CN107937528B - Glioma prognosis marker hsa _ circ _0125365 and application - Google Patents
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Abstract
The invention belongs to the technical field of biology, and discloses a glioma prognosis marker hsa _ circ _0125365 and application thereof. Namely, the reagent for detecting the expression quantity of circRNA hsa _ circ _0125365 derived from glioma tissues is used for preparing a prognostic preparation for glioma patients. The research proves that the patient with higher expression level of circRNA hsa _ circ _0125365 in glioma has higher postoperative survival rate. A prognostic decision is made for a patient with glioma by detecting the expression level of circRNA hsa _ circ _0125365 in the glioma tissue of the patient.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a circRNA marker for glioma prognosis, application of a reagent for detecting the marker in preparation of a glioma prognosis 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 search of glioma prognostic markers for carrying out prognostic analysis on patients, the improvement of the postoperative life quality of glioma patients, the corresponding selection of reasonable subsequent treatment schemes and the improvement of survival rate are research tasks 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 wide application of deep sequencing technology 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 by gene to form circRNA, and that they 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 potential as molecular markers of many diseases.
Disclosure of Invention
The first object of the present invention is: provides a circRNA marker hsa _ circ _0125365 from glioma tissue for prognosis of patients with glioma, and the sequence 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 glioma tissues in preparing glioma prognosis preparations.
It is a third object of the present invention to provide a glioma prognostic kit capable of determining the amount of hsa _ circ _0125365 in a glioma tissue.
The glioma prognosis kit contains a PCR primer for detecting the content of hsa _ circ _ 0125365. Preferred primers have the sequences shown in SEQ NO 2 and 3.
The glioma prognosis kit contains all reagents for extracting RNA from glioma tissues and carrying out reverse transcription and fluorescence quantitative PCR besides the primer of hsa _ circ _ 0125365. The method comprises the following steps:
(1) extracting total RNA from glioma tissue with reagent comprising RNA stabilizing solution, Trizol reagent, chloroform, isopropanol, and enzyme-free water;
(2) reagents for reverse transcription of hsa _ circ _0125365 into cDNA using total RNA as template, including reverse transcription buffer, deoxynucleotide triphosphates, RNase inhibitor, MMLV reverse transcriptase and random primers for hsa _ circ _ 0125365;
(3) reagents for real-time quantitative PCR of cDNA comprise a circRNA hsa _ circ _0125365 real-time fluorescent quantitative PCR specific primer, a GAPDH reference specific PCR primer, a real-time fluorescent quantitative SYBR dye and enzyme-free water.
The research of the invention proves that the circRNA hsa _ circ _0125365 derived from glioma tissues can be used for prognosis analysis of glioma patients, and the expression quantity of the circRNA hsa _ circ _0125365 derived from glioma tissues has correlation with the postoperative survival rate of the patients. Therefore, the method can be used for the prognostic analysis of glioma patients.
The applicant analyzes 26 glioma tissues and 12 normal brain tissues by fluorescent quantitative PCR and finds that the expression of hsa _ circ _0125365 is obviously different in the two tissues (P is less than 0.0001), and then performs survival curve analysis on 15 glioma patients, and finds that the circRNA hsa _ circ _0125365 derived from the glioma tissues is related to the survival rate of the patients, and the higher the content is, the higher the survival rate is. The method provides powerful technical support for prognosis analysis of glioma, is beneficial to improving the postoperative life quality of glioma patients, making a postoperative treatment scheme, improving survival rate and having profound clinical significance and popularization.
Drawings
FIG. 1 shows the difference in expression of hsa _ circ _0125365 in glioma tissues and normal brain tissues as analyzed by real-time fluorescent quantitative PCR;
FIG. 2 is a graph of ROC curves to analyze the specificity and sensitivity of brain tissue-derived hsa _ circ _0125365 to differences between glioma and normal brain tissue; hsa _ circ _0125365 was used as a biomarker with high diagnostic value for glioma (AUC 0.939, P0.040, sensitivity and specificity 90.0% and 78.3%, respectively).
FIG. 3 is a graph for the analysis of the dependence of the expression level of circRNA hsa _ circ _0125365 derived from glioma tissues on the survival rate of patients for a survival curve.
Detailed Description
The following examples are intended to further illustrate the invention without limiting it.
Example 1: preparation of reagent for detecting the expression level of circRNA hsa _ circ _0125365 for preparing a kit for prognosis of glioma patients (50 reactions)
50ml RNA stabilizing solution
2. Isopropanol 100ml
3. Chloroform 100ml
50ml Trizol (from Molecular Research Center Co., Ltd.)
5. 10ml of enzyme-free water
6.1 μ M random reverse transcription primer (Thermo Co., Ltd.) 50 μ l
7.5 × reverse transcription buffer (Thermo Co., Ltd.) 200ml
8.10mM deoxynucleotide triphosphate base (Thermo Co.) 100. mu.l
9.40U/. mu.l RNase inhibitor (Thermo Co.) 500. mu.l
10.200U/. mu.l MMLV reverse transcriptase (Thermo Co.) 50. mu.l
Premix Ex Taq (Thermo Co., Ltd.) 50. mu.l
12.10 μ M of real-time fluorescent quantitative PCR specific primers, 30 μ l, for circRNA hsa _ circ _0125365
circRNA hsa _ circ _0125365 forward primer: 5'-CCAAGCAGCTCACTACGATA-3' the flow of the air in the air conditioner,
circRNA hsa _ circ _0125365 reverse primer: 5'-CAAGCAGGTAGGAGATTCCA-3', respectively;
13.10 μ M GAPDH specific primer 30 μ l
The forward primer was set to 5'-ATCAAGATCATTGCTCCTCCTGAG-3' (wt.),
the reverse primer was 5'-CTGCTTGCTGATCCACATCTG-3'.
