Use of e33 region methylation levels in assessing medulloblastoma prognosis

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to application of E33 region methylation level in evaluation of medulloblastoma prognosis. The nucleotide sequence of the E33 region is shown in SEQ ID NO:1; the medulloblastoma comprises G3 subtype medulloblastoma and / or G4 subtype medulloblastoma. The application finds that the low methylation state of the E33 region is directly related to the prognosis of the medulloblastoma Group 3 / 4 patient, the high / low methylation level of the E33 region can be detected to effectively predict the clinical result of the patient, and the methylation level of the E33 region is detected to lay a foundation for early prognosis evaluation and personalized treatment strategy of the medulloblastoma Group 3 / 4 patient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and particularly relates to application of E33 region methylation level in evaluation of medulloblastoma prognosis. BACKGROUND

[0002] Medulloblastoma (MB) is the most common malignant central nervous system tumor in children, accounting for 8-10% of brain tumors in children. Early detection and early treatment can effectively improve the five-year survival rate of patients. Medulloblastoma is not a single disease, but a brain tumor composed of multiple different molecular subtypes. Currently recognized molecular subtypes of medulloblastoma mainly include four subtypes: WNT subgroup, Sonic Hedgehog subgroup, Group3, and Group4. These subtypes are different in treatment and prognosis, and correspond to different clinical strategies.

[0003] At present, the alternative solutions for medulloblastoma prognosis evaluation mainly include analysis methods based on gene mutation, copy number variation, traditional epigenetic markers (such as DNA methylation profile), RNA expression characteristics and specific molecular markers (such as Myc amplification). These alternative solutions can provide prognosis information to some extent, especially for different subtypes of medulloblastoma, but usually have the following limitations: (1) low prediction accuracy: single gene mutation or copy number variation is difficult to accurately reflect the biological complexity and heterogeneity of the tumor, and the specificity and sensitivity of the prediction result are low, especially for G3 and G4 subtype medulloblastoma with high molecular heterogeneity. (2) Lack of information on cell differentiation state: traditional DNA methylation profile and RNA expression characteristic analysis methods focus on the overall sample level, and it is difficult to capture the characteristics of different differentiation stages at the cell level, and the prognosis of medulloblastoma patients is often closely related to the cell differentiation state.

[0004] Otx2 It is known that E2F3 has a high expression in G3 subtype of medulloblastoma and is closely related to undifferentiated state and increased invasiveness. However, Otx2 The regulatory mechanism and specific epigenetic regulatory region of E2F3 have not been further clarified. SUMMARY

[0005] The purpose of the present application is to provide application of E33 region methylation level in evaluation of medulloblastoma prognosis, effectively predict the clinical outcome of medulloblastoma patients, evaluate the prognosis of medulloblastoma, and have high sensitivity and specificity.

[0006] The present application provides application of a substance for detecting E33 region methylation level in preparation of a product for evaluating medulloblastoma prognosis.

[0007] The nucleotide sequence of the E33 region is shown as SEQ ID NO: 1;

[0008] The medulloblastoma includes G3 subtype medulloblastoma and / or G4 subtype medulloblastoma.

[0009] Preferably, the prognosis includes one or more of a clinical outcome of the subject, a treatment effect of the subject, and a survival of the subject.

[0010] Preferably, the survival of the subject includes a survival period of the subject.

[0011] Preferably, the survival period of the subject includes one or more of a one-year survival rate, a three-year survival rate, and a five-year survival rate.

[0012] Preferably, the methylation level of the E33 region is significantly down-regulated in a high-risk medulloblastoma patient.

[0013] Preferably, the product includes: a kit, a gene chip, or a device containing a gene detection module for detecting the methylation level of the E33 region.

[0014] The application provides a prognosis evaluation device for medulloblastoma, comprising: a gene detection module and a prognosis evaluation module.

[0015] The medulloblastoma includes G3 subtype medulloblastoma and / or G4 subtype medulloblastoma.

[0016] The gene detection module is used for detecting a methylation rate of an E33 region; the nucleotide sequence of the E33 region is shown as SEQ ID NO: 1;

[0017] The prognosis evaluation module is used for determining a prognosis condition of a medulloblastoma patient according to the methylation rate of the E33 region, including: when the methylation rate of the E33 region is ≤1%, the patient is a high-risk patient, and the prognosis condition is not good; when the methylation rate of the E33 region is ≥20%, the patient is a low-risk patient, and the prognosis condition is good.

