CN107937528A - Glioma prognostic marker hsa_circ_0125365 and application - Google Patents
Glioma prognostic marker hsa_circ_0125365 and application Download PDFInfo
- Publication number
- CN107937528A CN107937528A CN201711453665.8A CN201711453665A CN107937528A CN 107937528 A CN107937528 A CN 107937528A CN 201711453665 A CN201711453665 A CN 201711453665A CN 107937528 A CN107937528 A CN 107937528A
- Authority
- CN
- China
- Prior art keywords
- circ
- glioma
- hsa
- samples
- circrna
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to biological technical field, discloses a kind of glioma prognostic marker hsa_circ_0125365 and application.The reagent for detecting the circRNA hsa_circ_0125365 expression quantity in samples of human glioma source is used to prepare the prognosis preparation of patients with gliomas.By studying the patient for confirming that circRNA hsa_circ_0125365 expression quantity is higher in glioma, possess the survival rates of higher.By detecting the expression of circRNA hsa_circ_0125365 in patients with gliomas samples of human glioma, so as to make Index for diagnosis to patients with gliomas.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of circRNA markers for glioma prognosis and detection
The reagent of the marker 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 service life 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 continuously improves 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, 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, and have
The features such as rich, stability, high conservative and Space-time speciality, there is the potentiality as many disease molecules markers.
The content of the invention
The present invention first purpose be：A kind of samples of human glioma source for patients with gliomas prognosis is provided
CircRNA marker hsa_circ_0125365, its sequence such as SEQ NO:Shown in 1.
Second object of the present invention is to provide detection circRNA markers expression quantity in samples of human glioma
Application of the reagent in glioma prognosis preparation is prepared.
Third object of the present invention is to provide a kind of glioma prognosis kit, can measure in samples of human glioma
The content of hsa_circ_0125365.
The glioma prognosis kit, the PCR primer containing detection hsa_circ_0125365 contents.It is preferred that primer
Sequence such as SEQ NO:Shown in 2 and 3.
The glioma prognosis kit, in addition to the primer of hsa_circ_0125365, also contains from samples of human glioma
Middle extraction RNA simultaneously 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 cDNA agents useful for same by hsa_circ_0125365 reverse transcriptions by template of total serum IgE, including reverse transcription buffering
It is random used in liquid, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases and hsa_circ_0125365
Primer；
(3) by cDNA real-time quantitative PCR agents useful for same, including circRNA hsa_circ_0125365 real time fluorescent quantitatives
PCR specific primers, GAPDH internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.
Present invention research confirms that the circRNA hsa_circ_0125365 in samples of human glioma source suffer from available for glioma
Person's prognostic analysis, circRNA hsa_circ_0125365 expression quantity and patient's survival rates in samples of human glioma source have
Correlation.Therefore, the prognostic analysis available for patients with gliomas.
Applicant carries out analysis with 12 normal cerebral tissues to 26 samples of human glioma by quantitative fluorescent PCR and finds,
Differential expressions of the hsa_circ_0125365 in both is obvious (P ＜ 0.0001), afterwards to wherein 15 patients with gliomas into
Row survivorship curve is analyzed, and finds the survival rate phase of the circRNA hsa_circ_0125365 and patient in samples of human glioma source
Close, content is higher, and survival rate is higher.This method provides strong technical support for the prognostic analysis of glioma, helps to carry
The postoperative life quality of high patients with gliomas, works out aftertreatment scheme, improves survival rate, has far-reaching clinical meaning and pushes away
Wide property.
Brief description of the drawings
Fig. 1 analyzes hsa_circ_0125365 tables in samples of human glioma and normal cerebral tissue for real-time fluorescence quantitative PCR
Up to difference；
Fig. 2 is that ROC curve analyzes the hsa_circ_0125365 in brain tissue source to glioma and normal cerebral tissue's difference
Specificity, sensitivity；Hsa_circ_0125365 has higher diagnostic value (AUC=as biomarker to glioma
0.939, P=0.040, susceptibility and specificity are respectively 90.0% and 78.3%).
Fig. 3 the circRNA hsa_circ_0125365 expression quantity in tracing analysis samples of human glioma source and patient for survival
The correlation of survival rate.
Embodiment
The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.
