CN107937527B - Glioma diagnosis marker circ1:43920404|43920928 and application - Google Patents
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- CN107937527B CN107937527B CN201711453662.4A CN201711453662A CN107937527B CN 107937527 B CN107937527 B CN 107937527B CN 201711453662 A CN201711453662 A CN 201711453662A CN 107937527 B CN107937527 B CN 107937527B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
The invention belongs to the technical field of biology, and discloses a glioma diagnosis marker circ1:43920404|43920928 and application thereof. The content of circ1:43920404|43920928 in the serum exosomes of the glioma patients is analyzed by a fluorescent quantitative PCR technology, and the expression level of circ1:43920404|43920928 in the serum exosomes of the glioma patients is obviously reduced compared with that of a normal control group (p is less than 0.0001), and ROC curve analysis shows that the expression level of circ1:43920404|43920928 in the serum exosomes of the glioma patients has higher diagnostic value on glioma, wherein AUC is 0.877, p is 0.003, and the sensitivity and the specificity are 85.7% and 100% respectively.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a serum exosome circRNA marker for glioma diagnosis, application of a reagent for detecting the marker in preparation of a glioma diagnosis preparation, and a kit.
Background
Glioma is a malignant tumor with the highest intracranial incidence rate, accounts for 40.49% of intracranial tumors, and is clinically treated by adopting a method combining operation and chemotherapy, but due to infiltration, low sensitivity to chemotherapeutic drugs and the like, postoperative recurrence of patients is often caused, and the life health of human beings is seriously threatened. And once diagnosed, most of the glioma is in middle and advanced stage, and the postoperative survival rate is not high. In addition, the prognosis is poor and the average life span does not exceed five years in patients with WHO3-4 grade, i.e., high grade, brain glioma. Therefore, finding glioma diagnosis markers to diagnose and analyze patients as soon as possible, selecting reasonable subsequent treatment schemes correspondingly, and improving survival rate is a research task to be solved urgently 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 circ1:43920404|43920928 for glioma diagnosis, the sequence of which is 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 circ1:43920404|43920928 in serum exosomes.
The glioma diagnostic kit contains a PCR primer for detecting the content of circ1:43920404| 43920928. 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 circ1:43920404|43920928 primer. The method comprises the following steps: (1) reagents required for extracting serum exosomes: total Exosome Isolation Reagent (from serum), available from Invitrogen corporation under the trademark 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: circ1:43920404|43920928 upstream and downstream primers, GAPDH internal reference upstream and downstream primers, SYBR dye, and enzyme-free water.
The invention has the beneficial effects that: circular RNA in a serum exosome of circ1:43920404|43920928 is found for the first time, and the circular RNA has high diagnostic value on glioma; through the research and application of the serum circRNA marker and the diagnosis kit, the diagnosis of glioma is more convenient and feasible, the clinical doctor can quickly and accurately master the illness state of the patient, the foundation is laid for improving the clinical treatment effect, and the help is provided for finding a novel micromolecule drug target with potential treatment value.
Drawings
FIG. 1 is a real-time fluorescent quantitative PCR analysis of the expression difference of circ1:43920404|43920928 in normal human serum exosomes and glioma patient serum exosomes;
FIG. 2 is a diagram showing the specificity and sensitivity of ROC curve analysis of serum exosome-derived circ1:43920404|43920928 in glioma diagnosis.
Detailed Description
The following examples are intended to further illustrate the invention without limiting it.
First, research object
Case groups were 30 glioma serum samples collected at tumor hospital, Hunan province, 2016 to 1-2017, 6 months. The control group was 12 healthy individuals contemporaneously screened for community disease, with case frequency matched by gender and age (+ -5 years). Samples for research are collected at the same period, and sampling, subpackaging and storing conditions are consistent.
Second, research method
(1) Preparation of serum exosomes: 200. mu.l of serum was centrifuged at 2000g for 30 minutes at room temperature, 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 IsolationReagent (from serum), cat. No. 4478360, Invitrogen) was added thereto and shaken gently upside down, and the mixture was incubated at 4 ℃ for 45 minutes. After completion of incubation, centrifugation was carried out at 10000g for 10 minutes at normal temperature, the supernatant was discarded, 200. mu.l of Trizol (MRC) was added to the pellet to resuspend the pellet, the suspension was transferred to a new 1.5ml tube, and Trizol was added to 1 ml.
