CN111500739B - Biomarker for detecting nasopharyngeal carcinoma prognosis and application thereof - Google Patents
Biomarker for detecting nasopharyngeal carcinoma prognosis and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and discloses a biomarker for detecting nasopharyngeal carcinoma prognosis and application thereof. The biomarker provided by the invention consists of one or three of miR-106a-5p, BTG3 and SOX 9. When the biomarkers consist of miR-106a-5p, BTG3 and SOX9, the nasopharyngeal carcinoma prognosis can be predicted quickly and accurately according to the quantitative expression amount scores of the three biomarkers. If the biomarker combination is applied to the preparation of a reagent or a tool for detecting the nasopharyngeal carcinoma prognosis, the prediction accuracy of the nasopharyngeal carcinoma patient prognosis can be obviously improved, and then a population with poor prognosis is screened out from a large number of nasopharyngeal carcinoma patients, so that the patients with better prognosis are prevented from being over-treated while being reasonably treated, the economic pressure and medical resource pressure of the patients and the society are relieved, the treatment cost is reduced, and the prognosis of the nasopharyngeal carcinoma patient is improved.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a biomarker for detecting nasopharyngeal carcinoma prognosis and application thereof.
Background
Nasopharyngeal carcinoma (NPC) is the most common malignant tumor of head and neck cancer, and the incidence rate in China is about 80% of the world, and the incidence rate is 15-30 persons/10 ten thousand persons. The existing treatment of nasopharyngeal carcinoma comprises various methods such as radiotherapy, chemotherapy, combination of radiotherapy and chemotherapy, targeted drugs and the like, and needs to be reasonably selected according to the actual condition of a patient. The prognosis of each nasopharyngeal carcinoma patient is one of the most clinically concerned issues, how to make different diagnosis plans according to their possible different prognosis conditions so as to obtain better treatment effect. At present, the approximate prognosis of a patient is judged and a treatment strategy is formulated clinically mainly through the clinical pathological stage of the patient when the patient is diagnosed, however, the prognosis of the patient in the same clinical pathological stage often has larger difference, so that a method for predicting the prognosis of nasopharyngeal carcinoma more accurately is urgently needed clinically. The discovery of a new prognosis marker for patients with nasopharyngeal carcinoma can accurately predict the prognosis of the patients, provide basis for further diagnosis and treatment of the patients, and has great significance for diagnosis and treatment of the nasopharyngeal carcinoma.
Disclosure of Invention
In view of the above, the present invention provides a biomarker for detecting nasopharyngeal carcinoma prognosis and the application thereof, wherein the biomarker can accurately determine the nasopharyngeal carcinoma patient prognosis.
In order to solve the technical problems, the invention provides a biomarker for detecting nasopharyngeal carcinoma prognosis, which consists of one or three of miR-106a-5p, miR-106a-5p target gene BTG3 and miR-106a-5p upstream transcription factor SOX9, wherein the sequence of miR-106a-5p is SEQ ID NO.1, the sequence of BTG3 is SEQ ID NO.2, and the sequence of SOX9 is SEQ ID NO. 3.
Preferably, the biomarker for detecting the prognosis of nasopharyngeal carcinoma is miR-106a-5p, and if the expression level of miR-106a-5p is judged to be high, the prognosis of nasopharyngeal carcinoma is poor.
Preferably, the biomarker for detecting nasopharyngeal carcinoma prognosis is miR-106a-5p target gene BTG3, and if the expression level of BTG3 is judged to be low, the nasopharyngeal carcinoma prognosis is poor.
Preferably, the biomarker for detecting the prognosis of nasopharyngeal carcinoma is miR-106a-5p upstream transcription factor SOX9, and if the expression level of SOX9 is judged to be high, the prognosis of nasopharyngeal carcinoma is poor.
Preferably, the biomarker for detecting the prognosis of nasopharyngeal carcinoma consists of miR-106a-5p, miR-106a-5p downstream target molecule BTG3 and miR-106a-5p upstream transcription factor SOX 9;
the nasopharyngeal carcinoma prognosis detection model is shown as the following formula:
S＝x1+x2+x3
if A is greater than or equal to 9, then x11 is ═ 1; if A < 9, then x1＝0；
If B is less than or equal to 7, then x21 is ═ 1; if B > 7, then x2＝0；
If C is greater than or equal to 8, then x31 is ═ 1; if C is less than 8, then x3＝0；
Wherein, S represents the prognosis score of nasopharyngeal carcinoma, S-3 represents the poor prognosis, S-2 represents the medium prognosis, S-1 represents the better prognosis, and S-0 represents the best prognosis; a represents the quantitative expression level of miR-106a-5 p; b represents the quantitative expression level of BTG 3; c represents the quantitative expression level of SOX9, x1Represents the prognostic score, x, corresponding to miR-106a-5p2Indicates the prognosis score, x, corresponding to BTG33Representing the corresponding prognostic score of SOX 9.
The invention also provides application of the biomarker for detecting nasopharyngeal carcinoma prognosis in preparation of a reagent or a tool for detecting nasopharyngeal carcinoma prognosis.
Compared with the prior art, the invention provides a biomarker for detecting nasopharyngeal carcinoma prognosis, and the biomarker consists of one or three of miR-106a-5p, BTG3 and SOX 9. When the biomarkers consist of miR-106a-5p, BTG3 and SOX9, the nasopharyngeal carcinoma prognosis can be predicted quickly and accurately according to the quantitative expression amount scores of the three biomarkers. If the biomarker combination is applied to the preparation of a reagent or a tool for detecting the nasopharyngeal carcinoma prognosis, the prediction accuracy of the nasopharyngeal carcinoma patient prognosis can be obviously improved, and then a population with poor prognosis is screened out from a large number of nasopharyngeal carcinoma patients, so that the patients with better prognosis are prevented from being over-treated while being reasonably treated, the economic pressure and medical resource pressure of the patients and the society are relieved, the treatment cost is reduced, and the prognosis of the nasopharyngeal carcinoma patient is improved.
Drawings
FIG. 1 is a line graph showing the survival rate of nasopharyngeal carcinoma patients with different miR-106a-5p expression levels;
FIG. 2 is a graph of a comparison of the detected fluorescence values of dual luciferase reporter genes;
FIG. 3 is a graph of correlation analysis of nasopharyngeal carcinoma tissue chip BTG3 and miR-106a-5p staining score;
FIG. 4 is a line graph showing the survival rate of nasopharyngeal carcinoma patients with different amounts of BTG3 expression;
FIG. 5 is a graph of a comparison of the detected fluorescence values of dual luciferase reporter genes;
FIG. 6 is a chromatine immunoprecipitation (Chip) experimental sonoelectrophoresis image;
FIG. 7 is a graph of correlation analysis of nasopharyngeal carcinoma tissue chip SOX9, miR-106a-5p staining score;
FIG. 8 is a line graph showing the survival rate of nasopharyngeal carcinoma patients with different SOX9 expression levels;
FIG. 9 is a line graph of survival analysis of miR-106a-5p in the TCGA database;
FIG. 10 is a line graph of a survival analysis of BTG3 in the TCGA database;
FIG. 11 is a line graph of the survival analysis of SOX9 in the TCGA database;
FIG. 12 is a ROC graph showing various methods for determining the prognosis of a nasopharyngeal carcinoma patient.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention and is not intended to limit the scope of the claims which follow.
All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.
To further illustrate the present invention, the following examples are provided to describe in detail a biomarker combination for predicting the prognosis of nasopharyngeal carcinoma patients and the application thereof.
Sample preparation
123 nasopharyngeal carcinoma tissue sample chips were prepared for Shanghai core ultra (OUTDO BIOTECH). Pathological diagnosis and staging of samples are according to 2018 AJCC staging manual (eighth edition) of common tumors, and the information of tissue chips is shown in Table 1.
