WO2024037389A1 - 一种认知障碍相关疾病生物标志物miR-32533及其应用 - Google Patents

一种认知障碍相关疾病生物标志物miR-32533及其应用 Download PDF

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WO2024037389A1
WO2024037389A1 PCT/CN2023/111859 CN2023111859W WO2024037389A1 WO 2024037389 A1 WO2024037389 A1 WO 2024037389A1 CN 2023111859 W CN2023111859 W CN 2023111859W WO 2024037389 A1 WO2024037389 A1 WO 2024037389A1
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mir
cognitive impairment
related diseases
disease
creb5
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French (fr)
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刘睿
李卓荣
曾利
赵凯悦
蔡中頔
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中国医学科学院医药生物技术研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the invention belongs to the field of biological detection technology, and specifically relates to a cognitive impairment-related disease biomarker miR-32533 and its application.
  • AD Alzheimer’s disease
  • PD Parkinson’s disease
  • VD vascular dementia
  • AD Alzheimer's disease
  • AD is the most common type of Alzheimer's disease.
  • AD is a progressive neurodegenerative disease that has an insidious onset and is highly related to age.
  • the clinical features include cognitive function decline and decreased learning and memory.
  • the main pathological mechanisms are the accumulation of extracellular ⁇ -amyloid (A ⁇ ) to form senile plaques and the excessive phosphorylation of intracellular microtubule-related Tau protein in neurons.
  • NFTs Neurofibrillary tangles
  • neuroinflammation and synaptic dysfunction ultimately lead to neuron death or loss.
  • the anti-AD therapeutic drugs approved by the FDA can only relieve mild and moderate AD symptoms, accompanied by a certain degree of adverse reactions. There is no drug that can reverse the pathological process of the disease and achieve a cure. At this stage, research on new drug targets and specific biodiagnostic markers is of great value for effectively treating AD, determining disease prognosis, and improving patients' quality of life.
  • Non-coding RNAs are abundantly expressed in the central nervous system and play an important epigenetic regulatory role in A ⁇ production, Tau protein phosphorylation, and endogenous apoptosis signaling pathways.
  • new ncRNAs with brain tissue specificity need to be further explored and studied.
  • the spatiotemporal characteristics, pathways and biological functions of ncRNAs in the pathological process of AD have not been systematically studied. Therefore, discovering new ncRNA molecules and elucidating the biological functions of abnormal ncRNAs in AD are of great significance for understanding the early molecular events of AD and proposing molecular mechanisms and drug discovery strategies at the surface level.
  • the purpose of the present invention is to provide a cognitive impairment-related disease biomarker miR-32533 and its application, design an early detection kit, effectively treat Alzheimer's disease (AD), determine the disease outcome and improve the patient's quality of life. .
  • the present invention provides a cognitive impairment-related disease biomarker, miR-32533, which includes the nucleotide sequence shown in SEQ ID NO.1.
  • the present invention also provides the application of miR-32533 described in the above technical solution in the preparation of drugs for treating diseases related to cognitive impairment.
  • the cognitive impairment-related diseases include Alzheimer's disease.
  • the treatment of cognitive impairment-related diseases includes one or more of the following I) to VI):
  • ADAM10 a non-amyloid metabolic pathway of ⁇ -amyloid protein
  • the present invention also provides a drug for treating diseases related to cognitive impairment, the active ingredients of which include miR-32533 and/or substances that promote the expression of miR-32533.
  • the substances include chemical small molecule drugs, nucleic acid drugs and antibody drugs.
  • the present invention also provides the application of miR-32533 described in the above technical solution in preparing one or more kits of the following a) to d):
  • Kits for monitoring the treatment status of people with cognitive impairment-related diseases
  • the cognitive impairment-related diseases include Alzheimer's disease.
  • the present invention also provides a cognitive impairment-related disease screening kit, which includes a reagent for detecting the miR-32533 described in the above technical solution and/or a reagent for detecting the expression level of CREB5.
  • the present invention also provides the application of the CREB5 gene as a target in the preparation of drugs for treating diseases related to cognitive impairment.
  • the cognitive impairment-related diseases include Alzheimer's disease.
  • the miR-32533 provided by the present invention including the nucleotide sequence shown in SEQ ID NO.1, has a high degree of stability and can resist the degradation of endogenous RNase. It is effective in AD model cells, AD model animals, and AD patients. It is significantly down-regulated in serum and can be used as a biomarker for detecting AD.
  • the present invention uses experimental techniques such as flow cytometry, MTS, Western blotting, gene overexpression and silencing to explore the spatiotemporal distribution, pathological effects and molecular mechanisms of miR-32533 in AD. It was clarified that miR-32533 can increase cell viability and reduce cell apoptosis; at the same time, miR-32533 can promote the expression level of ADAM10, the non-amyloid metabolic pathway of ⁇ -amyloid protein, and reduce the amyloid metabolic pathway BACE1 and PS1 of ⁇ -amyloid protein. The expression level of the protein inhibits ⁇ -amyloid (A ⁇ ) and exerts a neuroprotective effect.
  • a ⁇ ⁇ -amyloid
  • the present invention uses online databases (TargetScan, miRDB and Tarbase) to conduct bioinformatics analysis to further explore the occurrence of miR-32533 in the pathological process of AD.
  • online databases TiRScan, miRDB and Tarbase
  • miR-32533 specifically binds to CREB5 and specifically negatively regulates the expression level of CREB5 gene at the transcription and translation levels. Therefore, promoting the expression of miR-32533, reducing the expression level of CREB5, and inhibiting the production of A ⁇ can effectively prevent and treat AD.
  • Figure 1 is a heat map of the expression level of miR-32533 in the cerebral cortex of APP/PS1 mice and WT wild-type control mice detected by RNA high-throughput sequencing;
  • Figure 2 shows the results of RT-PCR detection of the expression level of miR-32533 in APP/PS1 mice and WT wild-type control mice;
  • Figure 3 shows the results of ROC curve analysis of the diagnostic and predictive value of miR-32533 in APP/PS1 mice
  • Figure 4 shows the results of RT-PCR detection of the expression level of miR-32533 in 5 ⁇ FAD mice
  • Figure 5 shows the results of RT-PCR detection of the expression level of miR-32533 in APPswe cells at different time points after copper ion treatment
  • Figure 6 shows the results of MTS detection of the effect of miR-32533 on the viability of APPswe cells induced by copper ions
  • Figure 7 shows the quantitative results of flow cytometry detection of miR-32533 on copper ion-induced apoptosis of APPswe cells
  • Figure 8 is a representative picture of flow cytometry detection of miR-32533 on copper ion-induced apoptosis of APPswe cells;
  • Figure 9 is a representative Western blot image of ADAM10, BACE1, PS1, APP, A ⁇ 1-42 and Bax after APPswe cells were transfected with miR-32533, miR-32533 inhibitor and irrelevant sequence control (NC/NCI);
  • Figure 10 shows the relative expression of representative proteins of ADAM10, BACE1, PS1, APP, A ⁇ 1-42 and Bax after APPswe cells were transfected with miR-32533, miR-32533 inhibitor and irrelevant sequence control (NC/NCI);
  • Figure 12 shows the correlation between the expression level of miR-32533 and the MMSE score
  • Figure 13 shows the ROC curve analysis of the diagnostic and predictive value of miR-32533 in AD patients and HAV healthy controls
  • Figure 14 shows the expression level of CREB5 in the cortex of 1, 3, 6, and 9-month-old APP/PS1 mice detected by RT-PCR;
  • Figure 15 shows the results of RT-PCR detection of the expression level of CREB5 in the cortex of 5 ⁇ FAD mice
  • Figure 16 shows the correlation between the expression levels of miR-32533 and CREB5 in the cerebral cortex of APP/PS1 mice;
  • Figure 17 shows APPswe cells transfected with miR-32533 mimics and miR-32533 inhibitor. CREB5 mRNA expression
  • Figure 18 is a representative image of the effects of miR-32533 overexpression and miR-32533 silencing on CREB5 protein expression levels in APPswe cells;
  • Figure 19 shows the quantification results of CREB5 protein expression levels in APPswe cells by miR-32533 overexpression and miR-32533 silencing
  • Figure 20 is a schematic diagram of the construction of dual-luciferase reporter plasmid wild-type pmirGLO-CREB5-3’UTR-WT and pmirGLO-CREB5-3’UTR-MUT mutant;
  • Figure 21 shows the fluorescence of HEK293 cells co-transfected with dual luciferase reporter gene detection of miR-32533 (or irrelevant sequence control NC) and pmirGLO-CREB5-3'UTR-WT (or pmirGLO-CREB5-3'UTR-MUT). The role of enzyme activity.
