Kit for detecting degree of methylation of mta1 associated with cerebral aneurysm and application thereof

Abstract

The application discloses a kit for detecting the degree of methylation of a gene related to cerebral aneurysm MTA1 and application thereof, characterized in that the kit comprises a pair of MTA1 gene DNA methylation-specific amplification primers and a gene DNA methylation-specific sequencing primer in the kit. MTA1 The specific amplification upstream primer has SEQ ID NO. 1, the specific amplification downstream primer has SEQ ID NO. 2, and the methylation-specific sequencing primer has SEQ ID NO. 3. MTA1 The region of the gene DNA detected by the sequence is a specific position at the front end of the gene, and the DNA methylation level is analyzed by a pyrosequencing technology. MTA1 The gene DNA methylation level is negatively correlated with the incidence of cerebral aneurysm; and the diagnostic kit can conveniently and quickly realize detection of cerebral aneurysm at a molecular level, has high detection efficiency and strong pertinence.

Description

Technical Field

[0001] This invention relates to the field of auxiliary diagnostic technology for cerebral aneurysms, and in particular to a method for detecting conditions related to the occurrence of cerebral aneurysms. MTA1 Kits for measuring methylation levels and their applications. Background Technology

[0002] Cerebral aneurysms are a cerebrovascular disease with an extremely high mortality rate. Rupture of cerebral arteries is the leading cause of death, with a mortality rate as high as 35% and a disability rate as high as 30%. Cerebral aneurysms have a very high latency period; they generally do not present with obvious clinical symptoms during their development. Patients usually discover they have a cerebral aneurysm incidentally when they seek medical attention after experiencing headaches or related symptoms. Furthermore, due to limitations in imaging technology and cost, patients often have poor awareness of their condition, so 80%-90% of unruptured aneurysms are discovered accidentally. Traditionally, the diagnosis of cerebral aneurysms relies mainly on magnetic resonance imaging (MRI) or cerebral angiography, but this often occurs in the middle or late stages of the disease, exhibiting a significant delay. Cerebral aneurysms are closely related to factors such as high blood pressure, stress, and fatigue; unhealthy lifestyle habits increase the risk of cerebral aneurysm occurrence and rupture. Once a cerebral aneurysm ruptures, it often endangers life or causes serious consequences, posing a significant threat to human life. Therefore, developing molecular markers that can predict cerebral aneurysms at an early stage is of great practical significance for their prevention and treatment.

[0003] Metastasis-associated protein 1 (MAP1) MTA1 )yes MTA MTA1, a founding member of the gene family, is primarily located in the cell nucleus and acts as a co-regulator to integrate DNA interacting factors into gene activity, thereby regulating gene expression. Recent research shows that MTA1 is an active regulator of angiogenesis, promoting angiogenesis. Researchers found that MTA1 expression in vascular endothelial cells promotes the formation of tubular structures. In various tumor cell lines, MTA1 expression levels are closely related to tumor tissue angiogenesis and invasion, while knockout... MTA1 Low expression levels can lead to impaired angiogenesis. The balance of angiogenesis-related regulatory factors in the human body plays a crucial role in the formation of cerebral aneurysms. Structural proteins and angiogenic factors exhibit different expression levels in normal blood vessel walls, ruptured aneurysm walls, and unruptured aneurysm walls. This suggests that MTA1 may play an important role in the occurrence and pathogenesis of cerebral aneurysms. Unhealthy lifestyle habits may alter DNA methylation levels, thereby changing... MTA1 Gene expression is involved in the pathological changes of cerebral aneurysms. Currently, there is no publicly available information, either domestically or internationally, regarding this. MTA1 Research reports on the association between gene DNA methylation and brain aneurysms. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a convenient, highly targeted, and highly accurate method for detecting cerebral aneurysms. MTA1 Reagent kits for gene methylation levels and their applications, among which MTA1 The degree of methylation is negatively correlated with the incidence of cerebral aneurysms, which can be detected by testing. MTA1 The degree of methylation can aid in the diagnosis of cerebral aneurysm occurrence.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a method for detecting cerebral aneurysms. MTA1 A kit for measuring gene methylation levels includes a pair of... MTA1 Gene methylation-specific amplification primers and one MTA1 Gene methylation-specific sequencing primers, wherein... MTA1 The nucleotide sequence of the upstream primer for gene methylation-specific amplification is shown in SEQ ID NO.1: 5'-GGTGGGGGGAGGTTTATTTG-3'; MTA1 The nucleotide sequence of the downstream primer for gene methylation-specific amplification is shown in SEQ ID NO.2: 5'-Biotin-CAACCCCAAAACTCCTACT-3'; MTA1 The nucleotide sequence of the gene methylation-specific sequencing primer is shown in SEQ ID NO.3: 5'-GGGGGAGGTTTATTTGTT-3'.