Embodiment 1：The reagent for preparing detection circRNA hsa_circ_0125365 expression quantity is used to prepare glioma trouble
The kit (50 secondary response) of person's prognosis
1.RNA stablizing solutions 50ml
2. isopropanol 100ml
3. chloroform 100ml
4.Trizol (coming from Molecular Research Center companies) 50ml
5. without enzyme water 10ml
6.1 μM of random 50 μ l of reverse transcriptase primer (Thermo companies)
7.5 × RT Buffer (Thermo companies) 200ml
100 μ l of 8.10mM triphosphoric acid bases deoxynucleotide (Thermo companies)
500 μ l of 9.40U/ μ l RNase inhibitors (Thermo companies)
10.200U/ μ l MMLV reverse transcriptases (Thermo companies) 50 μ l
50 μ l of 11.Premix Ex Taq (Thermo companies)
12.10 μM of 30 μ l of circRNA hsa_circ_0125365 real-time fluorescence quantitative PCRs specific primer
CircRNA hsa_circ_0125365 forward primers：5'-CCAAGCAGCTCACTACGATA-3',
CircRNA hsa_circ_0125365 reverse primers：5'-CAAGCAGGTAGGAGATTCCA-3'；
13.10 μM of 30 μ l of GAPDH specific primers
Forward primer is 5 '-ATCAAGATCATTGCTCCTCCTGAG-3 ',
Reverse primer is 5 '-CTGCTTGCTGATCCACATCTG-3 '.
Embodiment 2：CircRNA hsa_circ_0125365 are in the expression quantity of samples of human glioma and the relation of prognosis
1st, the preservation of samples of human glioma：Samples of human glioma to be measured is collected to deposit in the cryopreservation tube for filling RNA stablizing solutions,
Put spare to -80 DEG C of refrigerators.
2nd, the extracting of RNA 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 move to tube pipe, 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
Water mutually enters in new tube, and the isopropanol 0.5ml for adding precooling is mixed, and stands 20min on ice, and 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 centrifugation 5min, abandon supernatant, room temperature as far as possible
Dry 5-10min, adds DEPC water 10-20 μ l dissolvings RNA, -80 DEG C of preservations.
3rd, circRNA hsa_circ_0125365 reverse transcriptions：Use the Reverse Transcriptase kit of Thermo companies.20 μ l are inverse
The system of responsive transcription is as follows：
Reverse transcription first step condition：65 DEG C 5 minutes
| Component | Dosage/pipe |
| 5 × RT Buffer | 4μl |
| Triphosphoric acid base deoxynucleotide (10mM) | 2μl |
| RNA enzyme inhibitors (40U/ μ l) | 1μl |
| MMLV reverse transcriptases (200U/ μ l) | 1μl |
| The product of first step reverse transcription | 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 circRNA hsa_circ_0125365 specific primers of Han Heng biotechnologies (Shanghai) Co., Ltd. synthesis
Carry out real-time quantitative PCR：Reverse transcription product is first diluted 10 times, is mixed.20 μ l reaction systems are as follows：
The specific primer of PCR：
Forward primer：5'-CCAAGCAGCTCACTACGATA-3',
Reverse primer：5'-CAAGCAGGTAGGAGATTCCA-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-ΔΔCTCalibration：With 2-ΔΔCTRepresent the multiple for control group change, wherein △ CT=CTSample–CTInternal reference, △
△ CT=△ CTCase–△CTControl.This experimental data uses the analysis method of relative quantification, and GAPDH is as reference gene, glioma
The average value of patient △ CT is control.Data are analyzed using software GraPhPad Prism and SPSS.Applicant passes through glimmering
Fluorescent Quantitative PCR carries out analysis with 12 normal cerebral tissues to 26 samples of human glioma and finds that hsa_circ_0125365 is at both
In differential expression it is obvious (P ＜ 0.0001), see Fig. 1.
6th, the hsa_circ_0125365 in the ROC curve analysis brain tissue source of Fig. 2 is poor to glioma and normal cerebral tissue
Different specificity, sensitivity；Hsa_circ_0125365 has higher diagnostic value (AUC as biomarker to glioma
=0.939, P=0.040, susceptibility and specificity are respectively 90.0% and 78.3%).
7th, 16 patient's prognosis survivorship curve analyses are found, with circRNA hsa_circ_ in samples of human glioma
The patient of 0125365 low expression (subaverage) compares, circRNA hsa_circ_0125365 high tables in samples of human glioma
Up to patient survival's higher of (being higher than average value), difference has conspicuousness (P=0.119), sees Fig. 3.
Research shows that hsa_circ_0125365 can be as the specificity molecular marker of patients with gliomas prognosis above.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Glioma prognostic marker hsa_circ_0125365 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 568
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
uggaagcauc aguucacagc aguaaugcac acuguacaga uaagacaauu gaagcugcug 60
aagcccugcu ucauauggaa ucuccuaccu gcuugaggga uucaagaagu ccuguggaag 120
uguuuguucc uccuugugua ucaacuccag aauucaucca ugcugcuaug aggccagaug 180
ucauuacaga aacuguagug gaggugucaa cugaagaguc ugaacccaug gauaccucuc 240
cuauuccaac aucaccagau agccaugaac caaugaaaaa gaaaaaaguu ggccguaaac 300
caaagaccca gcaaucacca auuuccaaug ggucuccuga guuagguaua aagaagaaac 360
caagagaagg aaaaggaaac acaaccuauu ugugggaguu ucuuuuagau cuacuucaag 420
auaaaaauac uugucccagg uauauuaaau ggacucagag agaaaaaggc auauucaagc 480
ugguggauuc aaaggcuguc ucuaagcuuu ggggaaagca uaagaacaaa ccagacauga 540
acuaugaaac caugggacga gcuuugag 568
<210> 2
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 2
ccaagcagct cactacgata 20
<210> 3
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 3
caagcaggta ggagattcca 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)
1. a kind of glioma prognostic marker hsa_circ_0125365, its sequence such as SEQ NO:Shown in 1.