  • the present invention provides a cognitive impairment-related disease biomarker miR-32533, including the nucleotide sequence shown in SEQ ID NO. 1, specifically: 5'-UCUCUUCUGCUCUGUGUCACAGC-3'.
  • the nucleotide sequence shown in SEQ ID NO.1 of the present invention is the nucleotide sequence of the mature form of miR-32533.
  • the nucleotide sequence of the precursor of miR-32533 is preferably as shown in SEQ ID NO.2. Specifically For: 5'-UCUCUUCUGCUCUGUGUCACAGCAACAGAAAGCUGCACUGCUGAGGAACA-3'.
  • the present invention uses high-throughput sequencing technology to measure the expression profile of ncRNAs in the brains of 1-, 3-, 6-, and 9-month-old APP/PS1 mice, and screens to unearth miR-32533 with clear characteristic changes, which is highly stable and resistant to internal stress. Degradation of source RNase.
  • the present invention conducts multi-level expression verification in vivo and in vitro of miR-32533, which has tissue specificity, disease specificity, and high species conservation in the pathological process of AD, using miRNA real-time quantitative PCR (RT-PCR).
  • miR-32533 was significantly down-regulated in AD model cells, AD model animals, and the blood of AD patients; at the same time, receiver operating characteristic curve (ROC) analysis showed that miR-32533 was significantly down-regulated in AD patients and healthy controls. It has good clinical diagnostic value as a biomarker in blood. Therefore, there is a disease correlation between miR-32533 and AD and can be used as a diagnostic biomarker for AD.
  • ROC receiver operating characteristic curve
  • the present invention also provides the application of miR-32533 in preparing one or more kits of the following a) to d):
  • Kits for monitoring the treatment status of people with cognitive impairment-related diseases
  • the present invention discovered for the first time miR-32533 with a new sequence structure, and systematically explained its expression change characteristics and biological functions in the pathological processes of cognitive impairment-related diseases.
  • the expression level based on miR-32533 can reflect cognitive impairment-related diseases. Whether it occurs, using miR-32533 as a biomarker, through Detecting the relative expression of miR-32533 determines whether cognitive impairment-related diseases have occurred, which provides a new diagnostic idea for the diagnosis of cognitive impairment-related diseases.
  • using miR-32533 as a biomarker can also screen for cognitive impairment-related diseases. It can monitor the treatment status and prognosis of patients, determine the outcome of cognitive impairment-related diseases and improve the quality of life of patients.
  • Diseases related to cognitive impairment of the present invention include Alzheimer's disease (AD).
  • the present invention also provides an Alzheimer's disease screening kit, which detects the biomarker miR-32533 described in the above technical solution.
  • the present invention uses the biomarker miR-32533 as a detection target, provides a new idea for diagnosing cognitive impairment-related diseases, and is of great value for judging the prognosis of AD disease and improving the quality of life of patients.
  • the cognitive impairment-related diseases of the present invention preferably include Alzheimer's disease (AD).
  • the present invention also provides the application of biomarker miR-32533 in preparing drugs for treating cognitive impairment-related diseases.
  • the cognitive impairment-related diseases preferably include Alzheimer's disease.
  • the present invention in addition to the expression level of miR-32533 being able to reflect the occurrence of cognitive impairment-related diseases, it can also be used as a molecular target to treat cognitive impairment-related diseases.
  • the present invention first used APP/PS1 mice and control mice to conduct high-throughput sequencing analysis and found that the expression level of CREB5 in the pathological process of AD was negatively correlated with the expression of miR-32533 in APP/PS1 mice, and for the first time found that CREB5 and Diseases are related.
  • Bioinformatics software was used to predict and analyze the potential binding sites between CREB5's 3' untranslated regions (3'UTR) and miR-32533, and a dual-luciferase reporter gene was used to detect the specific binding of miR-32533 to CREB5. , found that the biomarker miR-32533 specifically negatively regulates the expression level of CREB5 at the transcription and translation levels.
  • the present invention also uses gene intervention methods to further explore the molecular mechanism of the miR-32533/CREB5 signal axis involved in AD in APPswe cells.
  • the present invention also provides a drug for treating Alzheimer's disease, the active ingredients of which include miR-32533 and/or substances that promote the expression of miR-32533.
  • the substances that promote the expression of miR-32533 preferably include chemical small molecule drugs, nucleic acid drugs and antibody drugs.
  • the medicine provided by the invention directly uses miR-32533 as an active ingredient, or uses a substance that promotes the expression of miR-32533 as an active ingredient, and can promote the expression level of miR-32533, reduce the expression level of CREB5, inhibit the production of A ⁇ , and achieve the treatment of AD. the goal of.
  • the present invention does not have strict requirements on the source of the substances that promote the expression of miR-32533. Any substances that can promote the expression of miR-32533, such as chemical small molecule drugs, nucleic acid molecules and antibody molecules, fall within the scope of the present invention.
  • the steps involved are routine steps, and the reagents used can be purchased routinely or prepared by oneself according to the product instructions.
  • APP/PS1 mice (recorded as APP/PS1mice, purchased from Zhishan (Beijing) Health Medical Research Institute Co., Ltd. (experimental animal license number: SCXK (Beijing) 2021-0010)) and wild mice (recorded as WT mice) were used as experimental materials.
  • APP/PS1 mice aged 1, 3, 6 and 9 months (denoted as APP/PS1mice, purchased from Zhishan (Beijing) Health Medical Research Institute Co., Ltd.) and wild-type controls aged 1, 3, 6 and 9 months were used.
  • Mice (denoted as WT control mice) were used as experimental materials.
  • RT-PCR technology was used to detect the expression levels of miR-32533 in different mice of different ages. The detection steps are as follows:
  • the Ultrapure RNA Kit ultrapure RNA extraction kit from Jiangsu Kangwei Century Biotechnology Co., Ltd. is used. The specific operation is as follows:
  • step (2) According to the ratio of adding 200 ⁇ L chloroform per 1 mL TRIzol, add 200 ⁇ L chloroform to the homogenized tissue in step (1), cover the tube cap, shake vigorously for 15 seconds, place at room temperature for 2 minutes, then centrifuge at 4°C for 10 minutes at 12000 rpm, and the sample Divide into 3 layers: lower red organic phase, middle layer, and upper colorless aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube.
  • step (6) Put the adsorption column back into the collection tube of step (5), empty it at 12,000 rpm for 2 minutes, discard the waste liquid, place the adsorption column at room temperature for a few minutes, and dry it thoroughly.
  • RNA solution Place the adsorption column in a new RNase-free centrifuge tube, add 30 ⁇ L RNase-free water to the middle of the adsorption column, place it at room temperature for 1 min, and centrifuge at 12000 rpm for 1 min. Collect the RNA solution and use a quartz plate on the Spark 20M multifunctional Determine the concentration and purity of the RNA on a microplate reader, and store the RNA at -80°C to prevent degradation.
  • Genomic DNA removal reaction Equilibrate all reagents to room temperature and set aside. Prepare a 20 ⁇ L reaction system according to Table 1 and incubate at 42°C for 2 minutes:
  • nucleotide sequence of the stem-loop reverse transcription primer is shown in SEQ ID NO.3, specifically: 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGTG-3’;
  • nucleotide sequence of the forward primer is shown in SEQ ID NO.4, specifically: 5'-CGCGTCTCTTCTGCTCTGTGT-3';
  • nucleotide sequence of the reverse primer is shown in SEQ ID NO.5, specifically: 5'-AGTGCAGGGTCCGAGGTATT-3';
  • ROC curve analysis was performed using GraphPad Prism Version 8.0 (GraphPad Prism Software, La Jolla, CA, USA). Using the expression level of miR-32533 as the independent variable and APP/PS1 mice and wild-type control (WT) mice of the same age as the dependent variables, the sample values were back-substituted, and sensitivity was used as the ordinate (true positive rate). , with 1-specificity as the abscissa (false positive rate), draw the fitting ROC curve of each indicator to determine the value of biomarkers in the blood as independent diagnosis and joint diagnosis. Determine sensitivity and specificity Degree, the area under curve (AUC), a comprehensive statistic, is used for quantitative analysis to evaluate the diagnostic accuracy of diagnostic biological indicators.
  • AUC area under curve
  • miR-32533 was significantly down-regulated in the cortex and hippocampus of 5 ⁇ FAD mice, further verifying that miR-32533 has good diagnostic value as a biomarker for Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • the Swedish-type human-mouse chimeric APP gene was stably transfected into human neuroblastoma SH-SY5Y cells to obtain APPswe cells.