TABLE 1 nasopharyngeal carcinoma tissue sample chip information
Example 1 (exploration of the prognostic relationship of the biomarker miR-106a-5p with nasopharyngeal carcinoma patients)
To explore the role of the biomarker miR-106a-5p in the progression of nasopharyngeal carcinoma, we performed In Situ Hybridization (ISH) experiments on the 123 tissue sample chips described above, comprising the following steps:
the method comprises the following steps: deparaffinization and hydration of tissue sample chips (this step is performed in a fume hood)
Soaking the tissue sample chip with xylene for 30min, and replacing the xylene every 10 min; and then fully soaking and washing the tissue sample chip soaked in the dimethylbenzene by using 100% ethanol, soaking the tissue sample chip in the 100% ethanol for 5 minutes, fully soaking and washing the tissue sample chip in 96% ethanol, soaking the tissue sample chip in the 96% ethanol for 5 minutes, fully soaking and washing the tissue sample chip in the 70% ethanol, soaking the tissue sample chip in the 70% ethanol for 5 minutes, and soaking the tissue sample chip in PBS for 5 minutes.
Step two: tissue sample chip digested by protease
The deparaffinized tissue sample chip is immersed in proteinase K (15 mug/mL) at 37 ℃ for 40 minutes, then fully soaked in PBS, fully soaked in 70% ethanol, soaked in 70% ethanol for 2 minutes, fully soaked in 96% ethanol, soaked in 96% ethanol for 2 minutes, fully soaked in 100% ethanol, soaked in 100% ethanol for 2 minutes, and finally taken out and placed in a clean place for air drying for 15 minutes.
Step three: hybridization (hybridization temperature 50 ℃ C.)
The tissue sample chip after protease digestion treatment was placed on a glass slide and 80. mu.L of hybridization solution was dropped, covered with a cover slip, and the slide was sealed with glue to prevent the sample from drying out, and then placed in a 50 ℃ hybridization apparatus for 1 hour.
Step four: developing film
After completion of hybridization, the mounting gel on the slide glass was removed, and the slide glass was incubated in 5 XSSC buffer for 5 minutes (room temperature), 1 XSSC buffer for 5 minutes (50 ℃ C.), 0.2 XSSC buffer for 5 minutes (room temperature), and PBS for 5 minutes (room temperature).
Step five: colour reaction
Spin-drying the washed slide glass, sealing in 1mL of sealing solution (1mL of sealing solution comprises 100 muL of 10 XRoche sealing solution and 900 muL of 1 Xmaleic acid buffer solution) for 15 minutes (room temperature), blotting the sealing solution with absorbent paper, incubating in a wet box with 1:800 anti-DIG-AP Fab fragments (overnight at 4 ℃), soaking in PBST at room temperature for 15 minutes, and replacing PBST every 5 minutes; slides were then incubated with 400. mu.L/slide of NBT/BCIP buffer (20. mu.L of Roche reagent buffer and 1mL of NBT/BCIP dilution per mL of NBT/BCIP buffer) for 24h at room temperature, protected from light.
The experimental results are as follows:
the results of the miR-106a-5p in situ hybridization experiments are judged by two pathologists without solving the section data, and the judgment standard is shown in Table 2. Final staining score was defined as the product of staining intensity and staining area: a staining score of 0-8 was defined as low expression of miR-106a-5p, and a staining score of 9-16 was defined as high expression of miR-106a-5 p.
TABLE 2 in situ hybridization result determination criteria
| Intensity of dyeing | Negative of | Weak positive | Moderate positive | Strong positive |
| Score of | 1 | 2 | 3 | 4 |
| Area of dyeing | 0-25％ | 26-50％ | 51-75％ | >75％ |
| Score of | 1 | 2 | 3 | 4 |
Further, 123 nasopharyngeal carcinoma tissue sample chips were scored according to the in situ hybridization result determination criteria, and the prognosis of nasopharyngeal carcinoma patients and the expression of the biomarker miR-106a-5p in different clinical stages of nasopharyngeal carcinoma patients were counted, with the results shown in Table 3.
TABLE 3 tissue sample chip miR-106a-5p score result and prognosis statistics
As can be seen from Table 3, the expression level of miR-106a-5p is higher in patients in clinical stage IV than in patients in clinical stages I-III, suggesting that the dysregulation of miR-106a-5p may be related to the terminal malignant progression of nasopharyngeal carcinoma.
Further, the correlation between miR-106a-5p expression and the prognosis of patients with nasopharyngeal carcinoma is analyzed based on the Kaplan-Meier method, and figure 1 is drawn, wherein figure 1 is a survival line graph of patients with nasopharyngeal carcinoma with different miR-106a-5p expression amounts, and figure 1 shows that the prognosis of patients with high miR-106a-5p expression is worse than that of patients with low miR-106a-5p expression.
Example 2 (demonstration that the biomarker BTG3 is a direct target for miR-106a-5 p)
The biological effective interaction of miR-106a-5p and BTG3 is proved by a luciferase reporter gene experiment, and the specific operation steps of the luciferase reporter gene experiment are as follows:
the method comprises the following steps: plasmid transfected nasopharyngeal carcinoma CNE2 cell
1. Grouping experiments:
group A: the plasmid was 106NC and PGL3-Mut
Group B: the plasmid was 106NC and PGL3-WT
Group C: the plasmid is 106Mimics and PGL3-Mut
Group D: the plasmid is 106Mimics and PGL3-WT
2. Inoculating CNE2 cells into a six-hole plate, setting 3 repeats per group, then injecting a culture medium into the six-hole plate for cell culture, and adding transfection reagents and corresponding plasmids according to groups for co-transfection when the cell density reaches 40% -50%.
The co-transfection process comprises the following steps: adding 5 mu L of Lip3000 transfection reagent into 1mL of basal medium, uniformly mixing, incubating for 5 minutes at room temperature, adding 1 mu g of corresponding plasmid and 200ng of renilla fluorescein plasmid according to groups, uniformly mixing, and incubating for 10 minutes at room temperature.
Step two: luciferase reporter gene detection
1. 48 hours after the cotransfection is finished, removing the cell basal medium in the six-well plate, washing the cells in the six-well plate by PBS, adding 100 mu L of 1 XPassive Lysis Buffer (the 1 XPassive Lysis Buffer is obtained by diluting 5 XPassive Lysis Buffer with water) into each well of the six-well plate to lyse the cells, transferring the cells into a 1.5mL centrifuge tube to be centrifuged, and taking the upper layer cell lysate to obtain corresponding A, B, C, D four groups of cell lysates.
2. Adding 100 mu L of LARII solution (the LARII solution is prepared by diluting and freeze-drying Luciferas Assay Substrate with 10mL of Luciferas Assay Buffer) into each well of a 96-well plate, adding 20 mu L of prepared cell lysate of different groups respectively, adding 3 repeat wells into each group, immediately putting into a fluorescence microplate reader to read a value a, then adding 100 mu L of premixed Stop & GLO solution (the Stop & GLO solution is prepared by mixing the Stop & GLO Substrate and the Stop & GLO Buffer in a ratio of 1: 50), immediately putting into the fluorescence microplate reader to read a value b, wherein the fluorescence value obtained in the experiment is the ratio of two readings, namely a/b, then drawing a graph 2 according to the obtained fluorescence value, and the graph 2 is a dual-luciferase reporter gene detection fluorescence value comparison graph.
The experimental results are as follows:
as can be seen from FIG. 2, 106Mimics significantly reduced luciferase activity in CNE2 cells transfected into the wild-type BTG3(PGL3-WT) plasmid, but did not significantly change luciferase activity in CNE2 cells transfected into the mutant BTG3(PGL3-Mut) plasmid. As can be seen from FIG. 2, BTG3 is a true target of miR-106a-5p in nasopharyngeal carcinoma cells.
Example 3 (biomarker BTG3 prognostic relation in nasopharyngeal carcinoma patients)
The experimental method comprises the following steps:
to explore the role of the biomarker BTG3 (i.e., miR-106a-5p target gene) in the progression of nasopharyngeal carcinoma, immunohistochemical experiments were also performed on 123 tissue sample chips against BTG3, comprising the following steps:
the method comprises the following steps: deparaffinization and hydration of tissue sample chips (this step is performed in a fume hood)
Soaking the tissue sample chip with xylene for 30min, and replacing the xylene every 10 min; and then fully soaking and washing the tissue sample chip soaked in the dimethylbenzene by using 100% ethanol, soaking the tissue sample chip in the 100% ethanol for 5 minutes, fully soaking and washing the tissue sample chip in 96% ethanol, soaking the tissue sample chip in the 96% ethanol for 5 minutes, fully soaking and washing the tissue sample chip in the 70% ethanol, soaking the tissue sample chip in the 70% ethanol for 5 minutes, and soaking the tissue sample chip in PBS for 5 minutes.
