A marker for diagnosis of moyamoya disease and application thereof

Abstract

The application belongs to the technical field of medical detection, and particularly relates to a marker for moyamoya disease diagnosis and application thereof. The marker for moyamoya disease diagnosis is miRNA, and the miRNA is hsa-miR-4433a-5p, the nucleotide sequence of which is shown as SEQ ID NO. 1. The study proves by experiments that the expression level of hsa-miR-4433a-5p in moyamoya disease patients is significantly higher than that in healthy people, and can be used as a marker for moyamoya disease diagnosis. Meanwhile, the marker for moyamoya disease diagnosis is applied to preparation of a kit, and the content of hsa-miR-4433a-5p in serum can be detected by real-time fluorescent quantitative PCR technology, which has the advantages of high sensitivity and strong accuracy, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a biomarker for the diagnosis of Moyamoya disease and its application. Background Technology

[0002] Moyamoya disease (MMD) is a chronic, progressive intracranial vascular stenosis or occlusion disorder of unknown cause. Its clinical features include progressive stenosis or occlusion of the distal internal carotid arteries bilaterally, accompanied by the formation of abnormal "smoke-like" collateral vessels at the base of the brain. The main clinical manifestations are transient ischemic attacks and ischemic strokes caused by large artery occlusion, and hemorrhagic strokes caused by rupture of the smoke-like vessels or aneurysms. It has a high rate of disability and mortality in adults. Currently, the diagnosis of MMD mainly relies on imaging findings, especially digital subtraction angiography (DSA). However, this is an invasive procedure with risks of radiation exposure and surgery, and it is not sensitive to subtle changes such as early intimal thickening. Other non-invasive methods, such as magnetic resonance angiography (MRI), can aid in diagnosis, but they still lack specific molecular-level indicators. By the time of diagnosis, patients often already have severe clinical symptoms, making early identification and intervention difficult. Therefore, the search for molecular biomarkers that can be minimally invasively detected in body fluids such as peripheral blood before the appearance of clinical symptoms or typical imaging changes is of urgent clinical significance for the early diagnosis, risk stratification, and prognostic assessment of Moyamoya disease.

[0003] Based on our preliminary study on the relationship between NPY in the serum and cerebrospinal fluid of patients with Moyamoya disease, this invention further focuses on miRNAs in extracellular vesicles. Extracellular vesicles are nanoscale vesicles actively secreted by cells. Their lipid bilayer structure effectively protects their contents, ensuring their stability in various body fluids and enabling intercellular communication across the blood-brain barrier. Molecules carried by extracellular vesicles, such as miRNAs, can accurately reflect the pathophysiological state of the source cells. miRNAs are a class of endogenous non-coding small RNAs whose expression profiles exhibit highly specific changes under different disease states and participate in the regulation of the occurrence and development of various diseases. Compared with traditional free molecules, vesicle-encapsulated miRNAs are more stable, more enriched, and have stronger source targeting, greatly improving their reliability as biomarkers. Therefore, extracellular vesicle-derived miRNAs have become potential ideal biomarkers for the early diagnosis, progression assessment, and prognostic monitoring of various diseases. This study aims to discover specific vesicular miRNAs related to Moyamoya disease to reveal its pathological mechanisms and provide novel minimally invasive detection targets for clinical use.

[0004] To achieve the above objectives, this invention provides a biomarker for the diagnosis of Moyamoya disease and its application. The biomarker is hsa-miR-4433a-5p, specifically expressed in serum extravesicular vesicles. Detecting the expression level of this biomarker using molecular biology techniques enables auxiliary diagnosis, early screening, and prognostic assessment of Moyamoya disease, providing a non-invasive, objective, and sensitive molecular detection method for clinical use. Summary of the Invention

[0005] The primary objective of this invention is to provide a biomarker for the diagnosis of Moyamoya disease.

[0006] A second objective of this invention is to provide a biomarker for the diagnosis of Moyamoya disease and its application in the preparation of a diagnostic kit for Moyamoya disease.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A biomarker for the diagnosis of Moyamoya disease, wherein the biomarker for the diagnosis of Moyamoya disease is a miRNA; the miRNA is hsa-miR-4433a-5p; the nucleotide sequence of hsa-miR-4433a-5p is shown in SEQ ID NO.1.

[0009] Furthermore, the miRNA is a miRNA from serum extracellular vesicles.

[0010] The above-described biomarker for the diagnosis of Moyamoya disease is used in the preparation of a diagnostic kit for Moyamoya disease.

