阅读说明：本技术 miRNA30簇作为阿尔茨海默病诊断标志物的应用 (Application of miRNA30 cluster as Alzheimer disease diagnostic marker ) 是由 刘睿 李卓荣 曾利 姜海伦 张俊霞 于 2020-04-08 设计创作，主要内容包括：本发明公开了miRNA30簇作为阿尔茨海默病诊断标志物的应用。本发明的miRNA 30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miRNA30簇的微小RNA标志物的引物和/或探针,检测其在人体中的相对表达量,本发明的miRNA30簇的微小RNA在阿尔茨海默病进程中表达显著升高,在转录和翻译水平负向调控ADAM10和SIRT1的表达,同时抑制ADAM10介导的非淀粉样蛋白生成途径和SIRT1去乙酰化作用介导的淀粉样蛋白降解途径,导致淀粉样蛋白在大脑过度聚集,促进AD的发生发展。因此miRNA 30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。(The invention discloses application of miRNA30 cluster as a diagnostic marker of Alzheimer's disease. The miRNA30 cluster microRNA has an effect on diagnosing and treating Alzheimer's disease, the relative expression amount of the miRNA30 cluster microRNA in a human body is detected by using primers and/or probes aiming at the microRNA markers of the miRNA30 cluster through AD model cells, AD model animals, natural aging animals and clinical blood samples, the expression of the miRNA30 cluster microRNA is remarkably increased in the process of Alzheimer's disease, the expressions of ADAM10 and SIRT1 are negatively regulated at the transcription and translation levels, and simultaneously, the non-amyloid generation pathway mediated by ADAM10 and the amyloid degradation pathway mediated by SIRT1 deacetylation are inhibited, so that amyloid is excessively aggregated in the brain, and the generation and development of AD are promoted. Therefore, the miRNA30 cluster microRNA can be used as a novel Alzheimer disease marker and used for the auxiliary diagnosis of Alzheimer disease.)

1. A miRNA biomarker for diagnosis and/or treatment of alzheimer's disease, wherein the miRNA biomarker is a miRNA30 cluster.

2. The miRNA biomarker for diagnosis and/or treatment of Alzheimer's disease of claim 1,

the miRNA30 cluster is selected from hsa-miR-30a, and the nucleotide sequence of the miRNA30 cluster is shown in SEQ ID NO. 1;

the miRNA30 cluster is selected from hsa-miR-30a-5p, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 2.

3. Use of a primer specific for the miRNA biomarker of claim 1 or 2 in the preparation of a kit.

4. The use of claim 3, wherein the kit is for providing a diagnosis of Alzheimer's disease, predicting the risk of developing Alzheimer's disease, or predicting the outcome of Alzheimer's disease in a patient suffering from or at risk of developing Alzheimer's disease.

5. The use of claim 3, wherein the primers are used to determine the expression level of the miRNA biomarker in the sample.

6. The use of claim 5, wherein the sample is serum.

7. The use of claim 5, wherein the expression level of the miRNA biomarker is the patient's expression level of the miRNA biomarker and a reference expression level for a healthy human miRNA biomarker.

8. The use of claim 5, wherein the determination of the expression level of the miRNA biomarker is a sequencing-based method, an array-based method, or a PCR-based method.

9. The use of the miRNA biomarker inhibitor of claim 1 in the preparation of a medicament for treating Alzheimer's disease.

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to application of miRNA30 cluster as an Alzheimer disease diagnosis marker.

Background

Currently, Alzheimer's Disease (AD) is a chronic nervous system degenerative Disease of cortex and hippocampus, which is clinically manifested by neurological symptoms such as progressive memory and cognitive dysfunction, emotional disorder, social difficulties, etc., and the main pathological mechanisms are senile plaque deposition caused by beta-Amyloid (β -Amyloid-beta peptide, Α β) aggregation, neurofibrillary tangles caused by Tau protein hyperphosphorylation, synaptic dysfunction, nerve cell inflammatory response, cerebral cortex atrophy, etc. The diagnosis and/or treatment of AD remains problematic and challenging due to the lack of efficient and accurate screening detection techniques, unclear pathogenesis, and slow progress in drug target research. Currently, clinically researched medicines and medicines on the market can only relieve mild and moderate symptoms of patients, and cannot fundamentally improve symptoms and cure diseases. Therefore, the search for reliable AD diagnosis markers and the elucidation of the pathogenesis of AD are the scientific problems to be solved urgently at present. Researches show that the gene mutations such as PSEN1, PSEN2, APP and the like cause rare Alzheimer disease, and familial AD with the number of attack less than 10 percent can be found through early gene screening; the genetic pathology of sporadic AD with an incidence of greater than 90% and the regulatory mechanisms of the associated signaling pathways are complex and have no related gene reports to date. In the research of sporadic AD, the change of the non-coding gene of the microRNA in transcriptomics has important significance for the prevention and treatment of AD and the discovery of clinical biomarkers.

