CN108165546A - A kind of miRNA biomarker, composition and application thereof - Google Patents
A kind of miRNA biomarker, composition and application thereof Download PDFInfo
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- CN108165546A CN108165546A CN201710180227.2A CN201710180227A CN108165546A CN 108165546 A CN108165546 A CN 108165546A CN 201710180227 A CN201710180227 A CN 201710180227A CN 108165546 A CN108165546 A CN 108165546A
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- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C12Q2600/00—Oligonucleotides characterized by their use
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The present invention relates to a kind of miRNA biomarkers, composition and application thereof.The miRNA biomarker can be applied in lung cancer detection reagent is prepared.A kind of composition is further related to, the composition includes at least one of miRNA and the forward primer of each miRNA and the sequence of reverse primer；The composition can be applied in lung cancer detection reagent.The miRNA of identification occurs have Close relation with lung cancer, biomarker can be used as to carry out screening and diagnosis to lung cancer, effective foundation is provided for clinical individual therapeutic intervention.
Description
Technical field
The invention belongs to biotechnology, specifically the present invention relates to biomarker, more particularly, to a kind of blood
Clear miRNA biomarker and its application in lung cancer detection.
Background technology
Lung cancer is current one of highest malignant tumour of morbidity and mortality in the world, and the patient of 60-70% is in first visit
When be late period, survival rate is only 16.1% within 5 years, and 5 years survival rates after I phase lung cancer therapy are up to 82%, therefore early screening
It is the important means for improving lung cancer survival rate.Currently used screening lung cancer means are low-dose spiral CT, but the technology is also deposited
In obvious shortcoming：(1) when the false positive rate height (the particularly pure intrapulmonary Ground-glass opacity of 5-10mm) and follow-up screening of screening
Between it is long, increase medical expense and the risk of radiation-actuate canceration.(2) lead to excessive traumatic biopsy and operation intervention.
MiRNA is the single-stranded tiny RNA of non-coding that a kind of length is about 20~24 nucleotide, be widely present in animals and plants and
In virus, target gene is regulated and controled in a manner that miRNA is sheared and inhibits protein translation, participates in the tune of various biological signal path
Section.Research shows that the miRNA more than half is positioned at region relevant with tumour generation and fragile site in the genome, with
The diagnosis of tumour, by stages, prognosis etc. it is closely related.
There are no the good miRNA of specificity and sensitivity so far to be adapted to do lung cancer detection marker, particularly
Blood serum designated object more lacks relevant evidence.Moreover, because the expression quantity of miRNA is relatively low in serum, find a kind of high sensitivity,
The easy to operate and low-cost method suitable for lung cancer detection is current serum miRNA urgently to be resolved hurrily applied to clinical detection
Problem, real-time fluorescence quantitative PCR are the main methods of current detection serum miRNA, and this method is quick, convenient, accuracy and spirit
Sensitivity can meet clinical demand.
In conclusion this field still needs a kind of quick, sensitive, easy to operate, low-cost lung cancer detection method.
Invention content
One of the objects of the present invention is to provide a kind of miRNA biomarkers, composition and application thereof.
First aspect present invention provides a kind of miRNA biomarker, and the miRNA biomarker includes one or more
It is a to be selected from following sequence：
miRNA-7：UGGAAGACUAGUGAUUUUGUUGU；
miRNA-17：CAAAGUGCUUACAGUGCAGGUAG；
miRNA-21：UAGCUUAUCAGACUGAUGUUGA；
miRNA-126：CAUUAUUACUUUUGGUACGCG；
miRNA-145：GUCCAGUUUUCCCAGGAAUCCC；
miRNA-146a：UGAGAACUGAAUUCCAUGGGUU；
miRNA-155：UUAAUGCUAAUCGUGAUAGGGGU；
miRNA-182：UUUGGCAAUGGUAGAACUCACACU；
miRNA-200b：CAUCUUACUGGGCAGCAUUGGA；
miRNA-205：UCCUUCAUUCCACCGGAGUCUG；
miRNA-210：CUGUGCGUGUGACAGCGGCUGA；
miRNA-221：AGCUACAUUGUCUGCUGGGUUUC.
In one preferred embodiment, the miRNA biomarker is Serum miRNA biomarker.
Second aspect of the present invention provide first aspect present invention miRNA biomarker prepare for detect and/or
Application in the reagent of diagnosing.
In one preferred embodiment, the reagent is the form of kit.