  • the APPswe cells specifically produce excessive A ⁇ and induce neurotoxicity.
  • copper ions are added, the overexpression of the endogenous APP gene is induced, and at the same time, it forms a chelate with the overproduced A ⁇ , inducing pathological changes such as A ⁇ toxicity, oxidative stress, and apoptosis, simulating the interaction between exogenous factors and endogenous factors.
  • pathological mechanism of AD induced by sex gene mutations.
  • APPswe cells were randomly divided into two groups: experimental group and control group.
  • the experimental group was treated with 300 ⁇ M copper ions, and the control group was not treated with any treatment.
  • the experimental group was randomly divided into experimental group 1, experimental group 2, experimental group 3 and experimental group 4, and the control group was divided into control group 1, control group
  • the experimental group 2, the control group 3 and the control group 4 were transiently transfected with liposomes with 100 nM miRNA-independent sequence negative control (NC: SEQ ID NO.
  • both experimental group 2 and control group 2 used liposomes to transiently transfect 100nM novel miR-32533 mimics (SEQ ID NO. 10, 5'-UCUCUUCUGCUCUGUGUCACAGC-3'); both experimental group 3 and control group 3 used Liposomes were transiently transfected with 100nM miRNA unrelated sequence negative control (NCI: SEQ ID NO.11, 5'-CAGUACUUUUGUGUAGUACAA-3'); both experimental group 4 and control group 4 were transiently transfected with 100nM novel miR -32533 inhibitor (SEQ ID NO. 12, 5'-GCUGUGACACAGAGCAGAAGAGA-3'). The treated cells in the experimental group and control group were incubated at 37°C for 24 h.
  • miR-32533 mimics and miR-32533 inhibitor were transfected into APPswe cells to construct a miR-32533 overexpression and knockdown AD in vitro cell model.
  • APPswe cells treated with 300 ⁇ M copper ions the results were similar to those of transfection.
  • overexpression of miR-32533 significantly increased the viability of APPswe cells, and silencing of miR-32533 significantly reduced the survival rate of APPswe cells.
  • Collect cells Take the cells treated in Step 1 Experimental Groups 1 to 4 and Control Groups 1 to 4, digest the cells with EDTA-free trypsin, centrifuge at 300g and 4°C for 5 minutes, and discard the supernatant;
  • Wash cells Wash cells twice with pre-cooled PBS, centrifuge at 300g and 4°C for 5 minutes each time, and discard the supernatant.
  • PVDF membrane is blocked in 5% skim milk at room temperature for 2h to 3h;
  • Exposure imaging ECL luminescent liquid was added dropwise to the PVDF membrane to react for 3 to 5 minutes, and then exposed on the Bio-Rad gel imager to obtain grayscale bands. Analyze the gray value of the band to calculate the relative expression level of the protein.
  • silencing of miR-32533 reduces the ratio of anti-apoptotic and apoptotic markers (Bcl-2/Bax), and up-regulation of miR-32533 expression significantly inhibits endogenous apoptosis signaling pathway markers.
  • the expression level and activity of miR-32533 significantly reduced the expression level of A ⁇ 1-42 and promoted
  • the expression level of ADAM10 protein in the non-amyloid metabolic pathway of ⁇ -amyloid protein inhibits the expression levels of BACE1 and PS1 in the ⁇ -amyloid metabolic pathway; silencing of miR-32533 significantly reduces the expression level of ADAM10 and promotes the expression of BACE1 and PS1.
  • miR-32533 is involved in the pathological process of A ⁇ generation and in AD-related neuronal damage. Therefore, overexpression of miR-32533 can significantly increase the vitality of APPswe cells, inhibit apoptosis-like degeneration, and exert a neuroprotective effect.
  • RT-PCR technology was used to detect the expression levels of miR-32533 in the serum of AD patients and the serum of healthy volunteers (HAV) of the same age. The detection steps as follows:
  • the serum RNA extraction kit (miRNeasy Serum) from the German company QIAGEN was used.
  • the specific experimental procedures are as follows:
  • Serum sample collection Use anticoagulant tubes to collect blood from AD patients and healthy controls respectively, centrifuge at 1000g for 15 minutes to collect the upper serum, aliquot 200 ⁇ L of each tube and freeze in a -80°C refrigerator;
  • step 1.2 of Example 2 the relative expression levels of miR-32533 in the blood of AD patients and the blood of healthy volunteers (HAV) of the same age were detected.
  • the MMSE score results evaluate the patient's cognitive function based on the Alzheimer's Disease Mini-Mental State Examination (MMSE).
  • the total score of the MMSE scale is 30 points, with 0 to 9 being classified as severe cognitive dysfunction, and 10 to 9 being classified as severe cognitive dysfunction. 20 is classified as moderate cognitive dysfunction, 21 to 26 is classified as mild cognitive impairment, and >27 is classified as normal cognitive function.
  • the sample values were back-substituted, with sensitivity as the ordinate (true positive rate) and 1-specificity as the abscissa. (false positive rate), draw the fitting ROC curve of each indicator, and judge the value of biomarkers in blood as independent diagnosis and joint diagnosis.
  • ROC curve was measured using the same step 2 as in Example 2, and the results are shown in Figure 13.
  • miR-32533 is significantly down-regulated in the pathological process of AD and has good clinical diagnostic value in the blood of AD patients.
  • TargetScan http://www.targetscan.org/
  • miRanda http://www.microrna.org/microrna/home.do
  • pmirGLO-CREB5-3'UTR-WT pmirGLO-CREB5-3'UTR-MUT reporter vector and miR-32533 were transfected into HEK293T cells
  • pmirGLO-CREB5-3' UTR-WT pmirGLO-CREB5-3'UTR-MUT reporter vector and NC were transfected into HEK293T cells as a control.
  • the dual-color luciferase reporter detection system Promega was used to detect firefly and Renilla fluorescence. The normalized relative reporter activity was derived from the enzyme activity.
  • the Dual Luciferase Reporter Assay Kit of Nanjing Novozan Biotechnology Co., Ltd. was used to detect the dual luciferase reporter gene.
  • the specific experimental procedures are as follows:
  • Cell lysis After HEK293 cells are treated, wash the cells twice with PBS, add 100 ⁇ L of 1 ⁇ cell lysis buffer (5 ⁇ Cell Lysis Buffer is diluted with deionized water to 1 ⁇ Cell Lysis Buffer), and lyse on a shaking table for 15 minutes; Centrifuge at 12000g for 2 minutes, and take the cell supernatant for detection;
  • Firefly luciferase activity detection Add 20 ⁇ L of cell lysis supernatant and 100 ⁇ L of luciferase substrate to a 96-well white plate, and immediately use the Spark 20M multifunctional microplate reader to detect the fluorescence, which is recorded as Firefly;
  • Renilla luciferase activity detection Add 100 ⁇ L of the newly prepared Renilla working solution to the reaction working solution in step (2), detect the fluorescence value of Renilla luciferase, and record it as Renilla;
  • Wt represents the relative fluorescence value after co-transfection of miR-32533 (recorded as novel miR-32533) and pmirGLO-CREB5-3'UTR-WT, which is the result of dual-luciferase reporter gene detection
  • Mut represents the relative fluorescence value after co-transfection of miR-32533 (denoted as novel miR-32533) and pmirGLO-CREB5-3'UTR-MUT, which is the result of dual-luciferase reporter gene detection.
  • the miR-32533 provided by the present invention can be used as a biomarker for detecting AD, can specifically bind to CREB5, reduce the expression level of CREB5, inhibit the production of A ⁇ , and can effectively prevent and treat AD.

