WO2021088317A1 - 一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物 - Google Patents

一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物 Download PDF

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WO2021088317A1
WO2021088317A1 PCT/CN2020/084626 CN2020084626W WO2021088317A1 WO 2021088317 A1 WO2021088317 A1 WO 2021088317A1 CN 2020084626 W CN2020084626 W CN 2020084626W WO 2021088317 A1 WO2021088317 A1 WO 2021088317A1
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expression
disease
mir
alzheimer
mirna
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刘睿
李卓荣
姜海伦
曾利
张俊霞
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中国医学科学院医药生物技术研究所
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Priority to US17/614,414 priority Critical patent/US20220259658A1/en
Publication of WO2021088317A1 publication Critical patent/WO2021088317A1/zh
Priority to US17/865,475 priority patent/US12006551B1/en

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  • the embodiments of the present invention relate to the field of biotechnology, in particular to a miRNA marker for diagnosis and/or treatment of Alzheimer's disease.
  • AD Alzheimer's Disease
  • a ⁇ hypothesis tau protein hyperphosphorylation hypothesis
  • cholinergic imbalance hypothesis are the main hypotheses in the pathological process of AD, and most of the drugs currently in clinical research are also based on the above hypotheses.
  • the prevention and treatment of AD mainly have the following problems: the lack of simple and non-invasive early screening methods; the pathological mechanism is not clear, and the lack of reliable drug research targets.
  • AD pathogenesis of AD
  • genes such as PSEN1, PSEN2, and APP in familial AD, which can be found through early genetic screening, while sporadic AD, which accounts for 95% of the total number of AD patients, has not been reported so far.
  • changes in related genes are of great significance to the prevention and treatment of AD and the discovery of clinical biomarkers.
  • the embodiment of the present invention provides a miRNA marker for diagnosis of Alzheimer's disease, so as to solve the problem that there is no diagnostic marker for Alzheimer's disease at the gene level in the prior art.
  • a miRNA marker for diagnosis and/or treatment of Alzheimer's disease characterized in that the miRNA marker is a miRNA23 cluster.
  • the miRNA23 cluster is selected from hsa-miR-23b, and its nucleotide sequence is shown in SEQ ID NO. 1;
  • the miRNA23 cluster is selected from hsa-miR-23b-3p, and its nucleotide sequence is shown in SEQ ID NO.2.
  • the embodiment of the present invention also provides an application of a primer in a preparation kit, and the primer is a primer for the miRNA marker described above.
  • the kit is used to provide diagnosis of Alzheimer's disease in patients suffering from or at risk of developing Alzheimer's disease, predicting the risk of developing Alzheimer's disease, or predicting the risk of Alzheimer's disease result.
  • the primer is used to determine the expression level of the miRNA marker in the sample.
  • the sample is serum.
  • the expression level of the miRNA marker is based on the patient's miRNA marker expression level and the healthy human miRNA marker reference expression level.
  • the determination of the expression level of the miRNA marker is a sequencing-based method, an array-based method, or a PCR-based method.
  • the embodiment of the present invention also provides an application of the above-mentioned miRNA marker agonist in the preparation of a medicine for the treatment of Alzheimer's disease.
  • the expression of miRNA 23 clusters of microRNAs is reduced in Alzheimer's disease, which reduces neuronal cell apoptosis by inhibiting GSK-3 ⁇ -mediated phosphorylation of tau protein.
  • the 23 microRNA clusters of miRNA are:
  • miRNA 23 cluster of microRNA is selected from the following characteristics: (a) miRNA microRNA, miRNA 23b microRNA is selected from hsa-miR-23b, and its sequence is shown in SEQ ID NO.1: cucaggugcucuggcugcuuggguuccuggcaugcugauuuugugacuuaagauuaaaaucacauugccagcaccacugacugca The default mature body (hsa-miR-23b-3p) sequence is shown in SEQ ID NO. 2: aucacauugccagggauuaccac; (b) modified miRNA microRNA derivatives; or 18-26 nt in length, functional and miRNA microRNA MicroRNAs or modified miRNA derivatives with the same or substantially the same RNA;
  • the embodiment of the present invention provides a preparation and a medicine, which are agonists of the microRNA in (1).
  • the embodiment of the present invention found that the 23 clusters of miRNA microRNAs play a role in the diagnosis and treatment of Alzheimer’s disease.