Example 2: relation between expression level of circRNA hsa _ circ _0125365 in glioma tissue and prognosis
1. Preservation of glioma tissues: collecting the glioma tissues to be detected, storing the glioma tissues in a freezing storage tube containing the RNA stable solution, and placing the glioma tissues in a refrigerator at the temperature of minus 80 ℃ for later use.
2. Extraction of RNA from tissues: taking a proper amount of specimen, adding liquid nitrogen into a mortar baked for 6-8h at 180 ℃, grinding the specimen into powder, adding 1ml of Trizol mortar specimen into the mortar, grinding the specimen into liquid, moving the liquid to a Tube, adding 200 mu l of chloroform into the Tube, shaking the Tube by hand for 15-30s, standing the Tube on ice for 15min, and centrifuging the Tube at 12000rpm at 4 ℃ 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 20min, and centrifuging at 12000rPm at 4 ℃ for 10 min; discarding supernatant, adding 1-2ml ethanol diluted with 75% DEPC water, mixing, centrifuging at 4 deg.C and 7500rPm for 5min, discarding supernatant, drying at room temperature for 5-10min, adding 10-20 μ l DEPC water to dissolve RNA, and storing at-80 deg.C.
3. circRNA hsa _ circ _0125365 reverse transcription: a reverse transcription kit from Thermo was used. The system for 20. mu.l reverse transcription reaction was as follows:
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 |
| Products of the first step reverse transcription | 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 quantitative PCR was performed using the primers specific to circRNA hsa _ circ _0125365 synthesized by Hantah Biotech (Shanghai) Ltd: firstly, the reverse transcription product is diluted by 10 times and mixed evenly. The 20. mu.l reaction was as follows:
specific primers for PCR:
a forward primer: 5'-CCAAGCAGCTCACTACGATA-3' the flow of the air in the air conditioner,
reverse primer: 5'-CAAGCAGGTAGGAGATTCCA-3' are provided.
GAPDH internal reference specific PCR primers:
the forward primer was set to 5'-ATCATCAGCAATGCCTCCT-3' (wt.),
the reverse primer was 5'-CATCACGCCACAGTTTCC-3'.
Real-time fluorescent quantitative PCR reaction program: 95 ℃ for 3 minutes, 40 cycles, 95 ℃ for 10 seconds, 60 ℃ for 30 seconds.
5、2-ΔΔCTCalibration: by 2-ΔΔCTRepresents the fold change for the control group, wherein △ CT ═ CTSample(s)–CTInternal reference，△△CT＝△CTCases of disease–△CTControlThe present experimental data was analyzed using the software GraPhPad Prism and SPSS, applicant analyzed 26 cases of glioma tissues and 12 cases of normal brain tissues by quantitative fluorescence PCR, and found that the expression difference between them was significant (P < 0) for hsa _ circ _0125365 (P < 0)0001), see fig. 1.
6. The ROC curve of fig. 2 analyzes the specificity and sensitivity of brain tissue-derived hsa _ circ _0125365 to differences between glioma and normal brain tissue; hsa _ circ _0125365 was used as a biomarker with high diagnostic value for glioma (AUC 0.939, P0.040, sensitivity and specificity 90.0% and 78.3%, respectively).
7. Prognostic survival curve analysis of 16 patients revealed a higher survival rate with high expression of circRNA hsa _ circ _0125365 (above mean) in glioma tissue compared to patients with low expression of circRNA hsa _ circ _0125365 (below mean) in glioma tissue with a significant difference (P ═ 0.119), as shown in figure 3.
The above studies indicate that hsa _ circ _0125365 can be used as a specific molecular marker for prognosis of glioma patients.
Sequence listing
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Claims (4)
1. Application of a reagent for detecting the expression quantity of hsa _ circ _0125365 in glioma tissues in preparing a glioma prognosis preparation, wherein the sequence of hsa _ circ _0125365 is shown as SEQ NO: 1.
2. The use of claim 1, wherein the reagent for detecting the expression of hsa _ circ _0125365 in glioma tissue comprises PCR primers for detecting the amount of hsa _ circ _ 0125365.
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, wherein the reagent for detecting the expression level of hsa _ circ _0125365 in glioma tissue contains all reagents for extracting RNA from glioma tissue and performing reverse transcription and fluorescence quantitative PCR, in addition to the primer of hsa _ circ _ 0125365.
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| Cell-Type Specific Features of Circular RNA Expression.;Salzman et al.;《PLOS Genetics》;20130930;第9卷(第9期);e1003777 * |
| Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.;Rybak-Wolf et al.;《Molecular Cell》;20151231;870-885 * |
| Identification of circular RNA signature in bladder cancer.;Yang et al.;《Journal of Cancer》;20170922;3456-3462 * |
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