Biomarker for diagnosis or prognosis of alzheimer's disease comprising mirna and uses thereof

Abstract

One aspect relates to: a marker composition for diagnosis or prognosis of Alzheimer's disease, comprising microRNA-214 (miRNA-214); a marker composition for diagnosis or prognosis of Alzheimer's disease, comprising an agent for measuring an expression level of miRNA-214; a diagnostic or prognostic kit; and a method for providing information for diagnosis or prognosis. According to one aspect, data analysis for the diagnosis of Alzheimer's disease can be made to measure the expression level of miRNA-214 in blood, and as a biomarker for the prognosis of Alzheimer's disease, the microRNA can be advantageously used to predict the progression rate and treatment result of diseases.

Description

Biomarkers for the diagnosis or prognosis prediction of Alzheimer's disease containing miRNAs and their uses

[0001] One example of the present invention relates to a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease comprising miRNA-214 (microRNA-214); a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease comprising a preparation for measuring the expression level of miRNA-214; a kit for diagnosis or prognosis prediction; and a method for providing information for diagnosis or prognosis prediction.

[0002] Alzheimer's disease (AD) is a type of neurodegenerative disease and the most common cause of dementia in the elderly. In most cases, Alzheimer's disease develops after the age of 65, but rarely, it can occur before then. In the United States, approximately 3% of the population aged 65–74, about 19% of the population aged 75–84, and about 50% of the population aged 85 or older suffer from the disease. In South Korea, where the population is aging rapidly, the market for Alzheimer's dementia treatments amounts to approximately 400 billion won, and it is projected to reach about 100 trillion won by 2050.

[0003] Currently, there is no definitive cure for Alzheimer's disease, and treatment is primarily carried out through symptomatic relief agents. To overcome the limitations of existing treatment methods, new antibody drugs targeting amyloid beta (Aβ) have been developed over the past decade and are reaching the commercialization stage. However, despite approval for clinical use, concerns have been raised that the therapeutic effects of these new drugs are limited to patients with early-stage Alzheimer's disease and may vary depending on race and genotype. In particular, in patients with severe and late-stage Alzheimer's disease, even if the novel antibodies bind well to Aβ, issues regarding the regulation of immune inflammation and phagocytic functions of microglia, which play a crucial role in the process of removing Aβ, are emerging.

[0004] Furthermore, diagnostic systems for Alzheimer's dementia are not yet sufficient. Existing diagnostic methods involve various procedures but have several drawbacks. Medical history interviews are susceptible to errors due to cognitive differences, structural brain imaging is costly, and nuclear brain imaging utilizes radioisotopes, which is also expensive. Measuring tau protein using cerebrospinal fluid is an invasive method that carries risks associated with fluid extraction. Therefore, there is an urgent need for the development of blood-based biomarkers that enable faster and earlier diagnosis, and can support risk assessment, early detection, prognosis, and management.

[0005] One aspect provides a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease comprising miRNA-214 (microRNA-214).

[0006] Another aspect provides a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease, comprising a preparation for measuring the expression level of miRNA-214 (microRNA-214).

[0007] Another aspect is to provide a kit for the diagnosis or prognosis prediction of Alzheimer's disease comprising the above-described composition.

[0008] Another aspect is to provide a method for providing information for the diagnosis or prognosis prediction of Alzheimer's disease, comprising the step of measuring the expression level of miRNA-214 in a biological sample derived from a subject.

[0009] To achieve the above objective, a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease is provided, comprising miRNA-214 (microRNA-214).

[0010] In one embodiment of the present invention, the miRNA-214 may be composed of the nucleotide sequence of SEQ ID NO. 1.

[0011] In another embodiment of the present invention, the miRNA-214 may inhibit the expression of NCKAP1 (Nck-associated protein 1).

[0012] In addition, the present invention provides a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease, comprising a preparation for measuring the expression level of miRNA-214 (microRNA-214).

[0013] In one embodiment of the present invention, the agent for measuring the expression level of the miRNA-214 may be a sense and antisense primer or probe that binds complementarily to the miRNA-214.

[0014] In another embodiment of the present invention, the miRNA-214 may be composed of the nucleotide sequence of SEQ ID NO. 1.

[0015] In another embodiment of the present invention, the miRNA-214 may be a blood-derived microRNA.

[0016] In another embodiment of the present invention, the prediction of the prognosis of Alzheimer's disease may be a prediction of the prognosis of cognitive function in patients with Alzheimer's disease.

[0017] In addition, the present invention provides a kit for diagnosing or predicting the prognosis of Alzheimer's disease comprising the above composition.

[0018] In addition, the present invention provides a method for providing information for the diagnosis or prognosis prediction of Alzheimer's disease, comprising the step of measuring the expression level of miRNA-214 in a biological sample derived from a subject.

[0019] In one embodiment of the present invention, the information providing method may determine that there is a high risk of developing Alzheimer's disease or a poor prognosis for Alzheimer's disease when the expression level of miRNA-214 is high.

[0020] In another embodiment of the present invention, the method may further include a step of determining that rapid cognitive decline is progressing when the expression level of miRNA-214 is high in the prognosis prediction of the Alzheimer's disease.

[0021] In another embodiment of the present invention, the miRNA-214 may be composed of the nucleotide sequence of SEQ ID NO. 1.