  • AD model cells AD model animals and natural aging animals, clinical blood samples, the use of microRNA markers targeting miRNA23 clusters Detected its expression in the human body, and found that the expression of miRNA 23 clusters of microRNAs is significantly reduced in the process of Alzheimer’s disease, so miRNA 23 clusters of microRNAs can be used as a new Alzheimer’s disease marker It is used in the auxiliary diagnosis of Alzheimer’s disease; the embodiment of the present invention found that the up-regulation of the 23 clusters of miRNA microRNA expression can negatively regulate the expression of GSK-3 ⁇ and inhibit tau protein phosphorylation and neuronal apoptosis; miRNA 23 clusters The down-regulation of the expression of microRNAs significantly increases the phosphorylation level of tau protein and promotes neuronal apoptosis.
  • the 23 clusters of miRNAs can negatively regulate the expression of GSK-3 ⁇ to play a neuroprotective role in the process of Alzheimer’s disease;
  • the function of miRNA 23 clusters of microRNAs has been studied systematically. Based on the above findings, miRNAs of 23 clusters of microRNAs can be used as new therapeutic targets for Alzheimer’s disease, and miRNAs of 23 clusters of microRNAs are used as Alzheimer’s. Targeted therapy of Murder's disease biomarkers provides new ideas.
  • Figure 1 shows the detection of the differential expression of miR-23b in brain tissues of AD model animals and wild-type animals by miRNA chip technology according to an embodiment of the present invention
  • Figure 2 shows the expression detection of miR-23b in the blood of AD model cells, AD model animals, natural aging animals, and AD patients according to an embodiment of the present invention
  • Figure 3 shows the effect of miR-23b on nerve cell viability and nerve cell apoptosis according to an embodiment of the present invention
  • FIG. 4 shows that miR-23b according to an embodiment of the present invention negatively regulates its expression by specifically targeting GSK-3 ⁇ mRNA and 3'UTR;
  • Figure 5 shows the effect of up-regulation or down-regulation of miR-23b expression on tau protein phosphorylation level and apoptosis-related proteins in an embodiment of the present invention
  • Figure 6 is an embodiment of the present invention overexpression of GSK-3 ⁇ can reverse the effect of miR-23b in regulating tau protein phosphorylation and neuronal cell apoptosis;
  • Fig. 7 shows the effect of miR-23b on the cognitive and memory functions of AD model animals according to an embodiment of the present invention
  • Fig. 8 is an example of the present invention to verify the changes of the GSK-3 ⁇ /p-tau and GSK-3 ⁇ /Bax/caspase-3 pathways regulated by miR-23b in the pathological process of AD.
  • the term "expression level” refers to a measured expression level compared with a reference nucleic acid (e.g., from a control), or a calculated average expression value (e.g., in RNA chip analysis).
  • a certain "expression level” can also be used as a result and determined by the comparison and measurement of several nucleic acids of interest disclosed below, and show the relative abundance of these transcripts with each other.
  • the expression level can also be evaluated relative to the expression in different tissues, patients on healthy controls, and so on.
  • sample or “biological sample” is a sample that is derived from or has been in contact with a biological organism.
  • biological samples are: cells, tissues, body fluids, biopsy samples, blood, urine, saliva, sputum, plasma, serum, cell culture supernatants, etc.
  • a “gene” is a nucleic acid segment that contains the information necessary to produce a functional RNA product in a controlled manner.
  • “Gene product” is a biological molecule produced by gene transcription or expression, such as mRNA or translated protein.
  • RNA is a short, naturally occurring RNA molecule, and should have a general meaning as understood by those skilled in the art.
  • a “miRNA-derived molecule” is a molecule obtained from a miRNA template chemically or enzymatically, such as cDNA.
  • array refers to an arrangement of addressable positions on a device (for example, a chip device). The number of locations can vary from a few to at least hundreds or thousands. Each position represents an independent reaction site.
  • Arrays include, but are not limited to, nucleic acid arrays, protein arrays, and antibody arrays.
  • Nucleic acid array refers to an array containing nucleic acid probes, such as oligonucleotides, polynucleotides, or larger portions of genes. The nucleic acids on the array are preferably single-stranded.
  • PCR-based method refers to a method involving polymerase chain reaction PCR. This is a method of exponentially amplifying nucleic acids "such as DNA or RNA” by using one, two or more primers to replicate enzymatically in vitro. For RNA amplification, reverse transcription can be used as the first step.
  • PCR-based methods include kinetic or quantitative PCR (qPCR), which are particularly suitable for analyzing expression levels.