In another preferred embodiment, the detection reagent is also comprising Cel-miRNA-39 as internal reference miRNA.
Third aspect present invention provides a kind of composition, and the composition includes miRNA biomarker and each miRNA
The sequence of corresponding forward primer and reverse primer,
Wherein, the miRNA biomarker includes one or more from the following sequence：
miRNA-7：UGGAAGACUAGUGAUUUUGUUGU；
miRNA-17：CAAAGUGCUUACAGUGCAGGUAG；
miRNA-21：UAGCUUAUCAGACUGAUGUUGA；
miRNA-126：CAUUAUUACUUUUGGUACGCG；
miRNA-145：GUCCAGUUUUCCCAGGAAUCCC；
miRNA-146a：UGAGAACUGAAUUCCAUGGGUU；
miRNA-155：UUAAUGCUAAUCGUGAUAGGGGU；
miRNA-182：UUUGGCAAUGGUAGAACUCACACU；
miRNA-200b：CAUCUUACUGGGCAGCAUUGGA；
miRNA-205：UCCUUCAUUCCACCGGAGUCUG；
miRNA-210：CUGUGCGUGUGACAGCGGCUGA；
miRNA-221：AGCUACAUUGUCUGCUGGGUUUC；
And wherein, the sequence of the corresponding forward primers of each miRNA and reverse primer is selected from the group：
Wherein V is A, G or C, and N is A, G, C or T.
In one preferred embodiment, the miRNA biomarker is Serum miRNA biomarker, and described
Composition as internal reference miRNA and/or includes the corresponding forward primer sequences of Cel-miRNA-39 also comprising Cel-miRNA-39
TCACCGGGTGTAAATCAGCTTG(Seq ID No.:And reverse primer sequences 13)
ACGACTCACTATAGGGCGAGCACAGAATTAATTTTTTTTTTTTTTTTVN(Seq ID No.:14, wherein V be A, G or
C, N are A, G, C or T).
Fourth aspect present invention provides the composition of third aspect present invention preparing for detecting and/or diagnosing
Purposes in reagent.
In one preferred embodiment, the reagent is the form of kit.
Fifth aspect present invention provides a kind of Primer composition for being used for detection and/or diagnosing, the Primer composition
Include one or more from the following primer：
Wherein V is A, G or C, and N is A, G, C or T.
In one preferred embodiment, the Primer composition further comprises as the Cel-miRNA-39's of internal reference miRNA
Detection primer, the detection primer are selected from forward primer TCACCGGGTGTAAATCAGCTTG (Seq ID No.:13) and reversely
Primer ACGACTCACTATAGGGCGAGCACAGAATTAATTTTTTTTTTTTTTTTVN (Seq ID No.:14, wherein V be A,
G, or C, N are A, G, C or T).
In another preferred embodiment, the primer in the Primer composition is fluorogenic quantitative detection primer, such as glimmering
Fluorescent Quantitative PCR detection primer.
The invention has the advantages that：
First, serum relative organization sample is easier to obtain, and compared with puncture, convenient sources performance is stablized, and belongs to noninvasive
(or minimally invasive) alleviates the pain of patient and the risk of complication caused by puncture, while solves tradition and puncture and can not draw materials
In the case of materials problem.
Secondly, present invention determine that miRNA combination occur with lung cancer it is closely related, can as biomarker to lung
Cancer carries out screening and diagnosis, and effective foundation is provided for clinical individual therapeutic intervention.
It is finally, quantitative accurate by detection of the Real-Time Fluorescent Quantitative PCR Technique to the serum levels miRNA of lung cancer patient,
It is convenient and reliable.
Present invention determine that miRNA combination and lung cancer occur it is closely related, can be used as biomarker to lung cancer patient
Screening and diagnosis are carried out, effective foundation is provided for clinical individual therapeutic intervention.Research for Sera of Lung Cancer miRNA from now on provides
Theoretical foundation, and the molecule diagnosis for lung cancer provides new approaches, has certain theory significance and potential practical value.
Specific embodiment
MiRNA can exist steadily in the long term in serum, therefore tumour-specific miRNA can be used as tumour-specific in blood
Marker.Analyze the expression of the special miRNA of lung cancer in blood, using miRNA as molecular marker progress lung cancer early screening,
Diagnosis and outcome prediction etc. contribute to the diagnosis for lung cancer, treatment to provide new approach, and the prevention and early diagnosis for lung cancer are early controlled and carried
For strong theory and practice foundation.