[0006] Furthermore, the aforementioned MTA1 The region where the gene DNA sequence was detected was within the CpG78 methylation island region Chr14 at the front of the gene: 105,420,312-105,420,399.

[0007] Furthermore, the specific locations of the 13 CpG sites within the Chr14 region (105,420,312-105,420,399) are as follows:

[0008] .

[0009] Furthermore, the aforementioned MTA1 The gene DNA sequence was analyzed using pyrosequencing technology. MTA1 DNA methylation levels were analyzed at 13 CpG sites within the gene.

[0010] Furthermore, the specific steps for determining the DNA methylation level are as follows:

[0011] (1) Bisulfite conversion: the sample DNA is subjected to bisulfite conversion using a bisulfite treatment kit;

[0012] (2) PCR reaction: 20 ng of the bisulfite-converted DNA sample is added to a Pyromark PCR kit, and a pair of MTA1 gene methylation-specific amplification primers is added for PCR amplification;

[0013] (3) Preparation for pyrosequencing;

[0014] (4) Pyrosequencing.

[0015] Further, the PCR amplification system in step (2) comprises: 34.8 microliters of double-distilled water; 10 microliters of 5x GC solution; 1 microliter of deoxyribonucleotide triphosphate, each at a concentration of 10 mM; 1 microliter of upstream primer, at a concentration of 50 pM per microliter; 1 microliter of downstream primer, at a concentration of 50 pM per microliter; 2 microliters of PCR reaction template; and 0.2 microliters of Taq enzyme, at a concentration of 5 U per microliter. The PCR amplification conditions are as follows: first, denaturation at 95°C for 3 min; then, annealing reaction at 94°C for 30 s, at 60°C for 30 s, and at 72°C for 1 min, for a total of 40 cycles; and then, extension reaction at 72°C for 5 min.

[0016] Further, step (3) is specifically as follows: 45 μl of annealing buffer containing 0.3 μM methylation-specific sequencing primer is pre-added to a PCR plate; the total amount of mixed agarose beads to be used is transferred to an Eppendorf tube; binding buffer is added to the agarose beads, so that the average volume of each sample is 50 μl, and the mixture is mixed; the mixture is added to the PCR product, 50 μl per sample; the PCR product is mixed at room temperature for 10 min, so that the magnetic beads are combined with the biotin; in a vacuum preparation workstation, 180 ml of high-purity water, 70% ethanol, washing buffer, and 120 ml of denaturing buffer are sequentially added to four sample plates; the pump of the vacuum preparation workstation is opened, and the vacuum preparation tool is washed in the high-purity water for 30 s; then, the vacuum preparation tool is moved to the PCR plate to grab the agarose beads; the PCR plate is taken out, and it is checked whether most of the magnetic beads are adsorbed on the vacuum preparation tool; the vacuum preparation tool is placed in 70% ethanol for 5 s; then, it is moved to the denaturing buffer for 5 s; and then, it is moved to the washing buffer for cleaning for 5-10 s; the pump is turned off; the vacuum preparation tool is placed in the plate containing the sequencing primer, shaken, and the agarose beads are released; the vacuum preparation tool is cleaned with high-purity water; the sample is placed on a heating plate, heated to 80°C for 2 min, and then cooled to room temperature, so that the pyrosequencing reaction can be performed.

[0017] Further, step (4) specifically involves sequencing the sample from step three using the Pyromark GoldQ24 kit on a Pyromark Q24 pyrosequencing instrument, and then performing methylation analysis on the results using Pyromark CpG software.

