CN107937533A - Glioma prognostic marker circ7:42148226 | 42148468 and application - Google Patents
Glioma prognostic marker circ7:42148226 | 42148468 and application Download PDFInfo
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- 206010018338 Glioma Diseases 0.000 title claims abstract description 57
- 208000032612 Glial tumor Diseases 0.000 title claims abstract description 45
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- 238000004393 prognosis Methods 0.000 claims abstract description 17
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- 238000002360 preparation method Methods 0.000 claims abstract description 4
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- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
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- 230000002980 postoperative effect Effects 0.000 description 3
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- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 3
- 229940048102 triphosphoric acid Drugs 0.000 description 3
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- 238000013211 curve analysis Methods 0.000 description 1
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Abstract
The invention belongs to biological technical field, discloses a kind of glioma prognostic marker circ7:42148226 | 42148468 and application.Detect the circRNAcirc7 in glioma source:42148226 | the reagent of 42148468 expression quantity is used to prepare the prognosis preparation of patients with gliomas.CircRNAcirc 7 in glioma is confirmed by studying:42148226 | the relatively low patient of 42148468 expression quantity, possesses the survival rates of higher.By detecting circRNAcirc7 in patients with gliomas samples of human glioma:42148226 | 42148468 expression, so as to make Index for diagnosis to patients with gliomas.
Description
Technical field
The invention belongs to biological technical field, be related to a kind of serum circRNA markers for glioma prognosis and
The reagent for detecting the marker is used to prepare the application of glioma prognosis preparation, also has kit.
Background technology
Glioma is the most common incidence of encephalic and the higher intracranial tumors of grade malignancy, at present to the treatment of glioma
Mainly based on surgery excision, then it is aided with chemicotherapy.But nonetheless the recurrence rate of patient is still very high, 2 annual survival rates are only
For 5%, malignant glioblastoma mean survival time (MST) is only 14 months or so.Cause glioma be difficult to the principal element cured it
First, glioma is presented invasive growth in itself, the boundary of tumour cell and normal cerebral tissue is very fuzzy and is difficult to operation
Excision completely；Further, since the fast breeding of glioma cell result in the formation of local tumor micro-environmental hypoxia, it is micro- in anoxic
The tumour cell survived under environment has stronger malignancy of tumor phenotype, is that glioma recurrence and postoperative chemicotherapy are resisted
Key factor.Therefore, find glioma prognostic marker and prognostic analysis is carried out to patient, to improve the postoperative life of patients with gliomas
Bioplasm amount, and rational successive treatment scheme is correspondingly selected, survival rate is improved, is that neuroscience field research urgently to be resolved hurrily is appointed
Business.
CircRNA is a kind of extensive and is diversely present in mammalian cell, has controlling gene expressional function
Endogenous non-coding RNA molecule, has covalence closed loop configuration, is widely present in various cells, and after
The current research hot spot 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 circRNA is formed by gene rearrangement, and they account for sizable ratio in all montage transcripts.In recent years
Gradually find also to contain substantial amounts of circRNA in excretion body, may play a significant role.At present, enriched since circRNA has
The features such as property, stability, high conservative and Space-time speciality, increasing work is just being played in terms of diagnosing tumor marker
With.
The content of the invention
The present invention first purpose be：A kind of CircRNA in the brain tissue source for patients with gliomas prognosis is provided
Marker circ7:42148226 | 42148468, its sequence such as SEQ NO:Shown in 1.
Second object of the present invention is to provide detection circRNA markers expression quantity in samples of human glioma
Application of the reagent in glioma prognosis preparation is prepared.
Third object of the present invention is to provide a kind of glioma prognosis kit, can measure in samples of human glioma
circ7:42148226 | 42148468 content.
The glioma prognosis kit, contains detection circ7:42148226 | the PCR primer of 42148468 contents.
It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.
The glioma prognosis kit, except circ7:42148226 | outside 42148468 primer, also contain from colloid
RNA is extracted in tumor tissue and carries out all reagents of reverse transcription and quantitative fluorescent PCR.Including：
(1) the extracted total RNA agents useful for same from samples of human glioma, including RNA stablizing solutions, Trizol reagents, three chloromethanes
Alkane, isopropanol, without enzyme water；
(2) it is template by circRNA circ7 using total serum IgE:42148226 | 42148468 reverse transcriptions are cDNA examinations used
Agent, including RT Buffer, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases and
circRNAcirc7:42148226 | random primer used in 42148468；
(3) by cDNA real-time quantitative PCR agents useful for same, including circRNA circ7:42148226 | 42148468 is real-time
Quantitative fluorescent PCR specific primer, GAPDH internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.
