CN107604071A - Glioma prognostic marker circ19:5604583 | 5604936 application - Google Patents

Abstract

The invention discloses a kind of glioma prognostic marker circ19:5604583 | 5604936 application, that is, detect the circRNA circ19 in brain tissue source:5604583 | 5604936 reagent is used for the prognosis preparation for preparing patients with gliomas.CircRNA circ19 in glioma are confirmed by studying:5604583 | the higher patient of 5604936 expression quantity, possess higher survival rates (P=0.013).By detecting circRNA circ19 in patients with gliomas samples of human glioma:5604583 | 5604936 expression, so as to make Index for diagnosis to patients with gliomas.

Description

Glioma prognostic marker circ19:5604583 | 5604936 application

Technical field

The invention belongs to biological technical field, is related to a kind of application of the circRNA marks for glioma prognosis.

Background technology

Glioma is also known as neurogliocytoma, including a variety of tumours for betiding neuroectodermal cells, country's report Account for 35.26%~60.96% (average 44.69%) of intracranial tumors.It is classified and still lacks unified opinion, on recent document often It is divided into two kinds of low classification and high-grade according to its grade malignancy.Low classification glioma accounts for the 10%~15% of intracranial tumors, including I~II grade the astrocytomas of one general pathologic classifications, pilocytic and fat cellular type astrocytoma, fibrous type and original Slurry type astrocytoma, slight human anaplastic astrocytoma, few branched oligodendrocyte knurl and ganglioglioma etc.；Life span is about 5 ~10 years, 5%~50% up to more than 10 years, and 30% case has the experience of pernicious upgrading.High-grade glioma is also known as pernicious glue Matter knurl, including III~IV grade of astrocytoma of general pathologic classifications, glioblastoma multiforme, pernicious anaplastic astrocytoma Cytoma or oligodendroglioma, medulloblastoma etc., life span are about 1 year, and more several months after the treatment recur, should in treatment Using composite treatment, including surgery excision, radiotherapy and chemotherapy etc., curative effect is difficult that satisfactory is a kind of common The disease of human health is endangered, is the problem of current neurosurgery and one of the important topic of medical research.Therefore, colloid is found Knurl prognostic marker carries out prognostic analysis to patient, and to improve the postoperative life quality of patients with gliomas, and correspondingly selection is closed The successive treatment scheme of reason, survival rate is improved, 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, 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, and have The features such as rich, stability, high conservative and Space-time speciality, there are the potentiality as many disease molecules marks.

The content of the invention

It is an object of the invention to provide detection CircRNA marks circ19:5604583 | 5604936 in brain tissue table Up to application of the reagent in glioma prognosis preparation is prepared of amount, CircRNA marker sequences such as SEQ NO:Shown in 1.

Circ19 in described detection brain tissue:5604583 | the reagent of 5604936 expression quantity is real-time fluorescence quantitative PCR Detection reagent.

Described real-time fluorescence quantitative PCR detection reagent includes carrying out the specific primer of real-time fluorescence quantitative PCR, sequence Such as SEQ NO:Shown in 2 and 3.

Described real-time fluorescence quantitative PCR detection reagent is kit,

Described kit, except circ19:5604583 | outside 5604936 primer, also contain and RNA is extracted from brain tissue And carry 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 template by circ19 using total serum IgE:5604583 | 5604936 reverse transcriptions are cDNA agents useful for same, including are reversed Record buffer solution, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases and circ19:5604583| Random primer used in 5604936；

(3) by cDNA real-time quantitative PCR agents useful for same, including circRNAcirc19:5604583 | 5604936 is glimmering in real time Fluorescent Quantitative PCR specific primer, GAPDH internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.

Applicant has found the circRNA in samples of human glioma source by quantitative fluorescent PCR and survivorship curve analysis circ19:5604583 | 5604936 is related to the survival rate of patient, and content is higher, and survival rate is higher.This method is pre- for glioma Post analysis provide strong technical support, are favorably improved the postoperative life quality of patients with gliomas, work out aftertreatment Scheme, survival rate is improved, there is far-reaching clinical meaning and generalization.

Brief description of the drawings

Fig. 1 is that real-time fluorescence quantitative PCR analyzes circ19:5604583 | 5604936 in normal cerebral tissue and glioma Differential expression；

The circ19 in Fig. 2 tracing analysis samples of human glioma sources for survival:5604583 | 5604936 expression height are to colloid The influence prognosis of knurl patient.

Embodiment

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

Embodiment 1 prepares detection circRNA circ19:5604583 | the reagent of 5604936 expression quantity is used to prepare colloid The kit (50 secondary response) of knurl patient's prognosis

1.RNA stablizing solutions 50ml

2. isopropanol 100ml

3. chloroform 100ml

4.Trizol 50ml

5. without enzyme water 10ml

6. 1 μM of random μ l of reverse transcriptase primer 50

7. 5 × RT Buffer 200ml

8. the μ l of 10mM triphosphoric acid bases deoxynucleotide 100

9. the μ l of 40U/ μ l RNase inhibitors 500

10. the μ l of 200U/ μ l MMLV reverse transcriptases 50

11.Premix Ex Taq 50μl

12. 10μM circRNA circ19:5604583 | the μ l of 5604936 real-time fluorescence quantitative PCR specific primer 30

CircRNA circ19:5604583 | 5604936 forward primers：5'-GTGCGAAGTGAAGAAGGAA-3',

CircRNA circ19:5604583 | 5604936 reverse primers：5'-TCTGTGGAGATGGCTGATG-3'；

13. 10 μM of μ l of GAPDH specific primers 30

Forward primer is 5 '-ATCATCAGCAATGCCTCCT-3 ',

Reverse primer is 5 '-CATCACGCCACAGTTTCC-3 '.