Step two: antigen retrieval, quenching, blocking
1. Heating 0.01 sodium citrate buffer solution in a water bath to boil, and heating in a tissue chip for 20 min. After the temperature was lowered to room temperature, the plate was washed with PBS solution for 5 minutes and 3 times.
2、3％H2O2Incubate for 10min at room temperature to eliminate endogenous peroxidase activity, wash with PBS solution, 3 min × 3 times.
3. Primary antibody blocking solution was incubated for 30 minutes at room temperature.
Step three: antibody binding
1. Primary anti-working solution prepared by diluting BTG3 antibody with primary anti-diluent 1:50 is added dropwise. Incubate at 4 ℃ overnight.
2. PBS solution rinse, 3 minutes × 3 times. The biotin-labeled secondary antibody was added dropwise and incubated at room temperature for 1 hour. PBS solution rinse, 3 minutes × 3 times.
Step four: colour reaction
1. The DAB staining agent develops color and the staining degree is controlled under a microscope. Running tap water for 10 minutes.
2. Hematoxylin stains the nucleus for two minutes, and hydrochloric acid alcohol differentiates. Running tap water for 15 minutes.
3. Dehydration, mounting, staining and scoring.
The tissue chip immunohistochemistry result of the biomarker BTG3 was judged by two pathologists without knowledge of the section data, and the judgment criteria were consistent with table 2. The final staining score was defined as the product of staining intensity and staining area. Staining scores from 0 to 7 were defined as low expression of BTG3, and staining scores from 8 to 16 were defined as high expression of BTG 3.
Further, 123 nasopharyngeal carcinoma tissue sample chips were scored according to the immunohistochemical determination standard of the biomarker BTG3, and the prognosis of nasopharyngeal carcinoma patients and the expression of the biomarker BTG3 in different clinical stages of nasopharyngeal carcinoma patients were counted, and the results are shown in Table 5.
TABLE 5
Correlation analysis is carried out on the staining scores of miR-106a-5p and BTG3, the analysis result is shown in figure 3, and as can be seen in figure 3, the expression content of BTG3 is in negative correlation with the expression of miR-106a-5p, which indicates that the abnormal expression of BTG3 is possibly related to the terminal malignant progression of nasopharyngeal carcinoma.
Further, based on the analysis of the correlation between BTG3 expression and the prognosis of patients with nasopharyngeal carcinoma by the Kaplan-Meier method, the results are shown in FIG. 4, and FIG. 4 shows that the prognosis of patients with low BTG3 expression is worse than that of patients with high BTG3 expression, contrary to the effect of the biomarker miR-106a-5p on the prognosis of patients with nasopharyngeal carcinoma.
Example 4 (demonstration that the biomarker SOX9 is an upstream gene of miR-106a-5 p)
The experimental method comprises the following steps:
to determine the upstream transcription factor for regulating miR-106a-5p, the sequence of the promoter of miR-106a-5p is analyzed by using a transcription factor binding site analysis website JASPAR (http:// JASPAR. jenerg. net), and it is determined that SRY-box transcription factor 9(SOX9) can be bound to-1472, indicating that SOX9 regulates the transcription of miR-106a-5 p.
Further, the interaction between the two is proved by luciferase reporter gene experiments and chromatin immunoprecipitation (ChIP), and the steps of the luciferase reporter gene experiments are as follows:
the method comprises the following steps: plasmid transfected nasopharyngeal carcinoma CNE2 cell
1. Grouping experiments:
group A: the plasmid is TF-NC and MIR106A promoter-NC
Group B: the plasmids are SOX9 and MIR106A promoter-NC
Group C: the plasmid is TF-NC
Group D: the plasmid is TF-NC and MIR106A promoter
Group E: the plasmids are SOX9 and MIR106A promoter
2. Inoculating CNE2 cells into a six-hole plate, setting 3 repeats per group, then injecting a culture medium into the six-hole plate for cell culture, and adding transfection reagents and corresponding plasmids according to groups for co-transfection when the cell density reaches 40% -50%.
The co-transfection process comprises the following steps: adding 5 mu L of Lip3000 transfection reagent into 1mL of basal medium, uniformly mixing, incubating for 5 minutes at room temperature, adding 1 mu g of corresponding plasmid and 200ng of renilla fluorescein plasmid according to groups, uniformly mixing, and incubating for 10 minutes at room temperature.
Step two: luciferase reporter gene detection
1. 48 hours after the cotransfection is finished, removing the cell basal medium in the six-well plate, washing the cells in the six-well plate by adopting PBS, then adding 100 mu L of 1 XPassive lysine Buffer (the 1 XPassive lysine Buffer is obtained by diluting 5 XPassive lysine Buffer with water) into each well of the six-well plate to crack the cells, transferring the cells into a 1.5mL centrifuge tube for centrifugation, and taking the upper layer cell lysate to obtain corresponding A, B, C, D, E five groups of cell lysates.
2. Adding 100 mu L of LARII solution (the LARII solution is prepared by diluting and freeze-drying Luciferas Assay Substrate with 10mL of Luciferas Assay Buffer) into each well of a 96-well plate, adding 20 mu L of prepared cell lysate of different groups respectively, adding 3 repeat wells into each group, immediately putting into a fluorescence microplate reader to read a value a, then adding 100 mu L of premixed Stop & GLO solution (the Stop & GLO solution is prepared by mixing the Stop & GLO Substrate and the Stop & GLO Buffer in a ratio of 1: 50), immediately putting into the fluorescence microplate reader to read a value b, wherein the fluorescence value obtained in the experiment is the ratio of two readings, namely a/b, and drawing a graph 5 according to the obtained fluorescence value, wherein the graph 5 is a dual-luciferase reporter gene detection fluorescence value comparison graph.
The chromatin immunoprecipitation experiment uses the chip kit of Merck Michibo company, and the specific operation steps are as follows:
the method comprises the following steps: CNE2 and 5-8F cells (1X 10)7) Cross-linking with 1% formaldehyde for 10min and sonicating to generate DNA fragments of 200 to 1000 bp.
Step two: the cross-linked DNA-protein is subjected to chromatin immunoprecipitation, and except for conventional input, negative control and positive control, an SOX9 antibody is added to the target protein to complete chromatin immunoprecipitation. The cross-linked DNA-protein was mixed with the antibody and incubated overnight at 4 ℃ with slow rotation.
Step three: 60 μ L of LProteinA + GAAgarose/Salmon sphere DNA was added and vortexed slowly at 4 ℃ for 60 minutes to precipitate the primary antibody-recognized corresponding complex.
Step four: all washing steps were completed according to the manufacturer's instructions and the resulting pellet was used for subsequent gel electrophoresis analysis.
Step five: and (3) carrying out PCR amplification on the precipitate obtained in the step three by using primers of miR-106a-5p promoter binding sites, taking DNA extracted from the total nuclear extract as a PCR positive control, and analyzing the PCR product by 2% agarose gel electrophoresis, wherein the analysis result is shown in FIG. 6.
The experimental results are as follows:
as can be seen from FIGS. 5 and 6, SOX9 directly binds to the predicted binding site of the miR-106a-5p promoter and transactivates miR-106a-5p, the biomarker SOX9 can be further determined to be the upstream gene of miR-106a-5 p.
