CN107557471B

CN107557471B - Glioma diagnosis marker Circ6:22020339|22020542 and application

Info

Publication number: CN107557471B
Application number: CN201711022778.2A
Authority: CN
Inventors: 李沛瑶; 武明花; 孟舒娟; 刘强; 陈琼; 余志斌; 张严
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: 2020-06-05
Anticipated expiration: 2037-10-27
Also published as: CN107557471A

Abstract

The invention discloses a glioma diagnosis marker Circ6:22020339|22020542 and application thereof, wherein the glioma diagnosis marker is found for the first time: exosome secreted by glioma cells has obvious enrichment effect on Circ6:22020339| 22020542; meanwhile, the expression level of Circ6:22020339|22020542 in serum exosome of glioma patients is obviously reduced compared with that of a control group (p is less than 0.0001), and ROC curve analysis shows that the expression level of the glioma has higher diagnostic value (AUC is 0.986, p is less than 0.0001, and the sensitivity and specificity are respectively 100% and 92.9%). Therefore, by detecting the expression level of the Circ6:22020339|22020542 in the serum exosome of the glioma patient, the glioma patient can be diagnosed quickly and early without wound.

Description

Glioma diagnosis marker Circ6:22020339|22020542 and application

Technical Field

The invention belongs to the technical field of biology, and relates to a serum circRNA marker Circ6:22020339|22020542 for glioma diagnosis, application of a reagent for detecting the marker in preparation of a glioma diagnosis preparation, and a kit.

Background

Brain glioma is the most common malignant neuroepithelial cell tumor in the cranium, accounts for 40.49% of intracranial tumors, and is one of the most common brain tumors in adults. From the time of diagnosis, the average life span of brain glioma patients does not exceed five years. At present, glioma treatment technologies are various, but due to the characteristics of high tumor infiltration and invasion speed, insensitivity to chemotherapy-radiotherapy mediated apoptosis and the like, the overall treatment effect is poor, the recurrence rate is high, and the prognosis is poor, so that the search for a new treatment target and a new prognosis index is very important. Glioma diagnosis is still in an empirical stage based on clinical, pathological and imaging information at present, and once diagnosis is carried out, most of glioma diagnosis is in middle and advanced stages, and the postoperative survival rate is not optimistic. Therefore, the research task of searching glioma diagnosis markers to screen high risk groups and correspondingly selecting reasonable subsequent treatment schemes to improve survival rate is urgent to solve in the field of neuroscience.

The circRNA is an endogenous closed circular non-coding RNA molecule which is different from linear RNA and widely and diversely exists in mammalian cells, has stable structure, is not easy to be influenced by RNA exonuclease, can regulate and control gene expression at multiple levels in the levels of transcription, translation and the like, widely exists in various cells, and has extremely high brain abundance. circRNA has recently become a new hotspot in the field of non-coding RNA. With the wide application of deep sequencing technology and the rapid development of biophysical and informatics technologies, it is found that transcripts of many exons in human can be nonlinearly reverse spliced or rearranged through gene to form circRNA, and the transcripts account for a considerable proportion of all spliced transcripts, have the characteristics of richness, stability, high conservation, space-time specificity and the like, and have the potential of serving as molecular markers of a plurality of diseases.

Disclosure of Invention

The first object of the present invention is: provides a serum exosome CircRNA marker for glioma diagnosis.

The main contents comprise: a serum exosome CircRNA marker Circ6:22020339|22020542 for glioma diagnosis has a sequence shown in SEQ NO 1.

The second purpose of the invention is to provide the application of the reagent for detecting the expression quantity of the CircRNA marker in a serum exosome in the preparation of a glioma diagnostic preparation.

It is a third object of the present invention to provide a glioma diagnostic kit capable of determining the content of Circ6:22020339|22020542 in serum exosomes.

The glioma diagnostic kit contains a PCR primer for detecting the content of Circ6:22020339| 22020542. Preferred primers have the sequences shown in SEQ NO 2 and 3.

The glioma diagnosis kit comprises all reagents for extracting exosome from serum, extracting RNA from exosome and performing reverse transcription and fluorescence quantitative PCR, except primers of Circ6:22020339| 22020542.

The method comprises the following steps:

(1) reagents required for extracting serum exosomes: total Exosome Isolation Reagent (fromservum), available from Invitrogen corporation under the trade designation 4478360;

(2) reagents required for the extraction of exosome RNAs: trizol reagent, trichloromethane, isopropanol, 75% ethanol and enzyme-free water;

(3) reagents required for reverse transcription: random Primer (Random Primer), enzyme-free water, 5 × reverse transcription buffer, base triphosphate deoxynucleotide, RNase inhibitor, MMLV reverse transcriptase;

(4) reagents required for fluorescent quantitative PCR: circumc 6:22020339|22020542 upstream and downstream primers, GAPDH internal reference upstream and downstream primers, SYBR dye, and enzyme-free water.