[0022] In another embodiment of the present invention, the biological sample may be one or more selected from the group consisting of tissue, cell, bone marrow, body fluid, and blood.

[0023] In another embodiment of the present invention, the expression level of miRNA-214 may be measured by one or more methods selected from the group consisting of next-generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RPA), microarray, and northern blotting.

[0024] The inventors intend to propose miRNA-214 as a biomarker for the diagnosis and progression of Alzheimer's dementia that can reflect microglia dysfunction, by confirming phagocytic dysfunction using a blood-derived microglia model of Alzheimer's disease patients, confirming an increase in phagocytic ability-related plasma-derived miRNA-214 expression, and analyzing the correlation with a decline in cognitive function.

[0025] Through the present invention, data analysis for the diagnosis of Alzheimer's disease is possible by measuring the expression level of miRNA-214 in the blood, and the microRNA can be usefully utilized as a biomarker for predicting the prognosis of Alzheimer's disease to predict the rate of disease progression and treatment results.

[0026] Figures 1A to 1E confirm the decrease in microglia phagocytic ability in Alzheimer's patients using a human-derived microglia model. Figure 1A shows a schematic diagram of the human blood-derived microglia modeling method, Figure 1B shows representative fluorescence images of bead and amyloid-beta oligomer pha

[0027] Figures 2A and 2B show the results of confirming the increased expression of blood miRNA-214-3p in a group of Alzheimer's patients, Figure 2A shows the qPCR results, and Figure 2B shows the ROS analysis graph.

[0028] Figures 3A to 3C show the results of confirming the correlation between the decrease in microglia phagocytic ability and the increase in the expression level of blood miRNA-214-3p in Alzheimer's patients. Figure 3A shows the results of the correlation analysis with the bead phagocytic ability of microglia according to the increase in the expression level of plasma-derived miRNA-214-3p, Figure 3B shows the results of the correlation analysis with the amyloid-beta oligomer phagomer phagocytic ability of microglia according to the increase in the expression level of plasma-derived miRNA-214-3p, and Figure 3C shows the results of the correlation analysis with the expression of NCKAP1 mRNA, a phagocytic ability factor, according to the increase in the expression level of plasma-derived miRNA-214-3p.

[0029] Figures 4A to 4C confirm the correlation between the decrease in cognitive function and the increase in blood miRNA-214-3p expression levels in a group of Alzheimer's patients. Figure 4A shows the MMSE, Figure 4B shows the CDR, and Figure 4C shows the results of the correlation analysis with the increase in plasma-derived miRNA-214-3p expression levels for the CDR-SB cognitive function indicators, respectively.

[0030] Figures 5A to 5D show the results of a longitudinal study conducted by following up the same subjects for a period of more than one year to confirm changes in the expression levels of blood miRNA-214-3p according to disease progression in a group of Alzheimer's patients. Figure 5A shows the results of an analysis of the correlation between changes in MMSE and changes in the phagocytic ability of patient-derived microglia according to changes in the CDR-SB cognitive function index, and Figure 5B shows the results of an analysis of the correlation between changes in MMSE and changes in the CDR-SB cognitive function index, and changes in plasma-derived miRNA-214-3p expression according to changes in the CDR-SB cognitive function index.

[0031] Figures 6A to 6C show the results of follow-up observations of Alzheimer's patients regarding disease progression, confirming an increase in miRNA-214-3p in the patient group exhibiting Rapid Cognitive Decliner (RD). The RD patient group exhibiting rapid cognitive decline showed characteristics of decreased amyloid-beta oligomer phagocytic activity (Figure 6A), decreased expression of the phagocytic factor NCKAP1 mRNA (Figure 6B), and increased expression of plasma-derived miRNA-214-3p (Figure 6C).

[0032] Figures 7A and 7B show the results of analyzing the expression levels of blood miRNA-214-3p according to the possession status of the apolipoprotein E 4 allele (APOE-ε4) in a group of Alzheimer's patients, confirming an increase in the expression level of plasma-derived miRNA-214-3p in the group of patients possessing homozygous APOE-ε4. Figure 7A shows a comparative analysis graph of the difference in expression of plasma-derived miRNA-214-3p according to the possession status of the APOE-ε4 allele in a group of Alzheimer's patients, and Figure 7B shows a graph of ROS curve analysis of the expression levels of plasma-derived miRNA-214-3p in the group of patients possessing homozygous APOE-ε4 and the group of patients possessing heterozygous APOE-ε4 compared to the group not possessing APOE-ε4.

[0033] Figures 8A to 8C show the results of confirming the decrease in major phagocytic factors and the increase in blood miRNA-214-3p expression in microglia at different stages of disease progression in 5xFAD Alzheimer's dementia animals. Figure 8A is the result of RNA-sequencing using brain microglia of 5xFAD Alzheimer's mice, Figure 8B is a graph analyzing the decrease in the expression of NCKAP1 mRNA, a major phagocytic factor, using brain microglia of 5xFAD Alzheimer's mice by qPCR, and Figure 8C is a graph comparing and analyzing the expression of miRNA-214-3p in the blood of 5xFAD Alzheimer's mice at different stages, confirming the increase in plasma-derived miRNA-214-3p as dementia progresses.