2. the reagent of marker expression quantity in samples of human glioma described in test right requirement 1 is preparing glioma prognosis preparation
In application.
3. a kind of glioma prognosis kit, it is characterised in that the hsa_circ_0125365 in samples of human glioma can be measured
Content.
4. glioma prognosis kit according to claim 3, it is characterised in that contain detection hsa_circ_0125365
The PCR primer of content.
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 hsa_circ_0125365
Primer outside, also contain and RNA extracted from samples of human glioma and carries out all reagents of reverse transcription and quantitative fluorescent PCR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711453665.8A CN107937528B (en) | 2017-12-28 | 2017-12-28 | Glioma prognosis marker hsa _ circ _0125365 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711453665.8A CN107937528B (en) | 2017-12-28 | 2017-12-28 | Glioma prognosis marker hsa _ circ _0125365 and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107937528A true CN107937528A (en) | 2018-04-20 |
| CN107937528B CN107937528B (en) | 2020-06-30 |
Family
ID=61940565
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711453665.8A Active CN107937528B (en) | 2017-12-28 | 2017-12-28 | Glioma prognosis marker hsa _ circ _0125365 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107937528B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109321657A (en) * | 2018-10-30 | 2019-02-12 | 徐州医科大学 | A kind of glioma prognostic marker and its application |
-
2017
- 2017-12-28 CN CN201711453665.8A patent/CN107937528B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| RYBAK-WOLF ET AL.: "Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.", 《MOLECULAR CELL》 * |
| SALZMAN ET AL.: "Cell-Type Specific Features of Circular RNA Expression.", 《PLOS GENETICS》 * |
| YANG ET AL.: "Identification of circular RNA signature in bladder cancer.", 《JOURNAL OF CANCER》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109321657A (en) * | 2018-10-30 | 2019-02-12 | 徐州医科大学 | A kind of glioma prognostic marker and its application |
| CN109321657B (en) * | 2018-10-30 | 2021-07-02 | 徐州医科大学 | Glioma prognosis marker and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107937528B (en) | 2020-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105177174A (en) | miRNA (microribonucleic acid) biomarkers and detection kit for colon cancer diagnosis | |
| CN113337613B (en) | Serum exosome tsRNA marker related to liver cancer, probe and application thereof | |
| CN104694623A (en) | Plasma miRNA marker for diagnosis of lung cancer and application | |
| CN108624693A (en) | Applications of the miR-577 in preparing diagnosis of nephropathy marker | |
| CN107557472A (en) | Diagnosis of glioma mark circ9:135881633 | 135883078 and application | |
| CN107937528A (en) | Glioma prognostic marker hsa_circ_0125365 and application | |
| CN107586844A (en) | Glioma prognostic marker Circ9:135881633 | 135883078 application | |
| CN107586848A (en) | Glioma prognostic marker circ8:127890589 | 127890998 and application | |
| CN110257514A (en) | A kind of new cancer of the esophagus blood miRNA marker and its application | |
| JPWO2019117270A1 (en) | How to help detect head and neck cancer | |
| CN107619869A (en) | Diagnosis of glioma and prognostic marker circ16:85633914 | 85634132 and application | |
| CN107988370A (en) | A kind of application of circRNA genes in diagnosing chronic granulocytic leukemia reagent is prepared | |
| CN107586849A (en) | The circ9 in brain tissue source:4117768 | 4118881 application | |
| CN107937530A (en) | Glioma prognostic marker hsa_circ_0125361 and application | |
| CN107604072A (en) | Glioma prognostic marker circ15:98707562 | 98708107 application | |
| CN107937536A (en) | Glioma prognostic marker circ19:47362476 | 47362693 and application | |
| CN107586846A (en) | Diagnosis of glioma mark Circ3:129880309 | 129880559 and application | |
| CN107604076A (en) | Diagnosis of glioma mark Circ6:4891713 | 4892379 and application | |
| CN107604074A (en) | Glioma prognostic marker circ15:101235082 | 101235577 and application | |
| CN107937538A (en) | Diagnosis of glioma marker circ1:201817088 | 201817285 and application | |
| CN104059987A (en) | Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit | |
| CN107937529A (en) | Diagnosis of glioma marker hsa_circ_0135404 and application | |
| CN107557441A (en) | Diagnosis of glioma mark Circ2:23823258 | 23823569 and application | |
| CN107619868A (en) | Glioma prognostic marker Circ3:129880309 | 129880559 application | |
| CN107937540A (en) | Diagnosis of glioma marker circ17:47618350 | 47619164 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |