CN107988375B - Biomarker and its application for predication of chemotherapy effect and diagnosis - Google Patents
Biomarker and its application for predication of chemotherapy effect and diagnosis Download PDFInfo
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- CN107988375B CN107988375B CN201810060233.9A CN201810060233A CN107988375B CN 107988375 B CN107988375 B CN 107988375B CN 201810060233 A CN201810060233 A CN 201810060233A CN 107988375 B CN107988375 B CN 107988375B
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- 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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- 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 one group for predication of chemotherapy effect and LncRNA biomarker and its application of diagnosis, belong to medical detection field.The curative effect of chemotherapeutics can be assessed and be predicted to the LncRNA biomarker comprising: ENST00000525217.1 and/or LNC_000361.
Description
Technical field
The present invention relates to one group for predication of chemotherapy effect and LncRNA biomarker and its application of diagnosis, belong to doctor
Treat detection field.
Background technique
Recent studies have shown that 90% subgenomic transcription is at non-coding RNA.Non-coding RNA is initially considered as " transcription
Noise ".However, more and more evidences show that these non-coding RNAs have important biology in many physiological processes
Effect.Non-coding RNA can be divided into short chain non-coding RNA (being less than 200bp) and long-chain non-coding RNA (being longer than 200bp).Long-chain
Non-coding RNA (LncRNA) can be divided into antisense long non-coding RNA, intron non-coding RNA (LincRNA), promoter correlation
LncRNA and non-translational region LncRNA.Previous result of study shows that LncRNA has in various kinds of cell and biological process
Important function, such as proliferation, cell cycle, chromosome remodeling and histone modification.In addition, their unconventionality expression swells with a variety of
Tumor is related, including breast cancer, gastric cancer, hepatocellular carcinoma and prostate cancer.
Some researches show that the lncRNA has apparent expression spectral difference between breast cancer tissue and the normal tissue of pairing
It is different；By the regulation expressed target gene, lncRNA can make tumour that more pernicious phenotype be presented.The discovery of Gupta team
One lncRNA (HOTAIR) expresses all apparent increases, and its table in primary tumor in breast cancer primary tumor and transfer stove
It is strong effective transfer and a dead predictive factor up to level；Another lncRNA (GAS5) is also proved in breast cancer tissue
Middle specificity low expression, while it can inhibit apoptosis and increment after high expression in breast cancer cell line.In recent years, more and more
Research confirm lncRNA as biomarker prediction tumor patient prognosis feasibility.
Summary of the invention
, first aspect present invention one group of LncRNA biomarker of offer, the LncRNA biomarker can be used as swollen
The biomarker of tumor medical diagnosis on disease, and can also assess and predict the curative effect of chemotherapeutics comprising:
ENST00000525217.1 and/or LNC_000361.
In the present invention, the ENST00000525217.1 sequence is referring to https: //lncipedia.org/db/
transcript/lnc-PGGHG-1:12；The LNC_000361 sequence is referring to https: //static-
Content.springer.com/esm/art%3A10.1186%2Fs12864-017-4145-0/MediaObjects/
12864_2017_4145_MOESM3_ESM.txt。
It is a discovery of the invention that after giving chemotherapy drug, when chemotherapeutics works, the LncRNA biology mark
Remember the obvious up-regulation of object expression, and it is invalid when, the LncRNA biomarker expresses no significant change or even faint decline.
In another embodiment, the chemotherapeutics includes: cis-platinum, carboplatin, epitrazide and oxaliplatin etc..
The second aspect of the present invention is to provide the detection kit of a kind of assessment and prediction chemotherapeutical medicine curative effect, the reagent
Box includes the primer for expanding ENST00000525217.1 and/or LNC_000361, and the primer sequence is as follows:
ENST00000525217.1 primer: positive: AGCTGAAAGATTCCCTGGGG is reversed:
AGACCAGCCCATGACCAAAA
LNC_000361 primer: positive: TGAACGAGATCAGTGCCCTT is reversed: TCTGGTTCGTGGATGATGCT.
The third aspect of the present invention is to provide the LncRNA biomarker answering in preparation/screening chemotherapeutics
With.
Detailed description of the invention
Fig. 1 is the expression of lncRNA marker (LNC_000361) in normal tissue and cancerous tissue
Fig. 2 is the expression of lncRNA marker (ENST00000525217.1) in normal tissue and cancerous tissue
Specific embodiment
Also the present invention further can be understood by embodiment, wherein the embodiment illustrates some preparations or user
Method.It is to be appreciated, however, that these embodiments do not limit the present invention.The change of the invention of currently known or further exploitation
Change is considered within the scope of the invention described herein and claimed below.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sam brook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring
HarborLaboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS
INMOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；
Theseries METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe,
CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；
METHODS INENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.),
AcademicPress, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119,
ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1LncRNA quantitative PCR experiment
It collects the frost flesh tissue sample of hospital cancer patient and its matches normal tissue.