[0034] The present invention will be described in detail below.

[0035] The present invention provides a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease comprising miRNA-214 (microRNA-214).

[0036] The term "miRNA-214" as used in this invention refers to one of the microRNAs that regulates the expression of specific genes. It primarily binds to mRNA to inhibit or activate the expression of specific genes, thereby influencing various physiological processes. In the blood of Alzheimer's patients, miRNA-214 interacts with the mRNA of the gene NCKAP1, which regulates the phagocytic function of microglia.

[0037] The term "Alzheimer's Disease (AD)" as used in this invention refers to a chronic, progressive neurological disease characterized by the gradual loss of cognitive function due to damage and degeneration of nerve cells. It primarily affects the elderly and is the most common cause of dementia; initially, a decline in memory occurs, followed by a gradual overall deterioration of language, judgment, and behavioral functions. The main characteristic of AD is structural damage to the brain caused by amyloid beta (Aβ) plaques and neurofibrillary tangles.

[0038] The term "diagnosis" as used in the present invention, in a broad sense, means determining the actual condition of a patient's disease in all aspects. The content of the determination includes the name of the disease, the etiology, the type of disease, the severity, the detailed aspects of the disease, and the presence or absence of complications. In the present invention, diagnosis is determining whether Alzheimer's disease has developed and the level of its progression.

[0039] The term "prognosis" as used in this invention refers to a prediction regarding the progression or recovery of the disease, and signifies a forecast or preliminary evaluation. In this invention, prognosis refers to the prognosis of cognitive function in patients with Alzheimer's disease, but is not limited thereto.

[0040] In one embodiment of the present invention, the miRNA-214 may be composed of the nucleotide sequence of SEQ ID NO. 1. In this case, the miRNA-214 may include a nucleotide sequence having sequence homology of 80% or more, preferably 90% or more, and more preferably 95%, 96%, 97%, 98%, or 99% or more with the nucleotide sequence represented by SEQ ID NO. 1.

[0041] In another embodiment of the present invention, the miRNA-214 may inhibit the expression of NCKAP1 (Nck-associated protein 1).

[0042] The term "NCKAP1 (Nck-associated protein 1)" used in this invention refers to a protein that plays a role in the reorganization of the actin cytoskeleton and cell migration within a cell. This protein plays a particularly important role in the process of phagocytosis in microglia, a process necessary to remove damaged neurons and harmful substances such as amyloid beta (Aβ) from the brain.

[0043]

[0044] In addition, the present invention provides a marker composition for the diagnosis or prognosis prediction of Alzheimer's disease, comprising a preparation for measuring the expression level of miRNA-214 (microRNA-214).

[0045] In one embodiment of the present invention, the agent for measuring the expression level of the miRNA-214 may be a sense and antisense primer or probe that binds complementarily to the miRNA-214.

[0046] The term "primer" as used in the present invention refers to a short gene sequence that serves as a starting point for DNA synthesis, and means an oligonucleotide synthesized for the purpose of use in diagnosis, DNA sequencing, etc. The primers can typically be synthesized and used with a length of 15 to 30 base pairs, but this may vary depending on the purpose of use, and can be modified by methylation, capping, etc., using known methods.

[0047] The term "probe" as used in the present invention refers to a nucleic acid capable of specifically binding to RNA of several to hundreds of bases in length, produced through enzymatic chemical separation, purification, or synthesis processes. The presence of RNA can be confirmed by labeling with radioisotopes, enzymes, or fluorescent dyes, and the probe can be designed and modified using known methods.

[0048] In another embodiment of the present invention, the miRNA-214 may be composed of the nucleotide sequence of SEQ ID NO. 1. In this case, the miRNA-214 may include a nucleotide sequence having sequence homology of 80% or more, preferably 90% or more, and more preferably 95%, 96%, 97%, 98%, or 99% or more with the nucleotide sequence represented by SEQ ID NO. 1.

[0049] In another embodiment of the present invention, the miRNA-214 may be a blood-derived microRNA.

[0050] In another embodiment of the present invention, the prediction of the prognosis of Alzheimer's disease may be a prediction of the prognosis of cognitive function in patients with Alzheimer's disease.

[0051] The term "cognitive function" as used in this invention refers to higher-order mental activities of the brain, including learning, memory, reasoning, problem-solving, and language ability. Cognitive function may progressively decline in neurodegenerative diseases such as Alzheimer's disease, and as the disease progresses, memory loss, impairment of language ability, weakening of judgment, and decline in spatial perception ability may occur.

[0052]

[0053] In addition, the present invention provides a kit for diagnosing or predicting the prognosis of Alzheimer's disease comprising the above composition.

[0054] The kit of the present invention may optionally include reagents necessary for carrying out a target amplification PCR reaction (e.g., PCR reaction), such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate. Additionally, the kit may include various polynucleotide molecules, reverse transcriptase, buffers and reagents, and antibodies that inhibit DNA polymerase activity. Furthermore, the optimal amount of reagent used in a specific reaction of the kit can be easily determined by a person skilled in the art who has acquired the disclosures in this specification. Typically, the kit may be manufactured as a separate package or compartment containing the aforementioned components, and the form of the kit may be any commercially available form.

