filtered out is verified, be retained in quantitative PCR and gene expression
Spectrum chip detection is expressed the change consistent gene peripheral blood characterizing gene as gastric cancer, filter out SLC6A6, HNRNPA3,
Six peripheral blood stomach cancer characteristic genes of ERBB2IP, NPEPPS, KRAS and MLL, these 6 peripheral blood stomach cancer characteristic gene orders
As shown in SEQ ID NO.1～SEQ ID NO.6.
8) assess the assortment of genes of above-mentioned 6 genes composition diagnostic effect to gastric cancer with logistic regression statistical analysis technique, calculate
The ROC AUC of this assortment of genes, sets up gastric cancer risk forecast model shown below:
Wherein, P is the risk probability that person under inspection gets a cancer of the stomach；b0To b6It is respectively corresponding Logic Regression Models parameter；ΔCt1To Δ Ct6
It is respectively the difference of 6 gastric cancer gene markers and the quantitative PCR period Ct value of reference gene；X be logistic regression logarithm seemingly
So than (log likelihood ratio).
Embodiment 2 utilizes whether the genetic marker analyte detection person under inspection of the early gastric cancer filtered out gets a cancer of the stomach
1. method step:
1) collection of sample peripheral blood sample to be detected: use PAXgeneTMBlood RNA Tube blood taking tube collects 2-3ml
The peripheral blood sample of patient.
2) extraction purification of total serum IgE in sample peripheral blood sample to be detected: use PAXgene Blood RNA Kit extracting examination
Agent box extracts and the total serum IgE in purification peripheral blood, and identifies with Agilent BioAnalyzer 2100 type mecroelectrophoresis analyser
The total serum IgE fragment integrity extracted and yield, by the purity of Nano1000 trace UV detector detection RNA sample.
3) reverse transcription reaction: use the High-Capacity cDNA Reverse Transcription of Life Techonolgy company
Kit Reverse Transcription box, use total serum IgE is template, with Olig (dT) as reverse transcriptase primer, carries out reverse transcription reaction synthesis cDNA.
4) fluorescence quantitative RT-RCR detection: according to 6 gene markers (SEQ ID NO.1～SEQ ID NO.6) and internal reference base
Because of the correlated series of GAPDH, design specific primer SEQ ID NO.7～SEQ ID NO.20 (wherein, primer SEQ ID NO.7～
SEQ ID NO.8 be used for the SLC6A6 gene marker shown in specific amplification SEQ ID NO.1, primer SEQ ID NO.9～
SEQ ID NO.10 be used for the HNRNPA3 gene marker shown in specific amplification SEQ ID NO.2, primer SEQ ID NO.11～
SEQ ID NO.12 be used for the ERBB2IP gene marker shown in specific amplification SEQ ID NO.3, primer SEQ ID NO.13～
SEQ ID NO.14 be used for the NPEPPS gene marker shown in specific amplification SEQ ID NO.4, primer SEQ ID NO.15～
SEQ ID NO.16 be used for the KRAS gene marker shown in specific amplification SEQ ID NO.5, primer SEQ ID NO.17～
SEQ ID NO.18 is used for the mll gene mark shown in specific amplification SEQ ID NO.6, primer SEQ ID NO.19～SEQ
ID NO.20 is used for specific amplification reference gene GAPDH), with this SEQ ID NO.7～SEQ ID NO.20 as primer, with
SEQ ID NO.21～SEQ ID NO.27 is that (wherein SEQ ID NO.21～SEQ ID NO.26 is respectively SEQ ID to fluorescent probe
The probe sequence of NO.1～SEQ ID NO.6 characterizing gene sequence, SEQ ID NO.27 is the probe sequence of reference gene GAPDH)
Or use and can utilize, with the SYBR Green dyestuff of pcr amplified fragment non-specific binding, the cDNA conduct that reverse transcription obtains
Amplification template, carries out real-time fluorescence quantitative RT-PCR reaction, it is thus achieved that these 6 gene markers mRNA in the blood sample of periphery
Relative amount.Table 1 below be classified as quantitative fluorescent PCR reaction system.
Table 1 quantitative fluorescent PCR reaction system
| Reagent | Concentration | Volume |
| Stomach cancer characteristic gene primer | 800nM | 2μL |
| Stomach cancer characteristic gene by fluorescence probe | 200nM | 0.5μL |
| Reference gene primer | 800nM | 2μL |
| Reference gene fluorescent probe | 200nM | 0.5μL |
| 2X PCR MasterMix | 12.5μL | |
| CDNA template | 2.67ng/μL | 7.5μL |
| Amount to | 25μL |
5) diagnosis of sample results to be detected: according to real-time fluorescence quantitative PCR detection SLC6A6, HNRNPA3, ERBB2IP,
This 6 gene markers mRNA relative amount in the blood sample of periphery of NPEPPS, KRAS and MLL, is built by embodiment 1
Vertical gastric cancer risk forecast model, calculates the logistic regression log-likelihood ratio X value of sample, and the testing result of X value >=0 is judged to sun
Property, i.e. patients with gastric cancer；< testing result of 0 is judged to feminine gender to X value, i.e. Healthy People.
