CN106929599A - MiRNA 6126 as pulmonary cancer diagnosis mark application - Google Patents
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Abstract
The invention discloses miRNA 6126 as pulmonary cancer diagnosis mark application.The invention provides the expression quantity of miRNA 6126 in detection sample to be tested excretion body material prepare diagnosis or sample to be tested described in auxiliary diagnosis whether the application in the product of lung cancer sample.Present invention also offers the expression quantity of miRNA 6126 in detection sample to be tested excretion body material prepare examination or sample to be tested described in auxiliary examination whether the application in the product of lung cancer sample.The experiment proves that, by the checking in the carcinogenic mouse blood excretion bodies of lung cancer cell line A549, it is determined that miRNA 6126 is expected to turn into the biomarker of clinical pulmonary cancer diagnosis.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of miRNA-6126 answering as pulmonary cancer diagnosis mark
With.
Background technology
Blood is found by methods such as genetic chip, high-flux sequence method and realtime quantitative inspections (qRT-PCR)
Diagnosis of the miRNA to NSCLC in liquid circulation has higher-value.Wherein Roth etc. has found the miR-361-3p and miR- in blood
625 help to recognize pernicious lung cancer from benign pulmonary disease.And the rise of miR-21 is diagnosis squamous cell lung carcinoma in blood
Reliabie mark, miR-200b-5p, miR-502-5p, miR-629, miR-17 and miR- in patients with lung adenocarcinoma blood
100 substantially increase compared with lung granuloma patient and healthy smoking person.Additionally, Rodriguez etc. has found lung tumors blood samples of patients
The miRNA contents such as the miR-122-5p that middle excretion body contains are apparently higher than BAL fluid.Munagala etc. has found lung
It is potential Biomarkers that miR-21 and miR-155 is raised for Lung Cancer Recurrence diagnosis in cancer excretion body.
Microrna (microRNA, miRNA), can used as a kind of tiny RNA of the non-coding of the unconventionality expression in some tumours
As diagnosing tumor and the biomarker of Index for diagnosis.However, dividing again due to obtaining tumor tissues by tissue biopsy
Analysing the method for its miRNA has traumatic, therefore limits its further application.Excretion body (exosomes) is a kind of diameter
In the subcellular fraction bilayer membrane granule of 50-90nm, wherein contain miRNA, and tumour can also constantly by excretion in growth course
Body is discharged into surrounding environment, therefore detection body fluid excretion body and excretion body RNA contribute to diagnosing tumor and Index for diagnosis.
Taylor etc. is proposed by detecting blood excretion body miRNA come diagnosis of ovarian cancer first, and is further used for judging prognosis.
The content of the invention
It is an object of the present invention to provide the use of the material of miRNA-6126 expression quantity in detection sample to be tested excretion body
On the way.
Material the invention provides miRNA-6126 expression quantity in detection sample to be tested excretion body is preparing diagnosis or auxiliary
Help the diagnosis sample to be tested whether the application in the product of lung cancer sample.
Present invention also offers detection sample to be tested excretion body in miRNA-6126 expression quantity material prepare examination or
Sample to be tested described in auxiliary examination whether the application in the product of lung cancer sample.
The expression quantity is relative expression quantity, is the expression quantity relative to reference gene, and reference gene is has-5.8s.
In above-mentioned application, the sample to be tested is in vitro serum.
In above-mentioned application, the material of miRNA-6126 expression quantity is included for expanding in the detection sample to be tested excretion body
The primer pair of miRNA-6126.
If expression of the miRNA-6126 in sample to be tested excretion body is significantly higher than the expression in non-lung cancer sample, treat
Test sample is originally or candidate is lung cancer sample, conversely, not being then or candidate is not.
Diagnosis is being prepared present invention also offers the material for detecting miRNA-6126 expression quantity in human serum excretion body to be measured
Or people to be measured described in auxiliary diagnosis whether the application in the product of patients with lung cancer.