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Abstract

一种认知障碍相关疾病生物标志物miR-32533及其应用。所述miR-32533包括如SEQ ID NO.1所示的核苷酸序列，可以作为检测认知障碍相关疾病，尤其是阿尔茨海默病(AD)的生物标志物，能够与CREB5基因特异性结合，降低CREB5基因的表达水平，抑制细胞外β-淀粉样蛋白(β-amyloid，Aβ)的生成，能够有效防治AD。

Description

一种认知障碍相关疾病生物标志物miR-32533及其应用
本申请要求于2022年08月17日提交中国专利局、申请号为CN202210983855.5、发明名称为“一种认知障碍相关疾病生物标志物miR-32533及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物检测技术领域，具体涉及一种认知障碍相关疾病生物标志物miR-32533及其应用。
背景技术
随着世界人口老龄化加重，认知障碍相关疾病与老年性痴呆发病率和死亡率逐年攀升，严重危害老年人的身心健康，给家庭和社会带来了沉重的负担。认知障碍相关疾病包括阿尔茨海默病(Alzheimer’s disease，AD)、帕金森病(Parkinson's disease，PD)、亨廷顿病(Huntington's disease)、血管性痴呆(vascular dementia,VD)等。其中，AD是老年性痴呆最常见的类型。AD是一种起病隐匿、与年龄高度相关的进行性神经退行性疾病。临床特征表现为学习和记忆下降的认知功能衰退，主要的病理机制为细胞外β-淀粉样蛋白(β-amyloid，Aβ)聚集形成老年斑、细胞内微管相关Tau蛋白过度磷酸化导致神经元纤维缠结(neurofibrillary tangles，NFTs)、神经炎症、突触功能障碍，最终导致神经元的死亡或者丢失。目前，FDA批准上市的抗AD治疗药物只能缓解轻、中度AD症状，伴随一定程度的不良反应，尚无药物可以逆转疾病的病理过程实现治愈。现阶段，研究新的药物靶点和特异性的生物诊断标志物，对于有效治疗AD、判断疾病转归及改善患者的生活质量具有重要价值。
非编码RNA(non-coding RNA，ncRNAs)在中枢神经系统大量表达，对Aβ生成、Tau蛋白磷酸化、以及内源性凋亡信号通路具有重要的表观遗传学调控作用。在认知障碍相关疾病中，具有脑组织特异性全新的ncRNAs有待进一步挖掘研究。同时，ncRNAs在AD病理进程中的时空特征、发生通路和生物学功能尚未进行系统地研究。因此，挖掘新的ncRNA分子，并阐释异常ncRNA在AD中的生物学功能，对理解AD的早期分子事件、提出表观层面的分子机理与药物发现策略具有重要意义。
发明内容
本发明的目的在于提供一种认知障碍相关疾病生物标志物miR-32533及其应用，设计早期检测试剂盒，有效治疗阿尔茨海默病(AD)、判断疾病转归及改善患者的生活质量。
本发明提供了一种认知障碍相关疾病生物标志物miR-32533，所述miR-32533包括如SEQ ID NO.1所示的核苷酸序列。
本发明还提供了上述技术方案所述的miR-32533在制备治疗认知障碍相关疾病的药物中的应用。
优选的，所述认知障碍相关疾病包括阿尔茨海默病。
优选的，所述治疗认知障碍相关疾病包括下述Ⅰ)～Ⅵ)中的一种或多种：
Ⅰ)促进β-淀粉样蛋白非淀粉样代谢途径ADAM10的表达水平；
Ⅱ)抑制β-淀粉样蛋白淀粉样代谢途径BACE1和PS1的表达水平；
Ⅲ)抑制β-淀粉样蛋白生成；
Ⅳ)发挥神经保护作用；
Ⅴ)提高细胞活力，降低细胞凋亡；
Ⅵ)降低CREB5的表达水平。
本发明还提供了一种治疗认知障碍相关疾病的药物，所述药物的有效成分包括miR-32533和/或促进miR-32533表达的物质。
优选的，所述物质中包括化学小分子药物、核酸药物和抗体药物。
本发明还提供了上述技术方案所述的miR-32533在制备下述a)～d)中的一种或多种试剂盒中的应用：
a)认知障碍相关疾病人群筛查的试剂盒；
b)认知障碍相关疾病人群诊断的试剂盒；
c)认知障碍相关疾病人群治疗状况监测的试剂盒；
d)认知障碍相关疾病人群预后监测的试剂盒。
优选的，所述认知障碍相关疾病包括阿尔茨海默病。
本发明还提供了一种认知障碍相关疾病筛查试剂盒，所述试剂盒包括检测上述技术方案所述miR-32533的试剂和/或检测CREB5的表达水平的试剂。
本发明还提供了CREB5基因作为靶点在制备治疗认知障碍相关疾病的药物中的应用。
优选的，所述认知障碍相关疾病包括阿尔茨海默病。
有益效果：
本发明提供的miR-32533，包括如SEQ ID NO.1所示的核苷酸序列，具有高度的稳定性、能够抵抗内源性RNA酶的降解，在AD模式细胞、AD模式动物、AD患者血清中显著下调，可以作为检测AD的生物标志物。
另外，本发明利用流式细胞术、MTS、蛋白免疫印迹、基因过表达和沉默等实验技术，探讨了miR-32533在AD中的时空分布、病理作用与分子机制。阐明了miR-32533能够提高细胞活力，降低细胞凋亡；同时，miR-32533能够促进β-淀粉样蛋白非淀粉样代谢途径ADAM10的表达水平，降低β-淀粉样蛋白淀粉样代谢途径BACE1和PS1的表达水平，抑制β-淀粉样蛋白(β-amyloid，Aβ)，发挥神经保护作用。并且，本发明利用在线数据库(TargetScan、miRDB以及Tarbase)进行生物信息学分析，进一步探究了miR-32533在AD病理过程中发 挥神经保护作用的药效和机制，发现miR-32533与CREB5特异性结合，并在转录和翻译水平特异性负性调控CREB5基因的表达水平。因此，促进miR-32533的表达，降低CREB5的表达水平，抑制Aβ的生成，能够有效防治AD。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为RNA高通量测序检测APP/PS1小鼠和WT野生型对照小鼠大脑皮层中miR-32533的表达水平的热图；
图2为RT-PCR检测miR-32533在APP/PS1小鼠和WT野生型对照小鼠中的表达水平的结果；
图3为ROC曲线分析miR-32533在APP/PS1小鼠中的诊断预测价值的结果；
图4为RT-PCR检测miR-32533在5×FAD小鼠中的表达水平的结果；
图5为RT-PCR检测铜离子处理后的不同时间点APPswe细胞中miR-32533的表达水平的结果；
图6为MTS检测miR-32533对铜离子诱导的APPswe细胞活力的影响的结果；
图7为流式细胞术检测miR-32533对铜离子诱导的APPswe细胞凋亡的量化结果；
图8为流式细胞术检测miR-32533对铜离子诱导的APPswe细胞凋亡的代表性图片；
图9为APPswe细胞转染miR-32533、miR-32533 inhibitor以及无关序列对照(NC/NCI)后ADAM10、BACE1、PS1、APP、Aβ1-42和Bax代表性蛋白免疫印迹图像；
图10为APPswe细胞转染miR-32533、miR-32533 inhibitor以及无关序列对照(NC/NCI)后ADAM10、BACE1、PS1、APP、Aβ1-42和Bax代表性蛋白相对表达量；
图11为miR-32533在AD患者血液和同龄健康志愿者(HAV)血液中的表达变化水平(n＝10，HAV；n＝11，AD患者)；
图12为miR-32533的表达水平与MMSE评分的相关性；
图13为ROC曲线分析miR-32533在AD患者以及HAV健康对照者中的诊断预测价值；
图14为RT-PCR检测1、3、6、9月龄APP/PS1小鼠皮层中CREB5的表达水平；
图15为RT-PCR检测5×FAD小鼠皮层中CREB5的表达水平的结果；
图16为APP/PS1小鼠大脑皮层中miR-32533与CREB5表达水平的相关性；
图17为miR-32533 mimics以及miR-32533 inhibitor转染APPswe细胞后 CREB5 mRNA的表达情况；
图18为miR-32533过表达以及miR-32533沉默对APPswe细胞中CREB5蛋白表达水平的代表性图像；
图19为miR-32533过表达以及miR-32533沉默对APPswe细胞中CREB5蛋白表达水平的量化结果；
图20为双荧光素酶报告基因质粒野生型pmirGLO-CREB5-3’UTR-WT以及pmirGLO-CREB5-3’UTR-MUT突变型的构建示意图；
图21为双荧光素酶报告基因检测miR-32533(或无关序列对照NC)与pmirGLO-CREB5-3’UTR-WT(或pmirGLO-CREB5-3’UTR-MUT)共转染对HEK293细胞的荧光素酶活性的作用。
具体实施方式
本发明提供了一种认知障碍相关疾病生物标志物miR-32533，包括如SEQ ID NO.1所示的核苷酸序列，具体为：5’-UCUCUUCUGCUCUGUGUCACAGC-3’。本发明SEQ ID NO.1所示的核苷酸序列为miR-32533成熟体的核苷酸序列，所述miR-32533的前体的核苷酸序列优选如SEQ ID NO.2所示，具体为：5’-UCUCUUCUGCUCUGUGUCACAGCAACAGAAAGCUGCACUGCUGAGGAACA-3’。