The invention has the beneficial effects that: the exosome secreted by the glioma cell is found to have obvious enrichment effect on the Circ6:22020339|22020542 for the first time, and meanwhile, the expression level of the Circ6:22020339|22020542 in the serum exosome of a glioma patient is found to be obviously reduced compared with that of a control group (p is less than 0.0001), and the ROC curve analysis shows that the exosome has higher diagnostic value on the glioma (AUC is 0.986, p is less than 0.0001, and the sensitivity and the specificity are respectively 100% and 92.9%). Therefore, the cyclic RNA has higher diagnosis and prognosis analysis values on glioma; by applying the cyclic RNA in glioma diagnosis and analysis, the glioma can be diagnosed more conveniently and accurately, a foundation is laid for a clinician to quickly and accurately master the state of an illness of a patient, the clinical treatment effect is improved, and help is provided for finding a novel micromolecular drug target with potential treatment value.

Drawings

FIG. 1 is a real-time fluorescent quantitative PCR analysis of the expression difference of Circ6:22020339|22020542 in glioma cells and exosomes secreted by the cells;

FIG. 2 is a real-time fluorescent quantitative PCR analysis of the difference of the expression of Circ6:22020339|22020542 in the exosomes of glioma serum and normal serum;

FIG. 3 shows the specificity and sensitivity of Roc analysis of serum exosome-derived Circ6:22020339|22020542 for early diagnosis of glioma.

Detailed Description

The following is intended to further illustrate the invention in connection with the embodiments, and not to limit the invention.

First, research object

1. The two glioma cell lines are U251 and U87 cell lines, and the six glioma primary cells are 1216, 1125, 1124B, 1124C, 0128C and 1104 respectively, which are provided by the institute of tumor of the basic medical college of the university of the Central and south China. Serum samples from 2.40 patients with glioma were provided by Xiangya Hospital, and 15 normal serum samples were healthy individuals who were concurrently undergoing community disease screening. Samples for research are collected at the same period, and sampling, subpackaging and storing conditions are consistent.

Second, research method

1. Extraction of RNA from cells, exosomes secreted by cells, glioma/normal serum exosomes

A. Extraction of cellular RNA

After the 8 cells were grown to the appropriate density, the medium was decanted, 1ml Trizol reagent was added to the dish and lysed on ice for 15 minutes. After the cleavage was completed, the Tube was transferred to a 1.5ml enzyme-free Tube, centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was transferred to a new Tube. Adding 200 μ l/ml of Trizol into Tube, shaking by hand for 15-30s, standing on ice for 5min, and centrifuging at 4 deg.C and 12000rpm for 15 min; carefully taking the upper water phase into a new tube, adding 0.5ml/ml of precooled isopropanol, uniformly mixing, standing in a refrigerator at the temperature of-20 ℃ for 20min, and centrifuging at the temperature of 4 ℃ at 12000rpm for 10 min; discarding the supernatant, adding 1-2ml ethanol diluted with 75% DEPC water, mixing, centrifuging at 7500rpm at 4 deg.C for 5min, discarding the supernatant, drying at room temperature for 5-10min, and adding 10 μ l enzyme-free water to dissolve RNA. Storing at-80 deg.C. Refrigerator temperatures were recorded daily by the experimenter.

B. Extraction of RNA from exosomes secreted by cells

After the above eight cells were cultured in a petri dish to a suitable density (the serum used in the medium had to be removed in advance of the exosomes), about 15ml of the supernatant medium was collected in an ultrafiltration tube (millipore corporation) and centrifuged at 4500g for 1h at 4 ℃. The ultrafiltrate after centrifugation was collected in a 1.5ml enzyme-free Tube, added with an ExoQuick-TC reagent (SBI) and shaken upside down, and allowed to stand at 4 ℃ overnight for precipitation. After overnight centrifugation at 1500g for 30min at room temperature, the supernatant was discarded, centrifugation at 1500g again for 5min and the supernatant was blotted dry. The precipitate is an exosome secreted by the cells in the supernatant of the culture medium. The pellet was resuspended with 1ml Trizol reagent, lysed on ice for 15 minutes and extracted as in A after lysis.

C. Extraction of RNA from glioma/Normal serum exosomes

Collecting 5ml of elbow venous blood by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, slightly shaking the anticoagulation tube after blood collection to uniformly mix the anticoagulation agent with the blood, standing for 24 hours at 4 ℃, and centrifuging for 5 minutes at 3000g normal temperature. The supernatant was removed by pipetting 200. mu.l with a micropipette and dispensed into a new 600. mu.l centrifuge tube and stored at-80 ℃ until use. Refrigerator temperatures were recorded regularly by the experimenter on a daily basis.

