MiRNA marker composition for AD early diagnosis and application thereof

Abstract

The invention discloses a miRNA (micro Ribonucleic Acid) marker composition capable of being used for early diagnosis of AD (Alzheimer's disease). The miRNA marker composition comprises hsa-miR-107 and hsa-miR-20a. The hsa-miR-107 and the hsa-miR-20a can be jointly applied to preparation of a product for AD early diagnosis, and a noninvasive or minimally invasive efficient diagnosis model which is high in detection rate, good in repeatability, easy to operate and suitable for AD early diagnosis is provided for AD early diagnosis, so that a scientific basis is provided for AD early screening, intervention and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a miRNA marker composition for early diagnosis of AD and an application thereof. Background Art

[0002] Alzheimer's disease (AD) is a common progressive neurodegenerative disease characterized by memory loss and cognitive decline. Due to the complex pathogenesis of AD, early diagnosis is difficult. Studies have shown that AD patients may experience a latent period of up to 20 to 30 years from the onset of pathological changes to the onset of obvious clinical symptoms. However, current diagnostic technologies for AD, including cognitive function tests, neuroimaging examinations, and Aβ and Tau biomarker detection, still have significant limitations in early detection and screening of high-risk populations.

[0003] Cognitive function tests (such as MMSE and MoCA) are commonly used clinical tools to judge the severity of the disease by evaluating the patient's memory, language ability, and executive function. However, these testing methods have the disadvantages of being highly subjective and highly dependent on the patient's cooperation, and are usually unable to accurately diagnose in the early stages of the disease.

[0004] Imaging examinations include magnetic resonance imaging and positron emission tomography. Magnetic resonance imaging (MRI) can observe brain atrophy, but brain atrophy is usually not significant until the middle and late stages of AD, so its early diagnostic value is limited. Positron emission tomography (PET) assists diagnosis by detecting the deposition of β-amyloid protein (Aβ) or Tau protein in the brain, but it is expensive and requires the use of radioactive tracers, making it difficult to be widely used for screening.

[0005] Detection of Aβ42, total Tau protein (t-Tau) and phosphorylated Tau protein (p-Tau) levels in cerebrospinal fluid is currently a relatively mature biomarker detection method. However, cerebrospinal fluid collection requires lumbar puncture, which is an invasive procedure with low patient acceptance and is difficult to promote in primary medical institutions. In contrast, blood testing has become a research hotspot in recent years due to its non-invasiveness and convenience, but due to the extremely low concentrations of Aβ and Tau proteins in the blood and their susceptibility to interference from other diseases or physiological states, the sensitivity and specificity of its detection are still insufficient.

[0006] Exosomes are nanoscale vesicles secreted by cells. They are widely present in body fluids such as blood and cerebrospinal fluid. They can carry biomolecules such as miRNA, proteins and sugar chains, reflecting the state of their source cells. Studies have shown that exosomes are widely present in nerve cells in various parts of the central nervous system, including neurons and glial cells. These exosomes can smoothly pass through the blood-brain barrier, enter the peripheral circulatory system from brain tissue, and appear in peripheral blood. Exosomes have gradually become a new direction for AD biomarker research. Summary of the invention

[0007] The object of the present invention is to provide a miRNA marker composition that can be used for early diagnosis of AD, so as to solve at least one of the above-mentioned technical problems.

[0008] According to one aspect of the present invention, a miRNA marker composition for early diagnosis of AD is provided, which comprises hsa-miR-107 and hsa-miR-20a.

[0009] In order to solve the problems of insufficient sensitivity and specificity, strong invasiveness, low patient acceptance and high cost in the early diagnosis of AD, the present invention has conducted a lot of research based on blood as the detection sample, and through a large number of screening and verification experiments, it is found that the ratio of the expression levels of hsa-miR-107 and hsa-miR-20a in the serum glycosylated exosomes of the subjects can be used to distinguish between early AD patients (mild cognitive impairment, MCI) and healthy people, and the combined detection of the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosomes can be used to distinguish between MCI and healthy people. The detection rate of MCI is significantly higher than the detection rate of protein markers such as Aβ1-42 / 40, p-Tau217, p-Tau231, GFAP and other protein markers for AD patients. hsa-miR-107 and hsa-miR-20a can be combined as miRNA markers for the early diagnosis of AD.

[0010] According to another aspect of the present invention, there is provided use of a product for detecting miRNA expression levels in the preparation of a product for early diagnosis of AD, wherein the miRNA includes hsa-miR-107 and hsa-miR-20a.