[0055] The above kit may be a single composition, or the individual components of the miRNA-214 expression level measuring agent may be stored individually for mixing before use. As such, the components of the miRNA-214 expression level measuring agent within the kit of the present invention may be packaged separately, or may be packaged as one or more mixtures of any combination of components. Each individual component or one or more mixtures may be placed in different containers, or the entire kit may be contained in a single container.

[0056]

[0057] In addition, the present invention provides a method for providing information for the diagnosis or prognosis prediction of Alzheimer's disease, comprising the step of measuring the expression level of miRNA-214 in a biological sample derived from a subject.

[0058] The term “biological sample” as used herein is a broad concept that includes all types of specimens derived from humans or animals. Specifically, biological samples include, but are not limited to, blood, serum, plasma, saliva, urine, cerebrospinal fluid, gastric secretions, mucosal samples, peritoneal samples, nasal secretions, sputum, pharyngeal exudate, etc.

[0059] Extracting DNA from the above biological sample can be performed according to methods commonly used in the industry, or using a commercially available DNA extraction kit.

[0060] In one embodiment of the present invention, the information providing method may determine that there is a high risk of developing Alzheimer's disease or a poor prognosis for Alzheimer's disease when the expression level of miRNA-214 is high.

[0061] In another embodiment of the present invention, the method may further include a step of determining that rapid cognitive decline is progressing when the expression level of miRNA-214 is high in the prognosis prediction of the Alzheimer's disease.

[0062] In another embodiment of the present invention, the miRNA-214 may be composed of the nucleotide sequence of SEQ ID NO. 1. In this case, the miRNA-214 may include a nucleotide sequence having sequence homology of 80% or more, preferably 90% or more, and more preferably 95%, 96%, 97%, 98%, or 99% or more with the nucleotide sequence represented by SEQ ID NO. 1.

[0063] In another embodiment of the present invention, the biological sample may be one or more selected from the group consisting of tissue, cell, bone marrow, body fluid, and blood.

[0064] In another embodiment of the present invention, the expression level of miRNA-214 may be measured by one or more methods selected from the group consisting of next-generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RPA), microarray, and northern blotting.

[0065] The term "Next generation sequencing (NGS)" as used in this invention refers to a technology capable of simultaneously analyzing multiple DNA or RNA sequences at high speed, and is used for genomic research, gene expression analysis, diagnosis, etc. The NGS includes the processes of sample preparation, sequencing, and data analysis, and can be performed by various known methods and platforms.

[0066] The term "Polymerase Chain Reaction (PCR)" as used in this invention refers to a method for enzymatically amplifying a specific DNA sequence, which is widely used in diagnosis, genetic research, and molecular biological analysis. The PCR can be implemented by repeating the process of amplifying a desired DNA sequence using primers and polymerases after denaturing DNA at high temperatures.

[0067] The term "Reverse Transcription Polymerase Chain Reaction (RT-PCR)" as used in the present invention refers to a method of first reverse transcribing RNA into cDNA and then amplifying and analyzing it using a polymerase chain reaction, which is used for gene expression analysis, infectious agent detection, etc. The RT-PCR can amplify a desired gene sequence by generating cDNA from RNA according to a known method and then performing PCR.

[0068] The term "Real-time Polymerase Chain Reaction (Real-time PCR)" as used in this invention refers to a method for quantitatively analyzing DNA concentration by monitoring the DNA amplification process in real time, which is useful for diagnosis and measuring gene expression levels. The Real-time PCR measures the amount of amplified DNA in real time using fluorescent labels, and various fluorescent probes and chemical substances may be used according to known methods.

[0069] The term "RNase Protection Assay (RPA)" used in the present invention refers to a method used to quantitatively measure the presence and amount of specific RNA, which involves detecting protected RNA by digesting it with RNase after binding to a radiolabeled RNA probe. The RPA is used for gene expression studies and mRNA stability studies, and various modifications are possible according to known methods.

[0070] The term "microarray" as used in this invention refers to a method of immobilizing numerous DNA or RNA sequences on a small substrate and simultaneously analyzing gene expression based thereon, which is utilized to discover biological markers and compare expression levels. The microarray can be constructed using various known platforms, and results can be detected through fluorescent or radioactive labeling.

[0071] The term "northern blotting" as used in the present invention is a method for detecting the presence and size of specific RNA molecules, which is a technique in which RNA separated by electrophoresis is transferred to a membrane and then analyzed through hybridization with a radioactive or fluorescently labeled probe. The above-mentioned northern blotting is widely used in gene expression studies and RNA-level analysis, and precise detection is possible through various known modifications.

[0072]

[0073] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0074]

[0075]

[0076] Examples

[0077] Example 1: Experimental materials and experimental methods

[0078] 1. Recruitment of Alzheimer's patients and IRB approval

[0079] For the present invention, patients with memory impairment who visited the Department of Neurology at Hanyang University Hospital in Korea between 2019 and 2022 were recruited. Diagnostic criteria for Alzheimer's disease were defined according to the 2011 National Institute on Aging-Alzheimer's Association (NIA-AA) workgroup reports. Demographic information, AD risk factor profiles, and medical history were collected from all participants using questionnaires, electronic medical records, and laboratory test management systems, and blood and cerebrospinal fluid samples were collected from men and women with AD and from controls without AD.