本发明利用高通量测序技术测定1、3、6、9月龄APP/PS1小鼠脑内ncRNAs表达谱，筛选挖掘出具有明确特征变化的miR-32533，具有高度的稳定性、能够抵抗内源性RNA酶的降解。本发明对AD病理过程中具有组织特异性、疾病特异性、高度物种保守性的miR-32533进行体内外多水平的表达验证，利用miRNA实时荧光定量PCR(Real-time Quantitative PCR,RT-PCR)明确了miR-32533在AD模式细胞、AD模式动物、AD患者血液中显著下调；同时，利用受试者工作特征曲线(receiver operating characteristic curve，ROC)分析显示miR-32533在AD患者以及健康对照者血液中作为生物标志物表现出良好的临床诊断价值。因此，miR-32533与AD存在疾病关联性，可以作为AD诊断生物标志物。
本发明还提供了miR-32533在制备下述a)～d)中的一种或多种试剂盒中的应用：
a)认知障碍相关疾病人群筛选的试剂盒；
b)认知障碍相关疾病人群诊断的试剂盒；
c)认知障碍相关疾病人群治疗状况监测的试剂盒；
d)认知障碍相关疾病人群预后监测的试剂盒。
本发明首次发现具有全新序列结构的miR-32533，系统的阐释了其在认知障碍相关疾病病理过程中的表达变化特征和生物学功能，基于miR-32533的表达量能够反映认知障碍相关疾病是否发生，以miR-32533作为生物标志物，通过 检测miR-32533的相对表达量判断认知障碍相关疾病是否发生，为诊断认知障碍相关疾病提供了新的诊断思路；同时，以miR-32533作为生物标志物还可以筛选患认知障碍相关疾病的人群，并且能够监测患者治疗状况和预后状况，判断认知障碍相关疾病转归及改善患者的生活质量。本发明所述认知障碍相关疾病包括阿尔茨海默病(AD)。
本发明还提供了一种阿尔茨海默病筛查试剂盒，所述试剂盒检测上述技术方案所述的生物标志物miR-32533。本发明以所述生物标志物miR-32533为检测靶点，为诊断认知障碍相关疾病提供了新的思路，对判断AD疾病转归及改善患者的生活质量具有重要价值。本发明所述认知障碍相关疾病优选包括阿尔茨海默病(AD)。
本发明还提供了生物标志物miR-32533在制备治疗认知障碍相关疾病的药物中的应用。
在本发明中，所述认知障碍相关疾病优选包括阿尔茨海默病。
在本发明中，除miR-32533的表达量能够反映认知障碍相关疾病发生外，还可以作为分子靶点治疗认知障碍相关疾病。本发明首先利用APP/PS1小鼠与对照小鼠进行高通量测序分析，发现在AD病理过程中CREB5的表达水平与APP/PS1小鼠中miR-32533表达呈负相关，并首次发现CREB5与疾病存在关联性。利用生物信息学软件预测分析CREB5的3’非翻译区(3’untranslated regions，3’UTR)与miR-32533潜在的结合位点，利用双荧光素酶报告基因检测miR-32533与CREB5特异性结合，发现生物标志物miR-32533在转录和翻译水平特异性负调控CREB5的表达水平。本发明同时利用基因干预手段在APPswe细胞进一步探究miR-32533/CREB5信号轴线参与AD的分子机制。蛋白免疫印迹显示miR-32533通过促进β-淀粉样蛋白非淀粉样代谢途径ADAM10的表达水平，降低β-淀粉样蛋白淀粉样代谢途径BACE1和PS1的表达水平，抑制Aβ生成，缓解Aβ诱导的神经毒性，改善AD的病理表征。因此，通过促进miR-32533的表达水平，降低CREB5的表达水平，抑制Aβ的生成，能够实现治疗AD的目的。
本发明还提供了一种治疗阿尔茨海默病的药物，所述药物的有效成分包括miR-32533和/或促进miR-32533表达的物质。
在本发明中，所述促进miR-32533表达的物质优选包括化学小分子药物、核酸药物和抗体药物。本发明提供的药物直接以miR-32533为活性成分，或者以促进miR-32533表达的物质为活性成分，能够促进miR-32533的表达水平，降低CREB5的表达水平，抑制Aβ的生成，实现治疗AD的目的。本发明对所述促进miR-32533表达的物质的来源没有严格要求，任何能够促进miR-32533表达的物质例如化学小分子药物、核酸分子和抗体分子均属于本发明的保护范围。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的一种认知 障碍相关疾病生物标志物miR-32533及其应用的技术方案进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
在本发明具体实施例中，如无特殊说明，涉及的步骤均为常规步骤，使用的试剂均可常规购买或者按照产品说明书自行配制得到。
实施例1
1.以APP/PS1小鼠(记为APP/PS1mice，购自至善(北京)健康医学研究院有限公司(实验动物许可证号：SCXK(京)2021-0010))和野生小鼠(记为WT mice)为实验材料，分别取1、3、6和9月龄APP/PS1小鼠和野生小鼠的大脑皮层，利用高通量测序技术检测不同年龄段不同小鼠大脑皮层的ncRNAs表达谱，发现在相同年龄段的APP/PS1小鼠和野生小鼠的大脑皮层存在差异表达的非编码RNA(如图1所示)，记为miR-32533，测定其核苷酸序列如SEQ ID NO.1所示(成熟体)，具体为，5’-UCUCUUCUGCUCUGUGUCACAGC-3’；miR-32533的前体序列(记为Pre-miR-32533)如SEQ ID NO.2所示，具体为：5’-UCUCUUCUGCUCUGUGUCACAGCAACAGAAAGCUGCACUGCUGAGGAACA-3’。以上步骤委托上海生工生物工程公司进行。
实施例2
miR-32533在阿尔茨海默病(AD)模式动物中的表达变化
1.RT-PCR检测
以1、3、6和9月龄的APP/PS1小鼠(记为APP/PS1mice，购自至善(北京)健康医学研究院有限公司)，1、3、6和9月龄的野生对照小鼠(记为WT control mice)为实验材料，利用RT-PCR技术检测不同龄段不同小鼠miR-32533的表达水平，检测步骤如下：
1.1总RNA提取
采用江苏康为世纪生物科技有限股份有限公司的Ultrapure RNA Kit超纯RNA提取试剂盒，具体操作如下：
(1)取上述动物组织分别剪碎，放入EP管中，每30mg组织加入1mL TRIzon Reagent，后反复吹打几次，使样本充分裂解，室温放置5min，使蛋白核酸复合物完全分离，放入匀浆仪进行匀浆处理。
(2)按照每1mL TRIzol加入200μL氯仿的比例，向步骤(1)匀浆处理后的组织中加入200μL氯仿，盖好管盖，剧烈振荡15s，室温放置2min后4℃，12000rpm离心10min，样品分为3层：下层红色有机相，中间层，上层无色水相，将上层水相转移到一个新的RNase-Free离心管中。
(3)水相溶液中加入等体积的70％RNase-free水配制的乙醇，颠倒混匀，全部加入到装入收集管的吸附柱中，12000rpm离心20s，倒掉收集管中的废液，将吸附柱重新放回收集管中。
(4)吸附柱中加入700μL Buffer RW1，12000rpm离心20s，倒掉收集管 中的废液。
(5)吸附柱中加入500μL Buffer RW2，12000rpm离心20s，倒掉收集管中的废液，将吸附柱重新放回收集管中；并重复此操作一次。
(6)吸附柱重新放回步骤(5)的收集管中，12000rpm空离2min，弃废液，将吸附柱置于室温数分钟，彻底晾干。
(7)吸附柱置于一个新的RNase-free离心管中，向吸附柱的中间部位加入30μL RNase-Free水，室温放置1min，12000rpm离心1min，收集RNA溶液，使用石英板在Spark 20M多功能酶标仪上测定RNA的浓度和纯度，-80℃保存RNA，防止降解。
1.2 miRNA实时荧光定量PCR(Real-time Quantitative PCR,RT-PCR)
用miRNA 1st Strand cDNA Synthesis Kit试剂盒进行miRNA的逆转录，采用miRNA Universal SYBR qPCR Master Mix试剂盒进行qPCR实验，检测1、3、6和9月龄的APP/PS1小鼠，1、3、6和9月龄的野生对照小鼠组织中miR-32533的表达水平，具体操作步骤如下：
1.2.1 miRNA逆转录反应
(1)基因组DNA去除反应：所有试剂平衡至室温备用，按表1配制20μL的反应体系，42℃孵育2min：
表1基因组DNA去除反应的反应体系
(2)第一链cDNA合成，反应体系如下表2：
表2第一链cDNA合成反应体系
其中，茎环逆转录引物的核苷酸序列如SEQ ID NO.3所示，具体为：5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGTG-3’；
在PCR扩增仪中设置程序：25℃，5min；50℃，15min；85℃，5min；获 得的cDNA产物保存于-20℃或立即进行PCR反应。
1.2.2 RT-qPCR反应
按照表3配制20μL qPCR反应体系，每个样本设置3个复孔；
表3 qPCR反应体系
其中，正向引物的核苷酸序列如SEQ ID NO.4所示，具体为：5’-CGCGTCTCTTCTGCTCTGTGT-3’；反向引物的核苷酸序列如SEQ ID NO.5所示，具体为：5’-AGTGCAGGGTCCGAGGTATT-3’；
