CN107937537B - Glioma diagnosis marker circ7:42148226|42148468 and application - Google Patents
Glioma diagnosis marker circ7:42148226|42148468 and application Download PDFInfo
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- CN107937537B CN107937537B CN201711453884.6A CN201711453884A CN107937537B CN 107937537 B CN107937537 B CN 107937537B CN 201711453884 A CN201711453884 A CN 201711453884A CN 107937537 B CN107937537 B CN 107937537B
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Abstract
The invention belongs to the technical field of biology, and discloses a glioma diagnosis marker circ7:42148226|42148468 and application thereof. In the invention, the following are found for the first time: exosome secreted by glioma cells has obvious enrichment effect on circ7:42148226| 42148468; meanwhile, the expression level of circ7:42148226|42148468 in serum exosomes of glioma patients is obviously reduced compared with that of a control group (P is less than 0.0001), and ROC analysis shows that the expression level of the glioma has higher diagnostic value (AUC is 0.850, P is 0.001, and the sensitivity and the specificity are 75.0 percent and 96.0 percent respectively). Therefore, by detecting the expression level of circ7:42148226|42148468 in the serum exosome of the glioma patient, the early and rapid noninvasive diagnosis of the glioma patient can be made.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a serum circRNA marker circ7:42148226|42148468 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 tumor of the central nervous system, is one of the main diseases of the central nervous system, and is the first place of the incidence rate of intracranial primary tumors. Due to the characteristics of high invasion and high recurrence rate, the glioma has low cure rate and the life cycle of patients is short. The continuous development of neuroimaging technology and microsurgery technology has a great promotion effect on the development of neurosurgery, and the continuous and deep molecular biological research provides a wide prospect for the gene therapy of tumors, but the survival rate of patients with brain glioma is not obviously improved. 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 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 ring 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 circRNA, 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 circRNA and may play an important role. At present, as circRNA has the characteristics of richness, stability, high conservation, space-time specificity and the like, the circRNA plays an increasingly large role in the aspect of tumor diagnosis markers.
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 circ7:42148226|42148468 for glioma diagnosis, the sequence of which is shown in SEQ NO 1. The circRNA is located on chromosome 7 of human and has a total length of 243 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 circ7:42148226|42148468 in serum exosomes.
The glioma diagnostic kit contains a PCR primer for detecting the content of circ7:42148226| 42148468. 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 circ7:42148226|42148468 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: 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: circ7:42148226|42148468 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 first time, the circ7:42148226|42148468 in the exosome generated by the glioma cell is obviously increased compared with the intracellular concentration, and the extracellular exosome has good enrichment effect on the circular RNA. Serum exosomes of glioma patients had a significant down-regulation of circ7:42148226|42148468 (P <0.0001) compared to normal serum exosome controls. ROC curve analysis shows that circ7:42148226|42148468 has high diagnostic value (AUC is 0.850, and the sensitivity and specificity are 75.0% and 96.0% respectively) on glioma as a biomarker.
Drawings
FIG. 1 is a real-time fluorescent quantitative PCR analysis of the expression difference of circ7:42148226|42148468 in glioma cells and exosomes secreted by the cells;
FIG. 2 is a real-time fluorescent quantitative PCR analysis of the expression difference of circ7:42148226|42148468 in glioma serum and normal serum exosomes;
FIG. 3 shows the specificity and sensitivity of Roc analysis of serum exosome-derived circ7:42148226|42148468 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; the six glioma primary cells are 1104, 1216, 1125, 1124B, 1124C and 0128C, which are all provided by the institute of tumor of the basic medical college of the university of Zhongnan.
Serum samples from 2.40 patients with glioma were provided by Xiangya Hospital, and 19 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 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 chloroform 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 of precooled isopropanol, uniformly mixing, standing on ice for more than 20min, and centrifuging at 4 ℃ and 12000rpm for 10 min; discarding supernatant, adding 1ml ethanol diluted with 75% DEPC water, mixing, centrifuging at 4 deg.C and 7500rpm for 5min, discarding supernatant as much as possible, drying at room temperature for 5-10min, adding 10 μ l enzyme-free water to dissolve RNA, and storing at-80 deg.C.