[0011] In some embodiments, the product for detecting miRNA expression levels is a product for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples, which can be a product for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples based on any known technology in the art that can detect miRNA expression levels in samples, and can be a commercially available product or a self-prepared product.

[0012] Based on blood samples, by separating and extracting glycosylated exosomes in serum, detecting the expression levels of hsa-miR-107 and hsa-miR-20a in glycosylated exosomes, and diagnosing or assisting in the diagnosis of early AD according to the ratio of the expression levels of the two miRNAs, an efficient diagnostic model suitable for the early diagnosis of AD with strong detection stability, high sensitivity, low or non-invasiveness, good repeatability, low cost and simple operation can be provided or constructed, thereby providing a scientific basis for the early screening, intervention and treatment of AD.

[0013] In some embodiments, the product for detecting the expression level of hsa-miR-107 and hsa-miR-20a in a serum glycosylated exosome sample can be a reagent, a kit, a chip and / or an instrument for detecting the expression level of miRNA in a serum glycosylated exosome sample by at least one of the following methods: nucleic acid sequencing, nucleic acid hybridization or nucleic acid amplification.

[0014] In some embodiments, the product for detecting the expression level of hsa-miR-107 and hsa-miR-20a in a serum glycosylated exosome sample can be a reagent, a kit, a chip and / or an instrument for detecting the expression level of miRNA in a serum glycosylated exosome sample by at least one of the following methods: high-throughput sequencing, in situ hybridization, gene chip or fluorescence quantitative PCR.

[0015] According to another aspect of the present invention, provided is the use of a composition comprising a serum glycosylated exosome extraction reagent and a reagent for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in a serum glycosylated exosome sample in the preparation of a product for early diagnosis of AD.

[0016] A serum glycosylated exosome extraction reagent and a reagent for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples are combined to prepare a product for early diagnosis of AD. Thus, early diagnosis of AD can be achieved by only collecting blood samples from patients. This is less invasive and simple to operate, and can significantly improve patient compliance and clinical applicability.

[0017] According to a fourth aspect of the present invention, a product for early diagnosis of AD is provided, which comprises a serum glycosylated exosome extraction reagent and a real-time fluorescence quantitative PCR reagent for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples.

[0018] In some embodiments, the serum glycosylated exosome extraction reagent can directly adopt an existing commercial extraction reagent for extracting serum glycosylated exosomes, or can be prepared by itself.

[0019] In some embodiments, the serum glycosylated exosome extraction reagent comprises a lectin-magnetic carrier coupling complex, a washing solution and an elution solution.

[0020] In some embodiments, the lectin-magnetic carrier coupled complex includes a magnetic carrier and a lectin coupled to the outside of the magnetic carrier.

[0021] In some embodiments, the lectin includes any one or two or more of japanese lectin, peanut lectin, pea lectin, concanavalin A lectin, lentil lectin, wheat germ agglutinin, soybean lectin, kidney bean lectin, and snail lectin.

[0022] In some embodiments, the magnetic carrier includes any one or two or more of dextran magnetic beads, agarose magnetic beads, resin or epoxy resin magnetic beads, and polystyrene magnetic beads.

[0023] In some embodiments, the washing solution is a washing buffer or purified water without metal salt ions, which is used to wash and remove non-specifically bound, non-glycosylated exosomes and other impurities during the separation process.

[0024] In some embodiments, the eluent is a borate buffer containing dissolved sugar, which is used to elute the glycosylated exosomes specifically bound to the lectin-magnetic carrier coupling complex, so that the eluted exosomes have a complete appearance without damage or lysis.

[0025] In some embodiments, the serum glycosylated exosome extraction reagent can be GlyExo-Capture TM Extracellular vesicle extraction reagent.

[0026] In some embodiments, the real-time fluorescence quantitative PCR reagent used to detect the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples can be an existing commercially available reagent that can detect the expression level of miRNA in a sample by real-time fluorescence quantitative PCR technology, or can be prepared by itself.

[0027] In some embodiments, the composition of the real-time fluorescence quantitative PCR reagent for detecting the expression level of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples may include amplification primers for hsa-miR-107, amplification primers for hsa-miR-20a, and probes. The primers and probes can be designed and synthesized using methods known to those skilled in the art.

[0028] In some embodiments, the nucleotide sequence of the forward primer of hsa-miR-107 is shown as SEQ ID NO:1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO:4; the nucleotide sequence of the forward primer of hsa-miR-20a is shown as SEQ ID NO:2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO:4; the nucleotide sequence of the probe is shown as SEQ ID NO:3.

