CN107586846A - Diagnosis of glioma mark Circ3:129880309 | 129880559 and application - Google Patents
Diagnosis of glioma mark Circ3:129880309 | 129880559 and application Download PDFInfo
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Abstract
The invention belongs to biological technical field, discloses diagnosis of glioma mark Circ3:129880309 | 129880559 and application.In the present invention, find first：Excretion body secreted by glioma cell is to Circ3:129880309 | 129880559 have obvious enrichment；Find Circ3 in patients with gliomas serum excretion body simultaneously:129880309 | 129880559 expression (p significantly raised compared to control group<0.0001), ROC analyses then show it have higher diagnostic value to glioma (AUC=0.889, p=0.001, sensitivity and specificity are respectively 72.2% and 100%).Therefore by detecting Circ3 in patients with gliomas serum excretion body:129880309 | 129880559 expression, early stage, quick Noninvasive diagnosis can be made to patients with gliomas.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of serum circRNA marks for diagnosis of glioma
Circ3:129880309 | 129880559 and detect the mark reagent be used for prepare diagnosis of glioma preparation application,
Also kit.
Background technology
Glioma is the brain tumor disease that adult takes place frequently the most, accounts for the 40.49% of intracranial tumors.Since making a definite diagnosis,
The Patients with gliomas the average survival time life-span is no more than 5 years.For this evil high with inhereditary material correlation of diagnosis and treatment glioma
Property disease, it is necessary to it is horizontal in molecular biology, explore its pathogenesis in terms of hereditary information expression.Presently colloid
Though the diagnostic and therapeutic method of knurl is in and updates the stage, patients with gliomas survival rate is not significantly improved.Colloid
Knurl diagnosis is still within the experience sexual stage based on clinical, pathology and iconography information, and one after diagnosing, absolutely mostly
Number is middle and advanced stage, and postoperative survival rate allows of no optimist.Therefore, diagnosis of glioma mark is found to sieve people at highest risk
Look into, and correspondingly select rational successive treatment scheme, improve survival rate, be neuroscience field Task urgently to be resolved hurrily.
Circular rna is a kind of extensive and is diversely present in mammalian cell, has controlling gene expressional function
Endogenous non-coding RNA molecule, there is covalence closed loop configuration, be widely present in various cells, Ye Shiji
The current research focus of microRNA (miRNA) RNA families afterwards.In recent years, with the extensive use and life of deep sequencing technology
The fast development of thing physics and informatics technology, it has been found that the transcript of many extrons of the mankind non-linearly can reversely be cut
Connect or circular rna is formed by gene rearrangement, and they account for sizable ratio in all montage transcripts.In recent years
Also gradually find also to contain substantial amounts of circular rna in excretion body, may play a significant role.Excretion body refers to contain complexity
RNA and protein small film bubble (30-150nm), now, it refers in particular to plate-like vesica of the diameter in 40-100nm.Nineteen eighty-three, excretion
Body is found in sheep granulophilocyte first, and Johnstone is named as " exosome " within 1987.Research in recent years shows
Show, excretion body is the important molecule of cell and cell-cell communication, participates in many physiology and pathologic process.Not only wrapped in excretion body
Containing protein component, in addition to some RNA compositions, as Microrna (microRNA, miRNA), long-chain non-coding RNA and mRNA,
Circular rna (circular RNA, circRNA).These RNA entrained by excretion body are referred to as excretion body source RNA, have had
Whole sequential structure and bioactivity, it is expected to as liquid biopsy molecular marker, there is light on accurate medical development
Prospect.
The content of the invention
The present invention first purpose be：A kind of serum excretion body CircRNA marks for diagnosis of glioma are provided.
Main contents include：A kind of serum excretion body CircRNA marks Circ3 for diagnosis of glioma:
129880309 | 129880559, its sequence such as SEQ NO:Shown in 1.
Second object of the present invention is to provide the described CircRNA marks of detection expression quantity in serum excretion body
Application of the reagent in diagnosis of glioma preparation is prepared.
Third object of the present invention is to provide a kind of diagnosis of glioma kit, and it can be determined in serum excretion body
Circ3:129880309 | 129880559 content.
Described diagnosis of glioma kit, contain detection Circ3：129880309 | the PCR of 129880559 contents draws
Thing.It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.