Present invention research confirms the circRNAcirc7 in samples of human glioma source:42148226 | 42148468 can be used for glue
Matter knurl patient's prognostic analysis, the circRNA circ7 in samples of human glioma source:42148226 | 42148468 expression quantity and patient
Survival rates have correlation.Therefore, the prognostic analysis available for patients with gliomas.
Applicant carries out analysis with 19 normal cerebral tissues to 40 samples of human glioma by quantitative fluorescent PCR and finds,
circ7:42148226 | 42148468 differential expression in both is obvious (P=0.037), after wherein 12 gliomas are suffered from
Person carries out survivorship curve analysis, finds the circRNA circ7 in samples of human glioma source:42148226 | 42148468 and patient
Survival rate it is related (P=0.047), content is lower, and survival rate is higher.This method provides strong for the prognostic analysis of glioma
Technical support, help to improve the postoperative life quality of patients with gliomas, work out aftertreatment scheme, improve survival rate, tool
There are far-reaching clinical meaning and generalization.
Brief description of the drawings
Fig. 1 analyzes circ7 for real-time fluorescence quantitative PCR:42148226 | 42148468 in samples of human glioma and normal brain activity group
Knit middle differential expression.
Fig. 2 is the circ7 that ROC curve analyzes brain tissue source:42148226 | 42148468 pairs of gliomas and normal brain activity group
Knit specificity, the sensitivity of difference；circ7:42148226 | 42148468 examine glioma with higher as biomarker
(AUC=0.875, p=0.003, susceptibility and specificity are respectively 80% and 83.3%) for disconnected value.
The circRNA circ7 in Fig. 3 tracing analysis samples of human glioma sources for survival:42148226 | 42148468 expression
Measure the correlation with survival.
Embodiment
The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.
Embodiment 1 prepares detection circ7:42148226 | the reagent of 42148468 expression quantity is used to prepare patients with gliomas
The kit (50 secondary response) of prognosis
1.RNA stablizing solutions 50ml
2. isopropanol 100ml
3. chloroform 100ml
4.Trizol (coming from Molecular Research Center companies) 50ml
5. without enzyme water 10ml
6. 1 μM of random 50 μ l of reverse transcriptase primer (Thermo companies)
5 × RT Buffer 7. (Thermo companies) 200ml
8. 100 μ l of 10mM triphosphoric acid bases deoxynucleotide (Thermo companies)
9. 500 μ l of 40U/ μ l RNase inhibitors (Thermo companies)
200U/ μ l MMLV reverse transcriptases 10. (Thermo companies) 50 μ l
50 μ l of 11.Premix Ex Taq (Thermo companies)
12. 10μM circRNA circ7:42148226 | 42148468 real-time fluorescence quantitative PCR specific primer, 30 μ l
circRNAcirc7:42148226 | 42148468 forward primers：
5'-CTCTGTGATAAGTCTGTCCAGG-3',
circRNA circ7:42148226 | 42148468 reverse primers：
5'-GAGCCCTCTGTGCCGTAC-3'；
13. 10 μM of 30 μ l of GAPDH specific primers
Forward primer is 5 '-ATCATCAGCAATGCCTCCT-3 ',
Reverse primer is 5 '-CATCACGCCACAGTTTCC-3 '.
2 circRNA circ7 of embodiment:42148226 | 42148468 in the expression quantity of samples of human glioma and the pass of prognosis
System
1st, the preservation of samples of human glioma：Samples of human glioma to be measured is collected to deposit in the cryopreservation tube for filling RNA stablizing solutions,
Put spare to -80 DEG C of refrigerators.
2nd, the extracting of RNA in organizing：Appropriate sample is taken to add liquid nitrogen grinding mark in the mortar after 180 DEG C are toasted 6-8h
This, be ground to it is powdered after in mortar add 1ml Trizol mortar samples, be ground into it is liquid after with move to tube manage, in
Static cracking 15 minutes on ice.4 DEG C after cracking, 12000rpm centrifugation 10min, supernatant moves to new tube pipes.Chlorination
Imitative 200 μ l shake 15-30s in Tube, with hand, place 15min on ice, and 4 DEG C of 12000rpm centrifuge 15min；Carefully take upper strata
Water mutually enters in new tube, and the isopropanol 0.5ml for adding precooling is mixed, and stands 20min on ice, and 4 DEG C of 12000rpm centrifuge 10min；
Supernatant is abandoned, the water-reducible ethanol 1-2ml of 75%DEPC is added and mixes, 4 DEG C, 7500rpm centrifugation 5min, abandon supernatant, room temperature as far as possible
Dry 5-10min, adds DEPC water 10-20 μ l dissolvings RNA, -80 DEG C of preservations, refrigerator temperature is recorded by laboratory technician daily.
