CN106086178A - A kind of serum miRNA marker relevant to gastric cancer auxiliary diagnosis and application thereof - Google Patents
A kind of serum miRNA marker relevant to gastric cancer auxiliary diagnosis and application thereof Download PDFInfo
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Abstract
The present invention discloses a kind of serum miRNA marker relevant to gastric cancer auxiliary diagnosis and application thereof, and this mark is miR 10b 5p, miR 132 3p, miR 185 one or more in 5p, miR 20a 3p and miR 296 5p of 5p, miR 195.Serum miRNA as novel biomarker, has good stability, Wicresoft easily obtains, susceptiveness and the high feature of specificity.This kind of molecular marker develop by for the various diseases including tumor diagnosis and further treatment new direction is provided.This research draws have the gastric cancer serum miRNA label of clinical diagnosis potential by more targeted.Research confirms that this group miRNA is as the reliability of the noinvasive label of diagnosis of gastric cancer and repeatability.
Description
Technical field
The invention belongs to genetic engineering and oncology, relate to a kind of serum miRNA relevant to gastric cancer auxiliary diagnosis
Mark and application thereof.
Background technology
Gastric cancer (Gastric cancer, GC) is one of global incidence and the higher malignant tumor of mortality rate.In China,
The mortality rate of gastric cancer still occupies tumor second.Owing to patients with gastric cancer early clinical manifestation is without obvious specificity, examine in early days in China
Disconnected rate is relatively low, and medical patients with gastric cancer great majority are already belonging to late period.At present in Japan and Korea S, promote and with gastroscope be
The early gastric cancer examination project on basis.But owing to economic condition and crowd accept level, China not yet can launch this intermediate item.And
And owing to gastroscope detection depends on examiner's experience, and have certain wound risk, also result in and cannot promote.Clinical practice at present
Most commonly used tumor markers, such as carcinoembryonic antigen (CEA) and CA19-9 (CA19-9), also lacks certain susceptiveness
And specificity.Along with the development of the biotechnology such as genomics, protein science and metabolism group, increasing biological marker
Thing has been observed that or studies.The label of early diagnosis gastric cancer can point the day and await for it thus, it is found that new, thus promote that gastric cancer is early
Phase intervenes and treatment, extends the life cycle of patient.
Microrna (miRNAs) is the class length little non-coding RNA molecule at the nucleotide of about 22, and it is by turning
Regulate and control the various vital movement processes of wide participation after record, including tumor generation, attack and transfer etc..Research finds, miRNA
Expression have in tumor in various degree upper mediation lower, base can have been established as a kind of emerging tumor markers for it
Plinth.2008, Mitchell detected free miRNA in peripheral blood, found that it can be stable in the presence of in peripheral blood, and
And can be as the noinvasive mark of diagnosing tumour.There is now research and confirm that circulation miRNA is at gastric cancer, pulmonary carcinoma, breast carcinoma, knot
Potential diagnostic value in the tumors such as rectal cancer.But due to research method and the difference of including crowd in, cause result of study the completeest
Complete consistent.Therefore, this research and utilization Exiqon miRNA qPCR panel chip and absolute quantitation based on qRT-PCR
Method, by the research of the gastric cancer serum to large sample, it is intended to finds the serum miRNA to gastric cancer with potential diagnostic value.And
The expression in stomach organization and outside Peripheral Blood secreted these miRNA in body is verified, further to define itself and stomach
The relation of cancer.If being directed to the diagnostic kit of gastric cancer according to this kind of miRNA design, it will promote the diagnosis and treatment water of China's gastric cancer
Flat, also for providing thinking to studying further of gastric cancer in the future.
Summary of the invention
It is an object of the invention to provide a kind of serum miRNA marker relevant to gastric cancer auxiliary diagnosis.
Another object of the present invention is to provide above-mentioned serum miRNA marker and primer thereof preparing gastric cancer auxiliary diagnosis
Test kit and the application in the medicine of preparation treatment gastric cancer.
