CN107586843A

CN107586843A - Diagnosis of glioma mark circ7:100812747 | 100813208 and application

Info

Publication number: CN107586843A
Application number: CN201711022756.6A
Authority: CN
Inventors: 武明花; 冯紫阳; 李沛瑶; 佘晓玲; 孙英男; 刘长红; 刘涛
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2017-10-27
Filing date: 2017-10-27
Publication date: 2018-01-16

Abstract

The invention discloses diagnosis of glioma mark circ7:100812747 | 100813208 and application.In the present invention, find first：Excretion body secreted by glioma cell is to circ7:100812747 | 100813208 have obvious enrichment；Find circ7 in patients with gliomas serum excretion body simultaneously:100812747 | 100813208 expression is significantly raised compared to control group (p=0.0444), ROC curve analysis shows that it has higher diagnostic value (AUC=0.924 to glioma, p=0.001, susceptibility and specificity are respectively 83.3% and 87.5%).Therefore by detecting circ7 in patients with gliomas serum excretion body:100812747 | 100813208 expression, early stage, quick Noninvasive diagnosis can be made to patients with gliomas.

Description

Diagnosis of glioma mark circ7:100812747 | 100813208 and application

Technical field

The invention belongs to biological technical field, is related to a kind of serum circRNA marks for diagnosis of glioma circ7:100812747 | 100813208 and detect the mark reagent be used for prepare diagnosis of glioma preparation application, Also 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 life-span 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 updates 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 mark 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, there is covalence closed loop configuration, be widely present in various cells, Ye Shiji The current research focus 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 RNA and protein small film bubble (30-150nm), 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 shows 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, in addition to some RNA compositions, as Microrna (microRNA, miRNA), long-chain non-coding RNA and mRNA, Circular rna (circ μ lar RNA, circRNA).These RNA entrained by excretion body are referred to as excretion body source RNA, have had Whole sequential structure and bioactivity, it is 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 marks for diagnosis of glioma are provided.

Main contents include：A kind of serum excretion body circRNA marks circ7 for diagnosis of glioma: 100812747 | 100813208, its sequence such as SEQ NO:Shown in 1.

Second object of the present invention is to provide the described circRNA marks of detection 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, and it can be determined in serum excretion body Circ7:100812747 | 100813208 content.

Described diagnosis of glioma kit, contain detection circ7:100812747 | the PCR of 100813208 contents draws Thing.It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.

Described diagnosis of glioma kit, except circ7:100812747 | outside 100813208 primer, also contain from blood Excretion body is extracted in clear, 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：circ7:100812747 | 100813208 upstream and downstream primers, GAPDH internal references Upstream and downstream primer, SYBR dyestuffs, without enzyme water.

The beneficial effects of the present invention are：Find first：Circ7 in excretion body caused by glioma cell: 100812747 | 100813208 is significantly raised compared to IC, and cell excretion body has good richness to the circular rna Collect effect.Circ7 in the serum excretion body of patients with gliomas:100812747 | 100813208 compare than normal serum excretion body Group significantly up-regulation (p=0.044).ROC curve analysis shows circ7:100812747 | 100813208 are used as biomarker pair Glioma has higher diagnostic value, and (AUC=0.924, p=0.001, susceptibility and specificity are respectively 83.3% He 87.5%).By application of the circular rna in diagnosis of glioma analysis, it can make it that the diagnosis of glioma is more convenient accurate Really, conditions of patients is quick and precisely grasped for clinician, is laid the foundation to improve clinical therapeutic efficacy, and it is potential to find to have The new small molecule drug targets of therapeutic value provide help.

Brief description of the drawings

Fig. 1 is that real-time fluorescence quantitative PCR analyzes circ7:100812747 | 100813208 in glioma cell and cell institute Differential expression in the excretion body of secretion；

Fig. 2 is that real-time fluorescence quantitative PCR analyzes circ7:100812747 | 100813208 in glioma serum and normal blood Differential expression in clear excretion body；

Fig. 3 is the circ7 that Roc analyzes serum excretion body source:100812747 | 100813208 pairs of glioma early diagnosis 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. one plant of glioma cell line is U251 cell lines, five plants of glioma primary cells be respectively 1216,1124C, 1124B, 1104,0128C, provided by institute of oncology of preclinical medicine institute of Central South University.

2. the serum sample of 40 patients with gliomas is provided by Xiang Ya hospitals, 15 normal serum samples are carried out for the same period The healthy individuals of community's disorder in screening.Sample for research is collected for the same period, sample, dispense, preservation condition it is consistent.