  • PCR-based methods can be used, for example, to detect the presence of a given mRNA, which reverse transcribes the complete mRNA library (the so-called transcriptome) into cDNA with the help of reverse transcriptase, and The presence of a given cDNA is detected with the help of corresponding primers.
  • This method is commonly referred to as reverse transcriptase PCR (rtPCR).
  • PCR-based method includes both end-point PCR applications and kinetic/real-time PCR techniques using special fluorophores or intercalating dyes that emit fluorescent signals as amplification targets Function and allows monitoring and quantification of the target.
  • the term "marker” or “biomarker” refers to an organism whose presence or concentration can be detected and associated with a known condition (such as a disease state) or clinical outcome (such as response to treatment).
  • a known condition such as a disease state
  • clinical outcome such as response to treatment.
  • Molecules such as nucleic acids, peptides, proteins, hormones, etc.
  • Example 1 Detection of the differential expression of miR-23b in brain tissues of AD model animals and wild-type animals by miRNA chip technology
  • the miRNA 23 clusters of microRNAs in the embodiment of the present invention are hsa-miR-23b.
  • the sequence is shown in SEQ ID NO.1: cucaggugcucuggcugcuuggguuccuggcaugcugauuugugacuuaa
  • hsa-miR-23b-3p The default mature body (hsa-miR-23b-3p) sequence is shown in SEQ ID NO. 2: aucacauugccagggauuaccac.
  • mature body (hsa-miR-23b-3p) miRNA reverse transcription primer SEQ ID NO. 3: gtcgtatcca gtgcagggtc cgaggtattcgcactggatacgacgtggta; quantitative PCR (qPCR) forward primer: SEQ ID NO.
  • the hippocampus and cortex tissues of 1, 3, 6 and 9-month-old APP/PS1 double transgenic mice and SAMP8 natural rapid aging mice were selected, and qPCR technology was used to detect the expression changes of miR-23b in the pathological process of AD.
  • Example 5 The effect of up-regulation of miR-23b expression on cell viability
  • the AD model cells were transfected with miRNA mimics to establish a cell model with up-regulated miR-23b expression, and the MTS colorimetric method was used to detect cell viability.
  • Example 6 The effect of up-regulation of miR-23b expression on cell apoptosis
  • the AD model cells were transfected with miRNA mimics to establish a cell model with up-regulated miR-23b expression, and flow cytometry was used to detect apoptosis.
  • miRNAs target gene prediction software-TargetScan and miRanda to predict the binding site of miR-23b and GSK3B, select a fragment of about 150 bp from the upstream and downstream of the binding site and clone it to the downstream of the luciferase reporter gene to construct a luciferase reporter Carrier Luc-GSK3B-WT; adopt the principle of changing the base arrangement to construct the mutant luciferase reporter carrier Luc-GSK3B-MUT, as shown in Figure 4, AB.
  • the wild-type or mutant-type reporter vector and miR-23b mimics were co-transfected into HEK293 cells.
  • the AD model cells were transfected with miRNA and mimics/inhibitor to establish a cell model with up-regulated or down-regulated miR-23b expression, qPCR technology was used to detect the content of GSK-3 ⁇ mRNA in the cell, and Western blot technology was used to detect the expression of GSK-3 ⁇ protein in the cell.
  • the AD model cells were transfected with miRNA mimics/inhibitor to establish a cell model with up-regulated or down-regulated miR-23b expression, and Western Blot technology was used to detect the phosphorylation of tau protein at Ser396 site and the expression of apoptosis-related proteins Bax and caspase-3 .
  • the expression of miR-23b was up-regulated, the expression of GSK-3 ⁇ decreased, the phosphorylation level of tau protein at Ser396 site decreased, and the apoptosis-related proteins Bax and caspase-3 The expression also decreased accordingly.
  • a cell model with up-regulated miR-23b expression was established by transfecting miRNA mimics with AD model cells, and the apoptosis rate was detected by flow cytometry, and the phosphorylation of tau protein at Ser396 site and the apoptosis-related protein Bax were detected by Western Blot technology.
  • the expression of Caspase-3 and Caspase-3, and the transfection method was used to over-express GSK-3 ⁇ , and the above indicators were tested again.
  • Example 11 The effect of miR-23b on the cognitive and memory function of AD model animals
  • 6-month-old APP/PS1 mice were selected and injected with miR-23b adeno-associated virus and negative control adeno-associated virus into the lateral ventricle.
  • Morris water maze method was used to detect the changes in learning and memory function of APP/PS1 mice.