2. result
Acquire 34 example gastric cancer, 28 example Healthy Peoples (Control) and 114 examples other malignant tumor patient in addition to gastric cancer
Peripheral blood sample, have detected 6 gene markers of early gastric cancer and reference gene GAPDH in peripheral blood with fluorescence quantitative RT-RCR
Relative expression quantity, calculate the logistic regression log-likelihood ratio X value of each sample, X value >=0 for positive test symbol, otherwise
For negative result.By testing result and pathology detection results contrast, show that 6 gene markers of the present invention are to early gastric cancer
Highly sensitive, the high specificity of examination, concrete testing result is shown in Fig. 1-2 and table 2 below, other malignant tumor include except gastric cancer with
Outer 30 example hepatocarcinoma, 33 example pulmonary carcinoma, 20 example nasopharyngeal carcinoma, 10 example breast carcinoma and 21 example carcinoma of prostate etc..
Sensitivity, specificity and the ROC AUC that gastric cancer is detected by table 2
Embodiment described above only have expressed embodiments of the present invention, and it describes more concrete and in detail, but can not therefore and
It is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for the person of ordinary skill of the art, do not taking off
On the premise of present inventive concept, it is also possible to make some deformation and improvement, these broadly fall into protection scope of the present invention.Therefore,
The protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. the gene marker for early gastric caacer examination, it is characterised in that described gene marker comprises: SEQ ID NO.1～
Peripheral blood stomach cancer characteristic gene order shown in SEQ ID NO.6.
2. the application in preparing early gastric caacer examination product of the gene marker described in claim 1.
Application the most according to claim 2, it is characterised in that described early gastric caacer examination product includes: use real-time quantitative
The detection of PCR, chip gene expression profile or the product of RNA sequence measurement examination gastric cancer.
Application the most according to claim 3, it is characterised in that the product of described real-time quantitative PCR examination early gastric cancer
Comprise the primer of peripheral blood stomach cancer characteristic gene order shown in specific amplified SEQ ID NO.1～SEQ ID NO.6.
Application the most according to claim 3, it is characterised in that described chip gene expression profile detection examination early gastric cancer
Product comprise: with peripheral blood stomach cancer characteristic gene order shown in SEQ ID NO.1～SEQ ID NO.6 hybridization probe.
6. the detection kit for early gastric caacer examination, it is characterised in that described test kit comprises specificity for SEQ ID
The primer of peripheral blood stomach cancer characteristic gene order shown in NO.1～SEQ ID NO.6.
Detection kit the most according to claim 6, it is characterised in that described test kit also comprises and pcr amplified fragment
The SYBR Green dyestuff of specific binding hydrolysis probes or non-specific binding.
Detection kit the most according to claim 6, it is characterised in that the sequence of described primer such as SEQ ID NO.7～
Shown in SEQ ID NO.18.
Detection kit the most according to claim 7, it is characterised in that the sequence of described hydrolysis probes such as SEQ ID NO.21～
Shown in SEQ ID NO.26.
10. the detection kit for early gastric caacer examination, it is characterised in that described test kit comprises specificity for SEQ
The probe of peripheral blood stomach cancer characteristic gene order shown in ID NO.1～SEQ ID NO.6.
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| CN106701986A (en) * | 2017-02-08 | 2017-05-24 | 泰山医学院 | Application of molecular marker in diagnosis and treatment of gastric carcinoma |
| CN110241220A (en) * | 2019-07-31 | 2019-09-17 | 华夏帮服科技有限公司 | For the peripheral blood open gene marker of breast cancer detection and its application |
| CN111540469A (en) * | 2020-05-29 | 2020-08-14 | 杭州广科安德生物科技有限公司 | Method for constructing mathematical model for in-vitro detection of gastric cancer and application thereof |
| CN114317740A (en) * | 2021-11-24 | 2022-04-12 | 博尔诚（北京）科技有限公司 | Marker and probe composition for gastric cancer screening and application of marker and probe composition |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701986A (en) * | 2017-02-08 | 2017-05-24 | 泰山医学院 | Application of molecular marker in diagnosis and treatment of gastric carcinoma |
| CN106701986B (en) * | 2017-02-08 | 2019-07-05 | 泰山医学院 | Application of the molecular marker in gastric cancer diagnosis and treatment |
| CN110241220A (en) * | 2019-07-31 | 2019-09-17 | 华夏帮服科技有限公司 | For the peripheral blood open gene marker of breast cancer detection and its application |
| CN110241220B (en) * | 2019-07-31 | 2022-11-01 | 青岛解码医学检验有限公司 | Peripheral blood transcriptional gene marker for breast cancer detection and application thereof |
| CN111540469A (en) * | 2020-05-29 | 2020-08-14 | 杭州广科安德生物科技有限公司 | Method for constructing mathematical model for in-vitro detection of gastric cancer and application thereof |
| CN114317740A (en) * | 2021-11-24 | 2022-04-12 | 博尔诚（北京）科技有限公司 | Marker and probe composition for gastric cancer screening and application of marker and probe composition |
| CN114317740B (en) * | 2021-11-24 | 2024-01-23 | 博尔诚（北京）科技有限公司 | Marker for gastric cancer screening, probe composition and application thereof |
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