A further object of the present invention is to provide for gastric cancer auxiliary diagnosis and the test kit for the treatment of and medicine.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of serum miRNA marker relevant to gastric cancer auxiliary diagnosis, this mark is miR-10b-5p
(UACCCUGUAGAACCGAAUUUGUG),miR-132-3p(UAACAGUCUACAGCCAUGGUCG),miR-185-5p
(UGGAGAGAAAGGCAGUUCCUGA),miR-195-5p(UAGCAGCACAGAAAUAUUGGC),miR-20a-3p
(ACUGCAUUAUGAGCACUUAAAG) one or more and in miR-296-5p (AGGGCCCCCCCUCAAUCCUGU).Should
Serum miRNA marker be preferably miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and
Two or more combination, more preferably miR-10b-5p, miR-132-3p, miR-185-in miR-296-5p
The combination that six kinds of miRNA of 5p, miR-195-5p, miR-20a-3p and miR-296-5p are formed.
The application in auxiliary diagnosis of gastric cancer of the above-mentioned serum miRNA marker.
The application in preparing gastric cancer auxiliary diagnosis test kit or treatment gastric cancer medicament of the above-mentioned serum miRNA marker.
A kind of primer of the serum miRNA marker relevant to gastric cancer auxiliary diagnosis, this primer comprise miR-10b-5p,
One or more miRNA's in miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p
Primer;Preferably comprise miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-in serum miRNA
The primer of two or more miRNA in 20a-3p and miR-296-5p;More preferably comprise miR-in serum miRNA
Six kinds of miRNA of 10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p draw
Thing.
Above-mentioned primer is at auxiliary diagnosis of gastric cancer or prepares the application in gastric cancer auxiliary diagnosis test kit.
A kind of gastric cancer auxiliary diagnosis test kit, containing miR-10b-5p, miR-132-in serum miRNA in this test kit
The primer of one or more miRNA in 3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p;Preferably
For containing miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-in serum miRNA
The primer of two or more miRNA in 296-5p;More preferably contain miR-10b-5p, miR-in serum miRNA
The primer of six kinds of miRNA of 132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p.
This test kit also includes the reagent that the reagent that round pcr is conventional is commonly used.
This test kit can also include that PCR reacts common agents, such as reverse transcriptase, buffer, dNTPs, MgCl2, DEPC
Water and Taq enzyme etc.;Standard substance and/or reference substance can also be contained.
Serum miRNA marker miR-10b-5p relevant to diagnosing gastric cancer involved in the present invention, miR-132-3p,
The sequence of every kind of miRNA in miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p is the most disclosed, but
It is combined each miRNA marker needing those skilled in the art to pay creative labor as gastric cancer auxiliary diagnosis mark
Dynamic.The amplimer of each miRNA marker all can be bought by market and obtain, the serum miRNA used in the embodiment of the present invention
The primer of mark is purchased from the specificity miRNA stem ring RT-PCR primer produced synthesized by the Rui Bo company of Guangzhou.
Specifically, the present invention solves the technical scheme of problem and includes: (1) sets up sample storehouse and the data of unified standard
Storehouse: gather standard compliant blood sample with S.O.P. (SOP), demographic data and clinic that systematic collection is complete provide
Material.(2) serum miRNA differential expression analysis of spectrum: analyze the serum miRNA of differential expression in gastric cancer and normal control population, and
Differential expression miRNAs is carried out the checking of further large sample multistage.(3) by multistage checking, these miRNA are specified
The ability of diagnosis of gastric cancer.(4) development of serum miRNA diagnostic kit: according to patients with gastric cancer and the difference in normal population serum
Different expression miRNA develops miRNAs diagnostic kit, it is achieved the noinvasive of patients with gastric cancer is assisted diagnosis.(4) these miRNA are analyzed
At stomach organization and secrete outward the expression in body, disclose the relation of these miRNA and gastric cancer, may be with this for developing in the future
The medicine of a little treatment gastric cancer relevant for miRNA provides foundation.