在实时荧光定量PCR仪中设置程序进行qPCR反应：预变性：95℃、5min；循环反应：95℃、10s，60℃、30s，95℃、15s，循环数40；融解曲线：60℃、60s；95℃，15s，利用2-ΔΔCT的方法计算miRNA的相对表达量，以U6作为内部参照，检测miR-32533在APP/PS1小鼠和WT野生型对照小鼠中相对表达水平，其中U6的茎环逆转录引物的核苷酸序列如SEQ ID NO.6所示，具体为：5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA-3’；扩增U6的正向引物的核苷酸序列如SEQ ID NO.7所示，具体为：5’-CAAATTCGTGAAGCGTTCCA-3’；扩增U6的反向引物的核苷酸序列如SEQ ID NO.8所示，具体为：5’-AGTGCAGGGTCCGAGGTATT-3’)
结果如图2所示，其中结果以均值±SEM(n＝3)计，*表示与WT control mice相比，APP/PS1mice的P<0.05，**表示与WT control mice相比，APP/PS1 mice的P＜0.01；M表示月份。
根据图2可以看出，miR-32533在相同年龄段的APP/PS1小鼠和WT野生型对照小鼠中的表达量具有显著的差异，miR-32533在APP/PS1小鼠皮层中显著下调。
2.ROC曲线检测
GraphPad Prism Version 8.0(GraphPad Prism Software,La Jolla,CA,USA)进行ROC曲线分析。以miR-32533的表达水平为自变量，以APP/PS1小鼠和同龄的野生型对照(WT)小鼠为因变量，对样本值进行回代，以敏感度作为纵坐标(真阳性率)，以1-特异度为横坐标(假阳性率)，绘制各指标拟合ROC曲线，判断血液中的生物标志物作为独立诊断及联合诊断的价值。判断灵敏度、特异 度，应用曲线下面积(Area under curve，AUC)这一综合统计量作定量分析，评估诊断生物指标诊断准确率，AUC越接近于1，说明诊断效果越好：①AUC介于0.5～0.7时有较低准确性。②AUC介于0.7～0.9时有一定准确性。③AUC在0.9以上时有较高准确性。④AUC＝0.5时，说明作用无效，无诊断价值。
结果如图3所示。根据图3可以看出，在APP/PS1小鼠以及WT对照小鼠中miR-32533作为阿尔茨海默病(AD)生物标志物表现出良好的诊断价值(AUC：0.857，P<0.01vs.WT)，可作为诊断阿尔茨海默病(AD)的标志物。
3.验证实验
以5×FAD小鼠(5×FAD mice，中国人民解放军军事科学院惠赠)和野生对照小鼠(记为WT control mice)的皮层和海马体为实验材料，采用上述步骤1的方式进行RT-PCR检测，检测结果如图4，其中结果以均值±SEM(n＝3)计，*表示与WT control mice相比，5×FAD mice的P<0.05，**表示与WT control mice相比，5×FAD mice的P＜0.01；Cortex为皮层，Hippocampus为海马体。
根据图4可以看出，miR-32533在5×FAD小鼠皮层和海马体中显著下调，进一步验证了miR-32533作为阿尔茨海默病(AD)生物标志物表现出良好的诊断价值。
实施例3
miR-32533在阿尔茨海默病(AD)模式细胞中的表达变化
1.细胞培养
(1)在人神经母细胞瘤的SH-SY5Y细胞中稳定转染瑞典型人鼠嵌合型APP基因，得到APPswe细胞，该APPswe细胞具体产生过量的Aβ，诱导神经毒性。当加入铜离子后，诱导内源性APP基因过度表达，同时与过度生成的Aβ形成螯合物，诱导Aβ毒性、氧化应激、凋亡等病理学变化，模拟了外源性因素与内源性基因突变共同诱导的AD病理机制。
(2)将步骤(1)APPswe细胞培养于含有2mM L-谷氨酰胺，400μM G418以及10％FBS的DMEM/F12培养基中。将APPswe细胞随机分为实验组和对照组两组，其中实验组用300μM铜离子处理，对照组不进行任何处理。随机抽取检测实验组铜离子处理后的不同时间点APPswe细胞中miR-32533的表达水平，结果如图5所示，其中结果以均值±SEM(n＝3)计，***表示与不添加铜离子相比，P＜0.001。
根据图5可以看出，miR-32533在不同时间点铜离子处理的APPswe细胞中呈时间依赖性显著下调。
(3)将步骤(1)APPswe细胞培养于含有2mM L-谷氨酰胺，400μM G418以及10％FBS的DMEM/F12培养基中。将APPswe细胞随机分为实验组和对照组两组，其中实验组用300μM铜离子处理，对照组不进行任何处理。实验组随机分为实验1组，实验2组，实验3组和实验4组，对照组分为对照1组、对 照2组、对照3组和对照4组，实验1组和对照1组均利用脂质体瞬时转染100nM miRNA无关序列阴性对照(negative control，NC：SEQ ID NO.9，5’-UUGUACUACACAAAAGUACUG-3’)；实验2组和对照2组均利用脂质体瞬时转染100nM novel miR-32533 mimics(SEQ ID NO.10，5’-UCUCUUCUGCUCUGUGUCACAGC-3’)；实验3组和对照3组均利用脂质体瞬时转染100nMmiRNA无关序列阴性对照(negative control，NCI：SEQ ID NO.11，5’-CAGUACUUUUGUGUAGUACAA-3’)；实验4组和对照4组均利用脂质体瞬时转染100nM novel miR-32533 inhibitor(SEQ ID NO.12，5’-GCUGUGACACAGAGCAGAAGAGA-3’)。实验组和对照组处理后的细胞在37℃孵育24h。
2.细胞活力检测
取步骤1实验1～4组和对照1～4组处理的细胞，吸除细胞上清液，每孔加入90μLDMEM/F12空白培养基和10μLMTS，37℃孵育2h，多功能酶标仪检测490nm波长处的OD值，细胞存活率(％)＝(OD490实验组-OD490空白组)/(OD490对照组-OD490空白组)×100％，结果如图6所示，其中结果以均值±SEM(n＝3)计，*表示与转染无关序列(NC/NCI)的对照组相比，P＜0.05。
根据图6可以看出，利用miR-32533 mimics和miR-32533 inhibitor转染到APPswe细胞中构建miR-32533过表达和敲低AD体外细胞模型，在300μM铜离子处理的APPswe细胞中，与转染无关序列(NC/NCI)对照组相比，miR-32533的过表达显著提高APPswe细胞活力，miR-32533的沉默显著降低APPswe细胞的存活率。
3.流式细胞术检测
采用南京诺唯赞公司Annexin V-PE/7-AAD凋亡检测试剂盒，具体实验操作如下：
(1)收集细胞：取步骤1实验1～4组和对照1～4组处理的细胞，用不含EDTA的胰酶消化细胞，300g、4℃离心5min，弃上清；
(2)洗涤细胞：预冷的PBS洗涤细胞两次，每次均在300g、4℃离心5min，弃上清。
(3)细胞重悬：加入100μL 1×Binding Buffer，轻轻吹匀至单细胞悬液；
(4)细胞染色：加入5μL AnnexinV-PE和5μL 7-AAD染液，避光室温孵育10min；加入400μL 1×Binding Buffer，轻轻混匀。
(5)检测：步骤(4)获得的样品在1h内用流式细胞仪(BD Biosciences，Franklin Lakes，NJ，USA)检测。检测结果如图7～8，其中结果以均值±SEM(n＝3)计，*表示与转染无关序列(NC/NCI)的对照组相比，P＜0.05。
根据图7～8可以看出，过表达miR-32533显著降低APPswe细胞早期凋亡率、晚期凋亡率和总凋亡率。miR-32533的沉默则表现出相反的效应，即提高 APPswe细胞的凋亡率。
4.蛋白免疫印迹(Westernblot)
4.1蛋白提取
4.2蛋白定量与变性
4.3 Westernblot实验
(1)SDS-聚丙烯酰胺凝胶的配制；
(2)蛋白上样和电泳：每孔上样20μg～60μg蛋白，电泳过程为恒压模式，80V、120min或者80V、60min，100V、40min；待蛋白样品中的溴芬蓝接近分离胶底部，停止电泳；
(3)转膜(湿转)：PVDF膜在无水甲醇中活化1min，超纯水中浸泡5min，转膜液中平衡10min。含蛋白样品的分离胶于转膜液中平衡10min。按照由负极到正极依次为海绵-滤纸-分离胶-PVDF膜-滤纸-海绵依次叠放，避免气泡，恒流模式，390mA冰上转膜70min；
(4)封闭：PVDF膜在5％脱脂牛奶中室温封闭2h～3h；
(5)一抗孵育：利用5％脱脂牛奶或者5％BSA按照1:1000的比例配制目标抗体，根据目的蛋白分子量和对应分子量的蛋白Marker裁膜，4℃摇晃孵育过夜；
(6)洗膜和二抗孵育：含有0.1％Tween-20的Tris缓冲盐溶液(Tris-buffered saline and Tween-20，TBST)摇晃洗膜5次，每次6min。配制一抗相应种属的辣根过氧化酶(Horseradish peroxidase，HRP)偶联的二抗，室温孵育1h；TBST缓冲液，洗膜5次，每次6min，去除非特异性结合；
(7)曝光成像：ECL发光液滴加至PVDF膜上反应3～5min，于Bio-Rad凝胶成像仪进行曝光，得到灰度条带。分析条带的灰度值计算蛋白相对表达水平。