Described diagnosis of glioma kit, except Circ3：129880309 | outside 129880559 primer, also contain from blood
Excretion body is extracted in clear, by extracting RNA in excretion body and carrying out all reagents of reverse transcription and quantitative fluorescent PCR.
Including：
(1) reagent needed for serum excretion body is extracted：Total Exosome Isolation Reagent(from
Serum), can be bought by Invitrogen companies, article No. 4478360；
(2) reagent needed for excretion body RNA is extracted：Trizol reagents, chloroform, isopropanol, 75% ethanol, without enzyme water；
(3) reagent needed for reverse transcription：Random primer (Random Primer), without enzyme water, 5 × RT Buffer, three phosphorus
Soda acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases；
(4) reagent needed for quantitative fluorescent PCR：Circ3：129880309 | 129880559 upstream and downstream primers, GAPDH internal references
Upstream and downstream primer, SYBR dyestuffs, without enzyme water.
The beneficial effects of the present invention are：Find first：Excretion body secreted by glioma cell is to Circ3:
129880309 | 129880559 have obvious enrichment；Find Circ3 in patients with gliomas serum excretion body simultaneously:
129880309 | 129880559 expression (p significantly raised compared to control group<0.0001), ROC analyses then show that its is right
Glioma has higher diagnostic value, and (AUC=0.889, p=0.001, sensitivity and specificity are respectively 72.2% He
100%).By application of the circular rna in diagnosis of glioma analysis, it can make it that the diagnosis of glioma is more convenient accurate
Really, conditions of patients is quick and precisely grasped for clinician, is laid the foundation to improve clinical therapeutic efficacy, and it is potential to find to have
The new small molecule drug targets of therapeutic value provide help.
Brief description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR analyzes Circ3:129880309 | 129880559 in glioma cell and cell institute
Differential expression in the excretion body of secretion；
Fig. 2 is that real-time fluorescence quantitative PCR analyzes Circ3:129880309 | 129880559 in glioma serum and normal blood
Differential expression in clear excretion body；
Fig. 3 is the Circ3 that Roc analyzes serum excretion body source:129880309 | 129880559 pairs of glioma early diagnosis
Specificity, sensitivity.
Embodiment
The present invention is intended to further illustrate below in conjunction with embodiment, is not intended to limit the present invention.
First, research object
1. two plants of glioma cell lines are U251 cell lines and U87 cell lines, four plants of glioma primary cells are respectively
1216th, 1125,1124C, 0128C, provided by institute of oncology of preclinical medicine institute of Central South University.
The serum sample of 2.35 patients with gliomas is provided by Xiang Ya hospitals, and 15 normal serum samples are the same period to carry out society
The healthy individuals of area's disorder in screening.Sample for research is collected for the same period, sample, dispense, preservation condition it is consistent.
2nd, research method
1. RNA extracting in excretion body, glioma/normal serum excretion body secreted by cell, cell
A. the extracting of cell RNA
Treat that above-mentioned six kinds of cell length to suitable density, outwells Pei Ji, 1ml Trizol reagents added in culture dish, on ice
Cracking 15 minutes.Cracking moves to 1.5ml and managed without enzyme Tube after terminating, 4 DEG C, and 12000rpm centrifugation 10min, supernatant moves to new
Tube is managed.Chlorination imitates 200 μ l in Tube, shakes 15-30s with hand, places 15min on ice, 4 DEG C, 12000rpm is centrifuged
15min；Carefully upper strata aqueous phase is taken to enter in new tube pipes, the isopropanol 0.5ml for adding precooling is mixed, and stands 20min on ice, 4 DEG C,
12000rpm centrifuges 10min；Supernatant is abandoned, the water-reducible ethanol 1-2ml of 75%DEPC is added and mixes, 4 DEG C, 7500rpm centrifugations
5min, supernatant is abandoned as far as possible, drying at room temperature 5-10min, adds the no μ l of enzyme water 10 dissolvings RNA.- 80 DEG C of preservations, it is daily by laboratory technician
Record refrigerator temperature.
B. in the excretion body secreted by cell RNA extracting
Treat that above-mentioned six kinds of cells support that (serum used must remove excretion in advance in culture medium to suitable density in culture dish
Body), collect supernatant culture medium about 15ml and centrifuge 1h in 4 DEG C in super filter tube (millipore companies), 4500g.It is super after centrifugation
Filtrate is collected in 1.5ml and managed without enzyme Tube, turns upside down and shakes up after addition ExoQuick-TC reagents (SBI companies), 4 DEG C static
Overnight precipitation.30min is centrifuged after normal temperature 1500g overnight, discards supernatant, 1500g centrifuges 5min again, blots supernatant.It is heavy
It is cell excretion body secreted in culture medium supernatant to form sediment.Precipitation is resuspended with 1ml Trizol reagents, cracks 15 on ice
Minute, the same A of extraction step after cracking terminates.