200. mu.l of the serum was centrifuged at 2000g for 30 minutes at room temperature, and the supernatant was transferred to a new 600. mu.l centrifuge tube by a micropipette, 40. mu.l of an Exosome-extracting Reagent (Total Exosome Isolation Reagent (fromserum), cat # 4478360, Invitrogen) was added thereto and shaken gently upside down, and the mixture was incubated at 4 ℃ for 45 minutes. And after incubation, centrifuging at room temperature for 10 minutes at 10000g, and removing supernatant to obtain precipitate, namely the exosome in the serum. The pellet was resuspended by adding 200. mu.l Trizol (MRC Co.), and the suspension was transferred to a new 1.5ml tube and supplemented with Trizol to 1 ml. And (4) cracking on ice for 15min, and extracting the same as the step A after cracking is finished.

Preparation of CDNA

Reverse transcription was performed according to the instructions of the reverse transcription kit (Thermo Co.). The total Reaction volume was 20. mu.l (total RNA 10. mu.l, Random primer 1. mu.l, enzyme-free water 1. mu.l, 5 × Reaction Buffer 4. mu.l, RI 1. mu.l, RT 1.00. mu.l and 10mM dNTP 2. mu.l).

Composition (I)	Dosage/tube
		Random reverse transcription primer (1. mu.M)	1μl
RNA samples	10μl
		Enzyme-free water	To 12μl

Reverse transcription first step conditions: 5 minutes at 65 DEG C

Reverse transcription second step procedure: 5 minutes at 25 ℃, 60 minutes at 42 ℃ and 5 minutes at 70 ℃.

3. Real-time fluorescent quantitative PCR

The real-time quantitative PCR is carried out by adopting specific primers (the primer sequences are shown in SEQ NO:2 and 3) synthesized by Hanhengzheng biotechnology (Shanghai) Limited company: firstly, the reverse transcription product is diluted by 10 times and mixed evenly. The 20 μ L reaction was as follows:

composition (I)	Dosage/tube
		SYBR Premix Ex Taq	10μl
Primer (10. mu.M)	0.5μl
		cDNA products	5μl
Enzyme-free water	To 20μl

Real-time fluorescent quantitative PCR reaction program: 95 ℃ for 3 minutes, 40 cycles, 95 ℃ for 10 seconds, 60 ℃ for 30 seconds.

(6) And (3) data analysis: by using 2^-ΔCTRepresents the expression fold of Circ6:22020339|22020542 relative to the internal reference, wherein △ CT ═ CT_Sample(s)–CT_{Internal reference}. The experimental data was analyzed by a relatively quantitative analysis method using GAPDH as an internal reference gene (primer sequences shown in SEQ NO: 4 and 5) and using software GraphPad Prism and SPSS 17.0.

Third, research results

1. The concentration of Circ6:22020339|22020542 in an exosome generated by the glioma cell is obviously increased compared with that in the cell, and the exosome has a good enrichment effect on the circular RNA. The specific results are shown in FIG. 1. 2. A control group of normal serum exosomes of Circ6:22020339|22020542 was significantly down-regulated in serum exosomes of glioma patients (p < 0.0001). The specific data are shown in fig. 2. ROC curve analysis showed that Circ6:22020339|22020542 as biomarker had higher diagnostic value for glioma (AUC ═ 0.986, p <0.0001, sensitivity and specificity of 100% and 92.9%, respectively), and the detailed results are shown in fig. 3.

Sequence listing

<110> Hunan ya Hospital of Zhongnan university

<120> glioma diagnosis marker Circ6:22020339|22020542 and application

<160>5

<170>SIPOSequenceListing 1.0

<210>1

<211>204

<212>RNA

<213> Intelligent (Homo sapiens)

<400>1

gaucugcauu uacugcucaa ccacaucuaa uuugaugucc ucugcagauu uaaaaugugu 60

gccuucuuuu ccgucaccaa gucaucccug gguuacuacu gaacauccuu cucaauuccc 120

cccgacccau ggauggcugu ucuccauugu cuguuucacc agauguccuc aaaacaaaca 180

gacagaagaa ggaaguggcu aaug 204

<210>2

<211>18

<212>DNA

<213> Unknown (Unknown)

<400>2

ggctgttctc cattgtct 18

<210>3

<211>18

<212>DNA

<213> Unknown (Unknown)

<400>3

taacccaggg atgacttg 18

<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

1. The application of the reagent for detecting the expression quantity of the Circ6:22020339|22020542 in a serum exosome in the preparation of a glioma diagnostic preparation, wherein the sequence of the Circ6:22020339|22020542 is shown as SEQ NO 1.

2. The use of claim 1, wherein the reagent for detecting the expression level of the Circ6:22020339|22020542 in the serum exosome comprises a PCR primer for detecting the content of the Circ6:22020339|22020542 in the exosome.

3. The use of claim 2, wherein the primer has the sequence shown in SEQ ID Nos. 2 and 3.

4. The use according to claim 2 or 3, characterized in that it contains, in addition to the primers of Circ6:22020339|22020542, all reagents for the extraction of exosomes from serum, the extraction of RNA from exosomes and the reverse transcription and fluorescent quantitative PCR.