CN107604074A - Glioma prognostic marker circ15:101235082 | 101235577 and application - Google Patents
Glioma prognostic marker circ15:101235082 | 101235577 and application Download PDFInfo
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- CN107604074A CN107604074A CN201711056901.2A CN201711056901A CN107604074A CN 107604074 A CN107604074 A CN 107604074A CN 201711056901 A CN201711056901 A CN 201711056901A CN 107604074 A CN107604074 A CN 107604074A
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- 206010018338 Glioma Diseases 0.000 title claims abstract description 43
- 208000032612 Glial tumor Diseases 0.000 title claims abstract description 28
- 239000003550 marker Substances 0.000 title abstract description 5
- 238000004393 prognosis Methods 0.000 claims abstract description 19
- 230000014509 gene expression Effects 0.000 claims abstract description 16
- 210000005013 brain tissue Anatomy 0.000 claims abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000010839 reverse transcription Methods 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 9
- 241000282414 Homo sapiens Species 0.000 abstract description 8
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
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- 102100034343 Integrase Human genes 0.000 description 5
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 5
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
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- 229940048102 triphosphoric acid Drugs 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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Abstract
The invention belongs to biological technical field, discloses a kind of glioma prognostic marker circ15:101235082 | 101235577 and application, that is, detect the circRNA circ15 in brain tissue source:101235082 | 101235577 reagent is used for the prognosis preparation for preparing patients with gliomas.CircRNA circ15 in glioma are confirmed by studying:101235082 | the higher patient of 101235577 expression quantity, possess higher survival rates.By detecting circRNA circ15 in patients with gliomas samples of human glioma:101235082 | 101235577 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 marks for glioma prognosis and
The reagent for detecting the mark is used to prepare the application of glioma prognosis preparation, also has 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, find glioma prognostic marker and prognosis point is carried out to patient
Analysis, and the postoperative life quality of patients with gliomas is improved, and rational successive treatment scheme is correspondingly selected, improve existence
Rate, it is neuroscience field Task 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 circ15:101235082 | 101235577, 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
circ15:101235082 | 101235577 content.
Described glioma prognosis kit, contain detection circ15:101235082 | the PCR of 101235577 contents draws
Thing.It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.
Described glioma prognosis kit, except circ15:101235082 | outside 101235577 primer, also contain from
RNA is extracted in brain 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 circ15 using total serum IgE:101235082 | 101235577 reverse transcriptions are cDNA agents useful for same, including
RT Buffer, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases and
circ15:101235082 | random primer used in 101235577;
(3) by cDNA real-time quantitative PCR agents useful for same, including circ15:101235082 | 101235577 real-time fluorescences are determined
Measure PCR specific primers, GAPDH internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.
Present invention research confirms the circRNA circ15 in brain tissue source:101235082 | 101235577 can be used for glue
Matter knurl patient's prognostic analysis, the circRNA circ15 in brain tissue source:101235082 | 101235577 expression quantity and patient's art
Survival rate has correlation afterwards.Therefore, the prognostic analysis available for patients with gliomas.
Applicant has found the circRNA in samples of human glioma source by quantitative fluorescent PCR and survivorship curve analysis
circ15:101235082 | 101235577 is related to the survival rate of patient, and content is higher, and survival rate is higher.This method is glioma
Prognostic analysis provide strong technical support, be favorably improved the postoperative life quality of patients with gliomas, work out postoperative
Therapeutic scheme, survival rate is improved, there is far-reaching clinical meaning and generalization.
Brief description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR analyzes circ15:101235082 | 101235577 samples of human glioma with it is normal
Differential expression in brain tissue;
The circ15 in Fig. 2 tracing analysis samples of human glioma sources for survival:101235082 | 101235577 expression height are right
The influence prognosis of 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 circ15:101235082 | the reagent of 101235577 expression quantity is used to prepare
The kit (50 secondary response) of patients with gliomas 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
The μ l of 8.10mM triphosphoric acid bases deoxynucleotide 100
The μ l of 9.40U/ μ l RNase inhibitors 500
The μ l of 10.200U/ μ l MMLV reverse transcriptases 50
11.Premix Ex Taq 50μl
12.10μM circRNA circ15:101235082 | 101235577 real-time fluorescence quantitative PCR specific primers 30
μl
circRNA circ15:101235082 | 101235577 forward primers:5'-ACCACGGAAGAAATGGGA-3',
circRNA circ15:101235082 | 101235577 reverse primers:;5'-CAGATGTGTCAGAACCCTCA-
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 brain tissue sample circRNA circ15 of embodiment 2:101235082 | the detection of 101235577 expression quantity
1st, collect glioma to be measured or normal cerebral tissue is put into the cryopreservation tube for filling RNA stablizing solutions, put to -80 DEG C
Refrigerator is standby.