2nd, research method

1. RNA extracting in excretion body, glioma/normal serum excretion body secreted by cell, cell

A. the extracting of cell RNA

Treat that above-mentioned six kinds of cell length to suitable density, outwells Pei Ji, 1ml Trizol reagents added in culture dish, on ice Cracking 15 minutes.Cracking moves to 1.5ml and managed without enzyme Tube after terminating, 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, drying at room temperature 5-10min, adds the no μ l of enzyme water 20 dissolvings RNA.- 80 DEG C of preservations.Regularly remembered daily by laboratory technician Record refrigerator temperature.

B. in the excretion body secreted by cell RNA extracting

Treat that above-mentioned six 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.It is super after centrifugation Filtrate is collected in 1.5ml and 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 normal 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 is resuspended with 1ml Trizol reagents, cracks 15 on ice Minute, the same A of extraction step after cracking terminates.

C. in glioma/normal serum excretion body RNA extracting

Take the μ l of serum 200 to be centrifuged 30 minutes in 2000g normal temperature, supernatant liquor is extracted to 600 new μ l with micropipettor Centrifuge tube, add 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.Incubation terminates rear 10000g normal temperature 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 into 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 20 μ l (total RNA10 μ l, Random primer1 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)。

Composition	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

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.

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, mixed.20 μ L reaction systems are as follows：

Composition	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.(6) data Analysis：Using 2^-ΔΔCTRepresent the circ7 of glioma serum excretion body:100812747 | 100813208 relative to normal serum outside Secrete the expression multiple of body, wherein △ CT=CT_Sample–CT_{Internal reference}, Δ Δ CT=Δs CT_Glioma–ΔCT_Normally.This experimental data is using relative Quantitative analysis method, as reference gene, (primer sequence is shown in SEQ NO to GAPDH：4 and 5), data utilize software GraphPad Prism and SPSS 17.0 are analyzed.

3rd, result of study

1. circ7 in excretion body caused by glioma cell:100812747 | 100813208 compared to IC Significantly raised, cell excretion body has 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:100812747 | 100813208 compared to normal serum excretion Body control group significantly raises (p=0.0444).Specific data are as shown in Figure 2.ROC curve analysis shows circ7:100812747| 100813208 as biomarkers to glioma have higher diagnostic value (AUC=0.924, p=0.001, susceptibility and Specificity is respectively 83.3% and 87.5%), and detailed results are shown in Fig. 3.

Sequence table

<110>Xiangya Hospital, Central-South China Univ.

<120>Diagnosis of glioma mark circ7:100812747 | 100813208 and application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 362

<212> RNA

<213>Homo sapiens (Homo sapiens)

<400> 1

guacuaaggu cuacaucgac cccuucacuu augaagaccc uaaugaggcu gugagggaau 60

uugcaaaaga gaucgauguc uccuacguca agauugaaga ggugauuggu gcaggugagu 120

uuggcgaggu gugccggggg cggcucaagg ccccagggaa gaaggagagc uguguggcaa 180

ucaagacccu gaaggguggc uacacggagc ggcagcggcg ugaguuucug agcgaggccu 240

ccaucauggg ccaguucgag caccccaaua ucauccgccu ggagggcgug gucaccaaca 300

gcaugcccgu caugauucuc acagaguuca uggagaacgg cgcccuggac uccuuccugc 360

gg 362

<210> 2

<211> 18

<212> DNA

<213>Unknown (Unknown)

<400> 2

ggcaatcaag accctgaa 18

<210> 3

<211> 20

<212> DNA

<213>Unknown (Unknown)

<400> 3

gcaccaatca cctcttcaat 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

A kind of 1. serum excretion body circRNA marks Circ7 for diagnosis of glioma:100812747 | 100813208, its Sequence such as SEQ NO:Shown in 1.
2. the reagent of circRNA marks expression quantity in serum excretion body described in test right requirement 1 is preparing glioma Application in diagnostic preparation.
3. a kind of diagnosis of glioma kit, it is characterised in that the circ7 in serum excretion body can be determined:100812747| 100813208 content.
4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection circ7:100812747| The PCR primer of 100813208 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 circ7:100812747| Outside 100813208 primer, also contain and excretion body is extracted from serum, by extracting RNA in excretion body and carrying out reverse transcription and glimmering All reagents of Fluorescent Quantitative PCR.