CN107937532A - Diagnosis of glioma marker hsa_circ_0021827 and application - Google Patents

Diagnosis of glioma marker hsa_circ_0021827 and application Download PDF

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CN107937532A
CN107937532A CN201711453689.3A CN201711453689A CN107937532A CN 107937532 A CN107937532 A CN 107937532A CN 201711453689 A CN201711453689 A CN 201711453689A CN 107937532 A CN107937532 A CN 107937532A
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diagnosis
circ
glioma
hsa
excretion body
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CN107937532B (en
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武明花
周和诚
李沛瑶
冯剑波
付海娟
王维
蒋健晖
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Xiangya Hospital of Central South University
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Abstract

The invention belongs to biological technical field, discloses diagnosis of glioma marker hsa_circ_0021827 and application.In the present invention, find first：The expression of hsa_circ_0021827 is significantly raised compared to control group (p=0.025) in patients with gliomas serum excretion body, ROC analyses then show that it has higher diagnostic value (AUC=0.823 to glioma, p=0.003, susceptibility and specificity are respectively 70% and 90.9%).Therefore by detecting the expression of hsa_circ_0021827 in patients with gliomas serum excretion body, early stage, quick Noninvasive diagnosis can be made to patients with gliomas.

Description

Diagnosis of glioma marker hsa_circ_0021827 and application
Technical field
The invention belongs to biological technical field, is related to a kind of serum circRNA markers hsa_ for diagnosis of glioma Circ_0021827 and detect the marker reagent be used to prepare the application of diagnosis of glioma preparation, also have 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, diagnosis of glioma marker is found to sieve people at highest risk Look into, and correspondingly select rational successive treatment scheme, improve survival rate, be neuroscience field Task urgently to be resolved hurrily.
Circular rna 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 circular rna is formed by gene rearrangement, and they account for sizable ratio in all montage transcripts.In recent years Also gradually find also to contain substantial amounts of circular rna in excretion body, may play a significant role.Excretion body refers to contain complexity The small film bubble (30-150nm) of RNA and protein, now, it refers in particular to plate-like vesica of the diameter in 40-100nm.Nineteen eighty-three, excretion Body is found in sheep granulophilocyte first, and Johnstone is named as " exosome " within 1987.Research in recent years is shown Show, excretion body is the important molecule of cell and cell-cell communication, participates in many physiology and pathologic process.Not only wrapped in excretion body Containing protein component, further include some RNA components, as Microrna (microRNA, miRNA), long-chain non-coding RNA and mRNA, Circular rna (circular RNA, circRNA).These RNA entrained by excretion body are referred to as excretion body source RNA, have had Whole sequential structure and bioactivity, are expected to as liquid biopsy molecular marker, there is light on accurate medical development Prospect.
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 hsa_circ_ for diagnosis of glioma 0021827, 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 Hsa_circ_0021827 content.
The diagnosis of glioma kit, the PCR primer containing detection hsa_circ_0021827 contents.It is preferred that primer Sequence such as SEQ NO:Shown in 2 and 3.
The diagnosis of glioma kit, in addition to the primer of hsa_circ_0021827, also contains and is extracted from serum 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：Hsa_circ_0021827 upstream and downstream primers, GAPDH internal reference upstream and downstream are drawn Thing, SYBR dyestuffs, without enzyme water.
The beneficial effects of the present invention are：
Find that hsa_circ_0021827 compares normal serum excretion body pair in the serum excretion body of patients with gliomas first According to group significantly up-regulation (p=0.025).ROC curve analysis shows that hsa_circ_0021827 as biomarker to glioma With higher diagnostic value, (AUC=0.823, p=0.003, susceptibility and specificity are respectively 70% and 90.9%).By this Application of the circular rna in diagnosis of glioma analysis, can make it that the diagnosis of glioma is more convenient accurate, be that clinician is fast Fast accurate grasp conditions of patients, lays the foundation to improve clinical therapeutic efficacy, and new to find to be worth with potential treatment Small-molecule drug target provides help.
Brief description of the drawings
Fig. 1 analyzes hsa_circ_0021827 in glioma serum and normal serum excretion body for real-time fluorescence quantitative PCR In differential expression；
Fig. 2 is that Roc analyzes the specificity that the hsa_circ_0021827 in serum excretion body source early diagnoses glioma, 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
The serum sample of 40 patients with gliomas is provided by Xiang Ya hospitals, and 15 normal serum samples carry out community for the same period The healthy individuals of 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 glioma/normal serum excretion body
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 l Trizol (MRC are added in precipitation Company) precipitation is resuspended, suspension is moved to new 1.5ml tube manages, and mends Trizol to 1ml.15min, cracking knot are cracked on ice 4 DEG C of 12000rpm centrifugation 10min after beam, supernatant move to new tube pipes.Chlorination imitates 200 μ l in Tube, shakes 15- with hand 30s, places 5min on ice, and 4 DEG C of 12000rpm centrifuge 15min；Carefully take upper strata aqueous phase to enter in new tube, add the isopropyl of precooling Alcohol 0.5ml is mixed, and stands be more than 20min on ice, and 4 DEG C of 12000rpm centrifuge 10min；Supernatant is abandoned, adds the dilution of 75%DEPC water Ethanol 1ml mix, 4 DEG C of 7500rpm centrifuge 5min, abandon supernatant as far as possible, drying at room temperature 5-10min, it is molten to add no 20 μ l of enzyme water Solve RNA.- 80 DEG C of preservations.By the daily time recording refrigerator temperature of laboratory technician.
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 RNA10 μ l, Random primer 1 μ l, no 1 μ l, 5 × Reaction Buffer of enzyme water 4 μ l, RI 1 μ l, RT 1 μ l and 10mMdNTP 2μl)。
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
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 Guangzhou Ji Sai bioengineering Co., Ltd：2 and 3) into Row 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
Specific primer (equal 10 μM of upstream and downstream) 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 hsa_circ_0021827 of glioma serum excretion body relative to normal The expression multiple of serum excretion body, wherein △ CT=CTSample–CTInternal reference, Δ Δ CT=Δs CTGlioma–ΔCTNormally.This experimental data is adopted With the analysis method of relative quantification, as reference gene, (primer sequence is shown in SEQ NO to GAPDH：4 and 5), data utilize software GraphPadPrism and SPSS 17.0 are analyzed.
3rd, result of study
Hsa_circ_0021827 is notable compared to normal serum excretion body control group in the serum excretion body of patients with gliomas Raise (p=0.025).Specific data are as shown in Figure 1.ROC curve analysis shows that hsa_circ_0021827 as biomarker Thing has glioma higher diagnostic value, and (AUC=0.823, p=0.003, susceptibility and specificity are respectively 70% He 90.9%), detailed results are shown in Fig. 2.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Diagnosis of glioma marker hsa_circ_0021827 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 178
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
ggggcuagag agcugaagga gagccaguuu ccccaaaauu gcugcaguga gaagaggagu 60
uuguuacuuu aaacagaggc ugaagaaacu auagaauuag cagagaaagu ggagaaggua 120
gaggauggag uugcagacuc uacaggaggc ucuuaaagug gaaauucagg uucaccag 178
<210> 2
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 2
tcaggttcac cagggggcta 20
<210> 3
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 3
ctatagtttc ttcagcctct g 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)

