CN107937534B

CN107937534B - Glioma diagnosis marker circ1:29154696|29154910 and application

Info

Publication number: CN107937534B
Application number: CN201711453836.7A
Authority: CN
Inventors: 武明花; 汤颖; 李沛瑶; 赵春花; 刘洋; 刘庆; 袁健
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2020-06-30
Anticipated expiration: 2037-12-28
Also published as: CN107937534A

Abstract

The invention belongs to the technical field of biology, and discloses a glioma diagnosis marker circ1:29154696|29154910 and application thereof. In the present invention, the expression level of circ1:29154696|29154910 in serum exosomes of glioma patients was found to be significantly higher (P ═ 0.01) compared with that of the control group for the first time, and ROC analysis showed that it has higher diagnostic value for glioma (AUC ═ 0.911, P ═ 0.002, sensitivity and specificity of 75% and 98%, respectively). Therefore, by detecting the expression level of circ1:29154696|29154910 in the serum exosome of the glioma patient, the early and rapid noninvasive diagnosis of the glioma patient can be made.

Description

Glioma diagnosis marker circ1:29154696|29154910 and application

Technical Field

The invention belongs to the technical field of biology, and relates to a serum circRNA marker circ1:29154696|29154910 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 frequent brain tumor disease of adults, and accounts for 40.49 percent of intracranial tumors. From the time of diagnosis, the average life span of brain glioma patients does not exceed five years. In order to diagnose glioma, a malignant disease with a very high genetic material correlation, the pathogenesis of glioma must be explored at the molecular biological level from the aspect of genetic information expression. At present, the diagnosis and treatment methods of glioma are in the continuous improvement stage, but the survival rate of glioma patients is not obviously improved. Glioma diagnosis is still in an empirical stage based on clinical, pathological and imaging information, and once diagnosed, most of them are in middle and advanced stages, and the survival rate after surgery 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 circular RNA is a kind of endogenous non-coding RNA molecules which are widely and diversely present in mammalian cells and have the function of regulating gene expression, has a covalently closed circular structure, is widely present in various cells, and is also a latest research hotspot of an RNA family following microRNA (miRNA). In recent years, with the widespread use of deep sequencing technologies and the rapid development of biophysical and informatics technologies, it has been found that human transcripts of many exons can be non-linearly reverse spliced or rearranged through genes to form circular RNAs, and that they account for a significant proportion of all spliced transcripts. In recent years, it has been found that exosomes also contain a large amount of circular RNA and may play an important role. Exosomes refer to small membrane vesicles (30-150nm) containing complex RNAs and proteins, which today refer specifically to discoidal vesicles with diameters between 40-100 nm. In 1983, exosomes were first found in sheep reticulocytes, which were named "exosomes" by Johnstone in 1987. Recent studies have shown that exosomes are important molecules for cell-to-cell communication, and participate in many physiological and pathological processes. The exosome contains not only protein components but also some RNA components, such as micro RNA (miRNA), long non-coding RNA and mRNA, and circular RNA (circular RNA). The RNAs carried by the exosomes are collectively called exosome source RNAs, have complete sequence structures and biological activities, are expected to be used as liquid biopsy molecular markers, and have bright prospects in the development of precise medicine.

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 circ1:29154696|29154910 for glioma diagnosis, the sequence of which is shown in SEQ NO 1. The circRNA is located on chromosome 1 of human and has a total length of 215 bp.

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 circ1:29154696|29154910 in serum exosomes.

The glioma diagnostic kit contains a PCR primer for detecting the content of circ1:29154696| 29154910. 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 the exosome and performing reverse transcription and fluorescence quantitative PCR, except a circ1:29154696|29154910 primer.

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, namely Random Primer (Random Primer), enzyme-free water, 5 × reverse transcription buffer solution, base triphosphate deoxynucleotide, RNase inhibitor and MMLV reverse transcriptase;

(4) reagents required for fluorescent quantitative PCR: circ1:29154696|29154910 upstream and downstream primers, GAPDH internal reference upstream and downstream primers, SYBR dye, and enzyme-free water.

