CN107937527A - Glioma diagnosis marker circ1:43920404|43920928 and application - Google Patents
Glioma diagnosis marker circ1:43920404|43920928 and application Download PDFInfo
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- CN107937527A CN107937527A CN201711453662.4A CN201711453662A CN107937527A CN 107937527 A CN107937527 A CN 107937527A CN 201711453662 A CN201711453662 A CN 201711453662A CN 107937527 A CN107937527 A CN 107937527A
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- 208000032612 Glial tumor Diseases 0.000 title claims abstract description 31
- 206010018338 Glioma Diseases 0.000 title claims abstract description 31
- 238000003745 diagnosis Methods 0.000 title claims abstract description 23
- 239000003550 marker Substances 0.000 title claims abstract description 8
- 210000002966 serum Anatomy 0.000 claims abstract description 28
- 230000014509 gene expression Effects 0.000 claims abstract description 7
- 238000003753 real-time PCR Methods 0.000 claims abstract description 5
- 230000029142 excretion Effects 0.000 claims description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims description 13
- 238000010839 reverse transcription Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 210000001808 exosome Anatomy 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000013211 curve analysis Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
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- 102000004190 Enzymes Human genes 0.000 description 6
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- 239000006228 supernatant Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 102100034343 Integrase Human genes 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108700011259 MicroRNAs Proteins 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000003161 ribonuclease inhibitor Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 2
- 229940048102 triphosphoric acid Drugs 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 1
- 108091028075 Circular RNA Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000012350 deep sequencing Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 208000029824 high grade glioma Diseases 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000011614 malignant glioma Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
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Abstract
The invention belongs to the technical field of biology, and discloses a glioma diagnosis marker circ1:43920404|43920928 and application thereof. The content of circ1:43920404|43920928 in the serum exosomes of the glioma patients is analyzed by a fluorescent quantitative PCR technology, and the expression level of circ1:43920404|43920928 in the serum exosomes of the glioma patients is obviously reduced compared with that of a normal control group (p is less than 0.0001), and ROC curve analysis shows that the expression level of circ1:43920404|43920928 in the serum exosomes of the glioma patients has higher diagnostic value on glioma, wherein AUC is 0.877, p is 0.003, and the sensitivity and the specificity are 85.7% and 100% respectively.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of serum excretion body circRNA for diagnosis of glioma and indicates
Thing and detect the marker reagent be used to prepare the application of diagnosis of glioma preparation, also have kit.
Background technology
Glioma is the highest malignant tumour of encephalic incidence, accounts for the 40.49% of intracranial tumors, although clinically using hand
The method that art, chemotherapy combine is treated, but due to its wellability, to reasons such as chemotherapeutics hyposensitivities, frequently results in trouble
Person's postoperative recurrence, serious threat human life and health.And once make a definite diagnosis, most of is all glioma middle and advanced stage, post-operative survival rates
Rate is not high.In addition, in WHO3-4 grades i.e. High-grade Gliomas patient, prognosis is poor, and the average survival time service life is no more than 5 years.
Therefore, find diagnosis of glioma marker and diagnostic analysis is carried out to patient, make a definite diagnosis the state of an illness as early as possible, and correspondingly after selection reasonably
Continuous therapeutic scheme, improves survival rate, 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, 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 serum excretion body circRNA markers for diagnosis of glioma are provided.
Main contents include：A kind of serum excretion body circRNA markers circ1 for diagnosis of glioma:
43920404 | 43920928, its sequence such as SEQ NO:Shown in 1.
Second object of the present invention is to provide detection circRNA markers 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, it can be measured in serum excretion body
Circ1:43920404 | 43920928 content.
The diagnosis of glioma kit contains detection circ1:43920404 | the PCR primer of 43920928 contents.It is excellent
Select the sequence such as SEQ NO of primer:Shown in 2 and 3.
The diagnosis of glioma kit, except circ1:43920404 | outside 43920928 primer, also contain from serum
It is middle to extract excretion body, by extracting RNA in excretion body and carrying out all reagents of reverse transcription and quantitative fluorescent PCR.Including：(1) carry
Take reagent needed for serum excretion body：Total Exosome Isolation Reagent (from serum), can be by
Invitrogen companies buy, 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 × it is inverse
Transcription buffer, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases；(4) examination needed for quantitative fluorescent PCR
Agent：circ1:43920404 | 43920928 upstream and downstream primers, GAPDH internal references upstream and downstream primer, SYBR dyestuffs, without enzyme water.
The beneficial effects of the present invention are：Circ1 is found first:43920404 | in 43920928 this serum excretion bodies
Circular rna, and find that it has higher diagnostic value to glioma；Pass through serum circRNA markers and diagnostic reagent
The development and application of box, can make it that the diagnosis of glioma is more convenient and easy, and disease is quick and precisely grasped for clinician
Feelings, lay the foundation to improve clinical therapeutic efficacy, and to find that the new small molecule drug targets with potential treatment value carry
For helping.
Brief description of the drawings
Fig. 1 analyzes circ1 for real-time fluorescence quantitative PCR:43920404 | 43920928 in normal human serum excretion body and glue
Differential expression in matter knurl patients serum's excretion body；
Fig. 2 is the circ1 that ROC curve analyzes serum excretion body source:43920404 | 43920928 pairs of diagnosis of glioma
Specificity, sensitivity.
Embodiment
The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.
First, research object
Case group is the 30 glioma serum samples collected in Hunan Provincial Tumour Hospital in January, 2016 in June, 2017.
Control group carries out the healthy individuals 12 of community's disorder in screening for the same period, and frequency is carried out with case by gender and age (± 5 years old)
Matching.Sample for research is collected for the same period, sample, dispense, preservation condition it is consistent.
