CN107604075A - Glioma prognostic marker circ7:66286511 | 66286709 and application - Google Patents
Glioma prognostic marker circ7:66286511 | 66286709 and application Download PDFInfo
- Publication number
- CN107604075A CN107604075A CN201711056948.9A CN201711056948A CN107604075A CN 107604075 A CN107604075 A CN 107604075A CN 201711056948 A CN201711056948 A CN 201711056948A CN 107604075 A CN107604075 A CN 107604075A
- Authority
- CN
- China
- Prior art keywords
- circ7
- glioma
- circrna
- patients
- gliomas
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of glioma prognostic marker circ7:66286511 | 66286709 and application, that is, detect the circRNA circ7 in brain tissue source:66286511 | 66286709 reagent is used for the prognosis preparation for preparing patients with gliomas.Researcher has found circRNA circ7:66286511 | 66286709 in patients with gliomas brain tissue expression significantly lower (P<0.0001), circRNA circ7 in glioma:66286511 | the higher patient of 66286709 expression quantity, possess higher survival rates (P=0.005).Thus the present invention can be by detecting circRNA circ7 in patients with gliomas samples of human glioma:66286511 | 66286709 expression, so as to judge the prognosis situation of patients with gliomas.
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 is the most common malignant nerve epithelial cell tumour of encephalic, accounts for the 40.49% of intracranial tumors, is adult
One of most common brain tumor.Since making a definite diagnosis, the Patients with gliomas the average survival time life-span is no more than 5 years.Glioma at present
Treatment technology it is various, but because tumor-infiltrated invasion and attack speed is fast, and the spy such as Apoptosis is insensitive to chemicotherapy mediation
Property, overall treatment effect is bad, high recurrence rate, poor prognosis, therefore finds new therapy target and prognostic indicator is most important.Mesh
Preceding diagnosis of glioma is still within the experience sexual stage based on clinical, pathology and iconography information, and one after diagnosing,
The overwhelming majority is middle and advanced stage, and survival rates allow of no optimist.Therefore, new glioma prognostic marker is found to carry out patient
Survival analysis, to improve the postoperative life quality of patients with gliomas, and rational successive treatment scheme is correspondingly selected, improve life
Rate is deposited, is neuroscience field Task urgently to be resolved hurrily.
CircRNA is that one kind is different from linear rna, is present in extensively and diversely the envelope of the endogenous in mammalian cell
Loop-like non-coding RNA molecule, its Stability Analysis of Structures, it is not easy to be influenceed by RNA excision enzymes, can transcribing, after transcription, the level such as translation
Multi-level controlling gene expression, is widely present in various types of cells, brain abundance is high.CircRNA turns into non-volume in recent years
The new focus in code RNA fields.With the fast development of the extensive use of deep sequencing technology and biophysics and informatics technology,
It is found that the transcript of many extrons of the mankind can be formed by non-linearly reverse splicing or by gene rearrangement
CircRNA, and they account for sizable ratio in all montage transcripts, and with rich, stability, high conservative
With the feature such as 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 circ7:66286511 | 66286709, 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
circ7:66286511 | 66286709 content.
Described glioma prognosis kit, contain detection circ7:66286511 | the PCR primer of 66286709 contents.
It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.
Described glioma prognosis kit, except circ7:66286511 | outside 66286709 primer, also contain from brain group
Knit middle extraction RNA and carry 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 circ7 using total serum IgE:66286511 | 66286709 reverse transcriptions are cDNA agents useful for same, including inverse
Transcription buffer, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases and circ7:66286511|
Random primer used in 66286709；
(3) by cDNA real-time quantitative PCR agents useful for same, including circRNA circ7:66286511 | 66286709 is real-time
Quantitative fluorescent PCR specific primer, GAPDH internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.