[0080] The cognitive status of the participants was screened using global cognitive assessments [Clinical Dementia Rating (CDR), Clinical Dementia Rating - Sum of Boxes (CDR-SOB), Mini-mental Status Examination (MMSE) scores], and brain imaging was also performed. CDR, CDR-SOB, and MMSE scores were obtained through standard procedures administered to the participants and knowledgeable informants. The CDR-SOB is a simple sum of scores obtained from each of the six evaluated domains and provides additional information to the CDR global score in mild dementia. The MMSE is a test that briefly assesses cognitive function and is the most widely used tool in the world for dementia screening.

[0081] For apolipoprotein E (APOE) genotyping analysis, genomic DNA was isolated and purified from the QIA symphony DSP DNA Mini Kit (Qiagen GmbH, Hilden, Germany) using an automated QIA symphony SP system (Qiagen) in accordance with the manufacturer's instructions. APOE genotyping analysis was performed using the Real-Q APOE genotyping Kit (Biosewoom, Seoul, Korea) on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer's instructions. Individuals possessing one or two APOE ε4 alleles were defined as APOE ε4 carriers.

[0082] Consent was obtained for the future use of CSF and blood samples for research purposes. The study design was approved by the Institutional Review Board of Hanyang University Hospital (HYUH IRB 2018-08-020, 2021-02-034), and the study procedures were conducted in accordance with the World Medical Association's Declaration of Helsinki.

[0083] Clinical information by group of the participants is as follows. (Table 1)

[0084] [Table 1] Clinical Information by Group

[0085]

[0086] Data are presented as means (standard deviations). Values ​​in square brackets represent the percentage of the total number within each group. P-values ​​were determined using ANOVA, the Kruskal-Wallis test, and the chi-square test.

[0087] AD: Alzheimer's disease, CN: Control group, CSF: Cerebrospinal fluid, MCI: Mild cognitive impairment, MMSE: Mini-Mental State Examination, CDR-SOB: Total score of the Clinical Dementia Rating Scale.

[0088]

[0089] 2. Modeling of human microglia-like cells (iMGs) in peripheral blood mononuclear cells

[0090] To generate iMG, peripheral blood mononuclear cells (PBMCs) extracted from whole blood were used to differentiate monocytes into microglia-like cells. PBMCs were separated by density gradient centrifugation using Ficoll (GE Healthcare, Uppsala, Sweden) and cultured at 37°C in RPMI-1640 (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Invitrogen, Carlsbad, CA, USA) under 5% CO2. The next day, adherent cells (monocytes) were cultured for 21 days in RPMI-1640 Glutamax (Gibco) supplemented with 1% antibiotic / antimycotic, 10 ng / ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D Systems, Minneapolis, MN, USA), and 100 ng / ml recombinant human interleukin (IL)-34 (R&D Systems) to model iMG cells, which are human peripheral blood-derived microglia.

[0091]

[0092] 3. Evaluation of Microglia's Phagocytic Ability

[0093] To evaluate the phagocytic ability of microglia, iMGs were incubated with fibrillated Aβ42 or red fluorescent microspheres (L3030, Sigma-Aldrich) at 37°C for 2 hours. Amyloid Beta 1-42 peptide (Aβ42) was used in a conjugated form with HiLyte 488 (Anaspec, Fremont, CA) and initially underwent a fibrillation process following resuspension. Briefly, Aβ42 was resuspended in 10 nM NaOH (10% of the final volume) and then adjusted to a concentration of 1 mg / ml using PBS. The resuspended Aβ42 was frozen at 20°C and subjected to a fibrillation process overnight (12–14 hours) at 37°C prior to use. Prior to phagocytosis analysis, cells were washed three times with PBS to remove de-phagocytosed fluorescent microspheres, and after fixation, stained with Alexa Fluor 488 paroloidin (F-actin, 1:1,000; Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions. Images were acquired using a confocal microscope (TCS SP5, Leica, Wetzlar, Germany).

[0094]

[0095] 4. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

[0096] Total RNA was extracted using Trizol reagent (Invitrogen) and evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, ND-2000). cDNA was obtained from EcoDry ™ It was synthesized using a cDNA kit (Clontech, CA, USA). cDNA amplification was performed using Power SYBR Green PCR Master Mix and primers with Applied Biosystems Step One Plus ™The experiment was performed on a Life Technologies system, followed by heating at 95°C for 10 minutes, and then 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. A melting curve was generated to confirm amplification specificity. Relative quantification (RQ) levels were calculated using the 2-ΔΔCt method with GAPDH as the internal standard control. The reported results are based on three experiments performed independently in different cell batches. The primers used were NCKAP1 (Qiagen, PPH15666A, PPM26732A) and GAPDH (Qiagen, PPH00150F).