  • the hippocampal and cortical tissues of 1, 3, 6, and 9-month-old APP/PS1 double transgenic mice and 3, 6, and 9-month-old SAMP8 naturally rapid aging mice were extracted, and qPCR technology was used to detect miR-23b in hippocampus and cortex tissues. Using Western Blot technology to detect the expression level of related proteins in hippocampus and cortical tissues.
  • AD in the cortical tissue of APP/PS1 double transgenic mice aged 1, 6, and 9 months and the hippocampus tissue of 1, 3, 6, and 9 months of age, the expression level of miR-23b decreased significantly ;
  • Western Blot results showed that in the cortical tissues of APP/PS1 double transgenic mice at different ages, compared with wild-type mice, the expression of GSK-3 ⁇ in APP/PS1 mice at the age of 1, 6 months increased significantly, and the tau protein Phosphorylation levels increased significantly at different months of age, and the expression of apoptosis-related proteins Bax and caspase-3 increased; in hippocampus, the expression of p-Tau-S396, caspase-3, and Bax were particularly significant at the age of 1 and 9 months.
  • Examples 1 to 11 of the present invention show that the expression of miRNA 23 clusters of microRNAs is significantly reduced in the pathological process of Alzheimer’s disease, and the expression of GSK-3 ⁇ is negatively regulated to inhibit tau protein phosphorylation and Apoptosis of nerve cells delays the progression of the disease.
  • the 23 clusters of miRNAs are expected to become a new target for the diagnosis and treatment of Alzheimer's disease.

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Abstract

提供了一种用于阿尔茨海默病治疗和/或诊断的miRNA标志物，所述miRNA标志物为miRNA23簇。还提供了miRNA23簇的微小RNA在阿尔茨海默病诊断、治疗方面的应用。

Description

一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物
本申请要求于2019年11月06日提交中国专利局、申请号为2019110783208、申请名称为一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物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
本发明实施例涉及生物技术领域，具体涉及一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物。
背景技术