[0011] Furthermore, the diagnostic kit for Moyamoya disease includes a primer pair for detecting hsa-miR-4433a-5p; the upstream primer sequence of the primer pair for detecting hsa-miR-4433a-5p is shown in SEQ ID NO.2, and the downstream primer sequence is shown in SEQ ID NO.3.

[0012] Furthermore, the diagnostic kit for Moyamoya disease also includes primer pairs for detecting the internal reference gene miR-16-5p; and 2×SYBR Green qPCR premix.

[0013] Furthermore, the upstream primer sequence of the primer pair for detecting the internal reference gene miR-16-5p is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.5.

[0014] Furthermore, the 2× SYBR Green qPCR premix includes SYBR Green, qPCR buffer, dNTPs, and DNA polymerase.

[0015] Compared with the prior art, the main advantages of the present invention are:

[0016] (1) This invention, through experiments, first discovered that the expression level of hsa-miR-4433a-5p in the serum extravesicles of patients with moyamoya disease is significantly higher than that of healthy individuals, with clear targeting, and can serve as a novel molecular marker for the diagnosis of moyamoya disease. Furthermore, applying this molecular marker to the preparation of a reagent kit enables non-invasive detection of the hsa-miR-4433a-5p content in serum using real-time quantitative PCR technology, exhibiting advantages such as high sensitivity, strong specificity, and good reproducibility. This detection method is simple to operate and cost-effective, providing objective molecular evidence for early screening, auxiliary diagnosis, and prognostic assessment of moyamoya disease, and possesses significant clinical translational value and broad market application prospects.

[0017] (2) Early clinical manifestations of Moyamoya disease are nonspecific, and traditional imaging gold standards have limitations such as invasiveness and radiation exposure. This invention utilizes the natural protective effect of the extracellular vesicle lipid bilayer on miRNA to ensure the high stability of the biomarker in blood and other body fluids. Compared with traditional cerebrospinal fluid testing, serum testing is more minimally invasive and easier to repeat sampling; at the same time, this testing method is simple to operate, highly automated, has a short testing cycle, and is low in cost, making it more suitable for clinical application and large-scale early screening. Attached Figure Description

[0018] Figure 1 The relative expression levels of hsa-miR-4433a-5p, a molecular marker for the diagnosis of moyamoya disease, in healthy controls and patients with moyamoya disease;

[0019] Figure 2 The diagnostic value of the molecular marker hsa-miR-4433a-5p in moyamoya disease was analyzed using ROC curve analysis. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0021] Example 1

[0022] Isolation and purification of serum extravesicles

[0023] (1) Twenty patients with Moyamoya disease were selected for the experiment, and 20 healthy individuals matched for baseline characteristics such as age and sex were collected as the control group. Fasting peripheral blood was collected from the subjects in the morning using 5 mL vacuum blood collection tubes (containing coagulant and separating gel). After blood collection, the blood collection tubes were left upright at room temperature for 30 minutes to allow the blood to coagulate fully.

[0024] (2) Centrifuge the blood collection tube at 2000 rpm for 10 minutes at room temperature after allowing it to stand. Carefully aspirate the clear serum from the upper layer of the tube, avoiding contact with the separating gel. Immediately transfer the serum to a 1.5 mL centrifuge tube and centrifuge at 3000 rpm for 20 minutes at 4°C. After centrifugation, carefully aspirate the supernatant and transfer it to a new tube. Centrifuge the supernatant at 12000 rpm for 30 minutes at 4°C. Aspirate the supernatant again to obtain clear serum.

[0025] (3) Take 200 µL of the above-mentioned clear serum and separate extracellular vesicles from the serum sample according to the instructions of the GlyExo-Capture kit. Mix the serum with the pretreatment buffer at the specified ratio and incubate with the capture magnetic beads at 4 °C for 2 hours. Separate on a magnetic rack and discard the supernatant. Wash the magnetic beads 3 times with washing buffer and discard the washing buffer. Finally, elute the bound extracellular vesicles with 50 µL of specific elution buffer and collect the eluent.

[0026] (4) The purified extracellular vesicle suspension was aliquoted according to experimental requirements and immediately stored at -80℃ for subsequent RNA extraction.

[0027] Example 2

[0028] Extraction of extracellular vesicle RNA:

[0029] Extracellular vesicle miRNAs were extracted using the miRNeasy Mini Kit, specifically including the following steps:

[0030] (1) Take 200 μL of extracellular vesicle eluent prepared in Example 1, transfer it to a 1.5 mL enzyme-free centrifuge tube, add 1000 µL of QIAzol Lysis Reagent, vortex to mix for 15 s, and let stand at room temperature for 5 min.