(2) Extraction of RNA in exosomes: the resuspension was allowed to quiescently lyse on ice for 15 minutes. 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, 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 the supernatant, adding 1ml 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, adding 10 μ l enzyme-free water to dissolve RNA, and storing at-80 deg.C.
(3) Preparation of cDNA: reverse transcription was performed according to the instructions of the reverse transcription kit (Thermo Co.). The Reaction was performed 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
| Composition (I) | Dosage/tube |
| 5 Xreverse transcription buffer | 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 ℃.
(4) Real-time fluorescent quantitative PCR: real-time quantitative PCR was performed using a circ1:43920404|43920928 specific primer (see SEQ ID NOs: 2 and 3 of the sequence Listing) synthesized by Hanheng Biotech (Shanghai) Ltd: firstly, the reverse transcription product is diluted by 10 times and mixed evenly. The 20. mu.l reaction was as follows:
real-time fluorescent quantitative PCR reaction program: 95 ℃ for 3 minutes, 40 cycles, 95 ℃ for 10 seconds, 60 ℃ for 30 seconds.
(5) And (3) data analysis: by using 2-ΔΔCTCirc1:43920404|43920928, representing glioma serum exosomes, expressed fold relative to normal serum exosomes, where △ CT ═ CTSample(s)–CTInternal reference，ΔΔCT＝ΔCT–ΔCTIs normal. The experimental data were analyzed by a relatively quantitative method using GAPDH as the reference gene (primer sequences shown in SEQ NO: 4 and 5). DELTA.CTIs normalData were analyzed using software GraphPad Prism and SPSS 17.0 as the mean Δ CT for normal serum exosomes. Third, research results
The expression level of the serum exosome circ1:43920404|43920928 in case group was significantly down-regulated compared to the control group (p < 0.0001). The specific data are shown in fig. 1.
ROC curve analysis showed that circ1:43920404|43920928 as biomarker had higher diagnostic value for glioma (AUC ═ 0.877, p ═ 0.003, sensitivity and specificity of 85.7% and 100%, respectively). The detailed results are shown in FIG. 2.
Sequence listing
<110> Hunan ya Hospital of Zhongnan university
<120> glioma diagnosis marker circ1:43920404|43920928 and application
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>294
<212>RNA
<213> Intelligent (Homo sapiens)
<400>1
agugcaucgg auggcuucug gaaaucugug gccacucgag ugcccaagga gcccccugag 60
auucgaaucc ucaacccaua uuucauccag gaggccgccu ucacccucau uggccugccc 120
uucaacaaug gccucauggg ccgggggaac aucccuaccc uuggcagugu ggcagugacc 180
auggcacuac acggcuguga cgagguggca gucgcaggau uuggcuauga caugagcaca 240
cccaacgcac cccugcacua cuaugagacc guucgcaugg cagccaucaa agag 294
<210>2
<211>19
<212>DNA
<213> Unknown (Unknown)
<400>2
ctacacggct gtgacgagg 19
<210>3
<211>21
<212>DNA
<213> Unknown (Unknown)
<400>3
ggatgaaata tgggttgagg a 21
<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 (4)
1. The application of the reagent for detecting the expression quantity of circ1:43920404|43920928 in a serum exosome in preparing a glioma diagnostic preparation is that the sequence of the circ1:43920404|43920928 is shown as SEQ NO:1, and the expression level of c i r c1:43920404|43920928 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 circ1:43920404|43920928 in the serum exosome comprises PCR primers for detecting the content of circ1:43920404| 43920928.
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 1, 2 or 3, wherein the reagent for detecting the expression level of circ1:43920404|43920928 in the serum exosomes comprises all reagents for extracting exosomes from serum, extracting RNA from exosomes and performing reverse transcription and fluorescence quantitative PCR, in addition to the primers for detecting circ1:43920404| 43920928.
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| WO2009039390A2 (en) * | 2007-09-20 | 2009-03-26 | Naurex Inc. | The development of glycobiology-based therapeutics for the treatment of brain tumors |
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Non-Patent Citations (3)
| Title |
|---|
| Circular RNAs are abundant，conserved and associated with ALU repeats;Jeck WR et al.;《RNA》;20131231;第19卷(第3期);第141-157页 * |
| Glycosyltransferase gene expression profiles classify cancer types and propose prognostic subtypes;Jahanshah Ashkani et al.;《Scientific Reports》;20160520;第6卷;第4页第3段和图1B * |
| Jeck WR et al..Circular RNAs are abundant，conserved and associated with ALU repeats.《RNA》.2013,第19卷(第3期),第141-157页. * |
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