目前，阿尔茨海默病(Alzheimer’s Disease，AD)是一种以老年斑、神经纤维缠结、神经元丢失为主要病理特征的神经退行性疾病，主要表现为渐进性记忆障碍、认知功能障碍、人格改变及语言障碍等神经精神症状。现有研究表明，Aβ假说、tau蛋白过度磷酸化假说、胆碱能失衡假说是AD病理进程中主要的发病假说，目前处于临床研究的药物也大多基于以上假说。目前AD防治主要存在以下问题：缺乏简单无创的早期筛查手段；病理机制尚不清楚，缺乏可靠的药物研究靶点。因此寻求可靠的AD诊断标志物、阐明AD发病机制是目前防治AD亟待解决的科学问题。研究表明，家族性AD存在PSEN1、PSEN2、APP等基因的突变，可以通过早期的基因筛查发现，而占据AD患者总数95％的散发性AD至今没有相关的基因报道。研究散发性AD中，相关基因的变化对AD的防治及临床生物标志物的发现具有重要的意义。
发明内容
为此，本发明实施例提供一种用于阿尔茨海默病诊断的miRNA标志物，以解决现有技术中的在基因水平上，没有阿尔茨海默病诊断标志物的问题。
为了实现上述目的，本发明实施例提供如下技术方案：
一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物，其特征在于，所述miRNA标志物为miRNA23簇。
优选的，所述miRNA23簇选自hsa-miR-23b，其核苷酸序列如SEQ ID NO.1所示；
所述miRNA23簇选自hsa-miR-23b-3p，其核苷酸序列如SEQ ID NO.2所示。
本发明实施例还提供一种引物在制备试剂盒中的应用，所述引物是针对上述所述的miRNA标志物的引物。
优选的，所述试剂盒用于提供在患有或有风险形成阿尔茨海默病的患者中诊断阿尔茨海默病，预测形成阿尔茨海默病的风险，或预测阿尔茨海默病的结果。
优选的，所述引物用于测定样品中所述miRNA标志物的表达水平。
优选的，所述样品为血清。
优选的，所述miRNA标志物的表达水平是以患者的miRNA标志物表达水平与健康人miRNA标志物参照表达水平。
优选的，所述miRNA标志物的表达水平的测定是基于测序的方法、基于阵列的方法或基于PCR的方法。
本发明实施例还提供一种以上述所述miRNA标志物的激动剂在制备治疗阿尔茨海默病药物中的应用。
本发明实施例中，miRNA 23簇的微小RNA在阿尔茨海默病中表达降低，其通过抑制GSK-3β介导的tau蛋白磷酸化减少神经细胞凋亡。其中，miRNA 23簇的微小RNA为：
(1)miRNA 23簇的微小RNA选自如下特征：(a)miRNA类微小RNA，的miRNA 23b类微小RNA选自hsa-miR-23b，其序列如SEQ ID  NO.1：cucaggugcucuggcugcuuggguuccuggcaugcugauuugugacuuaagauuaaaaucacauugccag ggauuaccacgcaaccacgaccuuggc所示，其默认成熟体(hsa-miR-23b-3p)序列如SEQ ID NO.2：aucacauugccagggauuaccac所示；(b)经修饰的miRNA类微小RNA衍生物；或长度为18-26nt、功能与miRNA类微小RNA相同或基本相同的微小RNA或经修饰的miRNA衍生物；
本发明实施例提供一种制剂和药物，该制剂和药物为(1)中的微小RNA的激动剂。
本发明实施例具有如下优点：
本发明实施例发现miRNA 23簇的微小RNA在阿尔茨海默病诊断、治疗方面具有作用，通过AD模式细胞、AD模式动物和自然老化动物、临床血液样本，利用针对miRNA23簇的微小RNA标志物的引物，检测其在人体中的表达量，发现miRNA 23簇的微小RNA在阿尔茨海默病进程中表达显著降低，因此miRNA 23簇的微小RNA可以作为一种新的阿尔茨海默病标志物，用于阿尔茨海默病的辅助诊断；本发明实施例发现miRNA 23簇的微小RNA表达上调可负向调控GSK-3β的表达，抑制tau蛋白磷酸化和神经细胞凋亡；miRNA 23簇的微小RNA表达下调则明显升高tau蛋白磷酸化水平，促进神经细胞凋亡，miRNA 23簇的微小RNA通过负向调控GSK-3β的表达在阿尔茨海默病进程中发挥神经保护作用；本发明实施例对miRNA 23簇的微小RNA的功能研究深入系统，基于以上发现，miRNA 23簇的微小RNA可以作为阿尔茨海默病新的治疗靶点，以miRNA 23簇的微小RNA作为阿尔茨海默病生物标志物的靶向治疗提供了新的思路。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案，下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显 而易见地，下面描述中的附图仅仅是示例性的，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图引伸获得其它的实施附图。
图1为本发明实施例的miRNA芯片技术检测miR-23b在AD模式动物及野生型动物脑组织中的差异表达；
图2为本发明实施例的miR-23b在AD模式细胞、AD模式动物和自然老化动物、AD患者血液中的表达检测；
图3为本发明实施例的miR-23b对神经细胞活力及神经细胞凋亡的影响；
