CN107641654A - Glioma prognostic marker circ11:66639700|66640123 and application thereof - Google Patents
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Abstract
The invention discloses a glioma prognosis marker circ11:66639700|66640123 and application thereof, namely a reagent for detecting circRNA circ11:66639700|66640123 derived from brain tissues is used for preparing a prognosis preparation for a glioma patient. The research proves that the patient with higher circRNA circ11:66639700|66640123 expression level in glioma has higher postoperative survival rate. The prognosis of glioma patients is judged by detecting the expression level of circRNA circ11:66639700|66640123 in the glioma tissues of glioma patients.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of circRNA marks for glioma prognosis and detection
The reagent of the mark is used to prepare the application of glioma prognosis preparation, also has kit.
Background technology
Glioma (Glioma) is the most common tumour of encephalic, and it, which is fallen ill, accounts for more than the 50% of encephalic primary tumo(u)r, and
Treat one of maximum tumour of difficulty.According to WHO pathological grading standards in 1997, four can be divided into according to its grade malignancy glioma
Level, I grade：Fibrillary astrocytoma, it is apt to occur in 8~13 years old；II grade：Low potential malignancy astrocytoma (LGA), accounts for all nerves
The 25% of glioma, it is apt to occur in 30~40 years old；III grade：Regression type astrocytoma (AA), is easily converted into IV grade；IV grade：Multiform
Property glioblastoma (GBM), well send out the age be 45~60 years old.The treatment of glioma at present mainly uses surgery excision and putting
The conventional meanses such as treatment.But because glioma has the characteristics that obscure boundary is clear, local infiltration ability is strong, tumor growth rate is fast,
And then the problems such as tumor resection difficulty is high, and excision is not thorough, and Postoperative recurrent rate is high is result in, and surgery excision is to normal neuronal
Function will also result in damage；And due to the presence of blood-brain barrier so that many chemotherapeutics are difficult to reach treatment site；Radiotherapy
Therapeutic action to glioma is limited, and may cause the canceration of normal structure.So find effective treatment of glioma
Target spot is still current neurosurgeon problem urgently to be resolved hurrily.
CircRNA 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 circRNA is formed by gene rearrangement, and they account for sizable ratio in all montage transcripts, and have
The features such as rich, stability, high conservative and Space-time speciality, there are the potentiality as many disease molecules marks.
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
Mark circ11:66639700 | 66640123, its sequence such as SEQ NO:Shown in 1.
Second object of the present invention is to provide the examination of the described CircRNA marks expression quantity in brain tissue of detection
Application of the agent in glioma prognosis preparation is prepared.
Third object of the present invention is to provide a kind of glioma prognosis kit, can determine in brain tissue
circ11:66639700 | 66640123 content.
Described glioma prognosis kit, contain detection circ11:66639700 | the PCR primer of 66640123 contents.
It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.
Described glioma prognosis kit, except circ11:66639700 | outside 66640123 primer, also contain from brain
RNA is extracted in 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 circ11 using total serum IgE:66639700 | 66640123 reverse transcriptions are cDNA agents useful for same, including inverse
Transcription buffer, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases and circ11:66639700|
Random primer used in 66640123；
(3) by cDNA real-time quantitative PCR agents useful for same, including circRNAcirc11:66639700 | 66640123 is real-time
Quantitative fluorescent PCR specific primer, GAPDH internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.
Applicant had found in 22 patients with gliomas by quantitative fluorescent PCR and survivorship curve analysis, samples of human glioma
The circRNA circ11 in source:66639700 | 66640123 is related to the survival rate of patient, and content is higher, and survival rate is higher.
This method provides strong technical support for the prognostic analysis of glioma, is favorably improved the postoperative life matter of patients with gliomas
Amount, aftertreatment scheme is worked out, improve survival rate, there is far-reaching clinical meaning and generalization.
Brief description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR analyzes circ11:66639700 | 66640123 normal cerebral tissue with
Differential expression in glioma；
The circ11 in Fig. 2 tracing analysis samples of human glioma sources for survival:66639700 | 66640123 expression are high
The low influence prognosis to patients with gliomas.
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 circRNA circ11:66639700 | the reagent of 66640123 expression quantity is used to prepare glue
The kit (50 secondary response) of matter knurl patient's prognosis
1.RNA stablizing solutions 50ml
2. isopropanol 100ml
3. chloroform 100ml
4.Trizol 50ml
5. without enzyme water 10ml
6. 1 μM of random μ l of reverse transcriptase primer 50
7. 5 × RT Buffer 200ml
8. the μ l of 10mM triphosphoric acid bases deoxynucleotide 100
9. the μ l of 40U/ μ l RNase inhibitors 500
10. the μ l of 200U/ μ l MMLV reverse transcriptases 50
11.Premix Ex Taq 50μl
12. 10μM circRNA circ11:66639700 | 66640123 real-time fluorescence quantitative PCRs specificity
The μ l of primer 30
circRNA circ11:66639700 | 66640123 forward primers：
5'-AGTTTGAGGAGTATGGTCCG-3',
circRNA circ11:66639700 | 66640123 reverse primers：
5'-GCTGCCGTCTTGTCTTCTA-3'；
13. 10 μM of μ l of GAPDH specific primers 30
Forward primer is 5 '-ATCATCAGCAATGCCTCCT-3 ',
Reverse primer is 5 '-CATCACGCCACAGTTTCC-3 '.