[0018] The above-mentioned methods are used to detect brain aneurysms. MTA1 Application of a kit for measuring the degree of methylation in gene promoter regions in the preparation of reagents for detecting or assisting in the diagnosis of cerebral aneurysms.

[0019] The above-mentioned methods are used to detect brain aneurysms. MTA1 Application of a kit for measuring the degree of methylation in gene promoter regions in the preparation of drugs for screening cerebral aneurysms.

[0020] Compared with the prior art, the advantages of the present invention are: the present invention discloses for the first time a method for detecting cerebral aneurysms. MTA1 A kit for measuring the degree of methylation in gene promoter regions and its applications. MTA1 The degree of methylation in gene promoter regions is negatively correlated with the incidence of cerebral aneurysms. This is used to detect... MTA1 Gene methylation-based detection kits can conveniently and rapidly detect cerebral arteries at the molecular level, offering high detection efficiency and specificity. MTA1 Drugs targeting gene methylation levels represent an innovative application for the auxiliary diagnosis, detection, and screening of cerebral aneurysms.

[0021] In summary, this invention is based on the detection Figure 1 Gene methylation-based detection kits can conveniently and quickly detect cerebral aneurysms at the molecular level. They are highly efficient and targeted, which is beneficial for the early detection and timely treatment of cerebral aneurysms. Attached Figure Description

[0022] MTA1 for Figure 2 The specific location of the gene sequence being detected is the distribution of 13 CpG methylation sites within the region hg38, Chr14: 105,420,312-105,420,399;

[0023] MTA1 for Figure 3 Example of gene methylation level detection results. The percentages shown in the figure represent the methylation degree of the corresponding CpG sites. As shown in the figure, the methylation degrees of CpG1 to CpG13 are 32%, 11%, 28%, 10%, 15%, 19%, 17%, 18%, 32%, 31%, 27%, 18%, and 17%, respectively.

[0024] MTA1For unruptured brain aneurysm and control group Figure 4 Gene DNA methylation difference (***p<0.001);

[0025] MTA1 For ruptured brain aneurysm and control group Figure 5 Gene DNA methylation difference (***p<0.001, ns: p>0.05);

[0026] MTA1 For unruptured and ruptured brain aneurysm and control group Figure 6 Gene average DNA methylation difference (***p<0.001);

[0027] MTA1 For MTA1 Gene DNA methylation and sensitivity and specificity of brain aneurysm (unruptured brain aneurysm AUC = 0.97, ruptured brain aneurysm AUC = 0.80). DETAILED DESCRIPTION

[0028] The application will be described in further detail below with reference to the accompanying drawings.

[0029] Example 1

[0030] 1. Collection of research subjects

[0031] Volunteers were recruited to participate in the study, and according to the international diagnostic criteria for brain aneurysm, the volunteers were divided into unruptured brain aneurysm group, ruptured brain aneurysm group and control group, and the general conditions such as gender and age of the volunteers were recorded. At the same time, venous blood was taken, and general blood biochemical tests were performed, as follows:

[0032] From the neurosurgery department of a hospital, 84 patients with ruptured brain aneurysm confirmed by cerebral angiography (average age 56.33±8.70) and 84 patients with unruptured brain aneurysm (average age 57.06±10.51) were collected, and 84 normal people with no history of cardiovascular and cerebrovascular disease confirmed by CT were collected as control group (average age 55.10±9.30). The gender and age were strictly matched. The fasting blood of all research subjects was tested for blood lipids, blood glucose and other general biochemical indicators, and 3ml of venous blood was taken into an EDTA anticoagulant tube. The supernatant plasma and intermediate layer white blood cells were stored in a-80℃ low temperature refrigerator for 20 minutes, and were used for uniform extraction of genomic DNA.

[0033] 2. Extraction of genomic DNA

[0034] Genomic DNA was extracted from the whole blood samples obtained in the above steps using a Lab-Aid 820 fully automated nucleic acid extractor. The concentration of the extracted DNA was then measured using a nucleic acid and protein analyzer for analysis. MTA1 Detection of gene DNA methylation levels.