Disclosure of Invention

Therefore, the embodiment of the invention provides a miRNA biomarker for Alzheimer disease diagnosis and/or treatment, so as to solve the problem that no Alzheimer disease diagnosis marker exists at the gene level in the prior art.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

a miRNA biomarker for diagnosis and/or treatment of alzheimer's disease, said miRNA biomarker being a miRNA30 cluster.

In one embodiment of the invention, the miRNA30 cluster is selected from hsa-miR-30a, and the nucleotide sequence of the miRNA30 cluster is shown in SEQ ID NO. 1;

the miRNA30 cluster is selected from hsa-miR-30a-5p, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 2.

The invention also provides application of the primer in preparation of a kit, wherein the primer is a specific primer aiming at the miRNA biomarker.

In one embodiment of the invention, the kit is used to provide a diagnosis of alzheimer's disease, to predict the risk of developing alzheimer's disease, or to predict the outcome of alzheimer's disease in a patient suffering from or at risk of developing alzheimer's disease.

In one embodiment of the invention, the primers are used to determine the expression level of the miRNA biomarker in a sample.

In one embodiment of the invention, the sample is serum.

In one embodiment of the invention, the expression level of the miRNA biomarker is the expression level of the miRNA biomarker in the patient and a reference expression level of a healthy human miRNA biomarker.

In one embodiment of the invention, the determination of the expression level of the miRNA biomarker is a sequencing-based method, an array-based method or a PCR-based method.

The invention also discloses application of the miRNA biomarker inhibitor in preparation of a medicament for treating Alzheimer disease, and belongs to the protection scope of the invention.

In the invention, the expression of miRNA30 cluster microRNA in Alzheimer disease is remarkably increased, and the miRNA30 cluster microRNA is combined with 3' UTR of ADAM10 to inhibit an ADAM10 as alpha-secretase to generate a non-starch-like protein pathway of soluble Abeta;

the micro RNA of the miRNA30 cluster is combined with the 3' UTR of the SIRT1, so that the SIRT1 is inhibited to deacetylate the retinoic acid beta receptor and activate an amyloid degradation pathway of ADAM10 transcription; the miRNA30 cluster microRNA can activate the activity of beta-secretase in an NF-kB signal path and promote the generation of insoluble amyloid A beta by inhibiting the biological activity of SIRT 1. At the same time, micrornas of miRNA30 cluster inhibit SIRT 1-mediated deacetylation of hyperphosphorylated Tau protein, leading to neurofibrillary tangles, leading to the death of neural oligodendrocytes. Wherein, the micro RNA of the miRNA30 cluster is:

(1) the microRNA of the miRNA30 cluster is selected from the following characteristics: (a) miRNA30 a type micro RNA of miRNA type micro RNA is selected from hsa-miR-30a, and the sequence of the miRNA30 a type micro RNA is shown in SEQ ID NO. 1: gcgacuguaaacauccucgacuggaagcugugaagccacagaugggcuuucagucggauguuugcagcugc, and the default mature body (hsa-miR-30a-5p) sequence is shown in SEQ ID NO. 2: gaaggucagcuccuacaaaugu; (b) modified miRNA micro RNA derivatives; or microRNA or modified miRNA derivative with the length of 18-26nt and the function same as or basically same as miRNA microRNA; the embodiment of the invention provides a preparation and a medicament, and the preparation and the medicament are inhibitors of the micro RNA in the step (1).

The embodiment of the invention has the following advantages:

the miRNA30 cluster microRNA has an effect on the diagnosis and treatment of Alzheimer disease, the relative expression level of the miRNA30 cluster microRNA in a human body is detected by using a primer and/or a probe aiming at the miRNA30 cluster microRNA marker through AD model cells, AD model animals, naturally aged animals and clinical blood samples, and the expression of the miRNA30 cluster microRNA is remarkably increased in the process of Alzheimer disease, so that the miRNA30 cluster microRNA can be used as a novel Alzheimer disease marker and is used for the auxiliary diagnosis of Alzheimer disease.

The embodiment of the invention finds that the up-regulation of the micro RNA expression of the miRNA30 cluster can negatively regulate the expression of ADAM10 and SIRT1 at the same time, so that excessive deposition of Abeta and nerve cell apoptosis are caused; the micro RNA expression of the miRNA30 cluster is reduced, so that the deposition level of A beta is obviously reduced, the nerve cell apoptosis is reduced, and the micro RNA of the miRNA30 cluster aggravates the pathological process of AD by negatively regulating the expression of ADAM10 and SIRT 1.