图4为本发明实施例的miR-23b通过特异性靶向GSK-3βmRNA 3’UTR负向调控其表达；
图5为本发明实施例的miR-23b表达上调或下调对tau蛋白磷酸化水平和凋亡相关蛋白的影响；
图6为本发明实施例的过表达GSK-3β可逆转miR-23b调控tau蛋白磷酸化和神经细胞凋亡的作用；
图7为本发明实施例的miR-23b对AD模式动物认知与记忆功能的影响；
图8为本发明实施例的整体水平验证miR-23b调控的GSK-3β/p-tau、GSK-3β/Bax/caspase-3通路在AD病理进程中的变化。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭示的内容轻易地了解本发明的其他优点及功效，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例中，术语“表达水平”是指与来自参照核酸(例如来自对照)比较，或与计算的平均表达值(例如在RNA芯片分析中)比较 的测定表达水平。某个“表达水平”也可作为结果(result)并通过下文公开的几种感兴趣核酸的比较和测量来测定，并展示出这些转录物彼此的相对丰度。表达水平还可相对于不同组织，患者对健康对照等中的表达来评估。
在本发明的上下文中，“样品”或“生物样品”是源自生物学生物体或已经与生物学生物体接触的样品。生物样品的例子是：细胞、组织、体液、活组织检查样本、血液、尿液、唾液、痰液、血浆、血清、细胞培养上清液等。
“基因”是含有以受控方式产生功能性RNA产物必需的信息的核酸区段。“基因产物”是通过基因转录或表达产生的生物分子，例如mRNA或翻译蛋白。
“miRNA”是短的、天然存在的RNA分子，并且应当具有本领域技术人员所理解的一般含义。“源自miRNA的分子”是以化学方法或酶促方法自miRNA模板获得的分子，诸如cDNA。
本发明实施例中，术语“阵列”指装置(例如芯片装置)上可寻址位置的排列。位置的数目可从几个变化到至少几百或几千个。每个位置代表独立的反应位点。阵列包括但不限于核酸阵列、蛋白质阵列和抗体阵列。“核酸阵列”指含有核酸探针(诸如寡核苷酸、多核苷酸或基因的较大部分)的阵列。阵列上的核酸优选为单链的。
“基于PCR的方法”指包含聚合酶链式反应PCR的方法。这是通过利用一种、两种或更多种引物在体外酶促复制指数扩增核酸“例如DNA或RNA”的方法。对于RNA扩增，可使用逆转录作为第一步。基于PCR的方法包含动力学或定量PCR(qPCR)，其特别适宜于分析表达水平。当其实现表达水平的测定时，可以例如使用基于PCR的方法以检测给定mRNA的存在，其在逆转录酶的帮助下将完整的mRNA库(所谓的转录物组)逆转录为cDNA，并在相应引物的帮助下检测给定cDNA的存在。此方法通常称为逆转录酶PCR(rtPCR)。
本发明实施例中，术语“基于PCR的方法”包含端点PCR应用及应 用特殊的荧光团或嵌入染料的动力学/实时PCR技术两者，所述荧光团或嵌入染料发射荧光信号作为扩增靶标的函数，并允许靶标的监测和定量。
本发明实施例中，术语“标志物”或“生物标志物”指其存在或浓度可进行检测并与已知的状况(诸如疾病状态)或临床结果(诸如对治疗的响应)相关联的生物分子，例如核酸、肽、蛋白质、激素等。
实施例1、miRNA芯片技术检测miR-23b在AD模式动物及野生型动物脑组织中的差异表达
分别选取1、3、6及9月龄APP/PS1双转基因小鼠，提取小鼠脑组织RNA并对miRNA进行标记，本发明实施例的miRNA 23簇的微小RNA为hsa-miR-23b，其序列如SEQ ID NO.1所示：cucaggugcucuggcugcuuggguuccuggcaugcugauuugugacuuaa
gauuaaaaucacauugccagggauuaccacgcaaccacgaccuuggc。其默认成熟体(hsa-miR-23b-3p)序列如SEQ ID NO.2：aucacauugccagggauuaccac所示。其中，成熟体(hsa-miR-23b-3p)miRNA逆转录引物：SEQ ID NO.3：gtcgtatcca gtgcagggtc cgaggtattcgcactggatacgacgtggta；定量PCR(qPCR)正向引物：SEQ ID NO.4：cgatcacattgccagggat；反向引物：SEQ ID NO.5：agtgcagggtccgaggtatt。而后将样品与miRCURYTM LNA Array(v.18.0)芯片杂交，使用Axon GenePix 4000B芯片扫描仪扫描芯片，使用GenePix pro V6.0软件进行原始数据分析。如图1所示，与野生型小鼠相比，miR-23b在不同月龄APP/PS1小鼠脑组织中表达显著降低(mean±SEM，n＝3，fold change＞2)。
实施例2、miR-23b在AD模式细胞中的表达检测
构建Cu 2+诱导损伤APPswe细胞的AD细胞模型，采用qPCR技术检测miR-23b在AD细胞模型病理进程中的表达变化。如图2中，A所示，与对照组(0h)相比，Cu 2+损伤后12h、24h及36h，miR-23b的表达量均显著降低((mean±SEM)(n＝3)，**p＜0.01，***p＜0.001)。
