CN107557473A - Diagnosis of glioma mark circ6:79060478 | 79060818 and application - Google Patents

Abstract

The invention belongs to biological technical field, discloses a kind of diagnosis of glioma mark circ6:79060478 | 79060818 and application.By extracting RNA after separating Exosomes in patients with gliomas serum, reverse transcription simultaneously carries out fluorescent quantitative PCR technique to circ6 in patients with gliomas serum excretion body:79060478 | 79060818 content is analyzed, and finds circ6 in patients with gliomas serum excretion body:79060478 | 79060818 expressions substantially reduce (P=0.0005) compared to Normal group, ROC analyses then show that it has higher diagnostic value (AUC=0.946 to glioma, P=0.000, susceptibility and specificity are respectively 81.8% and 86.4%).

Description

Diagnosis of glioma mark circ6:79060478 | 79060818 and application

Technical field

The invention belongs to biological technical field, be related to a kind of serum circRNA marks for diagnosis of glioma and The reagent for detecting the mark is used to prepare the application of diagnosis of glioma preparation, also has kit.

Background technology

Most hypotype of glioma is high pernicious intracranial tumors, and since making a definite diagnosis, Patients with gliomas is averagely raw The life-span is deposited no more than 5 years.Meanwhile glioma is also most common intracranial tumors, accounts for 40% of intracranial tumors or so.At present Diagnosis of glioma is still within the experience sexual stage based on clinical, pathology and iconography information, the diagnosis of glioma and Treatment is generally not obvious progressive.For this malignant disease high with inhereditary material correlation of diagnosis and treatment glioma, Must be horizontal in molecular biology, explore its pathogenesis in terms of hereditary information expression.Presently glioma is examined Though disconnected and treatment method is in and updates the stage, patients with gliomas survival rate is not significantly improved.Diagnosis of glioma The experience sexual stage being still within based on clinical, pathology and iconography information, and one after diagnosing, the overwhelming majority is Middle and advanced stage, postoperative survival rate allow of no optimist.Therefore, find diagnosis of glioma mark and diagnostic analysis is carried out to patient, to the greatest extent The state of an illness early is made a definite diagnosis, and correspondingly selects rational successive treatment scheme, survival rate is improved, is that neuroscience field is urgently to be resolved hurrily Task.

CircRNA 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 circRNA is formed by gene rearrangement, and they account for sizable ratio in all montage transcripts.In recent years Gradually find also to contain substantial amounts of circRNA in excretion body, may play a significant role.At present, due to circRNA is rich, The features such as stability, high conservative and Space-time speciality, just played a greater and greater role in terms of diagnosing tumor mark.

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 circ6 for diagnosis of glioma: 79060478 | 79060818, 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 Circ6:79060478 | 79060818 content.

Described diagnosis of glioma kit, contain detection circ6:79060478 | the PCR primer of 79060818 contents. It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.

Described diagnosis of glioma kit, except circ6:79060478 | outside 79060818 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：circ6:79060478 | in 79060818 upstream and downstream primers, GAPDH internal references Anti-sense primer, SYBR dyestuffs, without enzyme water.

The beneficial effects of the present invention are：Circ6 is found first:79060478 | in 79060818 this serum excretion bodies Circular rna there is higher diagnostic value to glioma；By the development of serum circRNA marks and diagnostic kit and Using can make it that the diagnosis of glioma is more convenient and easy, contribute to clinician quick and precisely to grasp conditions of patients, to carry High clinical therapeutic efficacy lays the foundation, and to find that the new small molecule drug targets with potential therapeutic value provide help.

Brief description of the drawings

Fig. 1 is that real-time fluorescence quantitative PCR analyzes circ6:79060478 | 79060818 in normal human serum excretion body and glue Differential expression in matter knurl patients serum's excretion body；

Fig. 2 is the circ6 that Roc analyzes serum excretion body source:79060478 | 79060818 pairs of diagnosis of glioma it is special Property, sensitivity.

Embodiment

The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.

First, research object

Case group is 2015 to the 2017 27 glioma serum samples collected in Hunan Provincial Tumour Hospital.Control group The healthy individuals 11 of community's disorder in screening are carried out for the same period.Sample for research is collected for the same period, samples, dispenses, preserves Condition is consistent.