按照蛋白免疫印迹基本操作过程检测关联分子蛋白水平的变化，其中，一抗孵育时，利用5％脱脂牛奶或者5％BSA按照1:1000的比例配制目标抗体(ADAM10、BACE1、PS1、APP、Aβ1-42和Bax)；二抗孵育时配制一抗相应种属的辣根过氧化酶(Horseradish peroxidase，HRP)偶联的二抗，最后曝光成像：ECL发光液滴加至PVDF膜上反应3-5min，于Bio-Rad凝胶成像仪进行曝光，得到灰度条带。分析条带的灰度值计算蛋白相对表达水平，结果如图9～10所示，其中结果以均值±SEM(n＝3)计，*表示与转染无关序列(NC/NCI)的对照组相比，P＜0.05，**表示与转染无关序列(NC/NCI)的对照组相比，P＜0.01。
根据图9～10可以看出，miR-32533的沉默降低抗凋亡和凋亡标志物(Bcl-2/Bax)的比率，miR-32533表达上调则显著抑制内源性凋亡信号通路标志物的表达水平与活性；miR-32533的过表达显著降低Aβ1-42的表达水平，并促进 β-淀粉样蛋白非淀粉样代谢途径中ADAM10蛋白的表达水平，抑制β-淀粉样蛋白淀粉样代谢途径BACE1和PS1的表达水平；miR-32533的沉默则显著降低ADAM10的表达水平，促进BACE1和PS1的表达水平，从而促进Aβ1-42的过度生成。上述结果提示，miR-32533参与Aβ生成的病理过程，参与AD相关的神经元损伤。因此，miR-32533的过表达可以显著提高APPswe细胞的活力，抑制细胞凋亡样退变，发挥神经保护作用。
实施例4
miR-32533在阿尔茨海默病(AD)患者血液中的表达变化
1.RT-PCR检测
收集11个AD患者和10个同龄健康志愿者(HAV)血液，以此为实验材料，利用RT-PCR技术检测AD患者血清和同龄健康志愿者(HAV)血清miR-32533的表达水平，检测步骤如下：
1.1血液样本miRNA的提取
采用德国QIAGEN公司的血清RNA提取试剂盒(miRNeasy Serum)，具体实验操作如下：
(1)血清样本采集：使用抗凝管分别采集AD患者和健康对照者血液，1000g离心15min取上层血清，200μL每管分装冻放于-80℃冰箱；
(2)取200μL血清，加入1mL的QIAzol Lysis Reagent，颠倒混匀，室温放置5min；
(3)加入200μL氯仿，剧烈震荡30s，室温放置3min，12000g、4℃离心15min；
(4)吸取上层水相，加入1.5倍体积的无水乙醇，振荡混匀；
(5)吸取上层水相和无水乙醇混合液至RNeasy MinElute spin柱中，8000g离心30s，倒掉滤液，收集RNA；
(6)加入700μL RWT缓冲液，8000g离心30s，倒掉滤液；
(7)加入500μL RPE缓冲液，8000g离心30s，倒掉滤液；
(8)加入500μL 80％乙醇，8000g离心2min，倒掉滤液；
(9)RNA收集柱重新放置于收集管中，8000g离心5min，弃掉滤液和收集管；
(10)RNA收集柱转移至新的1.5mL离心管中，滴加14μL RNase-free水，8000g离心1min洗脱收集RNA。
(12)Spark 20M多功能酶标仪上测定RNA的浓度和纯度，置于-80℃保存，进行后续实验。
1.2 miRNA实时荧光定量PCR(Real-time Quantitative PCR,RT-PCR)
利用同实施例2步骤1.2，检测miR-32533在AD患者血液和同龄健康志愿者(HAV)血液的相对表达水平，结果如图11～12所示，其中结果以均值±SEM (n＝3)计，*表示与同龄健康志愿者(HAV)血液相比，P＜0.05。
根据图11～12可以看出，根据图3可以看出，miR-32533在AD患者血液中显著下调，且与AD患者MMSE评分呈显著正相关(R2＝0.453，P<0.05)。其中，MMSE评分结果根据阿尔茨海默病简易精神状态检查量表(MMSE)对患者认知功能进行评估，MMSE量表总分为30分，0～9分为重度认知功能障碍，10～20分为中度认知功能障碍，21～26分为轻度认知功能障碍，>27分为认知功能正常。
2.ROC曲线检测
以miR-32533的表达水平为自变量，以AD患者和HAV健康对照者为因变量，对样本值进行回代，以敏感度作为纵坐标(真阳性率)，以1-特异度为横坐标(假阳性率)，绘制各指标拟合ROC曲线，判断血液中的生物标志物作为独立诊断及联合诊断的价值。
利用同实施例2步骤2，测定ROC曲线，结果如图13所示。根据图13可以看出，miR-32533在AD患者以及HAV健康对照者血液中作为生物标志物表现出良好的诊断价值(AUC＝0.757，Sensitivity＝100％，Specificity＝20％)。结合图11～12，miR-32533在AD病理过程中显著下调，并且在AD患者血液中具有良好的临床诊断价值。
实施例5
miR-32533靶基因预测
1.利用生物信息学软件TargetScan(http://www.targetscan.org/)，miRanda(http://www.microrna.org/microrna/home.do)，TarBase(http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php？r＝tarbasev8％2Findex)进行miR-32533靶基因分析，发现CREB5与AD存在疾病关联性，具体的：
通过qRT-PCR检测发现，CREB5在APP/PS1小鼠大脑皮层中显著上调(图14，其中结果以均值±SEM(n＝3)计，*表示与WT control mice相比，APP/PS1mice的P<0.05，***表示与WT control mice相比，APP/PS1mice的P＜0.001；M为月)；在5×FAD小鼠大脑皮层中显著上调(图15，其中结果以均值±SEM(n＝3)计，*表示与WT control mice相比，5×FAD mice的P<0.05)，miR-32533与CREB5的表达水平呈显著负相关(图16，R2＝0.566，P<0.01)。同时，miR-32533可以显著降低CREB5在mRNA和蛋白质水平上的表达，而miR-32533的沉默可以显著促进CREB5 mRNA和蛋白的表达水平(图17～19，其中结果以均值±SEM(n＝3)计，*表示与转染无关序列(NC/NCI)的对照组相比，P＜0.05)。
2.构建双荧光素酶报告基因及检测步骤：
根据生物信息学软件Starbase、miRbase以及Targetscan预测miR-32533与CREB5的3’UTR匹配的DNA序列，应用Primer 5.0设计特异性引物，PCR扩 增获得DNA片段，将其插入pmirGLO双荧光素酶报告系统质粒(Promega)，构建pmirGLO-CREB5-3’UTR-WT质粒(记为WT)。将miR-32533与CREB5结合位点进行碱基突变，构建pmirGLO-CREB5-3’UTR-MUT质粒(记为MUT)，结果如图20所示。采用脂质体转染法，将pmirGLO-CREB5-3’UTR-WT(pmirGLO-CREB5-3’UTR-MUT)报告子载体和miR-32533转染至HEK293T细胞，同时将pmirGLO-CREB5-3’UTR-WT(pmirGLO-CREB5-3’UTR-MUT)报告子载体和NC转染至HEK293T细胞，作为对照，转染48h后，利用双色荧光素酶报告子检测系统(Promega)检测firefly和Renilla荧光素酶的活性，得出标准化的相对报告子活性。
双荧光素酶报告基因的检测采用南京诺唯赞生物科技股份有限公司Dual Luciferase ReporterAssay Kit试剂盒，具体实验操作如下：
(1)裂解细胞：HEK293细胞经处理后，用PBS洗涤细胞两次，加入100μL 1×细胞裂解液(5×Cell Lysis Buffer用去离子水稀释为1×Cell Lysis Buffer)，摇床上裂解15min；12000g离心2min，取细胞上清液用于检测；
(2)萤火虫荧光素酶活性检测：在96孔白板中加入20μL细胞裂解上清和100μL luciferase底物，立即利用Spark 20M多功能酶标仪检测荧光，记为Firefly；
(3)海肾荧光素酶活性检测：步骤(2)反应工作液中加入100μL现配制的Renilla工作液，检测Renilla luciferase的荧光值，记为Renilla；
(4)分析：计算Firefly/Renilla的比值作为相对荧光值，结果如图21所示，其中图21中结果以均值±SEM(n＝3)计，Wt组*表示与转染无关序列NC的对照组相比，P＜0.05；Wt表示miR-32533(记为novel miR-32533)与pmirGLO-CREB5-3’UTR-WT共转染后相对荧光值，即双荧光素酶报告基因检测结果；Mut表示miR-32533(记为novel miR-32533)与pmirGLO-CREB5-3’UTR-MUT共转染后相对荧光值，即双荧光素酶报告基因检测结果。
根据图21可以看出，miR-32533与pmirGLO-CREB5-3’UTR-WT共转染显著降低HEK293细胞中荧光素酶活性；而miR-32533与pmirGLO-CREB5-3’UTR-MUT共转染则无显著变化，表明miR-32533直接与CREB5特异性结合，并在转录和翻译水平特异性负性调控CREB5的表达水平。