1. a kind of diagnosis of glioma marker hsa_circ_0021827, 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 hsa_circ_0021827 in serum excretion body can be measured Content.
4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection hsa_circ_0021827 The PCR primer of content.
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 hsa_circ_0021827 Primer outside, also contain and excretion body extracted from serum, by extracting RNA in excretion body and carrying out reverse transcription and quantitative fluorescent PCR All reagents.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114214411A (en) * 2021-12-27 2022-03-22 上海解颐生物科技有限公司 circRNA marker for glioma diagnosis and glioma diagnosis kit
CN114277031A (en) * 2022-01-25 2022-04-05 上海纽仁生物医药科技有限公司 hsa _ circ _0006420 circular RNA and application thereof in glioma diagnosis and prognosis evaluation
CN114921553A (en) * 2022-06-14 2022-08-19 中国医科大学附属第一医院 Human brain glioma marker hsa _ circ _0009362 and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102729A1 (en) * 2008-02-11 2009-08-20 Historx, Inc. Association of biomarkers with patient outcome
WO2012089753A2 (en) * 2010-12-30 2012-07-05 Febit Holding Gmbh Complex sets of mirnas as non-invasive biomarkers for glioblastoma
WO2013175429A1 (en) * 2012-05-23 2013-11-28 Universite D'aix Marseille Mmp2 as a predictive biomarker of response to antiangiogenic therapy and survival after therapy in cancer patients

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102729A1 (en) * 2008-02-11 2009-08-20 Historx, Inc. Association of biomarkers with patient outcome
WO2012089753A2 (en) * 2010-12-30 2012-07-05 Febit Holding Gmbh Complex sets of mirnas as non-invasive biomarkers for glioblastoma
WO2013175429A1 (en) * 2012-05-23 2013-11-28 Universite D'aix Marseille Mmp2 as a predictive biomarker of response to antiangiogenic therapy and survival after therapy in cancer patients

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU MF.等: "The Identification of Key Genes and Pathways in Glioma by Bioinformatics Analysis", 《HINDAWI JOURNAL OF IMMUNOLOGY RESEARCH》 *
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》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114214411A (en) * 2021-12-27 2022-03-22 上海解颐生物科技有限公司 circRNA marker for glioma diagnosis and glioma diagnosis kit
CN114214411B (en) * 2021-12-27 2022-12-13 上海解颐生物科技有限公司 circRNA marker for glioma diagnosis and glioma diagnosis kit
CN114277031A (en) * 2022-01-25 2022-04-05 上海纽仁生物医药科技有限公司 hsa _ circ _0006420 circular RNA and application thereof in glioma diagnosis and prognosis evaluation
CN114921553A (en) * 2022-06-14 2022-08-19 中国医科大学附属第一医院 Human brain glioma marker hsa _ circ _0009362 and application thereof

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