实施例3、miR-23b在AD模式动物和自然老化动物中的表达检测
分别选取1、3、6及9月龄APP/PS1双转基因小鼠与SAMP8自然 快速老化小鼠的海马和皮层组织，采用qPCR技术检测miR-23b在AD病理进程中的表达变化。如图2中，B-C所示，在两种动物模型皮层或海马组织中，与同月龄对照组小鼠(WT control/SAMR1小鼠)相比，miR-23b的表达水平在1、3、6、9月著降低((mean±SEM)(n＝4)，**p＜0.01，***p＜0.001)。
实施例4、miR-23b在AD患者血液中的表达检测
获取临床上确诊为AD的患者血清，采用qPCR技术检测miR-23b在AD患者血清中的含量。如图2中，D所示，AD患者血液中miR-23b的含量与同龄健康志愿者相比显著降低((mean±SEM)(n＝5～7)，**p＜0.01)。
实施例5、miR-23b表达上调对细胞活力的影响
采用AD模式细胞转染miRNA mimics方法建立miR-23b表达上调的细胞模型，选用MTS比色法进行细胞活力检测。如图3中，A-B所示，miR-23b表达上调可缓解Cu 2+诱导损伤的作用，增加AD模式细胞的存活率((mean±SEM)(n＝3)，*p＜0.05，***p＜0.001)。
实施例6、miR-23b表达上调对细胞凋亡的影响
采用AD模式细胞转染miRNA mimics方法建立miR-23b表达上调的细胞模型，采用流式细胞技术进行细胞凋亡检测。如图3中，C-D所示，miR-23b表达上调能够明显抑制神经细胞早期凋亡和晚期凋亡((mean±SEM)(n＝5)，**p＜0.01)。
实施例7、miR-23b与GSK-3β的结合情况
使用miRNAs靶基因预测软件--TargetScan及miRanda预测miR-23b与GSK3B的结合位点，选取结合位点上下游约150个bp碱基的片段克隆到荧光素酶报告基因下游，构建荧光素酶报告载体Luc-GSK3B-WT；采用改变碱基排列的原则，构建突变型荧光素酶报告载体Luc-GSK3B-MUT，如图4中，A-B所示。分别将野生型或突变型的报告载体与miR-23b mimics共转染至HEK293细胞，36h后，利用双荧光素酶报告基因检测系统(Promega)检测firefly和Renilla荧光素酶的活性。 如图4中，C所示，Luc-GSK3B-WT与miR-23b mimics共转染后，miR-23b表达上调能显著降低荧光素酶活性，而识别位点突变后，miR-23b对荧光素酶活性的抑制丧失((mean±SEM)(n＝3)，***p＜0.001)。
实施例8、miR-23b对GSK-3β的调控作用
采用AD模式细胞转染miRNA mimics/inhibitor方法建立miR-23b表达上调或下调的细胞模型，采用qPCR技术检测细胞内GSK-3βmRNA的含量，Western Blot技术检测细胞内GSK-3β蛋白的表达量。如图4中，D-F所示，miR-23b表达上调可以同时在mRNA水平及蛋白水平上负向调控GSK-3β的表达((mean±SEM)(n＝3)，*p＜0.05，***p＜0.001)。
实施例9、miR-23b表达上调或下调对tau蛋白磷酸化水平和凋亡相关蛋白的影响
采用AD模式细胞转染miRNA mimics/inhibitor方法建立miR-23b表达上调或下调的细胞模型，采用Western Blot技术检测tau蛋白在Ser396位点的磷酸化情况及凋亡相关蛋白Bax、caspase-3的表达。如图5所示，与对照组(NCM)相比，miR-23b表达上调后，GSK-3β的表达下降，tau蛋白在Ser396位点的磷酸化水平降低，凋亡相关蛋白Bax及caspase-3的表达也相应下降。抑制miR-23b内源性表达后，与对照组(NCI)相比，GSK-3β的表达上调，tau蛋白在Ser396位点的磷酸化水平、凋亡相关蛋白Bax及caspase-3的表达增加((mean±SEM)(n＝6)，*p＜0.05，**p＜0.01，***p＜0.001)。
实施例10、过表达GSK-3β可逆转miR-23b调控tau蛋白磷酸化水平和神经细胞凋亡的作用
采用AD模式细胞转染miRNA mimics方法建立miR-23b表达上调的细胞模型，通过流式细胞技术检测细胞凋亡率，Western Blot技术检测tau蛋白在Ser396位点的磷酸化情况及凋亡相关蛋白Bax和caspase-3的表达，同时采用转染方法过表达GSK-3β，再次检测上述指标。如图6中，A-B所示，过表达GSK-3β使神经细胞凋亡率明显升高；如图6中， C-D所示，同时过表达GSK-3β可上调tau蛋白在Ser396位点的磷酸化水平，增加凋亡相关蛋白Bax及caspase-3的表达，逆转miR-23b的神经保护作用((mean±SEM)(n＝3～6)，*p＜0.05，**p＜0.01，***p＜0.001)。