B. Extraction of RNA from exosomes secreted by cells
After the above 8 cells were cultured in a petri dish to a suitable density (the serum used in the medium had to be removed beforehand for exosomes), about 15ml of the supernatant medium was collected in an ultrafiltration tube (millipore corporation) and centrifuged at 4500g at 4 ℃ for 1 h. 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
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. 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 9. mu.l, Random primer 1. mu.l, enzyme-free water 2. 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
| Composition (I) | Dosage/tube |
| 5 × reverse transcription buffer solution | 4μl |
| Base triphosphate deoxynucleotide (10mM) | 2μl |
| RNase inhibitor (40U/. mu.l) | 1μl |
| MMLV reverse transcriptase (200U/. mu.l) | 1μl |
| First step reverse transcription product | 12μl |
| Total volume | 20μl |
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 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-ΔΔCTCirc7:42148226|42148468, representing glioma serum exosomes, expressed fold relative to normal serum exosomes, where △ CT ═ CTSample(s)–CTInternal reference，ΔΔCT＝ΔCTGlioma–ΔCTIs 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. The concentration of circ7:42148226|42148468 in an exosome generated by the glioma cell is obviously increased compared with that in the cell, and the exosome of the cell has a good enrichment effect on the circular RNA. The specific results are shown in FIG. 1.
2. Serum exosomes of glioma patients had a significant down-regulation of circ7:42148226|42148468 (p <0.0001) compared to normal serum exosome controls. The specific data are shown in fig. 2. ROC curve analysis showed that circ7:42148226|42148468 as a biomarker had higher diagnostic value for glioma (AUC 0.850, sensitivity and specificity of 75.0% and 96.0%, respectively), and the detailed results are shown in fig. 3.
Sequence listing
<110> Hunan ya Hospital of Zhongnan university
<120> glioma diagnosis marker circ7:42148226|42148468 and application
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>243
<212>RNA
<213> Intelligent (Homo sapiens)
<400>1
gguagugggg cuccauguaa ccauuccugg gguccauggc aaacaccguc ccgcgguacg 60
gcacagaggg cuccgccacg uguggcaggg acccauggau cucuuucuug aucaaugagg 120
cccucucguc acucgauguu gaagguuccu cacugacuuu gcugagcccc uggacauucu 180
guggcugcau agugauugcg uuucuucucu cucugugaua agucugucca ggacuuucau 240
ccu 243
<210>2
<211>22
<212>DNA
<213> Unknown (Unknown)
<400>2
ctctgtgata agtctgtcca gg 22
<210>3
<211>18
<212>DNA
<213> Unknown (Unknown)
<400>3
gagccctctg tgccgtac 18
<210>4
<211>19
<212>DNA
<213> Unknown (Unknown)
<400>4
<210>5
<211>18
<212>DNA
<213> Unknown (Unknown)
<400>5
catcacgcca cagtttcc 18
Claims (4)
1. The application of the reagent for detecting the expression quantity of circ7:42148226|42148468 in a serum exosome in preparing a glioma diagnostic preparation, wherein the sequence of the circ7:42148226|42148468 is shown as SEQ NO:1, and the expression level of the circ7:42148226|42148468 in the serum exosome of a glioma patient is lower than that of a normal person.
2. The use of claim 1, wherein the reagent for detecting the expression level of circ7:42148226|42148468 in the serum exosome comprises PCR primers for detecting the content of circ7:42148226| 42148468.
3. The glioma diagnostic kit of claim 2 wherein the sequences of the primers are shown in SEQ NO 2 and 3.
4. The use according to claim 1, 2 or 3, wherein the reagent for detecting the expression level of circ7:42148226|42148468 in the serum exosome 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 for detecting circ7:42148226| 42148468.
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| CN201711453884.6A CN107937537B (en) | 2017-12-28 | 2017-12-28 | Glioma diagnosis marker circ7:42148226|42148468 and application |
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| CN107937537A CN107937537A (en) | 2018-04-20 |
| CN107937537B true CN107937537B (en) | 2020-09-11 |
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2017
- 2017-12-28 CN CN201711453884.6A patent/CN107937537B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Cell-type specific features of circular RNA expression;Julia Salzman et al.;《PLoS Genet》;20130905;第9卷(第9期);e1003777第1-15页 * |
| Hedgehog signalling in medulloblastoma，glioblastoma and neuroblastoma;Mehdi H.Shahi et al.;《Oncology Reports》;20080301;第19卷(第3期);第681页右栏第2段和678页左栏第6段 * |
| Julia Salzman et al..Cell-type specific features of circular RNA expression.《PLoS Genet》.2013,第9卷(第9期),e1003777第1-15页. * |
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