CN107653319B - Glioma diagnosis marker circ8:61680968|61684188 and application - Google Patents
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- C12N15/09—Recombinant DNA-technology
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
The invention belongs to the technical field of biology, and discloses a glioma diagnosis marker circ8:61680968|61684188 and application thereof. The first discovery is that: the expression level of circ8:61680968|61684188 in brain tissue of glioma patients is obviously increased compared with that of a control group (P is 0.0359), and an ROC curve shows that the glioma-resistant glioma. Therefore, by detecting the expression level of circ8:61680968|61684188 in the brain tissue of the high-risk population with glia, the early and quick diagnosis of a patient with glioma can be made, and a new direction is provided for the targeted therapy of the glioma.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a brain tissue circRNA marker 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. At present, the circular RNA plays an increasingly important role in tumor diagnosis markers due to the characteristics of richness, stability, high conservation, space-time specificity and the like. Meanwhile, precise medicine and glioma molecular typing are gradually the current focus, and compared with the traditional morphological typing, the molecular typing provides possibility for revealing cytological and genetic essence of glioma occurrence and development, so that the circular RNA is expected to play a greater role in the aspect, becomes a novel glioma molecular typing marker, and provides a new direction for targeted therapy of glioma.
Disclosure of Invention
The first object of the present invention is: provides a brain tissue circRNA marker for glioma diagnosis.
The main contents comprise: a brain tissue circRNA marker circ8:61680968|61684188 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 brain tissues in preparing glioma diagnostic preparations.
It is a third object of the present invention to provide a glioma diagnostic kit capable of determining the content of circ8:61680968|61684188 in brain tissue.
The glioma diagnostic kit contains a PCR primer for detecting the content of circ8:61680968| 61684188. Preferred primers have the sequences shown in SEQ NO 2 and 3.
The glioma diagnosis kit contains all reagents for extracting tissue RNA, reverse transcription and fluorescent quantitative PCR besides the primers of circ8:61680968| 61684188.
The preparation comprises (1) reagents required for extracting tissue RNA, namely Trizol reagent, trichloromethane, isopropanol, 75% ethanol and enzyme-free water, (2) reagents required for reverse transcription, namely Random Primer, enzyme-free water, 5 × reverse transcription buffer solution, triphosphate base deoxynucleotide, RNase inhibitor and MMLV reverse transcriptase which are all purchased from Thermo company, and (3) reagents required for fluorescence quantitative PCR, namely Primer pairs as shown in SEQ NO 2 and SEQ NO 3 sequences, Primer pairs for detecting GAPDH, SYBR dye and enzyme-free water.
The invention has the beneficial effects that: the difference expression of the circular RNA circ8:61680968|61684188 in glioma brain tissue and normal tissue is found for the first time, and the method has higher diagnostic analysis value 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 shows the difference between circ8:61680968|61684188 expression in glioma tissues and normal brain tissues analyzed by real-time fluorescent quantitative PCR;
FIG. 2 shows the specificity and sensitivity of Roc analysis of glioma-derived circ8:61680968|61684188 in 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
Tumor tissues and 15 normal brain tissues of 40 patients with glioma were provided by tumor hospitals in Hunan province.
1. Extraction of RNA from glioma/Normal tissue
0.1g of each of the tumor and normal brain tissues is taken, the tumor and normal brain tissues are washed by proper amount of normal saline and then put into a mortar (the mortar needs to be wrapped by tin foil paper in advance and baked in an oven at high temperature of 180 ℃ for 6-8 hours), proper amount of liquid nitrogen is added to grind the tissues into powder, 0.6ml of Trizol (MRC company) is added to continue grinding for a plurality of minutes, 0.4ml of Trizol is added again, the mixture in the mortar is transferred to a 1.5ml enzyme-free Tube and is cracked for 15 minutes on ice. 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 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 20 μ l non-enzyme water to dissolve RNA. The concentration and the quality of RNA are measured by spectrophotometry, the OD260/280 ratio is between 1.8 and 2.0, and the RNA is stored at the temperature of minus 80 ℃.
Preparation of cDNA
Reverse transcription was performed according to the instructions of the reverse transcription kit (Thermo Co.). The total volume of the reaction was 20. mu.l.
| Composition (I) | Dosage/tube |
| Random reverse transcription primer (1. mu.M) | 1μl |
| RNA samples | 1μg |
| Enzyme-free water | To12μl |
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 (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 |
| Specific primers (both upstream and downstream 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.
(6) And (3) data analysis: by using 2-ΔΔCTExpression fold of circ8:61680968|61684188 in brain tissue of glioma patients relative to normal brain tissue, wherein △ 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
The circ8:61680968|61684188 in glioma tumor tissue was significantly elevated compared to normal tissue (P ═ 0.0359), with specific results shown in fig. 1. The ROC curve shows that it has high diagnostic value for glioma (AUC 0.862, P0.002, sensitivity 85.7%, specificity 77.8%), and the detailed results are shown in fig. 2.
Sequence listing
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Claims (4)
1. The application of the reagent for detecting the up-regulation of the expression quantity of the Circ8:61680968|61684188 in brain tissues in the preparation of a glioma diagnostic preparation has the sequence of the Circ8:61680968|61684188 shown in SEQ NO 1.
2. The use of claim 1, wherein the reagent for detecting the up-regulation of the expression level of Circ8:61680968|61684188 in brain tissue comprises PCR primers for detecting the content of Circ8:61680968| 61684188.
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 up-regulation of the expression level of Circ8:61680968|61684188 in brain tissue comprises all reagents for tissue RNA extraction, reverse transcription and fluorescence quantitative PCR in addition to the primer for detecting the content of Circ8:61680968| 61684188.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009102729A1 (en) * | 2008-02-11 | 2009-08-20 | Historx, Inc. | Association of biomarkers with patient outcome |
| WO2012089753A2 (en) * | 2010-12-30 | 2012-07-05 | Febit Holding Gmbh | Complex sets of mirnas as non-invasive biomarkers for glioblastoma |
| EP2586875A1 (en) * | 2011-10-27 | 2013-05-01 | Institut du Cerveau et de la Moelle Epiniere-ICM | Non-invasive method for diagnosing and monitoring glioma |
| WO2015200823A1 (en) * | 2014-06-26 | 2015-12-30 | Institute For Systems Biology | Markers and therapeutic indicators for glioblastoma multiforme (gbm) |
-
2017
- 2017-10-27 CN CN201711024885.9A patent/CN107653319B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009102729A1 (en) * | 2008-02-11 | 2009-08-20 | Historx, Inc. | Association of biomarkers with patient outcome |
| WO2012089753A2 (en) * | 2010-12-30 | 2012-07-05 | Febit Holding Gmbh | Complex sets of mirnas as non-invasive biomarkers for glioblastoma |
| EP2586875A1 (en) * | 2011-10-27 | 2013-05-01 | Institut du Cerveau et de la Moelle Epiniere-ICM | Non-invasive method for diagnosing and monitoring glioma |
| WO2015200823A1 (en) * | 2014-06-26 | 2015-12-30 | Institute For Systems Biology | Markers and therapeutic indicators for glioblastoma multiforme (gbm) |
Non-Patent Citations (3)
| Title |
|---|
| Cell-Type Specific Features of Circular RNA Expression;Salzman J.等;《PLoS Genet 》;20130905;参见Circbase整合信息 * |
| Circular RNA profile in gliomas revealed by identification tool UROBORUS;Song XF.等;《Nucleic Acids Research》;20160211;表S5、S6 * |
| Salzman J.等.Cell-Type Specific Features of Circular RNA Expression.《PLoS Genet 》.2013, * |
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