[0018] Preferably, the prognosis evaluation device further comprises a calibration module; the calibration module is used for constructing a multi-factor COX regression model of the methylation rate of the E33 region and patient clinical data.

[0019] Preferably, the patient clinical data includes gender, age, subtype, and metastasis state.

[0020] Beneficial effects:

[0021] The application provides application of a substance for detecting E33 region methylation level in preparation of a medulloblastoma prognosis evaluation product; a nucleotide sequence of the E33 region is shown in SEQ ID NO: 1; and the medulloblastoma includes G3 subtype medulloblastoma and / or G4 subtype medulloblastoma. The application finds that a low methylation state of the E33 region is directly related to the prognosis of a medulloblastoma Group 3 / 4 patient, and by detecting the E33 region methylation level, the clinical result of the patient can be effectively predicted, the E33 region methylation level lays a foundation for early prognosis evaluation and individualized treatment strategy of the medulloblastoma Group 3 / 4 patient, and the E33 region methylation level is significantly down-regulated in a high-risk medulloblastoma patient. The innovative achievement is expected to improve the treatment response of the patient and finally improve the survival rate. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

[0023] Figure 1 For the enhancer interaction network between a specific enhancer (SE) unit and its corresponding gene;

[0024] Figure 2 For the number and connectivity of the enhancer network mode;

[0025] Figure 3 For the interaction network of the enhancer related to Otx2

[0026] Figure 4 For the survival curve of the medulloblastoma Group 3 subtype;

[0027] Figure 5 For the survival curve of the medulloblastoma Group 3 subtype and Group 4 subtype;

[0028] Figure 6 For the result of the COX regression model evaluation on the effectiveness of the E33 region methylation level as an independent prognosis index;

[0029] Figure 7 For the ROC curve of the E33 region methylation level evaluation on the prognosis of the medulloblastoma. DETAILED DESCRIPTION

[0030] The application provides application of a substance for detecting E33 region methylation level in preparation of a medulloblastoma prognosis evaluation product. The E33 region in the application is Otx2 ​

[0031] As an implementation form, the prognosis comprises one or more of a clinical outcome of the subject to be prognosed, a treatment effect of the subject to be prognosed, and survival of the subject to be prognosed; as another implementation form, the prognosis comprises a clinical outcome of the subject to be prognosed, a treatment effect of the subject to be prognosed, and survival of the subject to be prognosed. As an implementation form, the survival of the subject to be prognosed comprises one or more of one-year survival rate, three-year survival rate, and five-year survival rate; as another implementation form, the survival of the subject to be prognosed comprises one-year survival rate, three-year survival rate, and five-year survival rate.

[0032] The application constructs an enhancer interaction network between specific enhancer (SE) units and their corresponding genes, finds a direct correlation between the hypomethylation state of the E33 region and the prognosis of medulloblastoma Group 3 / 4 patients, and can effectively predict the clinical outcome of patients by detecting the methylation level of the E33 region. The methylation state of the E33 region is expected to serve as an independent prognostic indicator, providing a new perspective for the clinical management of medulloblastoma, and providing a novel biomarker for the field of tumor epigenetics. Detecting the methylation level of the E33 region lays a foundation for early prognosis evaluation and personalized treatment strategies for medulloblastoma Group 3 / 4 patients, and the methylation level of the E33 region is significantly down-regulated in high-risk medulloblastoma patients. This innovative achievement is expected to improve the treatment response of patients and ultimately improve the survival rate.

[0033] As an implementation form, the product comprises: a kit, a gene chip, or a device containing a gene detection module for detecting the methylation level of the E33 region.

[0034] The application provides a prognosis evaluation device for medulloblastoma, comprising: a gene detection module and a prognosis evaluation module; the medulloblastoma comprises G3 subtype medulloblastoma and / or G4 subtype medulloblastoma;

[0035] The gene detection module is used for detecting the methylation rate of the E33 region; the nucleotide sequence of the E33 region is shown in SEQ ID NO: 1;

[0036] The prognosis evaluation module is used for determining the prognosis of a medulloblastoma patient according to the methylation rate of the E33 region, comprising: when the methylation rate of the E33 region is ≤1%, the patient is a high-risk patient and the prognosis is not good; when the methylation rate of the E33 region is ≥20%, the patient is a low-risk patient and the prognosis is good.