[0097]

[0098] 5. Plasma miRNA Analysis

[0099] Plasma samples were obtained via venipuncture on an empty stomach on the morning of the same day. Plasma was centrifuged at 2,000 rpm for 20 minutes, then aliquoted into polypropylene tubes and stored at 80°C. Plasma miRNAs were isolated using the miRNeasy Serum / Plasma-derived Advanced Kit (Qiagen, Hilden, Germany), reverse transcribed using the miRCURY LNA RT kit (Qiagen), and contained cel-miR-39 as the spike-in. 250 ng of cDNA was used for RT-qPCR, which was performed on a Step One Plus system (Applied Biosystems) using miRCURY LNA SYBR Green PCR Master Mix and miRCURY LNA miRNA PCR primers (Qiagen). All procedures were performed according to the manufacturer's instructions. After evaluating the critical cycle (Ct) value, the relative expression level of miRNA was calculated using the ΔΔCt method with miR-103a-3p or U6 snRNA as a normalization control.

[0100]

[0101] 6. RNA sequencing of AD transgenic mice (APP / PS1 mice)

[0102] Transgenic mice overexpressing human amyloid precursor protein (APP) 695 [K670N / M671L (Sweden)] and presenilin-1 (PS1) (M146V) mutations were provided by GlaxoSmithKline (Harlow, UK). Since APP / PS1 mice exhibit sex differences in disease progression, only male mice were used in the experiment. Data analysis was performed at 3, 6, and 9 months of age, and C57BL / 6 mice of the same age and sex were used as wild-type (WT) mice. Animals were assigned to experimental groups using block randomization.

[0103] Total RNA was isolated from cerebral microglia of WT and APP / PS1 mice using Trizol reagent (Invitrogen, 15596026). RNA quality was evaluated using an Agilent 2100 bioanalyzer and an RNA 6000 Nano Chip (Agilent Technologies), and RNA quantification was performed using an ND-2000 spectrophotometer (Thermo Inc). For library preparation and sequencing, 1 μg of total RNA was prepared using the SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc). rRNA was removed using the RIBO COP rRNA removal kit (LEXOGEN, Inc). The rRNA-removed RNA underwent cDNA synthesis and cleavage according to the manufacturer's instructions. Indexing was performed using the Illumina index. Amplification was carried out via PCR, and the average fragment size was subsequently evaluated using an Agilent 2100 bioanalyzer (DNA High Sensitivity Kit). Quantification was performed using a library quantification kit with the StepOne Real-Time PCR System (Life Technologies, Inc). High-process sequencing was performed using the HiSeq 2500 (Illumina, Inc) with paired 100 sequencing. Then, Total RNA-Seq reads were mapped using TopHat software tools to obtain bam files (alignment files). Read counts mapped to transcriptome regions in the alignment files were extracted using bedtools and Bioconductor, utilizing the R statistical programming language. The alignment files were also used for transcriptome assembly, expression level estimation, and detection of differential expression of genes, linc RNA, or isoforms. FPKM (Fragment-Per-Million-Exon-Per-Kilobase) was used to determine the expression levels of gene regions.Percentile normalization was used for comparison between samples. Functional gene classification was performed using DAVID (http: / david.abcc.ncifcrf.gov / ).

[0104]

[0105] 7. Mouse plasma collection

[0106] Mouse blood was collected in sodium heparin-coated tubes via cardiac puncture at the time of death. Plasma was produced by centrifuging freshly collected blood at 15,493 × g at 4°C for 5 minutes, aliquoted, and stored at 80°C.

[0107]

[0108] 8. Statistical Analysis

[0109] Data were presented as mean ± standard error (SEM). The statistical significance of differences between groups was evaluated using Prism 9 (GraphPad Software, San Diego, CA) with one-way analysis of variance (ANOVA) including t-tests and post-hoc Tukey tests. Results were considered significant when *p < 0.05, **p < 0.01, and ***p < 0.001. For post-hoc analysis, p-values ​​were obtained using Pearson correlation coefficients.

[0110]

[0111] Example 2: Experimental Results

[0112] 1. Analysis of Microglia Function Decline and Increased miRNA-214-3p Expression in Alzheimer's Patient Group

[0113] (1) Confirmation of reduced microglia phagocytic ability in Alzheimer's patients using a human-derived microglia model

[0114] As a decrease in the phagocytic ability of microglia has been reported as a pathological characteristic in Alzheimer's patients, the inventors intended to verify Alzheimer's disease-related changes by reproducing this through a human-derived microglia model.

[0115] Figure 1 illustrates the decrease in microglia phagocytic activity observed in Alzheimer's patients using a human-derived microglia model. Figure 1A is a schematic diagram explaining the modeling method for human blood-derived microglia, and Figure 1B includes representative fluorescence images evaluating the phagocytic activity of beads and amyloid-beta oligomers using this model. Here, beads were used as a control to evaluate the general phagocytic function of microglia. Figure 1C is a graph quantifying the decrease in bead phagocytic activity of microglia in Alzheimer's patients compared to a normal group, and Figure 1D shows the quantified results indicating the decrease in amyloid-beta oligomer phagomer phagocytic activity. Finally, Figure 1E shows the results confirming the decrease in NCKAP1 mRNA expression, a phagocytic activity factor, in Alzheimer's patients through qPCR analysis. A significant decrease in phagocytic activity was observed in microglia of Alzheimer's patients using a human-derived microglia model, which is associated with reduced NCKAP1 expression and reflects the functional decline of microglia in Alzheimer's patients.

[0116]

[0117] (2) Increased expression of miRNA-214-3p in the blood of Alzheimer's patients confirmed

[0118] Based on the fact that the blood expression of miRNA-214-3p increases in Alzheimer's patients, the inventors intended to verify this using qPCR to confirm its potential as a diagnostic indicator.