Example 5 (biomarker SOX9 prognostic relation in nasopharyngeal carcinoma patients)
The experimental method comprises the following steps:
in order to explore the role of the upstream gene SOX9 of the biomarker miR-106a-5p in the development of nasopharyngeal carcinoma, an immunohistochemical experiment was also carried out on 123 tissue sample chips aiming at SOX9, and the method comprises the following steps:
the method comprises the following steps: deparaffinization and hydration of tissue sample chips (this step is performed in a fume hood)
Soaking the tissue sample chip with xylene for 30min, and replacing the xylene every 10 min; and then fully soaking and washing the tissue sample chip soaked in the dimethylbenzene by using 100% ethanol, soaking the tissue sample chip in the 100% ethanol for 5 minutes, fully soaking and washing the tissue sample chip in 96% ethanol, soaking the tissue sample chip in the 96% ethanol for 5 minutes, fully soaking and washing the tissue sample chip in the 70% ethanol, soaking the tissue sample chip in the 70% ethanol for 5 minutes, and soaking the tissue sample chip in PBS for 5 minutes.
Step two: antigen retrieval, quenching, blocking
1. Heating 0.01 sodium citrate buffer solution in a water bath to boil, and heating in a tissue chip for 20 min. After the temperature was lowered to room temperature, the plate was washed with PBS solution for 5 minutes and 3 times.
2. 3% H2O2 was incubated at room temperature for 10min to eliminate endogenous peroxidase activity, washed with PBS solution, 3 min X3 times.
3. Primary antibody blocking solution was incubated for 30 minutes at room temperature.
Step three: antibody binding
1. A primary anti-working solution prepared by diluting the SOX9 antibody with a primary anti-diluent 1:50 was added dropwise. Incubate at 4 ℃ overnight.
2. PBS solution rinse, 3 minutes × 3 times. The biotin-labeled secondary antibody was added dropwise and incubated at room temperature for 1 hour. PBS solution rinse, 3 minutes × 3 times.
Step four: colour reaction
1. The DAB staining agent develops color and the staining degree is controlled under a microscope. Running tap water for 10 minutes.
2. Hematoxylin stains the nucleus for two minutes, and hydrochloric acid alcohol differentiates. Running tap water for 15 minutes.
3. Dehydration, mounting, staining and scoring.
The experimental results are as follows:
the tissue chip immunohistochemistry result of biomarker SOX9 was judged by two pathologists without knowledge of section data, and the judgment criteria were in accordance with table 2. Final staining score was defined as the product of staining intensity and staining area: scores from 0 to 7 were defined as low expression of SOX9 and scores from 8 to 16 were defined as high expression of SOX 9.
Further, 123 nasopharyngeal carcinoma tissue sample chips were scored according to the immunohistochemical determination criteria of the biomarker SOX9, and the prognosis of nasopharyngeal carcinoma patients and the expression of the biomarker SOX9 in different clinical stages of nasopharyngeal carcinoma patients were counted, and the results are shown in table 6.
TABLE 6
Correlation analysis is carried out on the staining scores of miR-106a-5p and SOX9, and as can be seen from FIG. 7, the expression content of SOX9 is positively correlated with the level of MIR106A-5p, which indicates that the disorder of SOX9 may be correlated with the terminal malignant progression of nasopharyngeal carcinoma.
Further, based on the analysis of the correlation between the expression of SOX9 and the prognosis of the nasopharyngeal carcinoma patients by the Kaplan-Meier method, the analysis result is shown in FIG. 8, and it can be seen from FIG. 8 that the clinical result of the patients with low SOX9 expression is worse than that of the patients with high SOX9 expression, and the correlation is positively correlated with the influence of the biomarker miR-106a-5p on the prognosis of the nasopharyngeal carcinoma patients.
Example 6(TCGA test results)
In order to verify the reliability of the influence of the expression of the biomarkers miR-106a-5p, BTG3 and SOX9 on the prognosis results of patients with nasopharyngeal carcinoma, the prognostic significance of the biomarkers miR-106a-5p, BTG3 and SOX9 was evaluated by means of Kaplan-Meier analysis on 525 patients with head and neck carcinoma by means of a cancer genome map (TCGA; http:// www.cbioportal.org) database. The TCGA database results are respectively shown in FIG. 9, FIG. 10 and FIG. 11, and it can be seen from FIG. 9 that the higher the expression of miR-106a-5p, the worse the prognosis of the nasopharyngeal carcinoma patient; as can be seen from FIG. 10, the lower the expression of BTG3, the worse the prognosis of the nasopharyngeal carcinoma patients; as can be seen from FIG. 11, the higher the expression of SOX9, the worse the prognosis of nasopharyngeal carcinoma patients. From the graphs in FIGS. 9-11, it can be seen that miR-106a-5p and its target gene BTG3 have negative correlation with each other on the prognosis of patients with nasopharyngeal carcinoma; miR-106a-5p is positively correlated with the influence of upstream transcription factor SOX9 on the prognosis of patients with nasopharyngeal carcinoma. The evaluation result verifies the experimental result, which shows that the reliability and the accuracy of the prognosis prediction of the nasopharyngeal carcinoma patient are high by adopting the biomarkers miR-106a-5p, BTG3 and SOX 9.
Example 7 (biomarker combinations and prognosis for nasopharyngeal carcinoma patients)
The biomarker combination for prognosis of nasopharyngeal carcinoma consists of miR-106a-5p with a sequence of SEQ ID NO.1, a downstream target gene BTG3 of miR-106a-5p with a sequence of SEQ ID NO.2 and an upstream transcription factor SOX9 of miR-106a-5p with a sequence of SEQ ID NO. 3.
The prognosis biomarker combination is adopted to carry out a prognosis detection model of nasopharyngeal carcinoma, and the model is shown as the following formula:
S＝x1+x2+x3
if A is greater than or equal to 9, then x11 is ═ 1; if A < 9, then x1＝0；
If B is less than or equal to 7, then x21 is ═ 1; if B > 7, then x2＝0；
If C is greater than or equal to 8, x31 is ═ 1; if C is less than 8, then x3＝0；
Wherein, S represents the prognosis risk value of nasopharyngeal carcinoma, S-3 represents the prognosis is poor, S-2 represents the prognosis is medium, S-1 represents the prognosis is good, and S-0 represents the prognosis is best; a represents the quantitative expression level of miR-106a-5 p; b represents the quantitative expression level of BTG 3; c represents the quantitative expression level of SOX 9. x is the number of1Represents the prognostic score, x, corresponding to miR-106a-5p2Representing the prognostic score corresponding to BTG3，x3Representing the corresponding prognostic score of SOX 9.
Specifically, the pathological section is stained and scored by in-situ hybridization and immunohistochemical technology by using the tissue pathological section after the biopsy of the patient. According to examples 1, 3 and 5, the pathological section staining score (quantitative expression) of miR-106a-5p is determined as A, the pathological section staining score (quantitative expression) of the downstream target gene BTG3 of miR-106a-5p is determined as B, and the pathological section staining score (quantitative expression) of the upstream transcription factor SOX9 of miR-106a-5p is determined as C by using the above prognostic marker combination. In pathological sections of each patient, if A is more than or equal to 9, the score is 1, and if A is less than or equal to 8, the score is 0; if B is less than or equal to 7, the score is 1, and if B is more than or equal to 8, the score is 0; if the C is more than or equal to 8, the score is 1, and if the C is less than or equal to 7, the score is 0; the total score of each patient is obtained by adding the three scores, and the score of each patient can be divided into four scores of 0, 1, 2 and 3. The score is 3 minutes, the patient is considered to have poor prognosis, and among the existing 114 patients, 17 patients belong to the group, and the five-year survival rate is 64.7 percent; the score is 2 minutes, the prognosis of the patient is considered to be moderate, and among the existing 114 patients, 32 patients belong to the group, and the five-year survival rate is 81.25 percent; the score is 1 minute, the prognosis of the patient is considered to be better, and among the 114 existing patients, 27 patients belong to the group, and the five-year survival rate is 85.2%; the score was 0, and the patient was considered to have the best prognosis, with 38 out of our existing 114 patients in this group, with a five-year survival rate of 94.7%.