The present inventor gathers standard compliant blood sample with S.O.P. (SOP), the population that systematic collection is complete
Data, clinical data, and have employed Exiqon miRNA qPCR panel chip and qRT-PCR method etc..
The experimental technique studied specifically mainly includes following components:
1. research samples selection: just control, row operation and chemicotherapy intervention and confirm as the patient of gastric cancer through pathology.Just
Often comparison is the normal population checked UP in hospital.
2.Exiqon miRNA qPCR panel chip primary dcreening operation: utilize TRIZOL-LS reagent that serum mixing sample is carried out
RNA extracts, and carries out qRT-PCR operation acquisition primary dcreening operation result.
3. training set, checking collection: utilize AM1556 test kit (ABI company) that each serum sample is carried out RNA extraction is logical
Cross reverse transcription reaction and obtain cDNA sample, add PCR primer and SYBR Green fluorescent dye carries out PCR reaction.Pass through comparison
The Ct value of standard substance, draws the miRNA content in sample.
4. utilize TRIZOL-LS reagent to extract RNA, the ExoQuick test kit (SBI company) in gastric cancer and cancer beside organism
Extract with AM1556 test kit (ABI company) and outer secrete the RNA in body, by the method for qRT-PCR, detection miRNA tissue with
The differential expression in body is secreted outside and.
5. statistical analysis: use χ2Inspection, paired t-test and nonparametric rank test are compared miRNA expression and are existed
Difference in different seminar.The diagnostic value of serum miRNA is confirmed by calculating ROC curve analysis.
Seminar of the present invention carries out the expression analysis of system at present by the miRNA in the Peripheral Blood to Patients with Gastric Cancer,
Have now been found one group 6 gastric cancer serum microRNA label (miR-10b-5p, miR-132-with clinical diagnosis potential
3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p).
Beneficial effects of the present invention:
1. compared to traditional tumor markers, serum miRNA as novel biomarker, have good stability,
Wicresoft easily obtains, susceptiveness and the high feature of specificity.Developing for including tumor of this kind of molecular marker
The diagnosis of various diseases and further treatment provide new direction.
2. researcher passes through Exiqon miRNA qPCR panel chip and absolute quantitation method based on qRT-PCR, right
Differential expression miRNA in gastric cancer and normal control population's serum carries out checking tight, multistage and evaluation.Confirm this group
MiRNA is as the reliability of the noinvasive label of diagnosis of gastric cancer and repeatability.
3. researcher finds that miR-10b-5p and miR-296-5p expression in III phase and IV phase patients with gastric cancer is the highest
In I phase and the patients with gastric cancer of II phase.It addition, its expression in stomach organization is also consistent with expression in serum, it is shown that this group
Close relation between miRNA and gastric cancer.MiR-10b-5p, miR-195-5p, miR-20a-3p and miR-296-5p exist simultaneously
Secrete the expression in body outside serum and be also above normal control.These results by these miRNA of future studies for gastric cancer
Mechanism and offer new thinking treat for gastric cancer for these miRNA.
Accompanying drawing explanation
Fig. 1: experiment flow figure
Fig. 2: 6 miRNA of high expressed in gastric cancer serum
Fig. 3: the miRNA obtained is carried out ROC curve analysis.
The intersection that A: training set collects with checking;B: training set;C: checking collection.
Fig. 4: 6 miRNA expression in different TNM gastric cancer serum by stages
Fig. 5: 6 miRNA expression in stomach organization
Fig. 6: 6 miRNA secrete the expression in body outside Serum Obtained From Advance Gastric Cancer
Detailed description of the invention
Inventor have collected substantial amounts of patients with gastric cancer in 2013 to 2015 years from No.1 Attached Hospital, Nanjing Medical Univ
With the Venous serum sample of normal Check-up crowd, by the arrangement to sample data, therefrom have selected 203 example gastric cancer and 167 examples
The reality that the sample of normal control is verified as Exiqon miRNA qPCR panel chip primary dcreening operation and follow-up a series of qRT-PCR
Test sample.188 example stomach organizations and 28 example cancer beside organisms are the most also left and taken.At the beginning of selected patients serum's sample standard deviation comes from
Control, row operation and chemicotherapy intervention and confirm as the patient of gastric cancer through pathology.And the system acquisition population of these samples
Data, clinical data.
