CN107937537A - Glioma diagnosis marker circ7:42148226|42148468 and application - Google Patents
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- 206010018338 Glioma Diseases 0.000 title claims abstract description 43
- 208000032612 Glial tumor Diseases 0.000 title claims abstract description 39
- 238000003745 diagnosis Methods 0.000 title claims abstract description 22
- 239000003550 marker Substances 0.000 title claims abstract description 8
- 210000002966 serum Anatomy 0.000 claims abstract description 30
- 230000029142 excretion Effects 0.000 claims description 34
- 239000003153 chemical reaction reagent Substances 0.000 claims description 16
- 238000010839 reverse transcription Methods 0.000 claims description 8
- 238000003753 real-time PCR Methods 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 18
- 210000001808 exosome Anatomy 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
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- 238000001556 precipitation Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
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- 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
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 108091028075 Circular RNA Proteins 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
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- 238000000034 method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000523 sample Substances 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
- 241000282414 Homo sapiens Species 0.000 description 2
- 108700011259 MicroRNAs Proteins 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000003161 ribonuclease inhibitor Substances 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
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- 238000007405 data analysis Methods 0.000 description 1
- 238000012350 deep sequencing Methods 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
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- 230000008707 rearrangement Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 229940048102 triphosphoric acid Drugs 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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Abstract
The invention belongs to the technical field of biology, and discloses a glioma diagnosis marker circ7:42148226|42148468 and application thereof. In the invention, the following are found for the first time: exosome secreted by glioma cells has obvious enrichment effect on circ7:42148226| 42148468; meanwhile, the expression level of circ7:42148226|42148468 in serum exosomes of glioma patients is obviously reduced compared with that of a control group (P is less than 0.0001), and ROC analysis shows that the expression level of the glioma has higher diagnostic value (AUC is 0.850, P is 0.001, and the sensitivity and the specificity are 75.0 percent and 96.0 percent respectively). Therefore, by detecting the expression level of circ7:42148226|42148468 in the serum exosome of the glioma patient, the early and rapid noninvasive diagnosis of the glioma patient can be made.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of serum circRNA markers for diagnosis of glioma
circ7:42148226 | 42148468 and detect the marker reagent be used to prepare the application of diagnosis of glioma preparation, also
There is kit.
Background technology
Glioma is the most common malignant tumour of central nervous system, is one of principal disease of central nervous system,
Occupy the first place of intracranial primary tumor incidence.Since the characteristics of its high invasion, high relapse rate, make it that glioma cure rate is low,
Patient survival is short.The development of neuroimaging technology and the continuous progress of microsurgical technique to neurosurgery has risen huge
Facilitation, and deepening continuously for molecular biology research provides bright prospects but to brain colloid for the gene therapy of tumour
The survival of knurl is not significantly improved.Therefore, find diagnosis of glioma marker and examination, and phase are carried out to people at highest risk
Answer ground to select rational successive treatment scheme, improve survival rate, be 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 circ7 for diagnosis of glioma:
42148226 | 42148468, its sequence such as SEQ NO:Shown in 1.The circRNA is located on No. 7 chromosome of the mankind, total length
243bp。
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
Circ7:42148226 | 42148468 content.
The diagnosis of glioma 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 diagnosis of glioma kit, except circ7:42148226 | outside 42148468 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) 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：circ7:42148226 | in 42148468 upstream and downstream primers, GAPDH internal references
Anti-sense primer, SYBR dyestuffs, without enzyme water.
The beneficial effects of the present invention are：
Circ7 in excretion body caused by glioma cell is found first:42148226 | 42148468 compared to intracellular
Concentration is significantly raised, and cell excretion body has good concentration effect to the circular rna.In the serum excretion body of patients with gliomas
circ7:42148226 | 42148468 significantly lower (P compared to normal serum excretion body control group<0.0001).ROC curve is analyzed
Show circ7:42148226 | 42148468 as biomarkers to glioma have higher diagnostic value (AUC=0.850,
Sensitivity and specificity are respectively 75.0% and 96.0%., can be with by application of the circular rna in diagnosis of glioma analysis
So that the diagnosis of glioma is more convenient accurate, conditions of patients is quick and precisely grasped for clinician, to improve clinical treatment effect
Fruit lays the foundation, and to find that the new small molecule drug targets with potential treatment value provide help.
Brief description of the drawings
Fig. 1 analyzes circ7 for real-time fluorescence quantitative PCR:42148226 | 42148468 divide in glioma cell and cell
Differential expression in the excretion body secreted；
Fig. 2 analyzes circ7 for real-time fluorescence quantitative PCR:42148226 | 42148468 in glioma serum and normal serum
Differential expression in excretion body；
Fig. 3 is the circ7 that Roc analyzes serum excretion body source:42148226 | what 42148468 pairs of gliomas early diagnosed
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, U87 cell line,；Six plants of glioma primary cells are respectively 1104,
1216th, 1125,1124B, 1124C, 0128C, provide by institute of oncology of preclinical medicine institute of Central South University.