The ROC graph is used to evaluate the clinical accuracy of the prediction model of the present embodiment, and is shown in fig. 12, the abscissa is the false positive rate (1-specificity), the ordinate is the true positive rate (sensitivity), the area value (AUC value) under the ROC curve is calculated to be between 1.0 and 0.5, and in the case of AUC >0.5, the AUC is closer to 1, which indicates that the diagnosis effect is better. If A, B, C values are used alone to classify patients into two groups of high and low expression to determine their prognosis as described in examples 1, 3 and 5, the AUC values are 0.605, 0.618 and 0.645 respectively. If the prognosis is judged by using the above grouping of example 7, the AUC value is 0.693, which is better than before, and shows that the accuracy of predicting the prognosis of the patient is higher by comprehensively considering A, B, C values.
In conclusion, the biomarker combinations miR-106a-5p, BTG3 and SOX9 are closely related to development and prognosis of nasopharyngeal carcinoma, and can be used for assisting in predicting survival and prognosis of nasopharyngeal carcinoma patients, and the biomarker miR-106a-5p is high-expression, the nasopharyngeal carcinoma patients are judged to have poor prognosis when the biomarker BTG3 is low-expression, and the nasopharyngeal carcinoma patients are judged to have poor prognosis when the biomarker SOX9 is high-expression. The luciferase reporter gene experiment also proves that the biomarker BTG3 is a direct target of miR-106a-5p, the luciferase reporter gene experiment and a chromatin immunoprecipitation (ChIP) experiment prove that the biomarker SOX9 is an upstream gene of miR-106a-5p, and the application of the biomarker combination disclosed by the invention can be further proved to be capable of accurately judging the prognosis of patients with nasopharyngeal carcinoma. On the basis, the combined expression level of the miR-106a-5p, BTG3 and SOX9 biomarkers of the same patient is comprehensively considered at the same time to judge the prognosis, and the clinical accuracy is higher than that of the prognosis judged by independently expressing the three biomarkers.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> affiliated hospital of Nantong university
<120> biomarker for detecting nasopharyngeal carcinoma prognosis and application thereof
<130> 2020.06.15
<141> 2020-06-15
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> RNA
<213> Homo sapiens
<400> 1
aaaagugcuu acagugcagg uag 23
<210> 2
<211> 1542
<212> DNA
<213> Homo sapiens
<400> 2
actcgtgtgc gcgctcgtcc gcccgccagt cctctcaacg cgcgcttggc cgcccgacga 60
cgcgggagcc gcacgcgccg gacgaggctc gctgcgctcc ctgttgccca gcgcgggccc 120
gttgaggcgg agccctcagt tcccggccag gacacggtct gggccgccga atctccggcc 180
gaagagcggc ggcggcagcg gcgggaaaaa aatgaagaat gaaattgctg ccgttgtctt 240
ctttttcaca aggctagttc gaaaacatga taagttgaaa aaagaggcag ttgagaggtt 300
tgctgagaaa ttgaccctaa tacttcaaga aaaatataaa aatcactggt atccagaaaa 360
accatcgaaa ggacaggcct acagatgtat tcgtgtcaat aaatttcaga gagttgatcc 420
tgatgtcctg aaagcctgtg aaaacagctg catcttgtat agtgacctgg gcttgccaaa 480
ggagctcact ctctgggtgg acccatgtga ggtgtgctgt cgtagagatg gggtttcacc 540
atgttggcca gactgctctc aaactcctga cctcgtgatc cgcccgcctt ggcctcccaa 600
agcgctggat tacaggcgtg agccactgcg cccggcctcc tcctttttga ttatgtatgg 660
agagaaaaac aatgcattca ttgttgccag ctttgaaaat aaagatgaga acaaggatga 720
gatctccagg aaagttacca gggcccttga taaggttacc tctgattatc attcaggatc 780
ctcttcttca gatgaagaaa caagtaagga aatggaagtg aaacccagtt cggtgactgc 840
agccgcaagt cctgtgtacc agatttcaga acttatattt ccacctcttc caatgtggca 900
ccctttgccc agaaaaaagc caggaatgta tcgagggaat ggccatcaga atcactatcc 960
tcctcctgtt ccatttggtt atccaaatca gggaagaaaa aataaaccat atcgcccaat 1020
tccagtgaca tgggtacctc ctcctggaat gcattgtgac cggaatcact ggattaatcc 1080
tcacatgtta gcacctcact aacttcgttt ttgattgtgt tggtgtcatg ttgagaaaaa 1140
ggtagaataa accttactac acattaaaag ttaaaagttc ttactaatag tagtgaagtt 1200
agatgggcca aaccatcaaa cttattttta tagaagttat tgagaataat ctttcttaaa 1260
aaatatatgc actttagata ttgatatagt ttgagaaatt ttattaaagt tagtcaagtg 1320
cctaagtttt taatattgga cttgagtatt tatatattgt gcatcaactc tgttggatac 1380
gagaacactg tagaagtgga cgatttgttc tagcaccttt gagaatttac tttatggagc 1440
gtatgtaagt tatttatata caaggaaatc tattttatgt cgttgtttaa gagaattgtg 1500
tgaaatcatg tagttgcaaa taaaaaatag tttgaggcat ga 1542
<210> 3
<211> 12401
<212> DNA
<213> Homo sapiens
<400> 3
cagtcgaccc agcccccgag actccctcac gccgctccaa aaccaaaacg gagcccaaca 60
cgaagctggg tgaagccgta gcttgcagga gccagggaga tgcgctctgc ccgggacttc 120
ccgggtcctg ttgagacgga aaggatcgca gggaagacag gataattggt ctttaactct 180
gaccgttacc ttcgaaattg caccttacaa gcatgtcctc tatttccgtg gcttgatggg 240
gacactttct ctgattaaga tccggaataa tcacccaccc aatgggatct cttagaggga 300
agcgctggtt agaatccctc caggctgaga tgcaatcatc gccattgagc ctgccctccc 360
ctagggaaac cccacaaaac catcctgaga atctcagccc tctttggtcg gtacctgaga 420
ctgcatagac ctaaactgaa aggtgatcga aggggaggag cagttgctcg tgcggaggaa 480
gctcttgggg ccgaagtggc cccggccgag tatgtcgcaa aggaggcacc actgcgttga 540
gcgcttactg tatctttact tttaaatcac caaggcaaaa tcaccttcag ctaaaaatgc 600
ctgaaagact ttttaaaata gtgggagtgg gcgttagggg gaagagatgg cctaggaaat 660
tttccgcagg gcgttctagg gatgagttga gggaagccgc atgacgcgcg gctccccggt 720
gccacagcta aggacagata ttttcgcaaa acccagaatg aaaaaagagc acgctccctt 780
tgggagcgct gtccctttgg gactggggcc ttccactccc acccctcctt tttccctgat 840
cggctccgca gctccacgac aagccagctg gtctggtctc tgacttgggc tccggtccgt 900
acccccgggg cgccctgctg tgttacagcc gcccgacgcc cccagacccg gccaggtcac 960
cagggcagat tggaggttcg cgccccctca cccgacacct cccgtccacc tcatctttct 1020
ttttttcgaa aacaggaaag ggaagaaaac tgaaacgggc ctcttggtct gcagttttag 1080
