CN114438195A - Alzheimer's disease detection kit, storage medium and electronic device - Google Patents
Alzheimer's disease detection kit, storage medium and electronic device Download PDFInfo
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Abstract
The invention discloses an Alzheimer disease detection kit, a storage medium and electronic equipment; specifically discloses an application of a marker miR-206-3p in the preparation of a medicine for diagnosing or treating Alzheimer's disease; according to the invention, the disease development stage of the Alzheimer disease patient is diagnosed by detecting the content of miR-206-3p in blood of the subject, a certain early warning effect is provided for AD diagnosis, and the method has a value for early diagnosis of AD.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to an Alzheimer disease detection kit, a storage medium and electronic equipment.
Background
Alzheimer's Disease (AD) is a neurodegenerative disease with a hidden pathogenic process, the population of which is mainly concentrated above 65 years. In the future, high nursing cost caused by the increase of the number of patients will undoubtedly bring heavy economic burden to the society and families. However, in the context of a rapid increase in the number of patients, the etiology of AD is unknown and there are no effective therapeutic agents. Given the slow development of AD, many subtle pathological changes occur 10-20 years before the onset of disease symptoms, which would be an effective means to address AD if AD-related pathological changes could be detected and intervened early. Therefore, the search for biomarkers for diagnosing AD is not always slow.
The early clinical diagnosis standard of AD is to judge whether a subject suffers from AD according to characteristics such as memory, consciousness, cognition and the like, the indexes are easily interfered by non-relevant factors, the description of AD neuropathological change is neither specific nor sensitive, and the diagnosis of AD has no objectivity. Until 2011, the national institute of aging and alzheimer's disease association (NIA-AA) standardized pathological diagnosis of AD, primarily pathological tau proteins and a β as markers for detection. With the increased awareness of AD, more and more researchers now recognize that biomarkers should remain uniform throughout the course of the disease. The traditional markers a β and tau are too simple and require the introduction of more markers reflecting pathological changes, and such markers can be used to assess the progression of AD. Therefore, the NIA-AA was further modified in 2018 to include neurodegenerative biomarkers in the diagnosis of AD. To date, markers for AD diagnosis include two broad categories: a bodily fluid marker and an imaging marker. Humoral markers include pathological protein a β, Tau protein (including T-Tau and p-Tau), and neurodegenerative markers in cerebrospinal fluid and blood. Although it is considered that a neurodegenerative marker is also an important index providing a pathological stage, a specific neurodegenerative marker caused by AD is lacking, and thus, at present, a β and tau protein are mainly used. The reliability of the cerebrospinal fluid diagnosis result is high, the cerebrospinal fluid diagnosis method is a currently accepted method, and the cerebrospinal fluid diagnosis method is difficult to popularize due to the fact that trauma is caused by obtaining of the cerebrospinal fluid and the examination cost is high. Although peripheral blood is obtained without wound, the content of the included markers Abeta and tau is low, which brings higher requirements for the diagnosis technology. The detection of the imaging marker mainly comprises PET imaging and functional magnetic resonance imaging, and the diagnosis has high sensitivity and strong specificity, but is limited by technical and cost problems and is difficult to popularize. Therefore, the search for noninvasive biomarker detection with high specificity is a scientific problem which needs to be solved urgently at present.
BDNF is a neurotrophic factor expressed at high levels in the hippocampus and has important effects on synaptic plasticity and memory formation. Current clinical data indicate that the levels of BDNF in serum and cerebrospinal fluid of healthy subjects are higher than those of patients with Mild Cognitive Impairment (MCI) and dementia, and additional studies demonstrate that the levels of BDNF in serum are positively correlated with the levels of Α β 42 in cerebrospinal fluid, and that a decrease in the levels of BDNF in serum is correlated with the severity of medial temporal lobe atrophy and AD. These evidences suggest that BDNF has relevance to AD and has potential as a diagnostic AD marker, but because of the high sensitivity requirements for such detection, there are problems with hysteresis over gene detection.
Disclosure of Invention
The research provides a miRNA diagnostic marker for diagnosing Alzheimer disease by examples, so as to solve the problem that the existing diagnostic marker is insufficient, and provide reference for screening and early intervention of Alzheimer disease.