Examination is being prepared present invention also offers the material for detecting miRNA-6126 expression quantity in human serum excretion body to be measured
Or people to be measured described in auxiliary examination whether the application in the product of patients with lung cancer.
In above-mentioned application, the material of miRNA-6126 expression quantity is included for expanding in the detection human serum excretion body to be measured
Increase the primer pair of miRNA-6126.
In above-mentioned application, sequence 2 in single strand dna and sequence table of the primer pair as shown in sequence in sequence table 1
Shown single strand dna composition.
The serum is in vitro serum.
It is a further object to provide a kind of product.
The product that the present invention is provided, is material or the institute of miRNA-6126 expression quantity in above-mentioned detection sample to be tested excretion body
State the material of miRNA-6126 expression quantity in detection human serum excretion body to be measured.
The said goods are following 1) -4) in any one：
1) diagnosis or sample to be tested described in auxiliary diagnosis whether the product of lung cancer sample；
2) examination or auxiliary examination described in sample to be tested whether the product of lung cancer sample；
3) diagnosis or people to be measured described in auxiliary diagnosis whether the product of patients with lung cancer；
4) examination or auxiliary examination described in people to be measured whether the product of patients with lung cancer.
MiRNA-6126 is also the scope of protection of the invention as the application in pulmonary cancer diagnosis label.
The present invention in order to set up the method for carrying out pulmonary cancer diagnosis and Index for diagnosis biomarker using excretion body, using forever
Biochemical human bronchial epithelial cell line BEP2D and alpha-radiation induces the cell line BERP35T44-1 that the cell occurs canceration,
Excretion body is separated, and is obtained using miRNA chip technologies and is expressed bright in Cancerization cell BERP35T44-1 cell excretion bodies
The aobvious miRNA for increasing, is detected in 31 serum of clinical lung cancer patient, compared with normal population, miRNA-6126
Expression is significantly increased.MiRNA-6126 is that clinical pulmonary cancer diagnosis lay the foundation, and can be used to prepare diagnostic kit.
Brief description of the drawings
Fig. 1 is hsa-5.8s gene real-time amplification curve maps and product solubility curve figure.
Fig. 2 is miR-4530 gene real-time amplification curve maps and product solubility curve figure.
Fig. 3 is miRNA-6126 expression high in human lung cancer serum.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
The discovery of the miRNA related to lung carcinoma cell of embodiment 1
The human bronchial epithelial cell line BEP2D of immortalization is recorded in the following literature：, building iron prop, Xiang Xiaoqiong, Wu Dechang
.238Pu α particles induce the Primary Study lung cancer in China magazines of human bronchial epithelial cell line BEP2D conversion, 2000,3 (6)：
428-431.2；
Tumour cell BERP35T44-1 has seroresistance higher, and stronger anchors independent growths ability, increases faster
Speed is grown, and with certain transfer ability, is recorded in the following literature：Alpha-radiation induces human bronchial epithelial cell and dislikes
Property conversion tumor cells biological characteristic research；Welcome spring recklessly；Military Medical Science Institute, Chinese People's Liberation Army's military affairs in 2003
The Master's thesis of the Academy of Medical Sciences.
1. cell culture：BEP2D cells and BERP35T44-1 cell culture in LHC-8 serum free mediums, put 37C,
Secondary Culture in 5%CO incubators.
2. the extraction of excretion body：Collect 107The good BEP2D and BERP35T44-1 cells and supernatants of growth conditions, adopt
Excretion body is separated with multistep differential centrifugation：4 DEG C, 300g is centrifuged 10 minutes removal cells；4 DEG C, 2000g is centrifuged 20 minutes and 4
DEG C, 10000g is centrifuged 30 minutes removal cell fragments；Finally, 4 DEG C, the precipitation that 100000g ultracentrifugations are obtained for 60 minutes is i.e.
It is excretion body.