[0031] (2) Add 200µL of chloroform, tighten the cap, shake vigorously for 15 seconds, and let stand at room temperature for 2 min. Centrifuge at 12000rpm for 15 min at 4℃, and transfer the uppermost aqueous phase into a new tube.

[0032] (3) Add an equal volume of 70% ethanol, mix well with a pipette, and immediately proceed to the next step.

[0033] (4) Transfer 700 µL of the mixture to an RNeasy MinElute spin column, centrifuge at 8000 rpm for 15 s, and discard the flow. Repeat this step until all the mixture has passed through the column.

[0034] (5) Add 700µL Buffer RWT to the column, centrifuge at 8000 rpm for 15 s, and discard the flow. Add 500µL Buffer RPE to the column, centrifuge at 8000 rpm for 15 s, and discard the flow. Add another 500µL Buffer RPE to the column, centrifuge at 8000 rpm for 2 min, and thoroughly dry the column membrane.

[0035] (6) Place the column in a new 2 mL collection tube and centrifuge at 12,000 rpm for 2 min to remove residual ethanol. Transfer the column to a new 1.5 mL enzyme-free centrifuge tube, add 15 µL of RNase-free water to the center of the column membrane, incubate at room temperature for 1 min, and centrifuge at 12,000 rpm for 2 min to elute RNA. The collected eluent is the purified RNA, which can be used immediately or stored at -80℃.

[0036] Example 3

[0037] RNA is reverse transcribed into cDNA:

[0038] The RNA solution extracted in Example 2 was reverse transcribed into cDNA using a reverse transcription kit. The reverse transcription reaction system is shown in Table 1. Using RNA as a template, 5×ABScript III RT Mix, Nuclease-free dH2O, and reverse transcription primers were added, gently mixed, and centrifuged. The reaction program was set on a PCR instrument to complete the reverse transcription. The reverse transcription reaction conditions on the PCR instrument are shown in Table 2. After the reaction, the cDNA product was diluted 5-fold with nucleic acid-free water and immediately used for quantitative real-time PCR or stored at -20℃.

[0039] Table 1 Reverse transcription reaction system

[0040]

[0041] Table 2 Reverse transcription reaction conditions

[0042]

[0043] Example 4

[0044] Preparation of a reagent kit for the diagnosis of Moyamoya disease

[0045] A method for preparing a reagent kit for the diagnosis of Moyamoya disease, the method specifically comprising the following steps:

[0046] (1) Primers were designed based on the nucleotide sequence of hsa-miR-4433a-5p, which is shown in SEQ ID NO.1. The upstream primer sequence of the primer pair for detecting hsa-miR-4433a-5p is shown in SEQ ID NO.2, and the downstream primer sequence is shown in SEQ ID NO.3. The diagnostic kit for Moyamoya disease also includes a primer pair for detecting the internal reference gene miR-16-5p; the upstream primer sequence of the primer pair for detecting the internal reference gene miR-16-5p is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.5. Each primer pair was synthesized according to the sequences of SEQ ID NO.2-SEQ ID NO.5, and the final concentration of each primer pair was prepared to 0.2 μM with sterile water. The specific sequence information is shown in Table 3.

[0047] Table 3 Sequence List

[0048]

[0049] (2) Take 100 μL of 100× SYBR Green, 1.00 mL of 10× qPCR buffer, 400 μL of 10 mMdNTPs, and 100 μL of 5 U / μL DNA polymerase, and add them to 7.30 mL of nuclease-free water. Gently invert and mix (avoid vortexing) to obtain 10 mL of 2× SYBR Green qPCR premix.

[0050] (3) The working solutions of each primer pair prepared in step (1) and the 2× SYBR GreenqPCR premix prepared in step (2) are loaded into the kit to obtain a kit for the diagnosis of Moyamoya disease.