The tissue samples circRNA circ11 of embodiment 2:66639700 | 66640123 detection
1st, collect samples of human glioma to be measured to be put into the cryopreservation tube for filling RNA stablizing solutions, put standby to -80 DEG C of refrigerators.
2nd, RNA extracting 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 are cracked after terminating, and 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
Aqueous phase enters in new tube, and 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 of 7500rpm centrifuge 5min, abandon supernatant as far as possible, room temperature is done
Dry 5-10min, add DEPC water 10-20 μ l dissolvings RNA.Spectrophotometric measures RNA concentration and quality, OD260/280 ratios
Between 1.8-2.0, -80 DEG C of preservations.Refrigerator temperature is recorded by laboratory technician daily.
3、circRNA circ11:66639700 | 66640123 reverse transcriptions：Use the reverse transcription reagents of Thermo companies
Box.The system of 20 μ L reverse transcription reactions is as follows：
| Composition | 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
| 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.
4th, the circ11 of Han Heng bio tech ltd synthesis:66639700 | 66640123 specific primers carry out real
When quantitative PCR：Reverse transcription product is first diluted 10 times, mixed.20 μ L reaction systems are as follows：
QRT-PCR specific primer：
Forward primer：5'-AGTTTGAGGAGTATGGTCCG-3',
Reverse primer：5'-GCTGCCGTCTTGTCTTCTA-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.
5th, the measure of 2- Δs Δ CT indexs：This experimental data uses 30 patients with gliomas and 12 Normal Human Brain Tissues,
And the analysis method using relative quantification, GAPDH is as reference gene, the circRNA circ11 that qRT-PCR is measured:
66639700 | 66640123CT values obtain Δ CT with the CT values with tissue-derived GAPDH as difference, then Δ CT is compareed with Δ CT
Δ Δ CT (taking normal sample Δ CT average value to be compareed for Δ CT) is obtained as difference, data are entered using software GraphPad Prism
Row Welch check analyses.Analysis is found, with circRNA circ11 in samples of human glioma:66639700 | 66640123 with it is normal
The circRNA circ11 of brain tissue:66639700 | 66640123 expression quantity have difference (see Fig. 1), and difference has conspicuousness (P
=0.0292).
6th, found by 30 patients with gliomas follow-up statistics used by experiment, 8 patients in follow-up by
The either number of changing or other reasonses being shut down in mobile phone not contacting, the patients with gliomas that can finally get in touch with or family members are 22,
This 22 patients or family members receive follow-up follow-up evaluation.We inquired in detail these patients or family members' First episode when
Between, treatment, recurrence status and death time etc., follow up time is 1-42 months.In selected patients with gliomas, choosing
The expression value for taking quantitative fluorescent PCR to analyze is normative reference, and that be higher than median after the arrangement of acquired results descending is circ11:
66639700 | 66640123 high expression, totally 15, receive follow-up as 11, other are circ11:66639700|66640123
Low expression, totally 15, receive follow-up as 11.Through Kaplan-Meier survival analysises, circ11:66639700|66640123
The life cycle of height expression patient is compared with circ11:66639700 | patient's length of 66640123 low expressions, good prognosis.Difference has statistics
Learn meaning (P=0.009).
7th, research shows more than, CircRNA circ11:66639700 | 66640123 can be as patients with gliomas prognosis
Specificity molecular marker.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Glioma prognostic marker circ11:66639700 | 66640123 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 424
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
gcucuuguca ggauggugaa gcuguucauc ggaaaccugc cccgggaggc uacagagcag 60
gagauucgcu cacucuucga gcaguauggg aaggugcugg aaugugacau cauuaagaau 120
uacggcuuug ugcacauaga agacaagacg gcagcugagg augccauacg caaccugcac 180
cauuacaagc uucauggggu gaacaucaac guggaagcca gcaagaauaa gagcaaaacc 240
ucaacaaagu ugcauguggg caacaucagu cccaccugca ccaauaagga gcuucgagcc 300
aaguuugagg aguauggucc ggucaucgaa ugugacaucg ugaaagauua ugccuucgua 360
cacauggagc gggcagagga ugcaguggag gccaucaggg gccuugauaa cacagaguuu 420
caag 424
<210> 2
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 2
agtttgagga gtatggtccg 20
<210> 3
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 3
gctgccgtct tgtcttcta 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. CircRNA marks circ11 in brain tissue source for glioma prognosis:66639700 | 66640123, Its sequence such as SEQ NO:Shown in 1.
- 2. the reagent of CircRNA marks expression quantity in brain tissue described in test right requirement 1 is preparing glioma prognosis Application in preparation.
- 3. a kind of glioma prognosis kit, it is characterised in that the circ11 in brain tissue can be determined:66639700| 66640123 content.
- 4. glioma prognosis kit according to claim 3, it is characterised in that contain detection circ11:66639700| The PCR primer of 66640123 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 circ11:66639700| Outside 66640123 primer, also contain the extraction RNA from brain tissue and carry out all reagents of reverse transcription and quantitative fluorescent PCR.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2017
- 2017-10-27 CN CN201711024924.5A patent/CN107641654A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106434928A (en) * | 2016-10-08 | 2017-02-22 | 东南大学 | CircRNA markers for early diagnosis of lung adenocarcinoma and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| NCBI: ""Homo sapiens RNA binding motif protein 4 (RBM4), transcript variant 3, 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》 * |
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