Applicant to 40 patients with gliomas tumor tissues and 15 normal human's brain tissue quantitative fluorescent PCRs by having found
circ7:66286511 | 66286709 in both notable difference express (P<0.0001).And to receiving 18 patients of follow-up
Carry out the circRNA circ7 that survivorship curve analysis finds samples of human glioma source:66286511 | 66286709 with the life of patient
Rate correlation is deposited, its content is higher, and survival rate is higher (P=0.005).This method provides strong for the prognostic analysis of glioma
Technical support, the postoperative life quality of patients with gliomas is favorably improved, works out aftertreatment scheme, improved survival rate, have
Far-reaching clinical meaning and generalization.
Brief description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR analyzes circ7:66286511 | 66286709 in normal cerebral tissue and glue
Differential expression in matter knurl；
The circ7 in Fig. 2 tracing analysis samples of human glioma sources for survival:66286511 | 66286709 expression height are to glue
The influence prognosis of matter knurl patient.
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 circ7:66286511 | the reagent of 66286709 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 random μ l of reverse transcriptase primer 50
7.5 × RT Buffer 200ml
The μ l of 8.10mM triphosphoric acid bases deoxynucleotide 100
9.40U/ the μ l of 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 circ7:66286511 | the μ l of 66286709 real-time fluorescence quantitative PCR specific primer 30
circRNA circ7:66286511 | 66286709 forward primers：5'-CAGTGATTGCTCCTATGC-3',
circRNA circ7:66286511 | 66286709 reverse primers：5'-CTGGCTTGATTACTTGGT-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 circ7 of embodiment 2:66286511 | 66286709 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.
By the daily time recording refrigerator temperature of laboratory technician.
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/ml Trizol shake 15-30s in Tube, with hand, place 5min on ice, and 4 DEG C of 12000rpm centrifuge 15min；It is small
The heart takes upper strata aqueous phase to enter in new tube, and the isopropanol 0.5ml/ml Trizol for adding precooling are mixed, and -20 DEG C of refrigerators are stood
20min, 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 centrifuges 5min, abandons supernatant as far as possible, drying at room temperature 5-10min, adds DEPC water 10-20 μ l dissolvings RNA.- 80 DEG C of guarantors
Deposit.Temperature is recorded by laboratory technician daily.
3、circRNA circ7:66286511 | 66286709 reverse transcriptions：Use the Reverse Transcriptase kit of Thermo companies.
The system of 20 μ L reverse transcription reactions is as follows：
| Composition | Dosage/pipe |
| Random reverse transcriptase primer (1 μM) | 1μl |
| RNA samples | 2μl |
| 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 circ7 of Han Heng bio tech ltd synthesis:66286511 | 66286709 specific primers carry 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'-CAGTGATTGCTCCTATGC-3',
Reverse primer：5'-CTGGCTTGATTACTTGGT-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 circ7 that qRT-PCR is measured:66286511 | 66286709CT values are made with the CT values with tissue-derived GAPDH
Difference obtains Δ CT, then by Δ CT and Δ CTControlObtaining Δ Δ CT as difference, (average value for taking normal sample Δ CT is Δ CTControl), number
Welch check analyses are carried out according to using software GraphPad Prism.Analysis is found, with circRNA in samples of human glioma
circ7:66286511 | 66286709 with the circRNA circ7 of normal cerebral tissue:66286511 | 66286709 expression measurers
Variant (see Fig. 1), difference have conspicuousness (P<0.0001).
6th, found by 40 patients with gliomas follow-up statistics used by experiment, 22 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 18,
This 18 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 circ7:
66286511 | 66286709 high expression, totally 20, receive follow-up as 9, other are circ7:66286511 | 66286709 is low
Expression, totally 20, receive follow-up as 9.Through Kaplan-Meier survival analysises, circ7:66286511 | 66286709 high tables
Up to patient life cycle compared with circ7:66286511 | patient's length of 66286709 low expressions, good prognosis.Difference is statistically significant
(P=0.005).