cggagtcggg attccacagc cctgagtggc acatgccggt caacttccca aagtcgggct 1140
cccgtgtggg gagaaataca cacgcaggaa tgcacaagca tcgcgtgttc gcaactgtcg 1200
ctgggaggtc tggcggctgt gatgggacat gcactcactc gggcaacacg tccacaggtg 1260
acatctattc gatcagtcaa cagatattta ttgcgcacct acgacgctat ctggtacttt 1320
ccttttcccc ttagctgttg ctgtccagcc cagcgccagt ccagagacgg gcttaccggc 1380
cgctcccttc cacaagaaac tcgtgtccgg cgtgaagagc ggacgataaa cactctccaa 1440
gcccagcgaa atgtactaat tacaggagcg gctgggagcc cgcgacacgg ggacctgtcc 1500
ctccagggat aggagtcttc tacctgcaag ggacccggcc tggggaactg gaatagacaa 1560
ggggtaaccc cttctcccat cacctctaat gagaacattg tccggggctg agatatttgg 1620
gggacgccgc tgctcacccc accaaacttt ccctagggac agaaaagcgt ccctaccgaa 1680
aacggattta aacaccgtcc ccacttttgc taaggaagtt tggataggga ggttgtggtc 1740
cttaattcgc caggtaaaag gaaccccatc agctcgcatc cctcaccccc atcctgagca 1800
ctgagtgttt gcgagaaact cgccctgcgc cagcaactct gggctccggt ctcagctccc 1860
gcgtagacgc ggaggagaga ttggcggccc ccgctgggga aattgagccc ggcgccggtc 1920
cgggacgtgc aagaggggga aagggggaga tcccagacct cggtttcccg agggcttccc 1980
tggggagcca ctcgcacctc tgtgtctccg ctcccggcgg cagacctcgc ggcgcttgct 2040
caagagcggg tatccgaaag cacgtcccga acacggagaa gcgccctccg tctcctgcct 2100
cgtgacctgc gccctttccc tccctcaccg catcagatgg aggaccctgg ctcctttcgc 2160
ccctggcaaa atgctctagc caacactggg tcaacagcct tagtttccag atctgtaacc 2220
tgaacatcag gagcgggttc ctttccttct acctgtgtct gaggtcctct ggtcggggac 2280
tgcgcgcgtc ttcccaggtc agggaggagg ggtcggggcg acctgagaaa gacacaacga 2340
agcgaatgga gcccctgaat ggggaaaggg gagtggacac caggaggccc ctgcagaaat 2400
gcaggaccga tccactcgcg agctgacaat gccacgcgct agacttggcc tgacggtggc 2460
aacgcttggc acctggggga taggggcctt ctcctggcct cagtccccaa attctgcgca 2520
gaatagaacg gctggcatct caagtgctct tttttcaccc ggatctctac agacacaagt 2580
tccctcactg agctgcgcag aggtggatcc gcacaggagg gaatatcgcg taggaattcc 2640
tgcaaatata aatctcagca gcaaagcagt ccagattgac tggaacacac cacccccagc 2700
caccaccatc caagttttcc ctggttctcg gagttggaaa cagtttctgg gcttttggcg 2760
ccccctctgg atacagatga aaactgcagt ccatttttaa gtcgcctgtt cataagtttt 2820
caggaagatg taaaattcaa aaatgcttgc atcaaatcaa cgggaaatgt ataaatcccc 2880
gtcgtttatg atgtaaaact ctacatatta gtatcttatt gcataattgc attaaaagta 2940
tatttataca tatacgtata tatcttgaca tatatttagt gaaatattcc aaaatatctt 3000
aaagtcatag ccctcttcac tgactttatt ccagcaaaca tggtaattac aatatcatac 3060
aactaagtac agacgacctg gctaaaatgt ctgcccgatg gtctccgagt ctcctgaatc 3120
aggtgtctga gaattacaca gttatactgt acacacaatg cccttctacc cgggacacag 3180
aaataggtcc acactacgcg gacttttttc tcctaggaaa ggacgatgct gttcttacac 3240
tttctgaaag taatcacaga gccctggata cgaagctatt gtatgcaaat ctcttaaatt 3300
tgtaaacgag ctatagggca ccagaaacat cccatttgaa gaagttacat tcgttaaaaa 3360
aaaaatgctg ttgaacaagg ctgtaactta ctaccttatg aagagttcca tttcctataa 3420
gaaatgtcgg gcttcttccg aagacaacga gagaaaacaa gattttaaga aattctcccg 3480
gaaggacatt gatttggatc ttgtgatagt gtcctcactt cgcaaattag aaagggaaaa 3540
aaaaactagt ttttattatg atgtgggccg attcaccaca acaataattt aattgaggcg 3600
aatttttgca agagcccaaa agggtggggg ggggggggga gtttaaaatt aagagtttcc 3660
caatgctgtg cgtttatttg ggattctgaa agcacagaac ccgcaagcga ccaagacttt 3720
tcttctatcc cagagcagat agctccgcac ttacccaacc tggctctaag catttcgtgt 3780
aaacacaaag gttgtgctca aatcacactt gaaatacatg agagacacca ccaatgcctc 3840
ccccagaact cccaactaca tgcaggtctg aaccgaccgg actagagtca gaagcaccgg 3900
cgcttcaccc cttcaggtgt ttcgtaaatg ccagcaaaag caggcaagca gcatgactcc 3960
gccagagtgg agcgttttgt ctgcggtggt gcccatttgt ttggtctttt acaaaccaag 4020
tgaccggcct gggcctcgcg gcccgggaca gccgcattgg caaacttcta tctctcaaag 4080
ccagagcagt tagcaaactc tcccccagac agggcgactc ggctgacgtt tttgacccgg 4140
ccaggaggca aagaccaaaa cgtcagagca gtagccctgt tactgaggag cgtcggcagg 4200
gtcgcgggta gagggggctg gagaatgact tgtcagagct caaggtcgat gtggcgcggg 4260
gcggcctcga gagcgccggg ctcctgcgtg gccacggccg ccgctgccaa ccttcgcggg 4320
gacttagctt tgctttccat tgactccctt tgcaaaagcg cagcagaatc ctgaccagcc 4380
gcaccagccc cggcgaaccc gagcatgtta atctatttat atggattatt acggaggaac 4440
agcgggcgtt gagtcaccaa aacatttgct tcaaaagact atttctaagc acttttgcag 4500
gcaggcaggc tcgctccagg cgcgtaaact cggctacgca ttaagaagcg gctgcttttc 4560
gaatactgca aactccagct aagtccccgg tgccgcggag agagcagtga aaagaaatgt 4620
cggaggtggg ggtagatcct agtctagaca cacacacttg cgcgcacaca cacacacaca 4680
cacacaagat tcgcgcggag aaggcactaa aattctggca ttccgagagt acgacaaact 4740
tacacacttg gaagtcccgg gtcccccgcc ttccccgcag caccccccgc ccccccaccc 4800
taccgtccgc cctttggctg cgatcccctc ccctctcctc ccctcccgcc tcgtcaccca 4860
gcccagtgcc acaatcctcc tccctcccca aaatcgggtc caatcagctg cctgccaacc 4920
ctgggactgc tgtgctgtga ttggcgggtg gctctaaggt gaggcggagt atttattaaa 4980
gagaccctgg gctgggagtt ggagagccga aagcggagct cgaaactgac tggaaacttc 5040
agtggcgcgg agactcgcca gtttcaaccc cggaaacttt tctttgcagg aggagaagag 5100
aaggggtgca agcgccccca cttttgctct ttttcctccc ctcctcctcc tctccaattc 5160
gcctcccccc acttggagcg ggcagctgtg aactggccac cccgcgcctt cctaagtgct 5220
cgccgcggta gccggccgac gcgccagctt ccccgggagc cgcttgctcc gcatccgggc 5280
agccgagggg agaggagccc gcgcctcgag tccccgagcc gccgcggctt ctcgcctttc 5340
ccggccacca gccccctgcc ccgggcccgc gtatgaatct cctggacccc ttcatgaaga 5400
tgaccgacga gcaggagaag ggcctgtccg gcgcccccag ccccaccatg tccgaggact 5460
ccgcgggctc gccctgcccg tcgggctccg gctcggacac cgagaacacg cggccccagg 5520
agaacacgtt ccccaagggc gagcccgatc tgaagaagga gagcgaggag gacaagttcc 5580
ccgtgtgcat ccgcgaggcg gtcagccagg tgctcaaagg ctacgactgg acgctggtgc 5640
ccatgccggt gcgcgtcaac ggctccagca agaacaagcc gcacgtcaag cggcccatga 5700
acgccttcat ggtgtgggcg caggcggcgc gcaggaagct cgcggaccag tacccgcact 5760
tgcacaacgc cgagctcagc aagacgctgg gcaagctctg gaggtaggac ccggcggggg 5820
cggcgcggca gggtgggcat cgcggcggct gggggcgctg gtcagggctg atttgccccg 5880