C. in glioma/normal serum excretion body RNA extracting
Ulnar vein blood 5ml is adopted using EDTA anticoagulant tubes, anticoagulant tube is gently shaken after blood sampling makes anti-coagulants be mixed with blood, and 4
DEG C stand 24 hours after 3000g normal temperature centrifuge 5 minutes.200 μ l upper serums are extracted with micropipettor to dispense to 600 new μ l
In centrifuge tube, -80 DEG C save backup.By the daily time recording refrigerator temperature of laboratory technician.
Take the above-mentioned μ l of serum 200 to be centrifuged 30 minutes in 2000g normal temperature, supernatant liquor is extracted to new with micropipettor
600 μ l centrifuge tubes, add 40 μ l excretion bodies extracts reagent (Total Exosome Isolation Reagent (from
Serum), article No.：4478360, Invitrogen companies) gently turning upside down shakes up, and 4 DEG C are incubated 45 minutes.After incubation terminates
10000g normal temperature centrifuges 10 minutes, discards supernatant, and gained precipitation is the excretion body in serum.200 μ l are added in precipitation
Precipitation is resuspended in Trizol (MRC companies), and suspension is moved into new 1.5ml tube manages, and mends Trizol to 1ml.Crack on ice
15min, the same A of extraction step after cracking terminates.
It is prepared by 2.cDNA
Reverse transcription reaction is carried out according to Reverse Transcriptase kit (Thermo companies) specification.Reaction cumulative volume is 20 μ l (total
10 μ l, Random primer of RNA 1 μ l, no 1 μ l, 5 × Reaction Buffer of enzyme water 4 μ l, RI 1 μ l, RT 1 μ l and
10mM dNTP 2μl)。
| Composition | Dosage/pipe |
| Random reverse transcriptase primer (1 μM) | 1μl |
| RNA samples | 10μl |
| Without enzyme water | To 12μl |
Reverse transcription first step condition：65 DEG C 5 minutes
| Composition | Dosage/pipe |
| 5 × RT Buffer | 4μl |
| Triphosphoric acid base deoxynucleotide (10mM) | 2μl |
| RNase inhibitor (40U/ μ l) | 1μl |
| MMLV reverse transcriptases (200U/ μ l) | 1μl |
| First step PCR product | 12μl |
| Cumulative volume | 20μl |
Reverse transcription second step program：25 DEG C 5 minutes, 42 DEG C 60 minutes, 70 DEG C 5 minutes.
3. real-time fluorescence quantitative PCR
(primer sequence is shown in SEQ NO to the specific primer synthesized using Han Heng biotechnologies (Shanghai) Co., Ltd.：2 and 3)
Carry out real-time quantitative PCR：Reverse transcription product is first diluted 10 times, mixed.20 μ L reaction systems are as follows：
Real-time fluorescence quantitative PCR response procedures：95 DEG C 3 minutes, 40 circulation, 95 DEG C 10 seconds, 60 DEG C 30 seconds.
(6) data analysis：Using 2-ΔΔCTRepresent Circ3:129880309 | 129880559 relative to internal reference expression times
Number, wherein △ CT=CTSample–CTInternal reference, △ △ CT=△ CT- △ CTControl.This experimental data uses the analysis method of relative quantification,
As reference gene, (primer sequence is shown in SEQ NO to GAPDH：4 and 5), using normal serum excretion body △ CT average value as △
CTControl, data are analyzed using software GraphPad Prism and SPSS 17.0.
3rd, result of study
1. Circ3 in excretion body caused by glioma cell:129880309 | 129880559 compared to IC
Significantly raised, cell excretion body has good concentration effect to the circular rna.Concrete outcome is as shown in Figure 1.