[0035] Example 2

[0036] DNA methylation level assay

[0037] This study used pyrosequencing technology to... Figure 1 The gene's anterior region hg38, Chr14 contains 13 CpG methylation sites (e.g., ...) within the region 105,420,312-105,420,399. Figure 1 DNA methylation level analysis was performed (as shown). MTA1 As shown MTA1 The gene sequence being detected is located in the region at the gene's front end Chr14: 105,420,312-105,420,399, containing 13 CpG methylation sites. Their specific locations are as follows:

[0038] .

[0039] This study used PyroMark Assay Design software for primer design. The PCR amplification primers and sequencing primers used in the experiment are as follows:

[0040] MTA1 The nucleotide sequence of the gene DNA methylation-specific forward primer (SEQ ID NO.1) is as follows: 5'-GGTGGGGGGAGGTTTATTTG-3';

[0041] MTA1 The nucleotide sequence of the gene DNA methylation-specific downstream primer (Reverse primer) SEQ ID NO.2 is as follows: 5'-Biotin-CAACCCCAAAACTCCTACT-3';

[0042] MTA1 Nucleotide sequence of DNA methylation-specific sequencing primer (SEQ ID)

[0043] NO.3 is as follows: 5'-GGGGGAGGTTTATTTGTT-3'.

[0044] Specific steps for determining DNA methylation levels:

[0045] First step: Sulfite conversion. The sample DNA was subjected to bisulfite conversion using QIAGEN EpiTect® Bisulfite Kits (Qiagen; #59104) according to the manufacturer's instructions, and the converted DNA was obtained by loading, washing, and eluting.

[0046] Second step: PCR reaction. The 20 ng of the converted DNA sample in the first step was added to the Pyromark PCR Kit (Qiagen; #978703), and the above-described pair of Figure 2 gene methylation-specific amplification primers were added to perform PCR amplification. The PCR amplification system (50 μΐ) contained: H2O 34.8 μΐ, 5x buffer GC (KAPA) 10 μΐ, dNTP (10 mM / each) 1 μΐ, Primer (up 50 pM / ul) 1 μΐ, Primer (down 50 pM / ul) 1 μΐ, Template 2 μΐ, Taq (5 U / μL) 0.2 μΐ. The PCR amplification conditions were: first denaturation at 95 °C for 3 min; then annealing reaction for 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min; and then extension reaction at 72 °C for 5 min.

[0047] Third step: Preparation for pyrosequencing: 45 μl annealing buffer (PyroMark Annealing Buffer; Qiagen; #979009) containing 0.3 μM of the above-mentioned methylation-specific sequencing primer was added in advance in the PCR plate; the total amount of mixed agarose beads (3 μl per sample) to be used was transferred to an Eppendorf tube; binding buffer (PyroMark Binding Buffer; Qiagen; #979006) was added to the agarose beads so that the average volume of each sample was about 50 μl, and the mixture was mixed; the above mixture was added to the PCR product (50 μl reaction volume) at 50 μl per sample; the PCR product was mixed at room temperature for 10 minutes to allow the magnetic beads to bind to the biotin; in the vacuum preparation workstation, 180 ml of high-purity water, 70% ethanol, washing buffer (PyroMark Wash Buffer; Qiagen; #979008) and 120 ml of denaturation buffer (PyroMark Denaturation Solution; Qiagen; #979007) were sequentially added to the four sample plates; the pump of the vacuum preparation workstation was opened, and the vacuum preparation tool was cleaned in high-purity water for 30 seconds; then the vacuum preparation tool (PyroMark Vacuum Prep Filter Probes; Qiagen; #979010) was moved to the PCR plate, and the agarose beads were grabbed (this operation was completed within three minutes after the magnetic beads combined with the PCR product); the PCR plate was taken out and checked to see if most of the magnetic beads were adsorbed on the vacuum preparation tool; the vacuum preparation tool was placed in 70% ethanol for 5 seconds; then moved to the denaturation buffer for 5 seconds; and then moved to the washing buffer for 5-10 seconds; the pump was turned off; the vacuum preparation tool was placed in the plate containing the sequencing primer, shaken, and the agarose beads were released (the sequencing primer can also be added last); the vacuum preparation tool was cleaned with high-purity water; the sample plate was placed on the heating plate and heated to 80°C for 2 minutes, and then cooled to room temperature, and the pyrosequencing reaction was performed.