2nd, research method

(1) preparation of serum excretion body：Take the μ l of serum 200 to be centrifuged 30 minutes in 2000g normal temperature, extracted with micropipettor Supernatant liquor adds 40 μ l excretion bodies extracts reagents (Total Exosome Isolation to 600 new μ l centrifuge tubes Reagent (from serum), article No. 4478360, Invitrogen companies) gently turning upside down shakes up, and 4 DEG C are incubated 45 points Clock.Incubation terminates rear 10000g normal temperature and centrifuged 10 minutes, discards supernatant, 200 μ l Trizol are added in precipitation makes precipitation weight It is outstanding, suspension is moved into new 1.5ml tube and managed, mends Trizol to 1ml.

(2) in excretion body RNA extracting：By above-mentioned re-suspension liquid in static cracking 15 minutes on ice.4 DEG C are cracked after terminating, 12000rpm centrifuges 10min, and supernatant moves to new tube pipes.Chlorination imitates 200 μ l in Tube, shakes 15-30s, ice with hand Upper placement 5min, 4 DEG C, 12000rpm centrifugations 15min；Carefully take upper strata aqueous phase to enter in new tube, add the isopropanol of precooling 0.5ml is mixed, and stands 20min on ice, 4 DEG C, 12000rpm centrifuges 10min；Supernatant is abandoned, adds the water-reducible ethanol of 75%DEPC 1-2ml is mixed, 4 DEG C, 7500rpm centrifugation 5min, is abandoned supernatant as far as possible, drying at room temperature 5-10min, is added the no μ l of enzyme water 10 dissolvings RNA。

(3) prepared by cDNA：Reverse transcription reaction is carried out according to miRNA Reverse Transcriptase kits (THERMO companies) specification.Instead Answer cumulative volume for 20 μ l (10 μ l, Random primer of total serum IgE 1 μ l, no μ l of 1 μ l, 5 × Reaction Buffer of enzyme water 4, μ l of 11 μ l and 10mM dNTP of μ l, RT of RI 2).

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

Reverse transcription second step program：25 DEG C 5 minutes, 42 DEG C 60 minutes, 70 DEG C 5 minutes.

(4) real-time fluorescence quantitative PCR：The circ6 of Han Heng bio tech ltd synthesis:79060478|79060818 Specific primer is (see sequence table SEQ NO：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：

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) data analysis：With 2^‐ΔΔCTRepresent the multiple for control group change, wherein △ CT=CT_Sample–CT_{Internal reference}, △ △ CT=△ CT-△ CT_Control(average value for taking normal sample △ CT is_Control).This experimental data uses the analysis method of relative quantification, GAPDH is as reference gene (see sequence table SEQ NO：4 and 5), data are successively carried out using software GraphPad Prism Welch is examined, and ROC curve analysis is carried out using software SPSS.

3rd, result of study

Case group serum excretion body circ6:79060478 | 79060818 expressions significantly lower (P=compared with control group 0.0005).Specific data are as shown in Figure 1.

ROC curve analysis display, circ6:79060478 | 79060818 are used as biomarker to glioma with higher (AUC=0.946, P=0.000, susceptibility and specificity are respectively 81.8% and 86.4%) to diagnostic value.Detailed results are shown in figure 2。

Sequence table

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

<120>Diagnosis of glioma mark circ6:79060478 | 79060818 and application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 250

<212> RNA

<213>Homo sapiens (Homo sapiens)

<400> 1

gugaaguauu acagacacuu agcaccugau cacuugcugc aaauauguca ucgacuagga 60

ccucuucuug aacaagaaau uccucaaagu guuccuggag uacaaacuuu auuaggagcu 120

ggaagacagu cuuuacuacg cacaaauaaa agcugcaagc auguugugug gaaaggaucu 180

gcucuggcug cguugcacug uggaagacca ccugagucac caguuaacua ugguagccca 240

cccagcauug 250

<210> 2

<211> 19

<212> DNA

<213>Unknown (Unknown)

<400> 2

aactatggta gcccaccca 19

<210> 3

<211> 20

<212> DNA

<213>Unknown (Unknown)

<400> 3

actccaggaa cactttgagg 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 1. serum excretion body circRNA marks circ6 for diagnosis of glioma:79060478 | 79060818, its sequence Row such as SEQ NO:Shown in 1.
2. 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. 3. a kind of diagnosis of glioma kit, it is characterised in that circ6 in serum excretion body can be determined:79060478| 79060818 content.
4. 4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection circ6:79060478| The PCR primer of 79060818 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 circ6:79060478| Outside 79060818 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.