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 5min on ice, 4 DEG C, 12000rpm centrifuges 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, 7500rpm centrifugation 5min, abandons supernatant, room temperature as far as possible
5-10min is dried, adds DEPC water 10-20 μ l dissolvings RNA.
3、circRNA circ15:101235082 | 101235577 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 circ15 of Han Heng bio tech ltd synthesis:101235082 | 101235577 specific primers are carried out
Real-time 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'-ACCACGGAAGAAATGGGA-3',
Reverse primer:5'-CAGATGTGTCAGAACCCTCA-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-ΔΔCTThe measure of index:This experimental data uses the analysis method of relative quantification, GAPDH as reference gene,
The circRNA circ15 that qRT-PCR is measured:101235082 | 101235577CT values and the CT with tissue-derived GAPDH
Value obtains Δ CT as difference, then by Δ CT and Δ CTControlObtaining Δ Δ CT as difference, (average value for taking normal sample Δ CT is Δ
CTControl), data carry out Welch check analyses using software GraphPad Prism.Analysis finds, and in samples of human glioma
circRNA circ15:101235082 | 101235577 with the circRNA circ15 of normal cerebral tissue:101235082|
101235577 expression quantity have difference (see Fig. 1), and difference has conspicuousness (P<0.0001).
6th, found by 30 patients with gliomas follow-up statistics used by experiment, 10 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 20,
This 20 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 circ15:
101235082 | 101235577 high expression, totally 10, other are circ15:101235082 | 101235577 low expressions, totally 10
Example.Through Kaplan-Meier survival analysises, circ15:101235082 | the life cycles of 101235577 high expression patients compared with
circ15:101235082 | patient's length of 101235577 low expressions, good prognosis.Difference is statistically significant (P=0.05).With
Upper research shows, circ15:101235082 | 101235577 can be as the specificity molecular marker of patients with gliomas prognosis.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Glioma prognostic marker circ15:101235082 | 101235577 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 496
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
aacauggucc aagacaauuc cugggaaagu ucaguucuuc ucaagugagg guucugacac 60
aucuguacca auuccaguag ugccacuacg ggguguggac gacuccuacc cgccccagaa 120
gaaguccuuc augaugcuca aguacaugca cgaccacuac uuggacaagu augaaugguu 180
uaugagagca gaugaugacg uguacaucaa aggagaccgu cuggagaacu uccugaggag 240
uuugaacagc agcgagcccc ucuuucuugg gcagacaggc cugggcacca cggaagaaau 300
gggaaaacug gcccuggagc cuggugagaa cuucugcaug ggggggccug gcgugaucau 360
gagccgggag gugcuucgga gaauggugcc gcacauuggc aagugucucc gggagaugua 420
caccacccau gaggacgugg aggugggaag guguguccgg agguuugcag gggugcagug 480
ugucuggucu uaugag 496
<210> 2
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 2
accacggaag aaatggga 18
<210> 3
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 3
cagatgtgtc agaaccctca 20
<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 circ15 in brain tissue source for glioma prognosis:101235082| 101235577, 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 circ15 in brain tissue can be determined:101235082| 101235577 content.
- 4. glioma prognosis kit according to claim 3, it is characterised in that contain detection circ15:101235082 | the PCR primer of 101235577 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 circ15:101235082 | outside 101235577 primer, also contain the extraction RNA from brain tissue and carry out all examinations of reverse transcription and quantitative fluorescent PCR Agent.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2667193A1 (en) * | 2012-05-23 | 2013-11-27 | Université d'Aix-Marseille | MMP2 as a predictive biomarker of response to antiangiogenic therapy and survival after therapy in cancer patients |
CN103981271A (en) * | 2014-05-26 | 2014-08-13 | 中南大学 | Application method of serum Exosomes derived long no-coding RNA LINC00470 |
-
2017
- 2017-10-27 CN CN201711056901.2A patent/CN107604074B/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2667193A1 (en) * | 2012-05-23 | 2013-11-27 | Université d'Aix-Marseille | MMP2 as a predictive biomarker of response to antiangiogenic therapy and survival after therapy in cancer patients |
CN103981271A (en) * | 2014-05-26 | 2014-08-13 | 中南大学 | Application method of serum Exosomes derived long no-coding RNA LINC00470 |
Non-Patent Citations (2)
Title |
---|
HONGPING XIA 等: "Loss of brain-enriched miR-124 enhances the stem-like traits and invasiveness of glioma cells", 《J BIOL CHEM》 * |
JECK WR 等: "Circular RNAs are abundant, conserved, and associated with ALU repeats", 《RNA》 * |
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