[0037] As an implementation manner, the prognosis evaluation device further comprises a calibration module; the calibration module is configured to construct a multi-factor COX regression model of the E33 region methylation rate and patient clinical data; the patient clinical data comprises gender, age, subtype, and metastasis state.

[0038] The application utilizes the above-mentioned prognosis evaluation device, and by detecting the methylation state of the E33 region, the prognosis of a tumor patient can be more accurately predicted, the feasibility and accuracy of clinical application are improved, the sensitivity is high, and the specificity is high.

[0039] In order to further illustrate the application, the application of the E33 region methylation level in evaluating the prognosis of medulloblastoma is described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0040] Example 1

[0041] 1. According to the prior art (doi: 10.1093 / bib / bbac508, doi: 10.1016 / j.devcel.2022.11.011), an enhancer interaction network (EIN) between specific enhancer (SE) units and their corresponding genes is constructed using the eNET algorithm Figure 1 .

[0042] 2. Each enhancer network is analyzed in depth to assess the number of network patterns and connectivity, thereby identifying three different network patterns: complex, multiple, and simple. In these networks, genes with the highest rank and most interactions (e.g. Celf4 and Otx2 ) are predicted to be key factors in maintaining cell identity and disease progression ( Figure 2 ). “Hub enhancers” are distinguished from other SE regions that co-regulate gene expression due to their higher chromatin interaction frequency. The “hub enhancer” network associated with Otx2 interacts extensively, and multiple core regions in this network exhibit a clear hypomethylation signature ( Figure 3 ). This finding indicates that the hypomethylation state of the E33 region, defined by WGBS (whole-genome bisulfite sequencing) data, is a newly discovered region that was not covered by previous methylation chip data.

[0043] 3. Survival analysis using patient clinical indicators and prognosis

[0044] (1) Collect tumor tissues of 56 patients with medulloblastoma Group 3 subtype (G3-MB), and precisely locate the E33 region by whole genome bisulfite sequencing (WGBS) technology. WGBS converts unmethylated C bases by bisulfite treatment to distinguish C bases with methylation modification, and combines high-throughput sequencing technology to determine whether the CpG site is methylated. The methylation rate of the E33 region in the tumor tissue of the patient is obtained in the following manner: first, calculate the methylation rate of a single CpG site: CpG methylation rate (%) = mC / (UmC + mC) x 100%; wherein, mC is the number of reads supporting methylation C, and UmC is the number of reads supporting unmethylated C; then average the methylation rate of the single CpG site in the region: E33 region methylation rate = Σ methylation rate of each CpG site / number of CpG in the region. According to the size of the methylation rate of the E33 region, sort from high to low, and select the top 20% of 11 patients as the low methylation group, and the bottom 20% of 11 patients as the high methylation group. Survival analysis is performed on the two groups of patients. The results show that the prognosis of patients with low methylation of medulloblastoma Group 3 subtype is usually poor (G3-MB) Figure 4 ), and the threshold value of the E33 region methylation rate of the low methylation group is 1%, and the threshold value of the E33 region methylation rate of the high methylation group is 20%.

[0045] (2) Collect tumor tissues of patients with medulloblastoma Group 3 subtype and tumor tissues of patients with medulloblastoma Group 4 subtype, and detect the E33 region methylation rate in the manner of step (1), and perform grouping and survival analysis. The threshold value of the E33 region methylation rate of the low methylation group (10 cases of medulloblastoma Group 3 subtype and 17 cases of medulloblastoma Group 4 subtype) is 1%, and the threshold value of the E33 region methylation rate of the high methylation group (11 cases of medulloblastoma Group 3 subtype and 16 cases of medulloblastoma Group 4 subtype) is 20%. The results show that the prognosis of patients with low methylation of medulloblastoma Group 3 subtype and Group 4 subtype is usually poor (G3-MB) Figure 5 ).