ccccgcctcc catcgcccgg gagttgccgt tccgggagcc ggcgggatgg ggttgggagt 5940
gggaatgggg tgtaactgtg gctcagagtt tgacaaagtt cttgggctgc tcgcggggac 6000
gcggaggagg ggggtggtaa gtggaagagg tgagggaggt agctggagga tggacgaaga 6060
ctggtgggag acggaaggag ggggctgcca gcctgctctc cagtcgcctg gaagctcaat 6120
cggggcgggg aagtgaaact tgcctccctc ctacccggcc tcttaaaact gcactctctc 6180
gtgcagcccc actgtccacg gagatggggc aagggagaaa ccgaggttgg aggagaccct 6240
tggcaggaac tgggaggcgg gaggagggag gctactggaa ataggtggga gtgtatggtg 6300
gggggtgaga attggggacc ttcttgcagc ttaagtaatt tgggggaaag ttttcaaagg 6360
gggttggggt tgggggcggt aagtcgagca gcaaaggcgt ttagggggca gcaccgggag 6420
tcgttttcat ctccagcgtt tccaaaatag aaatagaagg ggaggggagg gagggggcgg 6480
ggagtgaccg ctcaggtcag actgcaataa cttatttatt tatttatttt taagaaaagt 6540
tatgagctgt ggttgcaggc aggagggaag atggagttgt gtgcagagga agccgagtgg 6600
tctgggtcgc cgcctcctcc ccgccgacct gacagtttgg cggatttcac tgacccctct 6660
ccctcttttt ctctgtgccc cccgccccgc cccgagcaga cttctgaacg agagcgagaa 6720
gcggcccttc gtggaggagg cggagcggct gcgcgtgcag cacaagaagg accacccgga 6780
ttacaagtac cagccgcggc ggaggaagtc ggtgaagaac gggcaggcgg aggcagagga 6840
ggccacggag cagacgcaca tctcccccaa cgccatcttc aaggcgctgc aggccgactc 6900
gccacactcc tcctccggca tgagcgaggt gcactccccc ggcgagcact cgggtgagtc 6960
gcccctcgac cccaccggac aagctatctc cgtcccgcct ggcacacccc ctgccctccg 7020
cctgggagat tcttcgtggg gactttatgc ttcccgggag ggacacactg ccctttgcgc 7080
ccgtcccgct cccctctcta cccagagcct aagaggcatc caaacaacac acacacaaac 7140
acacacaccc caactcaatc ccagcatccg aagagattaa cttttttatt gggaggtaaa 7200
atgcccttaa cagccttaca agacctctcc cttcttctct gctcccccac cccaaaagca 7260
cacacagggc tcttacacaa gtagcaatta ggtcttccgg accctccggg ccccagaccc 7320
tcccctgata aaagggggct gtccagtgtg taccggcggg ttaatcattg ggcgacttat 7380
ctccggtgca gcgcgcctct tgcgcgggtg cgggccctta ttacacttta gcagcgaggg 7440
agggtccccg gagggtgcct aagactaggg cgtctgcaca gcccttgttg attttctcgt 7500
gcttgttctt ttattgtcca cagggcaatc ccagggccca ccgaccccac ccaccacccc 7560
caaaaccgac gtgcagccgg gcaaggctga cctgaagcga gaggggcgcc ccttgccaga 7620
ggggggcaga cagcccccta tcgacttccg cgacgtggac atcggcgagc tgagcagcga 7680
cgtcatctcc aacatcgaga ccttcgatgt caacgagttt gaccagtacc tgccgcccaa 7740
cggccacccg ggggtgccgg ccacgcacgg ccaggtcacc tacacgggca gctacggcat 7800
cagcagcacc gcggccaccc cggcgagcgc gggccacgtg tggatgtcca agcagcaggc 7860
gccgccgcca cccccgcagc agcccccaca ggccccgccg gccccgcagg cgcccccgca 7920
gccgcaggcg gcgcccccac agcagccggc ggcacccccg cagcagccac aggcgcacac 7980
gctgaccacg ctgagcagcg agccgggcca gtcccagcga acgcacatca agacggagca 8040
gctgagcccc agccactaca gcgagcagca gcagcactcg ccccaacaga tcgcctacag 8100
ccccttcaac ctcccacact acagcccctc ctacccgccc atcacccgct cacagtacga 8160
ctacaccgac caccagaact ccagctccta ctacagccac gcggcaggcc agggcaccgg 8220
cctctactcc accttcacct acatgaaccc cgctcagcgc cccatgtaca cccccatcgc 8280
cgacacctct ggggtccctt ccatcccgca gacccacagc ccccagcact gggaacaacc 8340
cgtctacaca cagctcactc gaccttgagg aggcctccca cgaagggcga agatggccga 8400
gatgatccta aaaataaccg aagaaagaga ggaccaacca gaattccctt tggacatttg 8460
tgtttttttg tttttttatt ttgttttgtt ttttcttctt cttcttcttc cttaaagaca 8520
tttaagctaa aggcaactcg tacccaaatt tccaagacac aaacatgacc tatccaagcg 8580
cattacccac ttgtggccaa tcagtggcca ggccaacctt ggctaaatgg agcagcgaaa 8640
tcaacgagaa actggacttt ttaaaccctc ttcagagcaa gcgtggagga tgatggagaa 8700
tcgtgtgatc agtgtgctaa atctctctgc ctgtttggac tttgtaatta tttttttagc 8760
agtaattaaa gaaaaaagtc ctctgtgagg aatattctct attttaaata tttttagtat 8820
gtactgtgta tgattcatta ccattttgag gggatttata catattttta gataaaatta 8880
aatgctctta tttttccaac agctaaacta ctcttagttg aacagtgtgc cctagctttt 8940
cttgcaacca gagtattttt gtacagattt gctttctctt acaaaaagaa aaaaaaaatc 9000
ctgttgtatt aacatttaaa aacagaattg tgttatgtga tcagttttgg gggttaactt 9060
tgcttaattc ctcaggcttt gcgatttaag gaggagctgc cttaaaaaaa aataaaggcc 9120
ttattttgca attatgggag taaacaatag tctagagaag catttggtaa gctttatcat 9180
atatatattt tttaaagaag agaaaaacac cttgagcctt aaaacggtgc tgctgggaaa 9240
catttgcact cttttagtgc atttcctcct gcctttgctt gttcactgca gtcttaagaa 9300
agaggtaaaa ggcaagcaaa ggagatgaaa tctgttctgg gaatgtttca gcagccaata 9360
agtgcccgag cacactgccc ccggttgcct gcctgggccc catgtggaag gcagatgcct 9420
gctcgctctg tcacctgtgc ctctcagaac accagcagtt aaccttcaag acattccact 9480
tgctaaaatt atttattttg taaggagagg ttttaattaa aacaaaaaaa aattcttttt 9540
tttttttttt tccaatttta ccttctttaa aataggttgt tggagctttc ctcaaagggt 9600
atggtcatct gttgttaaat tatgttctta actgtaacca gttttttttt atttatctct 9660
ttaatctttt tttattatta aaagcaagtt tctttgtatt cctcacccta gatttgtata 9720
aatgcctttt tgtccatccc ttttttcttt gttgtttttg ttgaaaacaa actggaaact 9780
tgtttctttt tttgtataaa tgagagattg caaatgtagt gtatcactga gtcatttgca 9840
gtgttttctg ccacagacct ttgggctgcc ttatattgtg tgtgtgtgtg ggtgtgtgtg 9900
tgttttgaca caaaaacaat gcaagcatgt gtcatccata tttctctgca tcttctcttg 9960
gagtgaggga ggctacctgg aggggatcag cccactgaca gaccttaatc ttaattactg 10020
ctgtggctag agagtttgag gattgctttt taaaaaagac agcaaacttt tttttttatt 10080
taaaaaaaga tatattaaca gttttagaag tcagtagaat aaaatcttaa agcactcata 10140
atatggcatc cttcaatttc tgtataaaag cagatctttt taaaaagata cttctgtaac 10200
ttaagaaacc tggcatttaa atcatatttt gtctttaggt aaaagctttg gtttgtgttc 10260
gtgttttgtt tgtttcactt gtttccctcc cagccccaaa ccttttgttc tctccgtgaa 10320
acttaccttt ccctttttct ttctcttttt tttttttgta tattattgtt tacaataaat 10380
atacattgca ttaaaaagaa agtggccctg tggatttatt cacccagttc tcctgttgga 10440
tgatttggca aatttgagca caaaagacat tgtggagtgc tgattgctgt gatgtttttg 10500
ttttttatca gcacctttgg caatcctaga aaacaagctg aggaaaagac tgtatctcca 10560
aaaatctagg cagaaaaatc ttgaaaagtg ccactccaat agatcacaca gaaaattaca 10620
tgtcagtagt tgctcactct ggcaaaaatg tttgttgtgg ttttcatgac ctcatgcttc 10680