With reference to flow chart (Fig. 1), randomly choosed from gastric cancer and normal control serum sample 30 example gastric cancer samples with
And 10 example normal controls, and 3 example gastric cancer serum mixing samples and 1 normal mixing sample (mixing it are mixed into respectively
Sample is converged the sample forming 2ml by 10 example 200ul serum samples).This 4 example mixing sample is carried out Exiqon miRNA
QPCR panel chip primary dcreening operation and analysis, concrete steps are with reference to the description of Exiqon miRNA qPCR panel chip:
1. serum extracting
Taking out blood serum sample, after sample thaws, 3000xg is centrifuged 5min and removes some fragments and some insoluble component.Transfer
Supernatant, in new 1.5ml pipe, after adding 750ul TRIZOL-LS, acutely shakes 5s.
2. two-phase laminated flow
After homogenate, sample hatches 5 minutes in 15 to 30 DEG C.The sample of the TRIZOL-LS reagent homogenate of every 1ml adds
The chloroform of 0.2ml, covers tightly lid.The most acutely vibration body is after 15 seconds, hatches 2 to 3 minutes for 15 to 30 DEG C.13,000g at 4 DEG C
Centrifugal 15 minutes.
3.RNA precipitates
Aqueous phase is transferred in new centrifuge tube.Aqueous phase mixes with isopropanol to precipitate RNA therein, adds the amount of isopropanol
For: the sample of 1ml TRIZOL-LS reagent homogenate adds the isopropanol of 0.5ml and the glycogen of 5ul.4 DEG C stand half an hour, allow
RNA separates out as far as possible.It is centrifuged 15 minutes in 4 DEG C of 13,000g.
4.RNA cleans
Remove supernatant, the sample of every 1ml TRIZOL-LS reagent homogenate add 75% (v/v) ethanol of at least 1ml,
Clean RNA precipitate.Standing 10 minutes, then 10000g is centrifuged 5 minutes at 4 DEG C.
The most again RNA precipitate is dissolved
Remove ethanol solution, air drying RNA precipitate 5-10 minute, add the water rifle without RNase and repeatedly blow and beat several
Secondary, then hatch 10 minutes for 55 to 60 DEG C.
6. measurement concentration:
Usually lead to~5 μ g RNA/50ml serum.
7.cDNA synthesizes
(1) dilution template ribonucleic acid: use DEPC water that 20-25ng template ribonucleic acid is diluted to 14ul (final concentration of 1.492-
1.786ng/μl)。
(2) reactant liquor is prepared: 5 × Reaction Buffer and DEPC water are placed in and are dissolved on ice, and shakes mixing.
Enzyme mix is placed in-20 DEG C of ice chests, flicks mixing and be placed on ice before using.Use after all reagent are all centrifugal.
(3) configuration reactant liquor: the reactant liquor in configuration following table
(4) mix and be centrifuged reagent: centrifugal, to ensure that all solution is thoroughly mixed after shaking or aspirating mixing reactant liquor
Uniformly.
(5) reverse transcription reaction and heat inactivation: by reactant liquor after 42 DEG C of incubations 60 minutes, in 95 DEG C of incubations 5 minutes to lose
Reverse transcriptase alive.
8.Real-Time PCR
Reagent:
Nuclease free water(Exiqon)
SYBRTM Green master mix(Exiqon)
CDNA template
ROX(Invitrogen)
miRNA PCR ARRAY(Exiqon)
Instrument:
ABI PRISM7900 system(Applied Biosystems)
(1) Real-time PCR reagent is prepared: by the cDNA template of preparation, DEPC water and SYBRTM Green master
Mix is placed in and dissolves 15-20 minute on ice.