3、circRNAcirc7:42148226 | 42148468 reverse transcriptions：Use the Reverse Transcriptase kit of Thermo companies.
The system of 20 μ l reverse transcription reactions is as follows：
| Component | Dosage/pipe |
| Random reverse transcriptase primer (1 μM) | 1μl |
| RNA samples | 2μg |
| Without enzyme water | To 12μl |
Reverse transcription first step condition：65 DEG C 5 minutes
| Component | 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 |
| The product of first step reverse transcription | 12μl |
| Cumulative volume | 20μl |
Reverse transcription second step program：25 DEG C 5 minutes, 42 DEG C 60 minutes, 70 DEG C 5 minutes.
4th, the circRNA circ7 of Han Heng biotechnologies (Shanghai) Co., Ltd. synthesis:42148226 | 42148468 is special
Property primer carry out real-time quantitative PCR：Reverse transcription product is first diluted 10 times, is mixed.20 μ l reaction systems are as follows：
The specific primer of PCR：
Forward primer：5'-CTCTGTGATAAGTCTGTCCAGG-3',
Reverse primer：5'-GAGCCCTCTGTGCCGTAC-3'.
GAPDH internal reference Specific PCR primers：
Forward primer is 5 '-ATCATCAGCAATGCCTCCT-3 ',
Reverse primer is 5 '-CATCACGCCACAGTTTCC-3 '.
Real-time fluorescence quantitative PCR response procedures：95 DEG C 3 minutes, 40 circulation, 95 DEG C 10 seconds, 60 DEG C 30 seconds.
5、2-ΔΔCTCalibration：With 2-ΔΔCTRepresent the multiple for control group change, wherein △ CT=CTSample–CTInternal reference, △
△ CT=△ CTCase–△CTControl.This experimental data uses the analysis method of relative quantification, and GAPDH as reference gene, (be shown in by primer
Sequence table SEQ NO:The average value of 4 and 5), patients with gliomas △ CT are control.Data using software GraphPad Prism and
SPSS is analyzed.
6. pair 12 patient's prognosis survivorship curve analyses are found, with circRNA circ7 in samples of human glioma:42148226
| the patient of 42148468 high expression (being higher than average value) compares, circRNA circ7 in samples of human glioma:42148226|
Patient survival's higher of 42148468 low expressions (subaverage), difference have conspicuousness (P=0.047), see Fig. 3.
Research shows above, circ7:42148226 | 42148468 can be as the specificity marker of patients with gliomas prognosis
Thing.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Glioma prognostic marker circ7:42148226 | 42148468 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 243
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
gguagugggg cuccauguaa ccauuccugg gguccauggc aaacaccguc ccgcgguacg 60
gcacagaggg cuccgccacg uguggcaggg acccauggau cucuuucuug aucaaugagg 120
cccucucguc acucgauguu gaagguuccu cacugacuuu gcugagcccc uggacauucu 180
guggcugcau agugauugcg uuucuucucu cucugugaua agucugucca ggacuuucau 240
ccu 243
<210> 2
<211> 22
<212> DNA
<213>Unknown (Unknown)
<400> 2
ctctgtgata agtctgtcca gg 22
<210> 3
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 3
gagccctctg tgccgtac 18
<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. glioma prognostic marker circ7:42148226 | 42148468, its sequence such as SEQ NO:Shown in 1.
- 2. the reagent of marker expression quantity in samples of human glioma described in test right requirement 1 is preparing glioma prognosis preparation In application.
- 3. a kind of glioma prognosis kit, it is characterised in that the circ7 in samples of human glioma can be measured:42148226| 42148468 content.
- 4. glioma prognosis kit according to claim 3, it is characterised in that contain detection circ7:42148226| The PCR primer of 42148468 contents.
- 5. glioma prognosis 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 glioma prognosis kit described in claim 3 or 4 or 5, it is characterised in that except circ7:42148226| Outside 42148468 primer, also contain the extraction RNA from samples of human glioma and carry out all examinations of reverse transcription and quantitative fluorescent PCR Agent.
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Non-Patent Citations (2)
| Title |
|---|
| JULIA SALZMAN ET AL.: "Cell-type specific features of circular RNA expression", 《PLOS GENET》 * |
| QIAOYU LI ET AL.: "The Hedgehog signalling pathway and its prognostic impact in human glioma", 《ANZ JOURNAL OF SURGERY》 * |
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