3. the electronic microscope photos of excretion body：1ml PBS are added in being precipitated to excretion body, taking 20 μ l after piping and druming dissolving is added dropwise in load
On sample copper mesh, be stored at room temperature 1 minute, liquid blotted from side with filter paper, then be added dropwise the μ l of 2% Salkowski's solution (PH6.8) 30 in
On copper mesh, room temperature negative staining 1 minute.Filter paper blots negative staining liquid, is baked 2 minutes under incandescent lamp, and photograph is observed under transmission electron microscope.
4. the extraction of excretion body RNA：500 μ l Trizol reagents are added in being precipitated to excretion body, and (U.S. Invitrogen is public
Department) total serum IgE is extracted, obtain BEP2D cell excretion body RNA and BERP35T44-1 cell excretion bodies RNA.
Total rna concentration is determined with ultramicrospectrophotometer.Purity is assessed with A260nm/A280nm ratios.In order to determine
It is internal reference from RNU6B, using the method for real-time fluorescence quantitative PCR in total serum IgE with the presence of miRNA in the total serum IgE for extracting
MiRNA detected.
5.miRNA array experiments：The excretion body of two kinds of cells of BEP2D, BERP35T44-1, totally 2 total serum IgE samples carry out
MiRNA array experiments.MiRNA array experiments are used the LC Sciences microRNA of people by LC Sciences companies
Microarray-Single chip gene expression profiles are completed.
The LC Sciences microRNA Microarray-Single chip gene expression profiles of employment (join river by Hangzhou
Biology information technology Co., Ltd provides) detection BEP2D cell excretion body RNA and BERP35T44-1 cell excretion body RNA, warp
Find that RNA mass is intact, is marked cross experiment after detection.The scanned instrument scanning of hybridization hybrid chip, data signal extraction,
LOWESS filterings are normalized and Differential expression analysis etc..Obtain human bronchial epithelial cell line BEP2D excretion body base
Because of express spectra and tumour cell BERP35T44-1 excretion body gene expression profiles.
Compare two gene expression profiles, as a result compared with human bronchial epithelial cell line BEP2D gene expression profile, tumour cell
The miRNA of BERP35T44-1 gene expression profile up-regulateds has 140, and expressing the miRNA for lowering has 169.Data display,
It is 8.78 times that miRNA-6126 raises multiple.
Application of the expression quantity of embodiment 2, miRNA-6126 in diagnosing patient
First, RNA is extracted
Serum of Patients with Lung Cancer is originated：Serum of Patients with Lung Cancer all by PLA General Hospital provide, headed by examine lung cancer, patient
Not yet received any treatment such as including operation and chemicotherapy.Diagnosis is clear and definite, and pathology understands.
Normal control serum origin：Normal control serum is all provided by PLA General Hospital MEC, is health
Crowd routinely haves a medical check-up acquisition.
The RNA of Serum of Patients with Lung Cancer excretion body RNA and normal control serum excretion body is extracted respectively.
1st, sample information is as follows：
Table 1
2nd, design of primers and checking
Neck ring reverse transcription primer and qPCR sense primers are designed using DNAMAN, anti-sense primer uses universal primer, purpose
Fragment length about 60bp or so, 60 DEG C of annealing temperature (sequence is shown in Table 2).Primer checking use and quantitative PCR identical condition, with
CDNA biased samples are template, and as a result melting curve is simple spike, and agarose gel electrophoresis is detected as single band, and PCR expands
Increase specificity preferably.Quantitative result is shown in Fig. 1, Fig. 2.
Table 2miR-4530 sequences and qPCR primer sequences
3rd, RT-PCR methods (reverse transcription synthesizes cDNA)
The RNA that will have been extracted takes out from -80 DEG C of refrigerators, is placed on and dissolves on ice, uses FastQuant RT Kit (with
GDNAase) (TIANGEN) synthesizes the first chain cDNA.
Again respectively with cDNA as template, relative quantification PCR is carried out with sense primer and anti-sense primer.