[0029] In some embodiments, the composition of the real-time fluorescence quantitative PCR reagent for detecting the expression level of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples may also include reagents such as buffer, polymerase, dNTP, ddH2O, fluorescent dyes, etc. required for real-time fluorescence quantitative PCR amplification. Among them, the buffer, polymerase, dNTP, ddH2O, fluorescent dyes, etc. reagents can all be obtained through commercial channels.

[0030] In the present invention, products for early diagnosis of AD include but are not limited to reagents, kits, chips, detection systems or instruments for diagnosing or assisting in diagnosing whether a subject suffers from AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 For hsa-miR-107 and hsa-miR-20a Box plot of CT; Figure 2 is a scatter plot of miRNA marker composition and p-Tau217; Figure 3 is the ROC curve of the miRNA marker combination; Figure 4 The miRNA marker composition, Aβ1-42 / 40 protein, p-Tau217 protein, p-Tau231 protein and GFAP protein provided by the present invention are used as detection markers to detect the AD patients in the sample. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific examples and drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the reagents used in the examples are conventional products that can be obtained commercially, and the experimental methods for the specific conditions not specified in the examples are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the kit and instrument manufacturers.

[0033] Example 1 1. Sample collection and processing Use vacuum blood collection tubes (coagulant / separation gel) to collect blood from the subjects, and centrifuge at 2000×g for 10 min at 10℃~30℃ within 6 hours after blood collection to separate serum. Serum samples after the above treatment should be used for testing immediately, or stored at -70℃ or below for no more than 12 months, and repeated freezing and thawing is strictly prohibited.

[0034] A total of 97 serum samples from AD patients and 40 serum samples from healthy people were collected. Among them, all AD patients had clear clinical diagnosis results (scale evaluation and / or plasma protein marker examination) and were MCI patients.

[0035] Utilizing GlyExo-Capture TM Extracellular vesicle extraction reagent (Beijing Kaijing Gene Technology Co., Ltd., GlyE01-24) was used to separate and extract glycosylated exosomes from serum samples.

[0036] 2. RT-qPCR (1) Using miRNeasy ® Mini Kit (Qiagen, 217004) was used to extract total RNA of glycosylated exosomes, and the first-strand cDNA was synthesized by reverse transcription using miRNA 1st Strand cDNA Synthesis Kit (by tailing A) (Nanjing Novogene, MR201-02). The reverse transcription reaction system is shown in Table 1 and the primers are shown in Table 4.

[0037] Table 1 Reverse transcription reaction system

[0038] Reaction procedure: 42℃ 15 min; 85℃ 1 min; 4℃ ∞.

[0039] (2) The miRNA qPCR reaction was performed on the ABI7500 real-time fluorescence quantitative PCR system using 2×Robustart Premix-UNG (Probe qPCR) (Zhuhai Baorui Biotechnology, M2161). The reaction system, reaction procedure and primer sequences are shown in Tables 2~4 respectively.

[0040] Table 2 miRNA qPCR reaction system

[0041] Table 3 miRNA qPCR reaction procedure

[0042] Table 4 Primer sequences

[0043] Note: The reverse transcription primer is a degenerate primer, where V = C or G or A, and N = C or G or A or T.

[0044] 3. Diagnostic efficacy verification and comparison (1) Comparison of test results between early AD patients and healthy subjects Calculate the expression of hsa-miR-107 and hsa-miR-20a for each sample CT, and then draw a box plot of early AD and healthy people, and use the rank sum test to determine whether there is a significant difference. The results are as follows Figure 1 As shown. Figure 1 The results showed that hsa-miR-107 and hsa-miR-20a There were significant differences in CT between patients with early AD and healthy subjects.

[0045] (2) Correlation between the miRNA marker composition of the present invention and the protein marker p-Tau217 The phosphorylated Tau217 protein assay kit (single molecule immunoassay) (AstraBio, R14050) was used to detect the p-Tau217 content in the serum sample, and then the correlation between the miRNA marker composition of the present invention and the protein marker p-Tau217 was calculated by the Pearson correlation coefficient. The results are as follows: Figure 2 As shown. Figure 2 The results show that there is a correlation between the miRNA marker composition of the present invention and the protein marker p-Tau217.