[0048] Fourth step: Pyrosequencing: The samples in the third step were sequenced on the PyroMark Q24 pyrosequencing instrument using the Pyromark Gold Q24 Reagents (Pyromark Gold Q24 Reagents; Qiagen; #978802), and then the results were analyzed for methylation using the PyroMark CpG software (the methylation level detection results are shown in Figure 2 ). MTA1 The gene methylation level detection results are shown in Figure 2 The percentage shown in the figure is the methylation degree of the corresponding CpG site, as MTA1The methylation levels of CpG1 to CpG13 were 32%, 11%, 28%, 10%, 15%, 19%, 17%, 18%, 32%, 31%, 27%, 18%, and 17%, respectively.

[0049] Example 3

[0050] Data analysis

[0051] The data were analyzed using GraphPad Prism 8. The primers designed in this study could detect MTA1 The methylation levels of 13 CpG sites in the Chr14: 105,420,312-105,420,399 region were detected. By comparing the case-control groups, we found that the methylation levels of Figure 3 CpG1 (p<0.001), CpG2 (p<0.001), CpG3 (p<0.001), CpG4 (p<0.001), CpG5 (p<0.001), CpG6 (p<0.001), CpG7 (p<0.001), CpG8 (p<0.001), CpG9 (p<0.001), CpG10 (p<0.001), CpG11 (p<0.001), CpG12 (p<0.001), CpG13 (p<0.001), and the average methylation level (p<0.001) were lower than those in the control group MTA1 ) in the unruptured cerebral aneurysm patients. Figure 4 CpG1 (p<0.001), CpG2 (p<0.001), CpG3 (p<0.001), CpG9 (p<0.001), CpG10 (p<0.001), CpG11 (p<0.001), and the average methylation level (p<0.001) were lower than those in the control group MTA1 ) in the ruptured cerebral aneurysm patients. MTA1 The results of the average methylation analysis showed that the average methylation level of the unruptured cerebral aneurysm patients Figure 5 was significantly lower than that of the ruptured cerebral aneurysm patients and the control group, and the methylation level gradually increased in the three groups (p<0.001, MTA1 ). ROC curve analysis showed that Figure 6 the methylation level of the unruptured cerebral aneurysm patients had an area under the curve (AUC) of 0.97 (p<0.001, Figure 6 ) with a sensitivity of 98.8% and a specificity of 90.4% at the optimal critical point. The AUC area of the ruptured cerebral aneurysm patients was 0.80 (p<0.001, MTA1 ) with a sensitivity of 90.4% and a specificity of 63.9% at the optimal critical point.

[0052] The above research results show that the blood of patients with unruptured cerebral aneurysms contains... MTA1 DNA methylation was significantly lower in patients with ruptured DNA than in healthy controls, and methylation levels showed a gradually increasing trend across the three groups. Therefore, MTA1 The detection of the degree of DNA methylation is expected to serve as a predictor for the early diagnosis of cerebral aneurysms.

[0053] In summary, the present invention provides a detection method. MTA1 A kit for determining the degree of DNA methylation and its application, particularly in the detection of cerebral aneurysms, offers advantages such as accuracy, reliability, flexibility, speed, and cost-effectiveness. This invention utilizes the aforementioned kit for... ​ Detection of DNA methylation levels can provide rapid and reliable insights for the auxiliary diagnosis, detection, or screening of drugs for cerebral aneurysms.

[0054] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A kit for detecting the degree of methylation of the MTA1 gene associated with the occurrence of cerebral aneurysms, characterized in that... The device includes a pair of MTA1 gene methylation-specific amplification primers and an MTA1 gene methylation-specific sequencing primer, wherein the nucleotide sequence of the upstream primer for MTA1 gene methylation-specific amplification is shown in SEQ ID NO.1: 5'-GGTGGGGGGAGGTTTATTTG-3'; and the nucleotide sequence of the downstream primer for MTA1 gene methylation-specific amplification is shown in SEQ ID NO.2: 5'-Biotin-CAACCCCAAAACTCCTACT-3'. The nucleotide sequence of the MTA1 gene methylation-specific sequencing primers is shown in SEQ ID NO.3: 5'-GGGGGAGGTTTATTTGTT-3'.