Relative quantification PCR system such as table 3：
Table 3
Relative quantification PCR reactions use Roche96 quantitative real time PCR Instruments carry out quantitative PCR：
Table 4
The expression quantity of genes of interest is with 2 in experimental group relative comparison group (has-5.8s) sample-ΔΔCtMethod is calculated, and is as a result seen
Table 5.
The expression quantity (relative expression quantity relative to has-5.8s) of genes of interest in each sample of table 5.
Statistical procedures are carried out with the data of Gram Pad software registers 5, as a result as shown in Figure 3.
Statistical procedures are carried out to the data of table 5 with Sas softwares, as a result as shown in following table 6-7：
Table 6 is 31 lung cancer patient group miRNA-6126 results
Table 7 is 5 Normal group miRNA-6126 results
Normality is unsatisfactory for, this example uses the rank test of Group Design quantitative data：Z=-3.4307, P=0.0006,
Two groups of difference are statistically significant.
Sequence table
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Claims (10)
1. detect the material of miRNA-6126 expression quantity in sample to be tested excretion body to be measured described in preparation diagnosis or auxiliary diagnosis
Sample whether the application in the product of lung cancer sample.
2. the material of miRNA-6126 expression quantity is preparing examination or is aiding in be measured described in examination in detection sample to be tested excretion body
Sample whether the application in the product of lung cancer sample.
3. application according to claim 1 and 2, it is characterised in that：MiRNA- in the detection sample to be tested excretion body
The material of 6126 expression quantity includes the primer pair for expanding miRNA-6126.
4. detect that the material of miRNA-6126 expression quantity in human serum excretion body to be measured is treated described in preparation diagnosis or auxiliary diagnosis
Survey people whether the application in the product of patients with lung cancer.
5. detect that the material of miRNA-6126 expression quantity in human serum excretion body to be measured is preparing examination or aiding in being treated described in examination
Survey people whether the application in the product of patients with lung cancer.
6. according to the application of claim 4 or 5, it is characterised in that：
The material of miRNA-6126 expression quantity is included for expanding miRNA-6126's in the detection human serum excretion body to be measured
Primer pair.
7. the application according to claim 3 or 6, it is characterised in that：
Single stranded DNA point in single strand dna and sequence table of the primer pair as shown in sequence in sequence table 1 shown in sequence 2
Son composition.
8. a kind of product, is miRNA- in the detection sample to be tested excretion body in any application in claim 1-7
The material of miRNA-6126 expression quantity in the material of 6126 expression quantity or detection people to be measured.
9. product according to claim 8, it is characterised in that：The product is following 1) -4) in any one：
1) diagnosis or sample to be tested described in auxiliary diagnosis whether the product of lung cancer sample；
2) examination or auxiliary examination described in sample to be tested whether the product of lung cancer sample；
3) diagnosis or people to be measured described in auxiliary diagnosis whether the product of patients with lung cancer；
4) examination or auxiliary examination described in people to be measured whether the product of patients with lung cancer.
10.miRNA-6126 is used as the application in pulmonary cancer diagnosis label.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109825575A (en) * | 2019-04-08 | 2019-05-31 | 首都医科大学附属北京胸科医院 | Auxiliary diagnosis miRNA marker lungy and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106471132A (en) * | 2014-06-18 | 2017-03-01 | 东丽株式会社 | The detection kit of pulmonary carcinoma or device and detection method |
| CN106755544A (en) * | 2017-03-10 | 2017-05-31 | 大连医科大学附属第医院 | MiRNA marker and application in a kind of serum excretion body related to adenocarcinoma of lung early diagnosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106471132A (en) * | 2014-06-18 | 2017-03-01 | 东丽株式会社 | The detection kit of pulmonary carcinoma or device and detection method |
| CN106755544A (en) * | 2017-03-10 | 2017-05-31 | 大连医科大学附属第医院 | MiRNA marker and application in a kind of serum excretion body related to adenocarcinoma of lung early diagnosis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825575A (en) * | 2019-04-08 | 2019-05-31 | 首都医科大学附属北京胸科医院 | Auxiliary diagnosis miRNA marker lungy and its application |
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