The invention preliminarily screens potential differential miRNAs (target AD pathological marker protein) on an AD model animal and an AD model cell, and then tests the feasibility of candidate differential miRNAs as the diagnosis of AD by clinically confirmed MCI, AD, PD and plasma samples of patients with mental diseases. According to the invention, the difference multiple of miRNA levels in blood plasma of a detector and normal human is expected to be used as an evaluation parameter of AD risk, so that the problems of low abundance of protein markers and lag of difference phenomena are avoided better.
In order to achieve the purpose, the invention is implemented according to the following technical scheme:
the application of the marker miR-206-3p in preparing the medicine for diagnosing or treating the Alzheimer disease.
Preferably, the mature nucleotide sequence of miR-206-3p is:
5'-UGGAAUGUAAGGAAGUGUGUGG-3'。
the invention also provides a kit for diagnosing Alzheimer's disease, which comprises a detection reagent for detecting the content of miR-206-3 p.
Preferably, the detection reagent comprises a primer for amplifying miR-206-3 p.
Preferably, the sequence of the primer is:
(upstream primer sequence) 5'-GGAATGTAAGGAAGTGTGTGGAAA-3', (downstream primer sequence) 5'-GTCCAGTTTTTTTTTTTTTTTCTCG-3' or (upstream primer sequence) 5'-CACGCATGGAATGTAAGGAAGT-3', (downstream primer sequence) 5'-CCAGTGCAGG GTCCGAGGT-3' or (upstream primer sequence) 5'-GGCCACATGCTTCTTTATATCCT-3', (downstream primer sequence) 5'-CCAAAACCACACACTTCCTTAC-3' or (upstream primer sequence) 5'-AGGCCACATGCTTCTTTATATCC-3', (downstream primer sequence) 5'-CCAAAACCACAC ACTTCCTTACAT-3' or (upstream primer sequence) 5'-CAGGCCACATGCTTCTTTATATC-3', (downstream primer sequence) 5'-CAAAACCACACACTTCCTTACA-3'.
Preferably, the detection reagent contains a probe for detecting the content of miR-206-3 p.
Preferably, the sequence of the probe is: 5 '-Digoxin-CCACACACUUCCUUACAUUCCA-Digoxin-3' or 5 '-Digoxin-ACACACUUCCUUACAUUCCA-Digoxin-3' or 5 '-Digoxin-CACACUUCCUUACAUUCCA-Digoxin-3'.
The present invention also provides a method of screening for a drug for treating alzheimer's disease; the method comprises the following steps:
step 1: treating an AD model animal or AD model cells with a candidate drug;
step 2: detecting the amount of miR-206-3p in the treated AD model animal or AD model cell;
and step 3: screening the medicines according to the content of the obtained miR-206-3 p; and if the content of the miR-206-3p in the candidate drug is reduced, the candidate drug is the target drug obtained by screening.
The present invention also provides a storage medium having computer-executable program code stored thereon, which when executed by one or more processors of a computer system, the computer system performs a method of diagnosing alzheimer's disease, the method comprising the steps of: step 1: obtaining the content T of miR-206-3p in a detection sample of a subject and the content N of miR-206-3p in a detection sample of a normal person; the subject is older than 55 years;
step 2: calculating R ═ T/N;
and step 3: if R is less than or equal to 1, the output is healthy when T is less than or equal to N, otherwise the output is unhealthy, and here, the output is unhealthy when T is greater than N, namely miR-206-3p is higher than a normal person or a set judgment threshold value. Unhealthy here refers to at least one of a plurality of conditions of unhealthy. In this case, N may be a miR-206-3p content value of a normal person detected simultaneously as a reference, or a judgment threshold obtained by a previous detection.
Further, step 3 is:
if R is less than or equal to 2.15, outputting that the subject is healthy; when the judgment value is adopted, a stricter judgment standard is adopted, and the issued result is more accurate, namely the unhealthy state is determined only when the T is far more than the N value;
if R is more than 2.15 and less than or equal to 4.23; outputting the subject as being in a stage of developing mild cognitive impairment;
if R is more than 4.23 and less than or equal to 5.7; outputting the subject as being in mild cognitive impairment to progress toward the AD stage;
if 5.7< R; the subject is output as having AD.