The invention has the beneficial effects that:

it was first found that circ1:29154696|29154910 was significantly upregulated in serum exosomes of glioma patients compared to normal serum exosome controls (p ═ 0.01). ROC curve analysis shows that circ1:29154696|29154910 has higher diagnostic value for glioma as a biomarker (AUC ═ 0.911, sensitivity and specificity of 75% and 98%, respectively). 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 circ1:29154696|29154910 in the serum of glioma patients and the exosomes of normal serum;

FIG. 2 shows the specificity and sensitivity of circ1:29154696|29154910 derived from serum exosomes 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

Serum samples from 40 patients with glioma were provided by Xiangya Hospital, and 15 normal serum samples were healthy individuals who were contemporaneously community disease screened. 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 glioma/Normal serum exosomes

200. mu.l of serum was centrifuged at 2000g for 30 minutes at room temperature, the supernatant was removed by a micropipette to a new 600. mu.l centrifuge tube, 40. mu.l of an Exosome-extracting Reagent (Total Exosome Isolation Reagent (from serum), cat. No. 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. Cleavage on ice for 15 min. After cleavage was complete, centrifugation was carried out at 12000rpm for 10min at 4 ℃ and the supernatant was transferred to a new tube. Adding 200 μ l chloroform into Tube, shaking by hand for 15-30s, standing on ice for 5min, centrifuging at 4 deg.C and 12000rpm for 15 min; carefully taking the upper water phase into a new tube, adding 0.5ml of precooled isopropanol, uniformly mixing, standing on ice for more than 20min, and centrifuging at 12000rpm for 10min at 4 ℃; discarding the supernatant, adding 1ml ethanol diluted with 75% DEPC water, mixing, centrifuging at 4 deg.C and 7500rpm for 5min, discarding the supernatant, drying at room temperature for 5-10min, and adding 10 μ l enzyme-free water to dissolve RNA. Stored at-80 ℃ and refrigerator temperatures were recorded daily by the laboratory. Preparation of cDNA

The Reaction was carried out in a total volume of 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. 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. mu.l reaction was as follows:

composition (I)	Dosage/tube
		SYBR Premix Ex Taq	10μl
Primer (10. mu.M)	0.5μl
		cDNA product (after dilution)	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.

4. And (3) data analysis: by using 2^-ΔΔCTCirc1:29154696|29154910, representing glioma serum exosomes, expressed fold relative to normal serum exosomes, where △ CT ═ CT_Sample(s)–CT_{Internal reference}，ΔΔCT＝ΔCT_Glioma–ΔCT_{Is normal}. 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. Serum exosomes of glioma patients had significant enrichment for circ1:29154696|29154910 and were significantly upregulated (p ═ 0.01) compared to normal serum exosome controls, with specific data as shown in fig. 1.

ROC curve analysis showed that circ1:29154696|29154910 as biomarker had higher diagnostic value for glioma (AUC 0.911, 75% and 98% sensitivity and specificity, respectively), and the detailed results are shown in fig. 2.

Sequence listing

<110> Hunan ya Hospital of Zhongnan university

<120> glioma diagnosis marker circ1:29154696|29154910 and application

<160>5

<170>SIPOSequenceListing 1.0

<210>1

<211>215

<212>RNA

<213> Intelligent (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

1. The application of the reagent for detecting the expression quantity of circ1:29154696|29154910 in a serum exosome in the preparation of a glioma diagnostic preparation, wherein the sequence of the circ1:29154696|29154910 is shown as SEQ NO: 1.

2. The use of claim 1, wherein the reagent for detecting the expression level of circ1:29154696|29154910 in the serum exosome comprises PCR primers for detecting the content of circ1:29154696| 29154910.

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

4. The use of claim 2 or 3, wherein the reagent for detecting the expression level of circ1:29154696|29154910 in the exosome in serum comprises all reagents for extracting exosome from serum, extracting RNA from exosome and performing reverse transcription and fluorescence quantitative PCR, in addition to the primers of circ1:29154696| 29154910.