[0119] Figure 2 shows an increase in miRNA-214-3p expression in the blood of the Alzheimer's patient group. Figure 2A shows the results of qPCR analysis of the increase in miRNA-214-3p expression in the blood of the Alzheimer's patient group compared to the normal group, and Figure 2B shows the ROS curves analyzing the expression levels of plasma-derived miRNA-214-3p in the normal group, mild cognitive impairment (MCI) group, and Alzheimer's (AD) patient group. It can be confirmed that plasma-derived miRNA-214-3p was increased in the AD patient group compared to the normal group and the MCI group. This suggests the potential of miRNA-214-3p as a diagnostic marker capable of distinguishing patients with Alzheimer's dementia. Since the expression of miRNA-214-3p in the blood of the Alzheimer's patient group was increased compared to the normal group and the MCI group, it was confirmed that it can be utilized as a potential biomarker for the diagnosis of Alzheimer's patients.

[0120]

[0121] (3) Analysis of the correlation between increased miRNA-214-3p expression and decreased microglia phagocytic ability in Alzheimer's patients

[0122] The inventors intended to determine whether miRNA-214-3p reflects changes in microglia function by analyzing the correlation between increased miRNA-214-3p expression observed in Alzheimer's patients and decreased phagocytic ability of microglia.

[0123] Figure 3 shows the correlation between increased expression of miRNA-214-3p in the blood and decreased microglia phagocytic activity in Alzheimer's patients. Figure 3A shows the correlation with general phagocytic activity (bead phagocytosis) following the increase in miRNA-214-3p, Figure 3B shows the correlation with amyloid-beta oligomer phagocytic activity, and Figure 3C shows the correlation with the expression of NCKAP1 mRNA, a phagocytic activity factor. Increased expression of plasma-derived miRNA-214-3p showed a high correlation with decreased microglia phagocytic activity in Alzheimer's patients, which demonstrates that miRNA-214-3p can be utilized as an indicator of changes in microglia function.

[0124]

[0125] (4) Confirmation of a correlation between increased miRNA-214-3p expression and cognitive decline in Alzheimer's patients

[0126] The inventors investigated the relationship between changes in miRNA-214-3p expression and cognitive decline in a group of Alzheimer's patients to determine whether miRNA-214-3p can be utilized as a biomarker reflecting changes in cognitive function.

[0127] Figure 4 illustrates the correlation between blood expression levels of miRNA-214-3p and indicators of cognitive decline in a group of Alzheimer's patients. Figure 4A shows the correlation with the cognitive function indicator MMSE, Figure 4B with CDR, and Figure 4C with CDR-SB. Through this, it was confirmed that increased miRNA-214-3p expression is a biomarker that can reflect cognitive decline in Alzheimer's patients. In other words, the high correlation between increased miRNA-214-3p expression and indicators of cognitive decline demonstrates the potential of this miRNA as an important indicator capable of predicting cognitive decline.

[0128]

[0129]

[0130] 2. Analysis of the association between miRNA-214-3p and the progression and characteristics of Alzheimer's disease

[0131] (1) Longitudinal study on changes in miRNA-214-3p expression according to disease progression in Alzheimer's patients

[0132] The inventors sought to verify the usefulness of this indicator by longitudinally tracking a group of Alzheimer's patients to determine how miRNA-214-3p expression changes with the progression of Alzheimer's disease.

[0133] Figure 5 shows changes in miRNA-214-3p expression according to disease progression in a group of Alzheimer's patients, based on the results of tracking the same study subjects for over one year. Figures 5A and 5B show the correlation between changes in the phagocytic ability of patient-derived microglia and cognitive function indicators (MMSE and CDR-SB), while Figures 5C and 5D show the correlation between changes in miRNA-214-3p expression and cognitive function indicators. This suggests that miRNA-214-3p and changes in the phagocytic ability of microglia can be utilized as biomarkers related to disease progression. In other words, it indicates that plasma-derived miRNA-214-3p expression and changes in the phagocytic ability of microglia are important indicators that can reflect the decline in cognitive function associated with disease progression in Alzheimer's patients.

[0134]

[0135] (2) Increased expression of miRNA-214-3p confirmed in patients with rapid cognitive decline among Alzheimer's patients

[0136] The inventors analyzed the expression of miRNA-214-3p in a group of Alzheimer's patients with rapidly declining cognitive function to determine whether it could reflect the characteristics of that patient group. Subjects with MMSE >16 and MMSE / yr ≤ -2.5, or MMSE ≤15 and MMSE / yr ≤ -5.5, or CDR-SB / yr ≥ 2.5 were analyzed as a group of patients showing rapid cognitive decline (Rapid Cognitive Decliner).

[0137] Figure 6 shows increased miRNA-214-3p expression in a group of patients with Rapid Cognitive Decliner (RD) exhibiting rapid cognitive decline. Figure 6A shows decreased amyloid-beta oligomer phagomer activity, Figure 6B shows decreased NCKAP1 mRNA expression, and Figure 6C shows increased miRNA-214-3p expression. This suggests that miRNA-214-3p is an indicator that can reflect the characteristics of the RD patient group, thereby presenting its potential as a biomarker related to RD decline.