(2) dilution cDNA template: cDNA template nuclease free water RT reaction obtained dilutes 110 times
(such as, in 20 μ l reactant liquors, adding 2180ul nuclease free water).
(3) all reaction reagents are mixed:
A., after PCR plate being simply centrifuged, sealer is removed.
B. the cDNA template that 110 times dilute is mixed according to 1:1 volume ratio with 2 × SYBR Green master mix.
C. it is inverted mixing reactant liquor and is centrifuged
D. mixed reaction solution is added each hole in plate
The most again PCR plate is sealed
(4) PCR plate simple, low temperature is centrifuged
(5) Real-time PCR amplification: carry out Real-time PCR amplification and dissolving according to the reaction condition in following table
Tracing analysis.
Real-time PCR cycle condition such as following table:
Data analysis: use Δ Δ Ct method
The subsidiary software of PCR instrument device is used to carry out primary data analysis, it is thus achieved that and original Cq value (Cp or Ct, according to instrument not
May be different with title).
It is proposed that use GenEx qPCR to analyze software (www.exiqon.com/mirna-pcr-analysis) logarithm
According to carrying out standard and deep data analysis.
A. the Δ Ct of each passageway related genes in each process group is calculated.
Δ Ct (group 1)=average Ct average of HK genes ' Ct for group 1 array
Δ Ct (group 2)=average Ct average of HK genes ' Ct for group 2 array
B. the Δ Δ Ct of each gene in 2 PCR Array (or two groups) is calculated.
Δ Δ Ct=Δ Ct (group 2)-Δ Ct (group 1)
Remarks: generally group 1 is comparison, and group 2 is experimental group.
C. by the differential expression of 2-Δ Δ Ct calculating group 2 with group 1 corresponding gene.
After chip primary dcreening operation, 58 differential expression miRNA in having obtained such as following table are (in 3 gastric cancer serum mixing samples
1.5 times of differences have been above it) relative to normal sample.
58 the differential expression miRNA obtained for primary dcreening operation, by training set and checking collection, use based on qRT-PCR
Absolute quantitation method is verified, concretely comprises the following steps:
1. serum RNA extracts: selecting ABI company serum RNA to extract test kit (AM1556), reference reagent box illustrates, often
Individual sample is drawn 200ul and is carried out extracting RNA, and finally dissolves with 100ul DEPC water.
The preparation of 2.cDNA:
1) 50 μ L reaction systems are used to carry out reverse transcription experiment
Above reaction system mixes, and after brief centrifugation, reacts with follow procedure:
2) again reaction system adds following reactant after above-mentioned reaction
3.qPCR
1) use 5 μ L reaction systems, test in the following proportions
Reaction system mixes, and after brief centrifugation, is positioned in real-time PCR, reacts with follow procedure:
Reaction adds solubility curve after terminating.
Data analysis: by the Ct value of the standard substance of comparison variable concentrations, can calculate acquisition every after converging into standard curve
The absolute concentration of the miRNA in individual sample.Utilize SPSS 16.0 software to carry out statistical analysis, obtained one group in training set and
Checking concentrate all be unanimously to 6 miRNA:miR-10b-5p of high expressed in gastric cancer serum, miR-132-3p, miR-185-5p,
MiR-195-5p, miR-20a-3p and miR-296-5p (concentrate P value both less than 0.05, Fig. 2 in training set and checking).By this
6 miRNA, can calculate the ROC curve of each sample.Such as Fig. 3, the molecular marker of these 6 miRNA compositions can be good at
Distinguish patients with gastric cancer and normal population.And analyzed by subgroup, find that miR-10b-5p and miR-296-5p is at III phase and IV
Expression in phase patients with gastric cancer is apparently higher than the patients with gastric cancer (Fig. 4) of I phase and II phase.
