CN107937534A - Diagnosis of glioma marker circ1:29154696 | 29154910 and application - Google Patents

Diagnosis of glioma marker circ1:29154696 | 29154910 and application Download PDF

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CN107937534A
CN107937534A CN201711453836.7A CN201711453836A CN107937534A CN 107937534 A CN107937534 A CN 107937534A CN 201711453836 A CN201711453836 A CN 201711453836A CN 107937534 A CN107937534 A CN 107937534A
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diagnosis
glioma
circ1
excretion body
serum
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CN107937534B (en
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武明花
汤颖
李沛瑶
赵春花
刘洋
刘庆
袁健
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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 circ1:29154696 | 29154910 and application.In the present invention, circ1 in patients with gliomas serum excretion body is found first:29154696 | 29154910 expression is significantly raised compared to control group (p=0.01), ROC analyses then show that it has higher diagnostic value (AUC=0.911 to glioma, P=0.002, sensitivity and specificity are respectively 75% and 98%).Therefore by detecting circ1 in patients with gliomas serum excretion body:29154696 | 29154910 expression, can make patients with gliomas early stage, quick Noninvasive diagnosis.

Description

Diagnosis of glioma marker circ1:29154696 | 29154910 and application
Technical field
The invention belongs to biological technical field, is related to a kind of serum circRNA markers for diagnosis of glioma circ1:29154696 | 29154910 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 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 (micro RNA, 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, With complete sequential structure and bioactivity, it is expected to, as liquid biopsy molecular marker, on accurate medical development have Bright 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 circ1 for diagnosis of glioma: 29154696 | 29154910, its sequence such as SEQ NO:Shown in 1.The circRNA is located on No. 1 chromosome of the mankind, total length 215bp。
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 Circ1:29154696 | 29154910 content.
The diagnosis of glioma kit, contains detection circ1:29154696 | the PCR primer of 29154910 contents. It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.
The diagnosis of glioma kit, except circ1:29154696 | outside 29154910 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：circ1:29154696 | in 29154910 upstream and downstream primers, GAPDH internal references Anti-sense primer, SYBR dyestuffs, without enzyme water.
The beneficial effects of the present invention are：
Circ1 in the serum excretion body of patients with gliomas is found first:29154696 | 29154910 compared to outside normal serum Body is secreted according to group significantly up-regulation (p=0.01).ROC curve is analysis shows that circ1:29154696 | 29154910 are used as biomarker Thing has glioma higher diagnostic value, and (AUC=0.911, sensitivity and specificity are respectively 75% and 98%).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 circ1 for real-time fluorescence quantitative PCR:29154696 | 29154910 patients with gliomas serum with it is normal Differential expression in serum excretion body；
Fig. 2 is the circ1 that ROC analyzes serum excretion body source:29154696 | what 29154910 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
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 is cracked on ice.Cracking knot 4 DEG C after beam, 12000rpm centrifugation 10min, supernatant moves to new tube pipes.Chlorination imitates 200 μ l in Tube, is shaken with hand 15-30s, places 5min, 4 DEG C, 12000rpm centrifuges 15min on ice；Carefully take upper strata aqueous phase to enter in new tube, add precooling Isopropanol 0.5ml is mixed, and stands be more than 20min on ice, 4 DEG C, 12000rpm centrifugations 10min；Supernatant is abandoned, adds 75%DEPC water Diluted ethanol 1ml is mixed, 4 DEG C, and 7500rpm centrifugation 5min, abandon supernatant, drying at room temperature 5-10min, adds no enzyme water 10 as far as possible μ l dissolve RNA.- 80 DEG C of preservations, refrigerator temperature is recorded by laboratory technician daily.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 10mM dNTP 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 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 circ1 of glioma serum excretion body:29154696 | 29154910 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. the serum excretion body of patients with gliomas is to circ1:29154696 | 29154910 have obvious enrichment, and compare Normal serum excretion body control group substantially raises (p=0.01), and specific data are as shown in Figure 1.
2.ROC tracing analysis shows circ1:29154696 | 29154910 as biomarkers to glioma have compared with (AUC=0.911, sensitivity and specificity are respectively 75% and 98%), and detailed results are shown in Fig. 2 for high diagnostic value.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Diagnosis of glioma marker circ1:29154696 | 29154910 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 215
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
gauuauaugc gucaggcagg agaagugacu uaugcagaug cucacaaggg acgcaaaaau 60
gaagggguga uugaauuugu aucuuauucu gauaugaaaa gagcuuugga aaaguuggau 120
ggaacugaag ucaaugggag aaaaaucaga uuaguugaag acaagccagg uuccagacga 180
cgccgguccu acuccagaag ccggagucau ucaag 215
<210> 2
<211> 22
<212> DNA
<213>Unknown (Unknown)
<400> 2
gattagttga agacaagcca gg 22
<210> 3
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 3
ttctcctgcc tgacgcata 19
<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 1. diagnosis of glioma marker circ1:29154696 | 29154910, its sequence such as SEQ NO:Shown in 1.
  2. 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. 3. a kind of diagnosis of glioma kit, it is characterised in that the circ1 in serum excretion body can be measured:29154696| 29154910 content.
  4. 4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection circ1:29154696| The PCR primer of 29154910 contents.
  5. 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. 6. according to the diagnosis of glioma kit described in claim 3 or 4 or 5, it is characterised in that except circ1:29154696| Outside 29154910 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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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
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》 *

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