agggcaaaag ctgtcccttg tggaggtcac agggaatatt agacctgaaa tcaggagctt 10740
gtcagtagac agaatgccag aggggtgact tgcttatctg gttttaatat ggcaaacttt 10800
ccgttcctat gaacacatac ccaagaaatg gagtatccac ttaaaaagca aaggaagcca 10860
gagaaaatca gtgtctacag ggaaccagag agaagcctgt cgtattaacc cattaaatga 10920
ttcagagcct tccagatttc tctgtagaga caatgaaagg ggatgatttt tctgctccct 10980
ccagtttaac tcattctaag cagacgcaaa gccattgtag aagaaacaag acctaatcct 11040
gttttccttg gccccagtta gatggggagt ttccaggttc agagaaacgt tcaggtcatt 11100
tttcattaaa tagatgaaat cactccttcc ctcacatggg cacacctccc catccccact 11160
cccacacccc agatgattgg agaagtgtct ctaaagagtc agtcaccttc accctctcag 11220
gctagtttta ctactgggcc tgtgtctctg gcagtcttaa ccagagtcca acctctctgg 11280
cattacctca cattgtctgt ttctgcttat tggattcctg gctgacatcc cacaccagga 11340
acatggcaac caggaaaatg gtagttatca aatcgaggca acattagtac tcggcctcct 11400
ttgcaaattc cttcgacaaa cattgattga gggcctgtga tctgcggggg actgtgcaaa 11460
tccttcccta ccccttcaga catcaaattc tgttccttgt ttgtgacata gccttccatg 11520
cgctgaggcc ctcccttctt acaaaatctc tatttttttt ttctcttgcc aacactcaca 11580
aagtcctttt taaaagtctc tgtggtgttc actaattctt gcctacctta ttcgttctaa 11640
tccctttgtc tattaaaatc aacacacacc attccactgc gactaccccc ttccctagca 11700
aatctgtatg tttgctagac tgttctccct tccagaaata tgctcttttt cccagtatgt 11760
ggcctttgcc aggacttctt gaagccaaaa tattctgttc ggacataaac ttgtgaaggg 11820
aactgaagca agttgttttg ctttcccgtg cctcactttc cccttttgct gagtatgttt 11880
ataattcctc ctacatagag cagggttgtt aatagaggag attttagcca gagcaatctg 11940
ggagttgact gcattggagt ctcctggtct gtacagcagt ccccctgcat tagaattcct 12000
gggagtaata gccaggaaaa agagtttcat aagctcccca agtggctctg atacacagta 12060
aaatgcaaga gacacaggag tgctgggaga gaattcctca gaatcccgct cacccccata 12120
tctatcagat ctagtcaaag cccaatatca ccagattata aaatattttg gaggaatttt 12180
tagtctaaca ccagaagcac agagcttatc accctggcaa cagcaacaca ttgactggaa 12240
acctaggaga gtcctccaca ccggaaagag tttgtatgta gatccagggt tatttgtttg 12300
tttgcactta aaataaaacc ttttcttttc taaccatttt aacaaaactg aaatttttag 12360
caaaagggaa taaaaataaa ctcatttaag aatcggtctt t 12401
Claims (3)
1. The biomarker for detecting nasopharyngeal carcinoma prognosis is characterized by consisting of miR-106a-5p, miR-106a-5p target gene BTG3 and miR-106a-5p upstream transcription factor SOX9, wherein the sequence of miR-106a-5p is SEQ ID NO.1, the sequence of BTG3 is SEQ ID NO.2, and the sequence of SOX9 is SEQ ID NO. 3.
2. Use of the biomarker for detecting nasopharyngeal carcinoma prognosis according to claim 1 in the preparation of a reagent or a tool for detecting nasopharyngeal carcinoma prognosis.
3. An application of a biomarker for detecting nasopharyngeal carcinoma prognosis in preparing a reagent or a tool for detecting nasopharyngeal carcinoma prognosis, wherein the biomarker is BTG3, and the sequence is SEQ ID NO. 2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642900A (en) * | 2006-01-05 | 2014-03-19 | 俄亥俄州立大学研究基金会 | Micrornarna-based methods and compositions for the diagnosis and treatment of solid cancers |
| CN111154870A (en) * | 2019-08-05 | 2020-05-15 | 江苏省肿瘤医院 | Biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005078139A2 (en) * | 2004-02-09 | 2005-08-25 | Thomas Jefferson University | DIAGNOSIS AND TREATMENT OF CANCERS WITH MicroRNA LOCATED IN OR NEAR CANCER-ASSOCIATED CHROMOSOMAL FEATURES |
| US20090136957A1 (en) * | 2007-09-15 | 2009-05-28 | Irena Ivanovska | Methods and compositions for regulating cell cycle progression via the miR-106B family |
| CN106119405B (en) * | 2016-08-31 | 2019-11-12 | 广州万德基因医学科技有限公司 | A kind of prognostic markers object of lung cancer, the method and application for using label prediction lung cancer for prognosis |
-
2020
- 2020-06-15 CN CN202010544426.9A patent/CN111500739B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642900A (en) * | 2006-01-05 | 2014-03-19 | 俄亥俄州立大学研究基金会 | Micrornarna-based methods and compositions for the diagnosis and treatment of solid cancers |
| CN105907842A (en) * | 2006-01-05 | 2016-08-31 | 俄亥俄州立大学研究基金会 | Methods for diagnosing breast cancer using microRNAs |
| CN111154870A (en) * | 2019-08-05 | 2020-05-15 | 江苏省肿瘤医院 | Biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation |
Non-Patent Citations (8)
| Title |
|---|
| Homo sapiens BTG anti-proliferation factor 3 (BTG3), transcript variant 1, mRNA;Wang L等;《GenBank》;20200512;Accession No.NM_001130914.2 * |
| Homo sapiens SRY-box transcription factor 9 (SOX9), RefSeqGene on chromosome 17;Wagner T等;《GenBank》;20200608;Accession No.NG_012490.1 * |
| MicroRNA-106a suppresses proliferation, migration, and invasion of bladder cancer cells by modulating MAPK signaling, cell cycle regulators, and Ets-1-mediated MMP-2 expression;Seung-Shick Shin等;《Oncology Reports》;20160811;第36卷(第4期);摘要、第2422页左栏第1段 * |
| MiR-106 b通过靶向BTG3调控非小细胞肺癌的增殖与细胞凋亡;史文博等;《解放军预防医学杂志》;20180430;第36卷(第4期);摘要 * |
| MIR106A-5p upregulation suppresses autophagy and accelerates malignant phenotype in nasopharyngeal carcinoma;Qingwen Zhu等;《Autophagy》;20200705;第17卷(第7期);第1667-1683页 * |
| Shu-biao Ye等.Tumor-derived exosomes promote tumor progression and T-cell dysfunction through the regulation of enriched exosomal microRNAs in human nasopharyngeal carcinoma.《Oncotarget》.2014,第5卷(第14期), * |
| SOX9 promotes nasopharyngeal carcinoma cell proliferation, migration and invasion through BMP2 and mTOR signaling;Bin Xiao等;《Gene》;20190726;第175卷;摘要 * |
| Tumor-derived exosomes promote tumor progression and T-cell dysfunction through the regulation of enriched exosomal microRNAs in human nasopharyngeal carcinoma;Shu-biao Ye等;《Oncotarget》;20140619;第5卷(第14期);摘要、第5446页左栏第2段至第5447页右栏第2段、表1 * |
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