One aspect of the present invention provides a kind of miRNA biomarker, and the miRNA biomarker includes one or more
Selected from following sequence：
miRNA-7：UGGAAGACUAGUGAUUUUGUUGU；
miRNA-17：CAAAGUGCUUACAGUGCAGGUAG；
miRNA-21：UAGCUUAUCAGACUGAUGUUGA；
miRNA-126：CAUUAUUACUUUUGGUACGCG；
miRNA-145：GUCCAGUUUUCCCAGGAAUCCC；
miRNA-146a：UGAGAACUGAAUUCCAUGGGUU；
miRNA-155：UUAAUGCUAAUCGUGAUAGGGGU；
miRNA-182：UUUGGCAAUGGUAGAACUCACACU；
miRNA-200b：CAUCUUACUGGGCAGCAUUGGA；
miRNA-205：UCCUUCAUUCCACCGGAGUCUG；
miRNA-210：CUGUGCGUGUGACAGCGGCUGA；
miRNA-221：AGCUACAUUGUCUGCUGGGUUUC.
In one preferred embodiment, the miRNA biomarker is obtained from serum.
The sequence of the corresponding forward primer of above-mentioned each miRNA and reverse primer is as follows：
Wherein V is A, G or C, and N is A, G, C or T.
One or more miRNA biomarkers of the present invention and/or its corresponding primer can be used for detecting and/or diagnosing
Lung cancer, it can also be used to the reagent for detecting lung cancer is prepared, such as the kit of detection such as diagnostic kit.Such examination
It is general also comprising internal reference miRNA in agent such as kit, such as can be by the use of Cel-miRNA-39 as internal reference miRNA, Cel-
The sequence of miRNA-39 is UCACCGGGUGUAAAUCAGCUUG, corresponding forward primer TCACCGGGTGTAAATCAGCTTG
(Seq ID No.:13), reverse primer ACGACTCACTATAGGGCGAGCACAGAATTAATTTTTTTTTTTTTTTTVN (Seq
ID No.:14, wherein V are A, G or C, and N is A, G, C or T).
It is, for example, possible to use one kind in miRNA biomarker sequence of the present invention, two kinds, three kinds, four kinds or more
A variety of and/or their corresponding primers or individually with these miRNA biomarkers are corresponding positive and/or reverse primer,
To detect or diagnosing or the reagent for being used to prepare detection or diagnosing.
Therefore, other aspects of the present invention also provide a kind of composition, the composition include miRNA biomarker and
The sequence of the corresponding forward primer of each miRNA and reverse primer.In addition the present invention also provides a kind of Primer composition, the primer sets
It closes object and includes the one or more of primer described in this specification, the Primer composition can be used for detection and/or diagnosing.
In addition aspect, the present invention also provides miRNA biomarker according to the present invention, composition according to the present invention, according to this
Application of the Primer composition of invention in preparing for the reagent (such as kit) of detection and/or diagnosing.
In the context of the present invention, the lung cancer of term " lung cancer " including form of ownership, including but not limited to Small Cell Lung Cancer,
Non- small small lung cancers, adenocarcinoma of lung, dermoid cancer, bronchial alveolar cells cancer etc..
All scientific and technical terms used in the present invention, it is unless otherwise indicated, usual with those skilled in the art
The meaning of understanding.Specific experiment operation sequencing etc., unless illustrating, using routine known to those skilled in the art
Operating technology and program.
It is detected or the step of diagnosing is as follows with the reagent of heretofore described detection or diagnosing：
1) serum sample collection and clinical grouping：Serum sample is acquired from hospital, simultaneity factor collects the pathology of patient
Data；
2) total serum IgE is extracted from tested sample serum, and adds in internal reference cel-miRNA-39；
3) add PolyA tails to each miRNA with PolyA polymerases；
4) can be used as detecting the miRNA of marker and internal reference cel-miRNA- using real time fluorescence quantifying PCR method detection
39。
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that following embodiment is only used
It is further described in the present invention, it is impossible to be interpreted as limiting the scope of the invention, person skilled in art can
Some nonessential modifications and adaptations are made to the present invention according to the invention described above content.