2. The kit for detecting the degree of MTA1 gene methylation associated with cerebral aneurysms according to claim 1, characterized in that: The region where the MTA1 gene DNA sequence was detected is within the CpG78 methylation island region Chr14:105,420,312-105,420,399 at the front of the gene.

3. The kit for detecting the degree of MTA1 gene methylation associated with cerebral aneurysms according to claim 2, characterized in that: The specific locations of the 13 CpG sites within the Chr14:105,420,312-105,420,399 region are as follows: 。 4. The kit for detecting the degree of MTA1 gene methylation associated with cerebral aneurysms according to claim 3, characterized in that: The DNA sequence of the MTA1 gene was analyzed using pyrosequencing technology to determine the DNA methylation level at 13 CpG sites within the MTA1 gene.

5. The kit for detecting the degree of MTA1 gene methylation associated with cerebral aneurysms according to claim 4, characterized in that... The specific steps for determining the DNA methylation level are as follows: (1) Bisulfite conversion: The sample DNA was converted to bisulfite using a bisulfite treatment kit; (2) PCR reaction: Take 20 ng of sulfite-converted DNA sample and add it to the Pyromark PCR kit, and add a pair of MTA1 gene methylation-specific amplification primers for PCR amplification; (3) Preparatory work for pyrosequencing; (4) Pyrosequencing.

6. The kit for detecting the degree of MTA1 gene methylation associated with cerebral aneurysms according to claim 5, characterized in that... The PCR amplification system in step (2) includes: 34.8 μL of double-distilled water; 10 μL of 5×GC solution; 1 μL of deoxyribonucleoside triphosphate, with a concentration of 10 mM for each reaction; 1 μL of upstream primer, with a concentration of 50 pM per μL; 1 μL of downstream primer, with a concentration of 50 pM per μL; 2 μL of PCR template; 0.2 μL of Taq enzyme, with a concentration of 5 U per μL; PCR amplification conditions: first denaturation at 95℃ for 3 min; then annealing reaction at 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 1 min, for a total of 40 cycles; and then extension reaction at 72℃ for 5 min.

7. The kit for detecting the degree of MTA1 gene methylation associated with cerebral aneurysms according to claim 5, characterized in that... Step (3) specifically involves adding 45 μl of annealing buffer containing 0.3 μM methylation-specific sequencing primers to the PCR plate; transferring the total amount of agarose beads to be mixed into an Eppendorf tube; adding binding buffer to the agarose beads to make an average volume of 50 μl per sample and mixing the mixture; adding the mixture to the PCR product, 50 μl per sample; mixing the PCR product at room temperature for 10 minutes to allow the magnetic beads to bind to biotin; and adding 180 ml of high-purity water, 70% ethanol, washing buffer, and 120 ml of denaturing buffer to the four sample plates in sequence in a vacuum preparation workstation. Turn on the pump of the vacuum preparation workstation and rinse the vacuum preparation tool in high-purity water for 30 seconds. Then, transfer the vacuum preparation tool to the PCR plate and pick up the agarose beads. Pick up the PCR plate and check if most of the magnetic beads have been adsorbed onto the vacuum preparation tool. Immerse the vacuum preparation tool in 70% ethanol for 5 seconds. Then, transfer it to denaturation buffer for 5 seconds. Next, transfer it to washing buffer for 5-10 seconds. Turn off the pump. Place the vacuum preparation tool into the plate containing the sequencing primers, shake it to release the agarose beads, and rinse the vacuum preparation tool with high-purity water. Place the plate containing the sample on a hot plate and heat it to 80°C for 2 minutes, then cool it to room temperature. The pyrosequencing reaction can then be performed.

8. The kit for detecting the degree of MTA1 gene methylation associated with cerebral aneurysms according to claim 5, characterized in that... Step (4) specifically involves sequencing the sample from step 3 using the Pyromark Gold Q24 kit on a Pyromark Q24 pyrosequencing instrument, and then performing methylation analysis on the results using Pyromark CpG software.

9. The use of any one of the kits in claims 1-8 in the preparation of reagents for detecting or assisting in the diagnosis of cerebral aneurysms.

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