根据上述实施例，本发明提供的miR-32533，可以作为检测AD的生物标志物，能够与CREB5特异性结合，降低CREB5的表达水平，抑制Aβ的生成，能够有效防治AD。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (11)

  1. 一种认知障碍相关疾病生物标志物miR-32533，其特征在于，所述miR-32533包括如SEQ ID NO.1所示的核苷酸序列。
  2. 权利要求1所述的miR-32533在制备治疗认知障碍相关疾病的药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述认知障碍相关疾病包括阿尔茨海默病。
  4. 根据权利要求2或3所述的应用，其特征在于，所述治疗认知障碍相关疾病包括下述Ⅰ)～Ⅵ)中的一种或多种：
    Ⅰ)促进β-淀粉样蛋白非淀粉样代谢途径ADAM10的表达水平；
    Ⅱ)降低β-淀粉样蛋白淀粉样代谢途径BACE1和PS1的表达水平；
    Ⅲ)抑制β-淀粉样蛋白生成；
    Ⅳ)发挥神经保护作用；
    Ⅴ)提高细胞活力，降低细胞凋亡；
    Ⅵ)降低CREB5的表达水平。
  5. 一种治疗认知障碍相关疾病的药物，其特征在于，所述药物的有效成分包括miR-32533和/或促进miR-32533表达的物质。
  6. 根据权利要求5所述的应用，其特征在于，所述促进miR-32533表达的物质包括化学小分子药物、核酸药物和抗体药物中的一种或多种。
  7. 权利要求1所述的miR-32533在制备下述a)～d)中的一种或多种试剂盒中的应用：
    a)认知障碍相关疾病人群筛查的试剂盒；
    b)认知障碍相关疾病人群诊断的试剂盒；
    c)认知障碍相关疾病人群治疗状况监测的试剂盒；
    d)认知障碍相关疾病人群预后监测的试剂盒。
  8. 根据权利要求7所述的应用，其特征在于，所述认知障碍相关疾病包括阿尔茨海默病。
  9. 一种认知障碍相关疾病筛查试剂盒，其特征在于，所述试剂盒包括检测权利要求1所述miR-32533的试剂和/或检测CREB5的表达水平的试剂。
  10. CREB5基因作为靶点在制备治疗认知障碍相关疾病的药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述认知障碍相关疾病包括阿尔茨海默病。
PCT/CN2023/111859 2022-08-17 2023-08-09 一种认知障碍相关疾病生物标志物miR-32533及其应用 WO2024037389A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103074423A (zh) * 2012-12-29 2013-05-01 深圳市第三人民医院 Creb5基因的用途
CN111956658A (zh) * 2020-07-28 2020-11-20 中国医学科学院医药生物技术研究所 miRNA 148簇作为诊断和/或治疗认知障碍相关疾病标志物的应用
CN113905739A (zh) * 2019-03-24 2022-01-07 株式会社阿丽浱欧 用于降低β淀粉样蛋白形成的组合物和方法及其组合物
CN115927583A (zh) * 2022-08-17 2023-04-07 中国医学科学院医药生物技术研究所 一种认知障碍相关疾病生物标志物miR-32533及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103074423A (zh) * 2012-12-29 2013-05-01 深圳市第三人民医院 Creb5基因的用途
CN113905739A (zh) * 2019-03-24 2022-01-07 株式会社阿丽浱欧 用于降低β淀粉样蛋白形成的组合物和方法及其组合物
CN111956658A (zh) * 2020-07-28 2020-11-20 中国医学科学院医药生物技术研究所 miRNA 148簇作为诊断和/或治疗认知障碍相关疾病标志物的应用
CN115927583A (zh) * 2022-08-17 2023-04-07 中国医学科学院医药生物技术研究所 一种认知障碍相关疾病生物标志物miR-32533及其应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
LEE CHANG YOUN, RYU IN SOO, RYU JIN-HYEOB, CHO HYUN-JEONG: "miRNAs as Therapeutic Tools in Alzheimer’s Disease", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL (MDPI), BASEL, CH, vol. 22, no. 23, 1 December 2021 (2021-12-01), Basel, CH , pages 13012, XP093140685, ISSN: 1422-0067, DOI: 10.3390/ijms222313012 *
LINLIN WANG, LIU RUI; ZHANG JUNXIA: "Research Progress of MicroRNA Related to Neurodegenerative Diseases", MEDICAL RECAPITULATE, YIXUE ZONGSHU, CN, vol. 23, no. 6, 20 March 2017 (2017-03-20), CN , pages 1041 - 1046, XP093140684, ISSN: 1006-2084, DOI: 10.3969/j.issn.1006-2084.2017.06.001 *
LIU SEN, FAN MIN, ZHENG QIANG, HAO SHENGWEI, YANG LONGJUN, XIA QINGRONG, QI CONGCONG, GE JINFANG: "MicroRNAs in Alzheimer's disease: Potential diagnostic markers and therapeutic targets", BIOMEDICINE & PHARMACOTHERAPY, ELSEVIER, FR, vol. 148, 1 April 2022 (2022-04-01), FR , pages 112681, XP093140688, ISSN: 0753-3322, DOI: 10.1016/j.biopha.2022.112681 *
NHO KWANGSIK, NUDELMAN KELLY, ALLEN MARIET, HODGES ANGELA, KIM SUNGEUN, RISACHER SHANNON L., APOSTOLOVA LIANA G., LIN KUANG, LUNNO: "Genome‐wide transcriptome analysis identifies novel dysregulated genes implicated in Alzheimer's pathology", ALZHEIMER'S & DEMENTIA, ELSEVIER, NEW YORK, NY, US, vol. 16, no. 9, 1 September 2020 (2020-09-01), US , pages 1213 - 1223, XP093140681, ISSN: 1552-5260, DOI: 10.1002/alz.12092 *
SWARBRICK S.; WRAGG N.; GHOSH S.; STOLZING ALEXANDRA: "Systematic Review of miRNA as Biomarkers in Alzheimer’s Disease", MOLECULAR NEUROBIOLOGY, SPRINGER US, NEW YORK, vol. 56, no. 9, 8 February 2019 (2019-02-08), New York, pages 6156 - 6167, XP036853048, ISSN: 0893-7648, DOI: 10.1007/s12035-019-1500-y *
ZHAO YUHAI, JABER VIVIAN, ALEXANDROV PETER N., VERGALLO ANDREA, LISTA SIMONE, HAMPEL HARALD, LUKIW WALTER J.: "microRNA-Based Biomarkers in Alzheimer’s Disease (AD)", FRONTIERS IN NEUROSCIENCE, FRONTIERS RESEARCH FOUNDATION, CH, vol. 14, 30 October 2020 (2020-10-30), CH , pages 585432, XP093140687, ISSN: 1662-453X, DOI: 10.3389/fnins.2020.585432 *

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