[0046] (3) ROC curve Mapping of hsa-miR-107 and hsa-miR-20a The ROC curve of CT is as follows Figure 3 According to the ROC curve, the AUC of the miRNA marker composition of the present invention for diagnosing early AD is 0.724, and the cutoff value determined by the maximum Youden index method is 6.03069.

[0047] (4) Comparison of detection rates Based on the above-mentioned sample cohort of 97 sera from early AD patients and 40 sera from healthy controls, the miRNA marker composition, Aβ1-42 / 40 protein, p-Tau217 protein, p-Tau231 protein, and GFAP protein provided by the present invention were used as detection markers to identify early AD patients in the samples, and the detection rate of each detection marker for early AD patients was calculated.

[0048] Detection method: After extracting glycosylated exosomes from serum samples, the hsa-miR-107 and hsa-miR-20a were obtained according to the method of "2. RT-qPCR verification" above. CT, then The CT value was used for judgment, with 6.03069 as the critical value for positive judgment; when it was less than 6.03069, the result was judged as positive; when it was greater than 6.03069, the result was judged as negative.

[0049] The levels of Aβ1-40 and Aβ1-42 in serum were detected using the β-amyloid 1-40 assay kit (single molecule immunoassay) (AstraBio, R14010) and the β-amyloid 1-42 assay kit (single molecule immunoassay) (AstraBio, R14020).

[0050] The content of p-Tau217 protein in serum was detected using the phosphorylated Tau217 protein assay kit (single molecule immunoassay) (AstraBio, R14050).

[0051] The content of p-Tau231 protein in serum was detected using the phosphorylated Tau231 protein assay kit (single molecule immunoassay) (AstraBio, R14100).

[0052] The content of GFAP protein in serum was detected using the glial fibrillary acid protein assay kit (single molecule immunoassay) (AstraBio, R14060).

[0053] The detection rates of hsa-miR-107 / hsa-miR-20a, Aβ1-42 / 40 protein, p-Tau217 protein, p-Tau231 protein, and GFAP protein in early AD patients in the sample cohort were as follows: Figure 4 shown.

[0054] Depend on Figure 4The results show that the detection rate of the miRNA marker composition provided by the present invention for early AD samples is 75.26%, which is significantly better than 28.87% of the protein indicators Aβ1-42 / 40, 58.76% of p-Tau217, 48.45% of p-Tau231 and 46.39% of GFAP, indicating that the miRNA marker composition provided by the present invention has high sensitivity and specificity for early diagnosis of AD.

[0055] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A miRNA marker composition for early diagnosis of AD, characterized in that: The miRNA marker composition includes hsa-miR-107 and hsa-miR-20a.

2. Use of a product for detecting miRNA expression levels in the preparation of a product for early diagnosis of AD, characterized in that: The miRNAs include hsa-miR-107 and hsa-miR-20a.

3. The use according to claim 2, characterized in that: The product for detecting miRNA expression levels is a product for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples.

4. The use according to claim 3, characterized in that: The product for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples is a reagent, a kit, a chip and / or an instrument for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples by at least one of the following methods: nucleic acid sequencing, nucleic acid hybridization or nucleic acid amplification.

5. The use according to claim 4, characterized in that: The product for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples is a reagent, a kit, a chip and / or an instrument for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples by at least one of the following methods: high-throughput sequencing, in situ hybridization, gene chip or fluorescence quantitative PCR.

6. Use of a composition comprising a serum glycosylated exosome extraction reagent and a reagent for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in a serum glycosylated exosome sample in the preparation of a product for early diagnosis of AD.

7. A product for early diagnosis of AD, characterized in that: It includes a serum glycosylated exosome extraction reagent and a real-time fluorescence quantitative PCR reagent for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples.

8. The product for early diagnosis of AD according to claim 7, characterized in that: The serum glycosylated exosome extraction reagent comprises a lectin-magnetic carrier coupling complex, a cleaning solution and an eluent.

9. The product for early diagnosis of AD according to claim 7 or 8, characterized in that: The real-time fluorescence quantitative PCR reagent for detecting the expression levels of hsa-miR-107 and hsa-miR-20a in serum glycosylated exosome samples comprises an amplification primer for hsa-miR-107, an amplification primer for hsa-miR-20a and a probe.

10. The product for early diagnosis of AD according to claim 9, characterized in that: The nucleotide sequence of the forward primer of hsa-miR-107 is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:4; the nucleotide sequence of the forward primer of hsa-miR-20a is shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:4; the nucleotide sequence of the probe is shown in SEQ ID NO:3.

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