Embodiment
The identification of one serum miRNA marker of embodiment
(1) sample collection
Collect 10 patients with lung cancer and the serum of 4 Healthy Peoples (normal control)；
(2) extraction of RNA：
RNA extractions are carried out using the miRNeasy Serum/Plasma Kit rapid extractions kit of Qiagen companies.Often
For part sample using 200 microlitres of blood plasma loadings, last elution volume is 50 microlitres.Extract after RNA sample through measured concentration and
The Ratio control sample quality of OD260/OD280, ratio obtain peak optimization reaction result between 1.8~2.0.
(3) PolyA tailings：
The RNA sample that 10 μ l is taken to extract adds in 1x108A copy cel-miRNA-39 adds in 5 × PAP of 4ul bufferings
Liquid adds in the PolyA polymerases (Life companies, 74225Y/Z) of 2-5U, spends RNA enzyme water and supplies to 25 μ l.37 DEG C of incubation 10-
It 20 minutes, is then incubated 10 minutes for 65 DEG C.
(4) reverse transcription reaction：
Take 10 μ l plus PolyA after product, add in the RT buffer solutions of 2 μ l, 2 μ ldNTPs (each 5mM), 20 μM reversed
Primer, the Omniscript (Qiagen companies, Cat No.205111) of 4U, spends RNA enzyme water and supplies to 20 μ l.37 DEG C, 1h；
85 DEG C, 5min；4 DEG C of refrigerations are for use.
(5) quantitative fluorescent PCR reacts：
Take 1 μ l reverse transcription products, 10 μ l 2 × SYBR Green Mix (Qiagen, Cat No.208054), 10 μM of forward directions
Primer, 10 μM of reverse primers, are eventually adding suitable H2O so that the reaction total volume that PCR reacts is 20 μ l.Amplification program is：
1) 95 DEG C, 2min；2) 95 DEG C, 5s；3) 60 DEG C, 10s；Repeat the 2) step to the 3) 40 cycles of step.
(6) calculating of each miRNA expression quantity
Each miRNA is corrected according to the amount of internal reference miRNA, more each miRNA is in lung cancer patient and normal population
Expression difference, with differential expression not less than 3 times for the positive, less than 3 times within be negative.
2 serum miRNA marker of embodiment is to the recall rate result of lung cancer
By each serum miRNA marker, (differential expression as described above is the positive not less than 3 times, less than 3 times with the interior of the body belonging to YIN
Property) testing result and write according to the Ministry of Public Health of China《Chinese the 6th fascicle primary of common cancer diagnosis and treatment specification
Lung bronchogenic carcinoma》In compare for goldstandard-pathological diagnosis result of diagnosing, calculate susceptibility and the spy of lung cancer detection
Different degree.
1. calculating and statistical method
Computational methods are as follows：
Statistical analysis is as follows：
(1) susceptibility：A/(A+C)
(2) specificity：D/(B+D)
2. each miRNA testing results
1)miRNA-7：
Sensitivity：80%
Specificity：100%
2)miRNA-17：
Sensitivity：70%
Specificity：100%
3)miRNA-21：
Sensitivity：50%
Specificity：100%
4)miRNA-126：
Sensitivity：70%
Specificity：100%
5)miRNA-145：
Sensitivity：60%
Specificity：100%
6)miRNA-146a：
Sensitivity：80%
Specificity：100%
7)miRNA-155：
Sensitivity：90%
Specificity：50%
8)miRNA-182：
Sensitivity：60%
Specificity：100%
9)miRNA-200b：
Sensitivity：80%
Specificity：75%
10)miRNA-205：
Sensitivity：50%
Specificity：75%
11)miRNA-210：
Sensitivity：80%
Specificity：100%
12)miRNA-221：
Sensitivity：70%
Specificity：75%
From result above as it can be seen that heretofore described serum miRNA marker and lung cancer generation are closely related, can be used for
As the marker of lung cancer detection, effective foundation is provided for pulmonary cancer diagnosis and interference treatment.
The specific embodiment of the present invention is illustrated, but the present invention is not limited thereto above, without departing from
Spirit of the invention, the present invention can also have various change.
。
Sequence table
<110>Shanghai Li Di Biotechnology Ltd.