Have detected these 6 miRNA after seminar further and secrete the expression of body, gastric cancer in stomach organization and serum China and foreign countries
Tissue extraction RNA utilizes TRIZOL, and secrete outward body extracting test kit is ExoQuick test kit (SBI company).200ul serum is carried
Take out outer secrete body 200ul DEPC water resuspended after, utilize AM1556 test kit (ABI company) externally to secrete body RNA and carry
Taking, step extracts process with serum RNA.
Using non parametric tests to analyze to find, miR-10b-5p and miR-296-5p expression in stomach organization is higher than
Cancer beside organism (Fig. 5).MiR-10b-5p, miR-195-5p, miR-20a-3p and miR-296-5p secrete in body outside gastric cancer serum
Expression also apparently higher than normal population (Fig. 6).
Test kit includes a collection of serum miRNA qRT-PCR primer, it is also possible to have the conventional examination needed for corresponding round pcr
Agent, such as: reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probe, RNase inhibitor, Taq enzyme etc., can basis
The concrete experimental technique used is selected, and these common agents are all well known to those skilled in the art, it can in addition contain there is standard
Product and comparison (such as normal person's sample etc. of quantitative markization).The value of this test kit is to have only to serum without other group
Tissue samples, by the expression contents of miRNA in the Fluorometric assay serum sample simplified most, assists this samples sources of diagnosis to suffer from
The probability suffering from gastric cancer of person.Serum miRNA is easy to detect, and quantitatively accurate, be greatly improved medical diagnosis on disease sensitivity and
Specificity, therefore puts into practice by this test kit, can help to instruct diagnosis and further individualized treatment.
Claims (8)
1. a serum miRNA marker relevant to gastric cancer auxiliary diagnosis, it is characterised in that this mark be miR-10b-5p,
One or more in miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p.
Serum miRNA marker the most according to claim 1, it is characterised in that this serum miRNA marker is miR-
In 10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p two kinds or two kinds with
On combination, preferably miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-
The combination that six kinds of miRNA of 296-5p are formed.
3. the application in auxiliary diagnosis of gastric cancer of the serum miRNA marker described in claim 1 or 2.
4. the serum miRNA marker described in claim 1 or 2 is preparing gastric cancer auxiliary diagnosis test kit or treatment gastric cancer medicament
In application.
5. the primer of a serum miRNA marker relevant to gastric cancer auxiliary diagnosis, it is characterised in that this primer comprises miR-
One or more in 10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p
The primer of miRNA;Preferably comprise miR-10b-5p in serum miRNA, miR-132-3p, miR-185-5p, miR-195-5p,
The primer of two or more miRNA in miR-20a-3p and miR-296-5p;More preferably comprise in serum miRNA
Six kinds of miRNA's of miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p
Primer.
6. the primer described in claim 5 at auxiliary diagnosis of gastric cancer or prepares the application in gastric cancer auxiliary diagnosis test kit.
7. a gastric cancer auxiliary diagnosis test kit, it is characterised in that in this test kit containing miR-10b-5p in serum miRNA,
One or more miRNA's in miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p
Primer;It is preferably containing miR-10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-in serum miRNA
The primer of two or more miRNA in 20a-3p and miR-296-5p;More preferably contain miR-in serum miRNA
Six kinds of miRNA of 10b-5p, miR-132-3p, miR-185-5p, miR-195-5p, miR-20a-3p and miR-296-5p draw
Thing.
Diagnostic kit the most according to claim 7, it is characterised in that also include the examination that round pcr is conventional in this test kit
The reagent that agent is conventional.
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CN107723366A (en) * | 2017-09-11 | 2018-02-23 | 朱伟 | A kind of serum miRNA marker related to cardia cancer auxiliary diagnosis and its application |
CN115537466A (en) * | 2019-04-30 | 2022-12-30 | 觅瑞实验室私人有限公司 | miRNA marker combination and kit for detecting gastric cancer |
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