Research shows above, circ7:66286511 | 66286709 can be as the specific molecular of patients with gliomas prognosis
Mark.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Glioma prognostic marker circ7:66286511 | 66286709 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 199
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
aguggagaga ucuacagacc aaguaaucaa gccagucaau guaggagcuc uaucaaaaug 60
gguugggaag auaccgccag auguuuuaca agacauggca gugauugcuc cuaugcuugc 120
caagcuugga uaugacccau augccaaccc accuaacuac ggaaaaccug aucccaaaau 180
uauugaaaac acucgaagg 199
<210> 2
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 2
cagtgattgc tcctatgc 18
<210> 3
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 3
ctggcttgat tacttggt 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. CircRNA marks circ7 in brain tissue source for glioma prognosis:66286511 | 66286709, 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 circ7 in brain tissue can be determined:66286511| 66286709 content.
- 4. glioma prognosis kit according to claim 3, it is characterised in that contain detection circ7:66286511| The PCR primer of 66286709 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 circ7:66286511| Outside 66286709 primer, also contain the extraction RNA from brain tissue and carry out all reagents of reverse transcription and quantitative fluorescent PCR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711056948.9A CN107604075A (en) | 2017-10-27 | 2017-10-27 | Glioma prognostic marker circ7:66286511 | 66286709 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711056948.9A CN107604075A (en) | 2017-10-27 | 2017-10-27 | Glioma prognostic marker circ7:66286511 | 66286709 and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107604075A true CN107604075A (en) | 2018-01-19 |
Family
ID=61084101
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711056948.9A Pending CN107604075A (en) | 2017-10-27 | 2017-10-27 | Glioma prognostic marker circ7:66286511 | 66286709 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107604075A (en) |
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 CN201711056948.9A patent/CN107604075A/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 tyrosylprotein sulfotransferase 1 (TPST1), 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》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | MicroRNA‐222 and MicroRNA‐146b are tissue and circulating biomarkers of recurrent papillary thyroid cancer | |
| Namkung et al. | Molecular subtypes of pancreatic cancer based on miRNA expression profiles have independent prognostic value | |
| CN106148529B (en) | LncRNA marker related to gastric cancer, special detection primer, detection method, kit and application thereof | |
| CN109182527B (en) | Interferon related kit for prognosis evaluation and chemotherapy effect prediction in glioma | |
| CN102876676A (en) | Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof | |
| CN101613748A (en) | A kind of method that detects serum marker of pancreatic cancer | |
| CN108192974A (en) | Applications of the long-chain non-coding RNA LINC00842 as biomarker in adenocarcinoma of lung prognosis detection preparation is prepared | |
| CN107586848A (en) | Glioma prognostic marker circ8:127890589 | 127890998 and application | |
| CN106755343A (en) | Cancer of pancreas Prognosis molecular marked compound | |
| CN107586844A (en) | Glioma prognostic marker Circ9:135881633 | 135883078 application | |
| CN110257514A (en) | A kind of new cancer of the esophagus blood miRNA marker and its application | |
| CN107619869A (en) | Diagnosis of glioma and prognostic marker circ16:85633914 | 85634132 and application | |
| CN108424962A (en) | The miRNA detection markers and its diagnostic kit of mesangial proliferative glomerulonephritis | |
| CN107604075A (en) | Glioma prognostic marker circ7:66286511 | 66286709 and application | |
| CN108753981A (en) | Application of the quantitative detection of HOXB8 genes in colorectal cancer Index for diagnosis | |
| CN107937528A (en) | Glioma prognostic marker hsa_circ_0125365 and application | |
| CN102424843B (en) | Application and detection kit of human miR-183/96/182 cluster | |
| CN107557441A (en) | Diagnosis of glioma mark Circ2:23823258 | 23823569 and application | |
| CN107604074A (en) | Glioma prognostic marker circ15:101235082 | 101235577 and application | |
| CN107937539A (en) | Glioma prognostic marker hsa_circ_0135404 and application | |
| CN107619868A (en) | Glioma prognostic marker Circ3:129880309 | 129880559 application | |
| CN107937536A (en) | Glioma prognostic marker circ19:47362476 | 47362693 and application | |
| CN107586849A (en) | The circ9 in brain tissue source:4117768 | 4118881 application | |
| CN107604072A (en) | Glioma prognostic marker circ15:98707562 | 98708107 application | |
| CN107937530A (en) | Glioma prognostic marker hsa_circ_0125361 and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180119 |