2nd, research method
(1) preparation of serum excretion body：Take 200 μ l of serum to be centrifuged 30 minutes in 2000g room temperature, extracted with micropipettor
Supernatant liquor adds 40 μ l excretion bodies extracts reagents (Total Exosome Isolation to 600 new μ l centrifuge tubes
Reagent (from serum), article No. 4478360, Invitrogen companies) gently turning upside down shakes up, and 4 DEG C are incubated 45 points
Clock.10000g room temperature centrifuges 10 minutes after incubation, discards supernatant, and 200 μ l Trizol are added in precipitation, and (MRC is public
Department) precipitation is resuspended, suspension is moved to new 1.5ml tube manages, and mends Trizol to 1ml.
(2) in excretion body RNA extracting：By above-mentioned re-suspension liquid in static cracking 15 minutes on ice.4 DEG C after cracking,
12000rpm centrifuges 10min, and supernatant moves to new tube pipes.Chlorination imitates 200 μ l in Tube, shakes 15-30s, ice with hand
Upper placement 5min, 4 DEG C of 12000rpm centrifuge 15min；Carefully take upper strata aqueous phase to enter in new tube, add the isopropanol of precooling
0.5ml is mixed, and stands be more than 20min on ice, and 4 DEG C of 12000rpm centrifuge 10min；Supernatant is abandoned, it is water-reducible to add 75%DEPC
Ethanol 1ml is mixed, 4 DEG C, 7500rpm centrifugation 5min, abandons supernatant as far as possible, drying at room temperature 5-10min, adds no 10 μ l of enzyme water dissolvings
RNA, -80 DEG C of preservations.
(3) prepared by cDNA：Reverse transcription reaction is carried out according to Reverse Transcriptase kit (Thermo companies) specification.Reaction is overall
Product for 20 μ l (4 1 μ l of μ l, RI of 10 μ l, Random primer of total serum IgE 1 μ l, no 1 μ l, 5 × Reaction Buffer of enzyme water,
2 μ l of RT1 μ l and 10mM dNTP).
| Component | 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
| 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 |
| First step reverse transcription 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.
(4) real-time fluorescence quantitative PCR：The circ1 of Han Heng biotechnologies (Shanghai) Co., Ltd. synthesis:43920404|
43920928 specific primers are (see sequence table SEQ NO：2 and 3) carry out real-time quantitative PCR：Reverse transcription product is first diluted 10
Times, mix.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.
(5) data analysis：Using 2-ΔΔCTRepresent the circ1 of glioma serum excretion body:43920404 | 43920928 phases
For the expression multiple of normal serum excretion body, wherein △ CT=CTSample–CTInternal reference, Δ Δ CT=Δs CT-Δ CTNormally.This experiment number
According to the analysis method using relative quantification, as reference gene, (primer sequence is shown in SEQ NO to GAPDH：4 and 5) Δ CTNormallyTo be normal
The Δ CT average values of serum excretion body, data are analyzed using software GraphPad Prism and SPSS 17.0.3rd, study
As a result
Case group serum excretion body circ1:43920404 | 43920928 expressions significantly lower (p compared with control group<
0.0001).Specific data are as shown in Figure 1.
ROC curve analysis shows that, circ1:43920404 | 43920928 are used as biomarker to glioma with higher
(AUC=0.877, p=0.003, susceptibility and specificity are respectively 85.7% and 100%) to diagnostic value.Detailed results are shown in figure
2。
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Diagnosis of glioma marker circ1:43920404 | 43920928 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 294
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
agugcaucgg auggcuucug gaaaucugug gccacucgag ugcccaagga gcccccugag 60
auucgaaucc ucaacccaua uuucauccag gaggccgccu ucacccucau uggccugccc 120
uucaacaaug gccucauggg ccgggggaac aucccuaccc uuggcagugu ggcagugacc 180
auggcacuac acggcuguga cgagguggca gucgcaggau uuggcuauga caugagcaca 240
cccaacgcac cccugcacua cuaugagacc guucgcaugg cagccaucaa agag 294
<210> 2
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 2
ctacacggct gtgacgagg 19
<210> 3
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 3
ggatgaaata tgggttgagg a 21
<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. diagnosis of glioma marker circ1:43920404 | 43920928, its sequence such as SEQ NO:Shown in 1.
- 2. the reagent of marker expression quantity in serum excretion body described in test right requirement 1 is preparing diagnosis of glioma preparation In application.
- 3. a kind of diagnosis of glioma kit, it is characterised in that the circ1 in serum excretion body can be measured:43920404| 43920928 content.
- 4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection circ1:43920404| The PCR primer of 43920928 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 circ1:43920404| Outside 43920928 primer, also contain and excretion body is extracted from serum, by extracting RNA in excretion body and carrying out reverse transcription and fluorescence All reagents of quantitative PCR.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009039390A2 (en) * | 2007-09-20 | 2009-03-26 | Naurex Inc. | The development of glycobiology-based therapeutics for the treatment of brain tumors |
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2017
- 2017-12-28 CN CN201711453662.4A patent/CN107937527B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009039390A2 (en) * | 2007-09-20 | 2009-03-26 | Naurex Inc. | The development of glycobiology-based therapeutics for the treatment of brain tumors |
Non-Patent Citations (2)
| Title |
|---|
| JAHANSHAH ASHKANI ET AL.: "Glycosyltransferase gene expression profiles classify cancer types and propose prognostic subtypes", 《SCIENTIFIC REPORTS》 * |
| JECK WR ET AL.: "Circular RNAs are abundant，conserved and associated with ALU repeats", 《RNA》 * |
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