The serum sample of 2.40 patients with gliomas is provided by Xiang Ya hospitals, and 19 normal serum samples carry out society for the same period
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. the extracting of RNA in excretion body, glioma/normal serum excretion body secreted by cell, cell
A. the extracting of cell RNA
Treat that above-mentioned 8 kinds of cell length to suitable density, outwells Pei Ji, 1mlTrizol reagents are added in culture dish, are split on ice
Solution 15 minutes.Move to 1.5ml after cracking to manage without enzyme Tube, 4 DEG C of 12000rpm centrifuge 10min, and supernatant moves to new
Tube is managed.Chlorination imitates 200 μ l in Tube, shakes 15-30s with hand, places 5min on ice, and 4 DEG C of 12000rpm centrifuge 15min；
Carefully upper strata aqueous phase is taken to enter in new tube, the isopropanol 0.5ml for adding precooling is mixed, and stands be more than 20min on ice, 4 DEG C
12000rpm centrifuges 10min；Supernatant is abandoned, the water-reducible ethanol 1ml of 75%DEPC is added and mixes, 4 DEG C of 7500rpm centrifuge 5min,
Supernatant is abandoned as far as possible, and drying at room temperature 5-10min, adds no 10 μ l of enzyme water dissolvings RNA, -80 DEG C of preservations.
B. in the excretion body secreted by cell RNA extracting
Treat that above-mentioned 8 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.After centrifugation super
Filtrate is collected in 1.5ml and is 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 room 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 1mlTrizol reagents are resuspended, crack 15 points on ice
Clock, the same A of extraction step after cracking terminates.
C. in glioma/normal serum excretion body RNA extracting
Take 200 μ l of serum to be centrifuged 30 minutes in 2000g room temperature, supernatant liquor is extracted to 600 new μ l with micropipettor
Centrifuge tube, adds 40 μ l excretion bodies extracts reagent (Total Exosome Isolation Reagent (from serum), goods
Number 4478360, Invitrogen companies) gently turning upside down shakes up, and 4 DEG C are incubated 45 minutes.10000g room temperature after incubation
Centrifugation 10 minutes, discards supernatant, and gained precipitation is the excretion body in serum.200 μ lTrizol (MRC are added in precipitation
Company) precipitation is resuspended, suspension is moved to new 1.5mltube manages, and mends Trizol to 1ml.15min, cracking knot are cracked on ice
The same A of extraction step after beam.
It is prepared by 2.cDNA
Reverse transcription reaction is carried out according to Reverse Transcriptase kit (Thermo companies) specification.Reaction cumulative volume is (total for 20 μ l
RNA9 μ l, Random primer1 μ l, no 2 μ l, 5 × Reaction Buffer4 μ l, RI1 μ l, RT1 μ l of enzyme water and
10mMdNTP2μl)。
| 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.
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, is mixed.20 μ L reaction systems are as follows：
| Component | Dosage/pipe |
| SYBR Premix Ex Taq | 10μl |
| Primer (10 μM) | 0.5μl |
| CDNA products (after dilution) | 5μl |
| Without enzyme water | To 20μl |
Real-time fluorescence quantitative PCR response procedures：95 DEG C 3 minutes, 40 circulation, 95 DEG C 10 seconds, 60 DEG C 30 seconds.
4. data analysis：Using 2-ΔΔCTRepresent the circ7 of glioma serum excretion body:42148226 | 42148468 is opposite
In the expression multiple of normal serum excretion body, wherein △ CT=CTSample–CTInternal reference, Δ Δ CT=Δs CTGlioma–ΔCTNormally.This experiment
Data use the analysis method of relative quantification, and as reference gene, (primer sequence is shown in SEQ NO to GAPDH：4 and 5), data utilize
Software GraphPad Prism and SPSS 17.0 is analyzed.
3rd, result of study
1. circ7 in excretion body caused by glioma cell:42148226 | 42148468 is bright compared to intracellular concentration
Aobvious rise, cell excretion body have good concentration effect to the circular rna.Concrete outcome is as shown in Figure 1.
2. circ7 in the serum excretion body of patients with gliomas:42148226 | 42148468 compare normal serum excretion body pair
(p is significantly lowered according to group<0.0001).Specific data are as shown in Figure 2.ROC curve is analysis shows that circ7:42148226|
42148468 have glioma as biomarker higher diagnostic value, and (AUC=0.850, sensitivity and specificity are respectively
For 75.0% and 96.0%), detailed results are shown in Fig. 3.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Diagnosis of glioma 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. diagnosis of glioma marker circ7:42148226 | 42148468, 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 circ7 in serum excretion body can be measured:42148226| 42148468 content.
- 4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection circ7:42148226| The PCR primer of 42148468 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 circ7:42148226| Outside 42148468 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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