The tissue samples circRNA circ19 of embodiment 2:5604583 | 5604936 detection

1st, collect samples of human glioma to be measured to be put into the cryopreservation tube for filling RNA stablizing solutions, put standby to -80 DEG C of refrigerators.

2nd, RNA extracting 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 with move to tube manage, in Static cracking 15 minutes on ice.4 DEG C are cracked after terminating, and 12000rpm centrifugation 10min, supernatant moves to new tube pipes.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 Aqueous phase enters in new tube, and the isopropanol 0.5ml for adding precooling is mixed, and stands 20min on ice, 4 DEG C, 12000rpm centrifuges 10min； Supernatant is abandoned, the water-reducible ethanol 1-2ml of 75%DEPC is added and mixes, 4 DEG C of 7500rpm centrifuge 5min, abandon supernatant as far as possible, room temperature is done Dry 5-10min, add DEPC water 10-20 μ l dissolvings RNA.Spectrophotometric measures RNA concentration and quality, OD260/280 ratios Between 1.8-2.0, -80 DEG C of preservations.Refrigerator temperature is recorded by laboratory technician daily.

3、circRNA circ19:5604583 | 5604936 reverse transcriptions：Use the Reverse Transcriptase kit of Thermo companies. The system of 20 μ L reverse transcription reactions is as follows：

Composition	Dosage/pipe
		Random reverse transcriptase primer (1 μM)	1μl
RNA samples	2μg
		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.

4th, the circ19 of Han Heng biotechnologies (Shanghai) Co., Ltd. synthesis:5604583 | 5604936 specific primers enter Row real-time quantitative PCR：Reverse transcription product is first diluted 10 times, mixed.20 μ L reaction systems are as follows：

QRT-PCR specific primer：

Forward primer：5'-GTGCGAAGTGAAGAAGGAA-3',

Reverse primer：5'-TCTGTGGAGATGGCTGATG-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.

5th, the measure of 2- Δs Δ CT indexs：This experimental data uses 30 patients with gliomas and 12 Normal Human Brain Tissues, And the analysis method of relative quantification, GAPDH is as reference gene, the circRNA circ19 that qRT-PCR is measured: 5604583 | 5604936CT values obtain Δ CT with the CT values with tissue-derived GAPDH as difference, then Δ CT is compareed into work with Δ CT Difference obtains Δ Δ CT (taking normal sample Δ CT average value to be compareed for Δ CT), and data are carried out using software GraphPad Prism Welch check analyses.Analysis is found, with circRNA circ19 in samples of human glioma:5604583 | 5604936 and normal brain activity group The circRNA circ19 knitted:5604583 | 5604936 expression quantity have difference (see Fig. 1), the statistically significant (P=of difference 0.0257)。

6th, found by 30 patients with gliomas follow-up statistics used by experiment, 8 patients in follow-up by The either number of changing or other reasonses being shut down in mobile phone not contacting, the patients with gliomas that can finally get in touch with or family members are 22, This 22 patients or family members receive follow-up follow-up evaluation.We inquired in detail these patients or family members' First episode when Between, treatment, recurrence status and death time etc., follow up time is 1-42 months.In selected patients with gliomas, choosing The expression value for taking quantitative fluorescent PCR to analyze is normative reference, and that be higher than median after the arrangement of acquired results descending is circ19: 5604583 | 5604936 high expression, totally 15, receive follow-up as 11, other are circ19:5604583 | 5604936 low tables Reach, totally 15.Receive follow-up as 11, through Kaplan-Meier survival analysises, circ19:5604583 | 5604936 high expression The life cycle of patient is compared with circ19:5604583 | patient's length of 5604936 low expressions, good prognosis.Statistically significant (the P of difference =0.013).

Research shows above, CircRNA circ19:5604583 | 5604936 can be as the special of patients with gliomas prognosis Property molecular marker.

Sequence table

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

<120>Glioma prognostic marker circ19:5604583 | 5604936 application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 263

<212> RNA

<213>Homo sapiens (Homo sapiens)

<400> 1

guugucgggg ccaaaguggu aacgaacgcc cgcagcccgg gggcucgaug cuauggauuc 60

gucaccaugu cgacaucuga cgaggcgacc aagugcauca gccaucucca cagaacugag 120

cugcauggac gaaugaucuc cguagagaag gccaaaaaug agccugcugg gaaaaagcuu 180

uccgacagaa aagagugcga agugaagaag gaaaaauuau cgagugucga cagacaucau 240

ucuguggaga ucaaaauuga aaa 263

<210> 2

<211> 19

<212> DNA

<213>Unknown (Unknown)

<400> 2

gtgcgaagtg aagaaggaa 19

<210> 3

<211> 19

<212> DNA

<213>Unknown (Unknown)

<400> 3

tctgtggaga tggctgatg 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 (3)

1. detect CircRNA marks circ19:5604583 | 5604936 in brain tissue expression quantity reagent prepare colloid Application in knurl prognosis preparation, its sequence such as SEQ NO:Shown in 1.

2. application according to claim 1, it is characterised in that circ19 in described detection brain tissue:5604583| The reagent of 5604936 expression quantity is real-time fluorescence quantitative PCR detection reagent.

3. application according to claim 2, it is characterised in that described real-time fluorescence quantitative PCR detection reagent include into The specific primer of row real-time fluorescence quantitative PCR, sequence such as SEQ NO:Shown in 2 and 3.