[0138]

[0139] (3) Confirmation of increased miRNA-214-3p expression according to APOE-ε4 allele status

[0140] Considering that carriers of the APOE-ε4 allele are more susceptible to the development of Alzheimer's disease, the inventors sought to confirm the association with genetic risk factors by analyzing miRNA-214-3p expression in them.

[0141] Figure 7 shows the change in miRNA-214-3p expression according to the possession of the APOE-ε4 allele. Figures 7A and 7B compared the change in miRNA-214-3p expression in the non-possessed group, heterozygous and homozygous APOE-ε4 possessed patient groups, and confirmed an increase in expression in the homozygous patient group.

[0142] Increased expression of miRNA-214-3p showed a significant correlation with APOE-ε4 allele possession status, suggesting that miRNA-214-3p can be a useful biomarker for selecting APOE-ε4 allele carriers.

[0143]

[0144] (4) Confirmation of increased miRNA-214-3p expression with disease progression in a 5xFAD animal model of Alzheimer's dementia

[0145] The inventors intended to confirm the expression patterns according to the progression of Alzheimer's pathology by observing how miRNA-214-3p expression changes according to the stage of disease progression through a 5xFAD mouse model.

[0146] Figure 8 shows the results confirming the decrease in major phagocytic factors in microglia and the increase in miRNA-214-3p expression in the blood according to disease progression in a 5xFAD Alzheimer's dementia animal model. Figure 8A shows a heatmap illustrating the decrease in gene expression during the engulfment stage of the phagocytic process according to the stage of Alzheimer's disease, obtained by performing RNA-sequencing on brain microglia of 5xFAD Alzheimer's mice. Figure 8B is a graph analyzing the decrease in NCKAP1 mRNA expression, a major phagocytic factor, in brain microglia of 5xFAD Alzheimer's mice using qPCR. Additionally, Figure 8C is a graph confirming that miRNA-214-3p expression increases in the blood of 5xFAD Alzheimer's mice according to dementia progression. This suggests that miRNA-214-3p may act as a significant biomarker in relation to the progression of Alzheimer's disease.

[0147]

[0148] As confirmed by the present embodiment, the close correlation between increased expression of miRNA-214-3p in the blood, decreased phagocytic activity of microglia, and cognitive decline in Alzheimer's disease patients suggests that miRNA-214-3p can serve as a biomarker reflecting the progression of Alzheimer's disease. In particular, according to the present invention, an increase in miRNA-214-3p expression can be utilized as an important indicator for diagnosing the disease state and progression rate of dementia patients, and can also provide useful diagnostic information for patient group segmentation, such as the presence of the APOE-ε4 genotype or rapid cognitive decline. Therefore, the miRNA-214-3p-based diagnostic method of the present invention, utilizing novel biomarkers targeting immune inflammation and phagocytic activity, can be evaluated as an efficient approach as a prognostic biomarker reflecting the progression of Alzheimer's disease, as well as a companion biomarker for expanding the application scope of various dementia treatments and selecting target patients.

Claims

1. A marker composition for the diagnosis or prognosis prediction of Alzheimer's disease comprising miRNA-214 (microRNA-214).

2. A marker composition according to claim 1, characterized in that the miRNA-214 consists of the nucleotide sequence of SEQ ID NO.

1.

3. A marker composition according to claim 1, wherein the miRNA-214 inhibits the expression of NCKAP1 (Nck-associated protein 1).

4. A marker composition for the diagnosis or prognosis prediction of Alzheimer's disease, comprising a preparation for measuring the expression level of miRNA-214 (microRNA-214).

5. A marker composition according to claim 4, wherein the agent for measuring the expression level of miRNA-214 is a sense and antisense primer or probe that binds complementarily to miRNA-214.

6. A marker composition according to claim 4, wherein the miRNA-214 is composed of the nucleotide sequence of SEQ ID NO.

1.

7. A marker composition according to claim 4, characterized in that the miRNA-214 is a blood-derived microRNA.

8. A marker composition according to claim 4, wherein the prediction of the prognosis of Alzheimer's disease predicts the prognosis of the cognitive function of a patient with Alzheimer's disease.

9. A kit for diagnosing or predicting the prognosis of Alzheimer's disease, comprising the composition of Claim 4.

10. A method for providing information for the diagnosis or prognosis prediction of Alzheimer's disease, comprising the step of measuring the expression level of miRNA-214 in a biological sample derived from a subject.

11. The information providing method of claim 10, wherein the information providing method determines that there is a high risk of developing Alzheimer's disease or a poor prognosis of Alzheimer's disease when the expression level of miRNA-214 is high.

12. A method for providing information according to claim 10, further comprising the step of determining that rapid cognitive decline is progressing when the expression level of miRNA-214 is high in the prognosis prediction of the Alzheimer's disease.

13. A method for providing information according to claim 10, wherein the miRNA-214 is composed of the nucleotide sequence of SEQ ID NO.

1.

14. A method for providing information according to claim 10, wherein the biological sample is one or more selected from the group consisting of tissue, cell, bone marrow, body fluid, and blood.

15. A method for providing information according to claim 10, wherein the expression level of miRNA-214 is measured through one or more methods selected from the group consisting of next-generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RPA), microarray, and northern blotting.

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