[0051] Experimental Example 1

[0052] Real-time quantitative PCR to verify the expression level of molecular markers:

[0053] Twenty cDNA samples from patients with Moyamoya disease prepared in Example 3 were used as the observation group, and 20 cDNA samples from healthy individuals were used as the control group. The cDNA template prepared in Example 3 was analyzed by real-time quantitative PCR using the diagnostic kit for Moyamoya disease prepared in Example 4 to quantify the expression level of the molecular marker hsa-miR-4433a-5p in serum extravesicles. The specific experimental steps are as follows:

[0054] Using cDNA as a template, primers for the molecular marker hsa-miR-4433a-5p and the internal reference gene miR-16-5p were added to the working solution along with 2×SYBR Green qPCR premix. 20 μL of reaction solution was prepared according to the quantitative PCR reaction system in Table 4. The quantitative PCR reaction procedure is shown in Table 5. Each sample was divided into three replicates. Using miR-16-5p as the internal reference gene, the relative expression level of the molecular marker hsa-miR-4433a-5p was calculated using the 2^(-ΔΔCt) method. Statistical analysis was performed on the quantitative PCR data. The relative expression levels of hsa-miR-4433a-5p in the serum of healthy controls and patients with moyamoya disease were obtained by plotting bar graphs. The results are shown in Table 5. Figure 1 As shown.

[0055] Table 4. Real-time PCR reaction system

[0056]

[0057] Table 5. Quantitative PCR reaction procedure

[0058]

[0059] Figure 1 This represents the relative expression level of the serum molecular marker hsa-miR-4433a-5p. Figure 1 It was found that the serum molecular marker hsa-miR-4433a-5p was expressed at low levels in the control group and at high levels in the serum of patients with Moyamoya disease. The expression level of hsa-miR-4433a-5p in serum can clearly distinguish Moyamoya disease patients from healthy individuals. Therefore, the serum molecular marker hsa-miR-4433a-5p can serve as a serum biomarker for Moyamoya disease patients and can provide new ideas and methods for the prediction and diagnosis of Moyamoya disease.

[0060] Experimental Example 2

[0061] ROC curves were used to assess the diagnostic value of serum biomarkers in patients with Moyamoya disease.

[0062] To further evaluate the diagnostic value of the molecular marker hsa-miR-4433a-5p for the diagnosis of Moyamoya disease, 100 samples were collected, including serum samples from 50 Moyamoya disease patients as the observation group and serum samples from 50 healthy individuals as the control group. Extracellular vesicles were extracted and cDNA was prepared according to the methods in Examples 1-3. The expression level of hsa-miR-4433a-5p was detected using the qPCR procedure in Example 1. The calculated relative expression level of hsa-miR-4433a-5p was used as a diagnostic indicator. Receiver operating characteristic (ROC) curves were plotted using statistical software, and the AUC value was calculated to evaluate the diagnostic value of the molecular marker hsa-miR-4433a-5p for Moyamoya disease. The results are as follows: Figure 2 As shown.

[0063] The results are as follows Figure 2 The figure shows the diagnostic value of the molecular marker hsa-miR-4433a-5p in moyamoya disease, analyzed using ROC curve analysis. Figure 2 The AUC value of the serum biomarker hsa-miR-4433a-5p in detecting Moyamoya disease patients was 0.903. An AUC above 0.7 indicates that the model has good classification ability; by detecting the expression level of hsa-miR-4433a-5p in serum, it can specifically distinguish Moyamoya disease patients from healthy individuals, thus achieving the goal of screening for Moyamoya disease.

[0064] In summary, this study used quantitative real-time PCR to detect and validate hsa-miR-4433a-5p in samples from patients and healthy individuals with Moyamoya disease. For the first time, it was found that hsa-miR-4433a-5p was significantly highly expressed in serum extravesicles in Moyamoya disease patients, validating the close association between hsa-miR-4433a-5p and the occurrence of Moyamoya disease. Therefore, hsa-miR-4433a-5p can serve as a potential biomarker for the diagnosis of Moyamoya disease, providing important reference for its early diagnosis.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. Use of a detection primer pair of hsa-miR-4433a-5p in the preparation of a kit for diagnosing moyamoya disease, characterized in that, The nucleotide sequence of hsa-miR-4433a-5p is shown in SEQ ID NO.1; hsa-miR-4433a-5p is a miRNA in serum extracellular vesicles; the upstream primer sequence of the primer pair is shown in SEQ ID NO.2, and the downstream primer sequence is shown in SEQ ID NO.

3.

2. Use according to claim 1, characterized in that, The diagnostic kit for Moyamoya disease also includes primer pairs for detecting the internal reference gene miR-16-5p; and 2× SYBR Green qPCR premix.

3. Use according to claim 2, characterized in that, The upstream primer sequence of the primer pair for detecting the internal reference gene miR-16-5p is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.

5.

4. Use according to claim 2, characterized in that, The 2× SYBR Green qPCR premix includes SYBR Green, qPCR buffer, dNTPs, and DNA polymerase.

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