2. Circ3 in the serum excretion body of patients with gliomas:129880309 | 129880559 compare normal serum excretion body
According to group significantly up-regulation (p<0.0001).Specific data are as shown in Figure 2.ROC curve analysis shows Circ3:129880309|
12988055 are used as biomarker to have higher diagnostic value (AUC=0.889, p=0.001, sensitivity and spy to glioma
The opposite sex is respectively 72.2% and 100%), and detailed results are shown in Fig. 3.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Diagnosis of glioma mark Circ3:129880309 | 129880559 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 251
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
guauuaaucu cuggagaaga cacauccaca guuagcacuu ucuucagaug cugacgcucg 60
gugaacaguu gccuuugguc acaagauuua gaagacacag uguccauccu cccagauugg 120
aucucuuuuu cauauggauc uucuguuucu augucuuuuu aaaaaauaac uuuuugggaa 180
accuuuugga uuacaacugu ucauccucac cuaugcaaag aaagggaagc uauugcuggg 240
auuuugagga g 251
<210> 2
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 2
actgttcatc ctcacctatg c 21
<210> 3
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 3
tgaccaaagg caactgttc 19
<210> 4
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 4
atcatcagca atgcctcct 19
<210> 5
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 5
catcacgcca cagtttcc 18
Claims (6)
- A kind of 1. serum excretion body CircRNA marks Circ3 for diagnosis of glioma:129880309 | 129880559, its Sequence such as SEQ NO:Shown in 1.
- 2. the reagent of CircRNA marks expression quantity in serum excretion body described in test right requirement 1 is preparing glioma Application in diagnostic preparation.
- 3. a kind of diagnosis of glioma kit, it is characterised in that the Circ3 in serum excretion body can be determined：129880309| 129880559 content.
- 4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection Circ3：129880309| The PCR primer of 129880559 contents.
- 5. diagnosis of glioma kit according to claim 4, it is characterised in that the sequence of primer such as SEQ NO:2 and 3 It is shown.
- 6. according to the diagnosis of glioma kit described in claim 3 or 4 or 5, it is characterised in that except Circ3：129880309| Outside 129880559 primer, also contain and excretion body is extracted from serum, by extracting RNA in excretion body and carrying out reverse transcription and glimmering All reagents of Fluorescent Quantitative PCR.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157792A (en) * | 2019-04-22 | 2019-08-23 | 中山大学孙逸仙纪念医院 | Serum excretion body has_circ_0004771 is preparing the application in alcohol dependence syndrome diagnostic reagent |
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| CN103993088A (en) * | 2014-05-26 | 2014-08-20 | 中南大学 | Application method of long non-coding RNA (ribonucleic acid) CASC2 originated from serum exosomes |
| US20150299702A1 (en) * | 2012-11-30 | 2015-10-22 | Aarhus Universitet | Circular rna for inhibition of microrna |
| CN106434928A (en) * | 2016-10-08 | 2017-02-22 | 东南大学 | CircRNA markers for early diagnosis of lung adenocarcinoma and application thereof |
| EP2510122B1 (en) * | 2009-12-08 | 2017-04-12 | Université Joseph Fourier | Use of mi-rnas as biomarkers for diagnosing gliomas |
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2017
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| EP2510122B1 (en) * | 2009-12-08 | 2017-04-12 | Université Joseph Fourier | Use of mi-rnas as biomarkers for diagnosing gliomas |
| US20150299702A1 (en) * | 2012-11-30 | 2015-10-22 | Aarhus Universitet | Circular rna for inhibition of microrna |
| CN103993088A (en) * | 2014-05-26 | 2014-08-20 | 中南大学 | Application method of long non-coding RNA (ribonucleic acid) CASC2 originated from serum exosomes |
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Non-Patent Citations (4)
| Title |
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| NCBI: ""Homo sapiens transmembrane and coiled-coil domain family 1 (TMCC1), transcript variant 1, mRNA"", 《GENBANK》 * |
| RYBAK-WOLF A等人: ""Circular RNAs in the Mammalian Brain Are Highly Abundant，conserved，and Dynamically Expressed"", 《MOLECULAR CELL》 * |
| SALZMAN J.等: ""Cell-Type Specific Features of Circular RNA Expression"", 《PLOS GENET》 * |
| SONG XF.等: ""Circular RNA profile in gliomas revealed by identification tool UROBORUS"", 《NUCLEIC ACIDS RESEARCH》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157792A (en) * | 2019-04-22 | 2019-08-23 | 中山大学孙逸仙纪念医院 | Serum excretion body has_circ_0004771 is preparing the application in alcohol dependence syndrome diagnostic reagent |
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Application publication date: 20180116 |