<120>A kind of miRNA biomarker, composition and application thereof
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Claims (10)
1. a kind of miRNA biomarker, the miRNA biomarker includes one or more from the following sequence：
miRNA-7：UGGAAGACUAGUGAUUUUGUUGU；
miRNA-17：CAAAGUGCUUACAGUGCAGGUAG；
miRNA-21：UAGCUUAUCAGACUGAUGUUGA；
miRNA-126：CAUUAUUACUUUUGGUACGCG；
miRNA-145：GUCCAGUUUUCCCAGGAAUCCC；
miRNA-146a：UGAGAACUGAAUUCCAUGGGUU；
miRNA-155：UUAAUGCUAAUCGUGAUAGGGGU；
miKNA-182：UUUGGCAAUGGUAGAACUCACACU；
miRNA-200b：CAUCUUACUGGGCAGCAUUGGA；
miRNA-205：UCCUUCAUUCCACCGGAGUCUG；
miRNA-210：CUGUGCGUGUGACAGCGGCUGA；
miRNA-221：AGCUACAUUGUCUGCUGGGUUUC.
2. miRNA biomarker as described in claim 1, which is characterized in that the miRNA biomarker is serum
MiRNA biomarker.
3. miRNA biomarker as claimed in claim 1 or 2 is in preparing for the reagent of detection and/or diagnosing
Purposes.
4. purposes as claimed in claim 3, which is characterized in that the reagent is the form of kit.
5. purposes as claimed in claim 3, which is characterized in that the reagent is also comprising Cel-miRNA-39 as internal reference
miRNA。
6. a kind of composition, the composition is comprising miRNA biomarker and the corresponding forward primers of each miRNA and reversely
The sequence of primer,
Wherein, the miRNA biomarker includes one or more from the following sequence：
miRNA-7：UGGAAGACUAGUGAUUUUGUUGU；
miRNA-17：CAAAGUGCUUACAGUGCAGGUAG；
miRNA-21：UAGCUUAUCAGACUGAUGUUGA；
miRNA-126：CAUUAUUACUUUUGGUACGCG；
miRNA-145：GUCCAGUUUUCCCAGGAAUCCC；
miRNA-146a：UGAGAACUGAAUUCCAUGGGUU；
miRNA-155：UUAAUGCUAAUCGUGAUAGGGGU；
miRNA-182：UUUGGCAAUGGUAGAACUCACACU；
miRNA-200b：CAUCUUACUGGGCAGCAUUGGA；
miRNA-205：UCCUUCAUUCCACCGGAGUCUG；
miRNA-210：CUGUGCGUGUGACAGCGGCUGA；
miRNA-221：AGCUACAUUGUCUGCUGGGUUUC；
And wherein, the sequence of the corresponding forward primers of each miRNA and reverse primer is selected from the group：
Wherein, V is A, G or C, and N is A, G, C or T.
7. composition as claimed in claim 6, which is characterized in that the miRNA biomarker is serum miRNA biology marks
Will object, and the composition is also corresponding as internal reference miRNA and/or comprising Cel-miRNA-39 comprising Cel-miRNA-39
Forward primer sequence TCACCGGGTGTAAATCAGCTTG (seq ID No.：And reverse primer sequences 13)
ACGACTCACTATAGGGCGAGCACAGAATTAATTTTTTTTTTTTTTTTVN(Seq ID No.：14, wherein V be A, G or
C, N are A, G, C or T).
8. purposes of the composition as claimed in claims 6 or 7 in preparing for the reagent of detection and/or diagnosing.
9. a kind of be selected from for detection and/or the Primer composition of diagnosing, the Primer composition comprising one or more
Following primer：
Wherein V is A, G or C, and N is A, G, C or T.
10. Primer composition as claimed in claim 9, which is characterized in that the Primer composition further comprises as internal reference
The detection primer of the Cel-miRNA-39 of miRNA, the detection primer are selected from forward primer TCACCGGGTGTAAATCAGCTTG
(seq D No.：And reverse primer ACGACTCACTATAGGGCGAGCACAGAATTAATTTTTTTTTTTTTTTTVN (Seq 13)
ID No.：14, wherein V are A, G or C, and N is A, G, C or T).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109828018A (en) * | 2019-02-27 | 2019-05-31 | 东南大学 | One kind being based on MoS2/Ti3C2MiRNA sensor and preparation method thereof and detection method |
| WO2021189390A1 (en) * | 2020-03-27 | 2021-09-30 | 深圳先进技术研究院 | Medicament for preventing/treating type 2 diabetes and uses |
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| WO2021189390A1 (en) * | 2020-03-27 | 2021-09-30 | 深圳先进技术研究院 | Medicament for preventing/treating type 2 diabetes and uses |
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