(1) RNA extracting and quantitative PCR:
Using Trizol from frozen tissue extracted total RNA, RNA is quantified with micro-volume spectrophotometer.
Reference gene selects hs-actin, and hs-actin and one group of LncRNA biomarker provided by the present invention draw
Object sequence is as follows:
Hs-actin primer: positive: TGCGCAGAAAACAAGATGAG is reversed: CACCTTCACCGTTCCAGTTT
ENST00000525217.1 primer: positive: AGCTGAAAGATTCCCTGGGG is reversed:
AGACCAGCCCATGACCAAAA
LNC_000361 primer: positive: TGAACGAGATCAGTGCCCTT is reversed: TCTGGTTCGTGGATGATGCT
(2) reverse transcription reaction
Reverse transcription is carried out using SuperScriptIIIFirst Strand Synthesis System kit, RNA rises
Beginning amount is 1 μ g, and the dosage of reverse transcription reaction component is as shown in table 2, reaction condition: being incubated for 5min at 65 DEG C, at least exists after the completion
1min is placed on ice；Reverse transcription reaction mixing liquid is added in products therefrom again, is incubated at 25 DEG C respectively after mixing
10min, it is incubated for 50min at 50 DEG C, is incubated for 5min at 85 DEG C, the dosage of reverse transcription reaction mixing liquid is as shown in table 3；After the completion
1 μ l RNaseH is added in each reaction products therefrom, cryo-conservation is spare after being incubated for 20min at 37 DEG C
(3) real-time quantitative PCR reacts
Quantitative PCR uses I master of LightCycler480SYBRGreen, and quantitative instrument is LightCycler480.Instead
Answer system as follows, each real-time quantitative PCR reaction and to complying three repetitions.
PCR system configures (20 μ l system)
| 2×SG Green qPCR Mix | 7.5μl |
| Forward Primer(10μM) | 0.25μl |
| Reverse Primer(10μM) | 0.25μl |
| cDNA | 1μl |
| Water,nuclease-free | 6μl |
| Total volume | 15μl |
The setting of PCR response procedures
(4) for statistical analysis to data using 17.0 software of SPSS.Data table in the form of mean+SD
Show, unless there are special explanation.The differential expression statistics that adenocarcinoma of lung matches lncRNA in normal lung tissue with it uses pairing sample
The method that this t is examined, all p values are bilateral, and p < 0.05 is considered statistically significant.
The result shows that: as depicted in figs. 1 and 2, compared with normally pairing tissue, the frost flesh tissue sample of cancer patient
Middle ENST00000525217.1 and/or LNC_000361 expression is significantly raised, shows ENST00000525217.1 and/or LNC_
000361 two kinds of LncRNA have reliable directive property, can be accurately used as the biomarker of tumour.
Therapeutic evaluation of the embodiment 2LncRNA marker to platinum-based chemotherapy drug
Cisplatin medicine is given to the patient that embodiment 1 is collected to treat, and the course for the treatment of 4 weeks, is obtained after the course for the treatment of by biopsy
Cancer patient's lesion tissue is taken, the LncRNA biomarker expression in tissue is measured according to the method for embodiment 1, so
It is for statistical analysis to data using 17.0 software of SPSS afterwards.
The result shows that: after chemotherapeutics administration, the ENST00000525217.1 and/or LNC_000361 of tumor tissues are equal
It is decreased significantly.Illustrate that ENST00000525217.1 and/or LNC_000361 has as assessment and prediction chemotherapeutical medicine curative effect
Biomarker ability, can be used to accurately prediction tumour medicine curative effect.
The content of present invention merely illustrates claimed some specific embodiments, one of them or more skill
Documented technical characteristic can be combined with arbitrary one or more technical solutions in art scheme, these are combined and obtain
Technical solution also in the application protection scope, the technical solution just as obtained from these are combined is disclosed in the present invention
It is specifically recorded in content the same.
Claims (3)
1. a kind of application of LncRNA in the biomarker of preparation prediction adenocarcinoma of lung chemotherapeutical medicine curative effect, the LncRNA are
ENST00000525217.1。
2. application according to claim 1, which is characterized in that the chemotherapeutics includes: cis-platinum, carboplatin, epitrazide and/
Or oxaliplatin.
3. expanding primer the answering in the kit of preparation prediction adenocarcinoma of lung chemotherapeutical medicine curative effect of ENST00000525217.1
With the primer sequence is as follows: positive: AGCTGAAAGATTCCCTGGGG, it is reversed: AGACCAGCCCATGACCAAAA.
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