实施例11、miR-23b对AD模式动物认知与记忆功能的影响
选取6月龄APP/PS1小鼠，侧脑室注射miR-23b腺相关病毒及阴性对照腺相关病毒，采用Morris水迷宫方法检测APP/PS1小鼠学习记忆功能的改变。如图7所示，与APP/PS1对照组小鼠相比，miR-23b注射组小鼠潜伏期明显缩短，在第五天时出现显著性差异；在空间探索实验中，miR-23b注射组小鼠在平台象限滞留的时间与穿越平台的次数均显著性升高((mean±SEM)(n＝9～10)，*p＜0.05，**p＜0.01，&p＜0.05，&&p＜0.01)。
实施例12、整体水平验证miR-23b调控的GSK-3β/p-tau、GSK-3β/Bax/caspase-3通路在AD病理进程中的变化
分别提取1、3、6、9月龄APP/PS1双转基因小鼠及3、6、9月龄SAMP8自然快速老化小鼠的海马和皮层组织，采用qPCR技术检测海马和皮层组织中miR-23b的含量，采用Western Blot技术检测海马和皮层组织中相关蛋白表达水平。
如图8中，A-D所示，在1、6、9月龄APP/PS1双转基因小鼠的皮层组织和1、3、6、9月龄海马组织中，miR-23b的表达水平均显著下降；Western Blot结果显示，在不同月龄APP/PS1双转基因小鼠的皮层组织中，与野生型小鼠相比，1、6月龄APP/PS1小鼠GSK-3β的表达显著增加，tau蛋白磷酸化水平在不同月龄均显著升高，凋亡相关蛋白Bax、caspase-3的表达增加；在海马组织中，尤其以1、9月龄p-Tau-S396、caspase-3、Bax表达显著增加，3月龄和6月龄p-Tau-S396表达显著增加，但caspase-3、Bax表达与野生型小鼠相比略有增加((mean±SEM)(n＝4)，*p＜0.05，**p＜0.01，***p＜0.001)。
如图8中，E-H所示，在3、6、9月龄SAMP8自然快速老化小鼠的皮层组织及3、9月龄的海马组织中，miR-23b的表达水平显著下调； Western Blot结果显示，在不同月龄SAMP8自然快速老化小鼠的皮层组织中，GSK-3β表达增加，tau蛋白在Ser396位点的磷酸化水平升高，凋亡相关蛋白Bax和caspase-3的表达增加，尤其以9月龄最为明显；在海马组织中，与野生型小鼠相比，GSK-3β的表达显著增加，tau蛋白磷酸化水平升高，凋亡相关蛋白Bax、caspase-3的表达也相应增加((mean±SEM)(n＝4)，*p＜0.05，**p＜0.01，***p＜0.001)。
本发明实施例1至实施例11的试验结果表明，miRNA 23簇的微小RNA在阿尔茨海默病病理进程中表达显著降低，并通过负向调控GSK-3β的表达，抑制tau蛋白磷酸化及神经细胞凋亡从而延缓疾病进程，miRNA 23簇的微小RNA有望成为阿尔茨海默病诊治的新靶点。
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Figure PCTCN2020084626-appb-000001
Figure PCTCN2020084626-appb-000002

Claims (9)

  1. 一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物，其特征在于，所述miRNA标志物为miRNA23簇。
  2. 如权利要求1所述的用于阿尔茨海默病诊断和/或治疗的miRNA标志物，其特征在于，
    所述miRNA23簇选自hsa-miR-23b，其核苷酸序列如SEQ ID NO.1所示；
    所述miRNA23簇选自hsa-miR-23b-3p，其核苷酸序列如SEQ ID NO.2所示。
  3. 一种引物在制备试剂盒中的应用，所述引物是针对权利要求1或2所述的miRNA标志物的引物。
  4. 如权利要求3所述的应用，其特征在于，所述试剂盒用于提供在患有或有风险形成阿尔茨海默病的患者中诊断阿尔茨海默病，预测形成阿尔茨海默病的风险，或预测阿尔茨海默病的结果。
  5. 如权利要求3所述的应用，其特征在于，所述引物用于测定样品中所述miRNA标志物的表达水平。
  6. 如权利要求5所述的应用，其特征在于，所述样品为血清。
  7. 如权利要求5所述的应用，其特征在于，所述miRNA标志物的表达水平是以患者的miRNA标志物表达水平与健康人miRNA标志物参照表达水平。
  8. 如权利要求5所述的应用，其特征在于，所述miRNA标志物的表达水平的测定是基于测序的方法、基于阵列的方法或基于PCR的方法。
  9. 一种以权利要求1所述miRNA标志物的激动剂在制备治疗阿尔茨海默病药物中的应用。
PCT/CN2020/084626 2019-11-06 2020-04-14 一种用于阿尔茨海默病诊断和/或治疗的miRNA标志物 WO2021088317A1 (zh)

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