[0046] 4. Multivariate analysis verification

[0047] Collect tumor tissues from 56 patients with medulloblastoma Group3 subtype and 80 patients with medulloblastoma Group4 subtype, and collect clinical data of patients such as gender, age, subtype and metastasis status, calculate the methylation rate of E33 region of patients according to the method of step 3, sort the methylation rate of E33 region from high to low, and take the top 20% and the bottom 20% respectively for grouping, combine with the clinical data to establish the Cox proportional hazards model, and evaluate the independent influence of these factors on the prognosis of patients. Through the maximum likelihood estimation method, the coefficient (β value) of each variable in the model is estimated, and then the hazard ratio (HR) of each variable is calculated, that is, the exponential result of the coefficient, so as to measure the degree of influence of each variable on the risk of patients. Then, the 95% confidence interval (CI) of the hazard ratio of each variable is calculated, which is obtained by multiplying HR by the positive and negative 1.96 times the coefficient standard error, which provides the uncertainty range of parameter estimation. At the same time, the P value of the coefficient of each variable is calculated to evaluate the statistical significance of its influence on the prognosis of patients; P A value less than 0.05 is generally considered statistically significant.

[0048] Based on the results of the above multivariate regression analysis, it can be seen that after considering multiple clinical variables such as gender, age and metastasis status, the methylation level of E33 region is the only independent variable that has significant predictive value for patient prognosis. The risk ratio (HR) of low methylation level is greater than 1, P the value is less than 0.05, indicating that as the methylation level of E33 region decreases, the survival risk of patients increases, that is, the survival time of patients may be shortened, and the survival rate may be reduced ( Figure 6 ). This finding highlights the importance of E33 region methylation status in clinical prognosis evaluation and may provide new biomarkers for future treatment strategies and interventions. Therefore, the methylation level of E33 region can be used as a powerful prognostic indicator to help clinicians better evaluate the treatment response and survival expectation of patients.

[0049] 5. Sensitivity and specificity analysis

[0050] Based on the conclusion of step 4, the sensitivity, false positive rate, specificity and AUC key performance indicators of the multivariate Cox model were calculated to comprehensively evaluate the predictive ability of the model. Finally, the model performance under different survival periods (1 year, 3 years, 5 years) was compared and analyzed to determine the prediction accuracy and stability of the model at different time points, and the results are shown in Figure 7 and Table 1.

[0051] Table 1 Sensitivity and specificity analysis results

[0052]

[0053] According to Figure 7 As can be seen from Table 1, the methylation level of E33 can effectively evaluate the prognosis of medulloblastoma Group 3 subtype and Group 4 subtype, and has high sensitivity and specificity.

[0054] As can be seen from the above, the methylation level of E33 region can effectively predict the clinical outcome of medulloblastoma patients and evaluate the prognosis of medulloblastoma, and has high sensitivity and specificity.

[0055] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which belong to the protection scope of the present application.

Claims

1. Application of substances that detect methylation levels in the E33 region in the preparation of products for assessing the prognosis of medulloblastoma; The nucleotide sequence of the E33 region is shown in SEQ ID NO:1; The medulloblastoma is a G3 subtype medulloblastoma and / or a G4 subtype medulloblastoma.

2. The application according to claim 1, characterized in that, The prognosis refers to the survival time of the subjects.

3. The application according to claim 2, characterized in that, The survival period of the subjects with the prognosis is one or more of the following: one-year survival rate, three-year survival rate, and five-year survival rate.

4. The application according to claim 1, characterized in that, The methylation level of the E33 region was significantly downregulated in high-risk medulloblastoma patients.

5. The application according to any one of claims 1 to 4, characterized in that, The product includes: a reagent kit, a gene chip, or a device containing a gene detection module for detecting the methylation level in the E33 region.

6. A prognostic assessment device for medulloblastoma, characterized in that, include: Genetic testing module and prognostic assessment module; The medulloblastoma is a G3 subtype medulloblastoma and / or a G4 subtype medulloblastoma; The gene detection module is used to detect the methylation rate of the E33 region; the nucleotide sequence of the E33 region is shown in SEQ ID NO:

1. The prognostic assessment module is used to determine the prognosis of medulloblastoma patients based on the methylation rate of the E33 region, including: when the methylation rate of the E33 region is ≤1%, the patient is a high-risk patient with a poor prognosis; When the methylation rate of the E33 region is ≥20%, the patient is considered low-risk and has a good prognosis.

7. The prognostic assessment device according to claim 6, characterized in that, The prognostic assessment device also includes a calibration module; the calibration module is used to construct a multivariate Cox regression model using the methylation rate of the E33 region and the patient's clinical data.

8. The prognostic assessment device according to claim 7, characterized in that, The patient's clinical data included gender, age, subtype, and metastatic status.

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