Based on the discovery that the miRNA30 cluster microRNA has a specific multi-target effect, the miRNA30 cluster microRNA can be used as a new Alzheimer disease treatment target, and a new thought is provided for targeted treatment by taking the miRNA30 cluster microRNA as an Alzheimer disease biomarker.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a diagram illustrating an embodiment of miRNA high-throughput chip technology for detecting differentially expressed microRNAs in AD pathological processes;

FIG. 2 is the detection of miR-30a expression level in blood of AD model cells, AD model animals, naturally aged animals and AD patients, wherein A, in the AD cell model, the miR-30a expression level is obviously increased compared with a control group; in the cortex of the AD animal model mouse, compared with a wild type mouse control group, the miR-30a expression quantity is obviously increased; in the hippocampus of an AD animal model mouse, the miR-30a expression quantity is obviously increased compared with a wild type mouse control group; d, in the serum of the AD patient, compared with a normal peer control group, the miR-30a expression level is obviously increased;

FIG. 3 is a graph of the effect of miR-30a on the viability of AD neural cells in an embodiment of the present invention;

FIG. 4 is a graph of the effect of miR-30a on amyloid (A β) according to an embodiment of the present invention;

FIG. 5 is an embodiment of the invention in which miR-30a specifically targets the mRNA 3' UTR of ADAM10 and SIRT 1; ADAM10 and miR-30a binding sequences are highly conserved in multiple species; SIRT1 and miR-30a binding sequences are highly conserved in multiple species; schematic construction of Luc-ADAM10-WT and Luc-ADAM10-MUT dual-luciferase reporter gene vectors; schematic construction diagrams of Luc-SIRT1-WT and Luc-SIRT1-MUT dual-luciferase reporter gene vectors; verifying the binding specificity of the miR-30a and the ADA10 target; verifying the binding specificity of the miR-30a and the SIRT1 target;

FIG. 6 is a diagram showing that miR-30a of the embodiment of the invention specifically and negatively regulates the expression of two specific targets of ADAM10 and SIRT1 at the transcription and translation levels; wherein, A, at the mRNA level, miR-30a negatively regulates the expression level of ADAM10 and SIRT 1; at the translation level, miR-30a negatively regulates the expression levels of ADAM10 and SIRT1 (Western blot); at the translation level, miR-30a negatively regulates the expression level of ADAM10 (relative quantification); at the translation level, miR-30a negatively regulates the SIRT1 expression level (relative quantification).

Detailed Description

Other advantages and features of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein it is to be understood that the invention is not limited to the specific embodiments disclosed, but is to be construed as limited only by the appended claims. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In embodiments of the invention, the term "expression level" refers to a measured expression level compared to a value from a reference nucleic acid (e.g., from a control), or compared to a calculated average expression value (e.g., in an RNA chip assay). A certain "expression level" can also be determined as a result (result) and by comparison and measurement of several nucleic acids of interest disclosed below, and exhibits the relative abundance of these transcripts with respect to each other. Expression levels can also be assessed relative to expression in different tissues, patients versus healthy controls, and the like.

In the context of the present invention, a "sample" or "biological sample" is a sample that originates from or has been contacted with a biological student object. Examples of biological samples are: cells, tissues, body fluids, biopsy samples, blood, urine, saliva, sputum, plasma, serum, cell culture supernatant, and the like.

A "gene" is a nucleic acid segment that contains the information necessary to produce a functional RNA product in a controlled manner. A "gene product" is a biological molecule, such as an mRNA or translated protein, produced by transcription or expression of a gene.

"miRNA" is a short, naturally occurring RNA molecule and should have a general meaning as understood by those skilled in the art. "miRNA-derived molecules" are molecules, such as cDNAs, that are chemically or enzymatically obtained from miRNA templates.

In embodiments of the present invention, the term "array" refers to an arrangement of addressable locations on a device (e.g., a chip device). The number of locations may vary from a few to at least several hundred or thousand. 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 comprising polymerase chain reaction PCR. This is a method for the exponential amplification of nucleic acids "such as DNA or RNA" by enzymatic replication in vitro using one, two or more primers. For RNA amplification, reverse transcription can be used as the first step. PCR-based methods include kinetic or quantitative PCR (qpcr), which is particularly suitable for analyzing expression levels. When it is effected the determination of the expression level, it is possible, for example, to use a PCR-based method for detecting the presence of a given mRNA, which reverse transcribes a complete mRNA pool (the so-called transcriptome) into cDNA with the aid of a reverse transcriptase, and detects the presence of a given cDNA with the aid of corresponding primers. This method is commonly referred to as reverse transcriptase pcr (rtpcr).

In embodiments of the invention, the term "PCR-based method" encompasses both endpoint PCR applications and kinetic/real-time PCR techniques employing specific fluorophores or intercalating dyes that emit a fluorescent signal as a function of the amplified target and allow for monitoring and quantification of the target.

In embodiments of the invention, the term "marker" or "biomarker" refers to a biological molecule, e.g., a nucleic acid, peptide, protein, hormone, etc., whose presence or concentration can be detected and correlated with a known condition (such as a disease state) or clinical outcome (such as response to treatment).