The present invention also provides an electronic device comprising: one or more processors; and a storage device storing one or more programs; when executed by the one or more processors, cause the one or more processors to implement a method for Alzheimer's disease diagnosis, the method comprising the steps of: step 1: obtaining the content T of miR-206-3p in a detection sample of a subject and the content N of miR-206-3p in a detection sample of a normal person; the subject is older than 55 years;
step 2: calculating R ═ T/N;
and step 3: if R is less than or equal to 1, outputting that the subject is healthy, otherwise, outputting that the subject is unhealthy. In this case, N may be a temporarily detected miR-206-3p content value of a normal person, or may be a judgment threshold obtained by a previous detection.
Further, step 3 is:
if R is less than or equal to 2.15, outputting that the subject is healthy;
if R is more than 2.15 and less than or equal to 4.23; outputting the subject as being in a stage of developing mild cognitive impairment;
if R is more than 4.23 and less than or equal to 5.7; outputting the subject as being in mild cognitive impairment to progress toward the AD stage;
if 5.7< R; the subject is output as having AD.
According to the invention, research shows that miR-206-3p participates in the regulation of the course of Alzheimer's disease, and the method is mainly realized through the following steps: in the pathogenesis process of the Alzheimer disease, the abnormally activated astrocytes express miR-206-3p at a high level, and the miR-206-3p expressed at the high level is transmitted to target cells through exosomes, directionally binds to a 3' UTR region of BDNF in the target cells, inhibits the expression of the BDNF, weakens the growth of neurons and maintains normal functions, and further accelerates the pathological development of AD.
The invention provides a miRNA marker for Alzheimer disease diagnosis, which comprises the following specific steps: firstly, qPCR (quantitative polymerase chain reaction) is used for detecting the level of miR-206-3p in primary astrocytes treated by oligomeric Abeta, in-situ hybridization is used for detecting abnormal change of miR-206-3p in brains of APP/PS1 transgenic mice, and by comparing the abnormal change of miR-206-3p in a normal group and a model group, miR-206-3p is preliminarily confirmed to have potential possibility as a pathological index; then, detecting the level of miR-206-3p in the plasma of 9-month-old APP/PS1 mice and littermate wild-type control mice on AD model animals to determine the feasibility of taking miR-206-3p in the plasma as an Alzheimer disease diagnostic marker; finally, clinically comparing the miR-206-3p level in the plasma of healthy volunteers with mild cognitive impairment patients, Alzheimer patients, Parkinson patients, schizophrenia patients, psychotic disorder patients, anxiety patients and depression patients respectively, if miR-206-3p is highly expressed in the plasma of AD patients and is increased along with the disease process in clinical data, the miR-206-3p can be used as a marker for AD diagnosis.
The invention has the beneficial effects that: the invention finds that the miR-206-3p level in AD model cells (astrocyte cells with the highest proportion in the brain), transgenic AD model mice (APP/PS1) and clinical AD patient plasma samples is obviously higher than that in a normal control group. Further clinical data show that the level of miR-206-3p in the plasma of patients with mild cognitive impairment is 4.23 times that of normal people, the level of miR-206-3p in the plasma of patients with dementia is 5.7 times that of normal people, and the fold difference is higher than that of mental diseases such as schizophrenia and mental disorder. The findings show that miR-206-3p has a bias in AD diagnosis, can provide a certain early warning effect for AD diagnosis, and has a potential value for AD early diagnosis.
Drawings
In order to clearly illustrate the specific embodiments of the present invention and certain detection techniques employed in the experiments, the following description of the embodiments and techniques employed will be made primarily by way of the accompanying drawings.
FIG. 1 is a graph of enrichment analysis of the expression levels of miRNAs in astrocyte exosomes in the in vitro AD model. Fig. 1A is a heat map of the results of differential miRNAs expression levels in the normal control group and AD model group, and fig. 1B is an analysis map of the enrichment of signaling pathways involving the target genes of the differential miRNAs.
Fig. 2 is a qPCR validation graph of differential miRNAs in exosomes.
FIG. 3 is a graph of miR-206-3p detection in astrocytes in an AD model in vitro.
FIG. 4 is a graph of miR-206-3p expression level in the cortex of APP/PS1 mouse.
FIG. 5 is a graph of miR-206-3p expression level in APP/PS1 mouse hippocampus.
FIG. 6 is a graph of in situ hybridization detection of miR-206-3p expression level in APP/PS1 brain slices. The strong fluorescence signal in panel A indicates high miR-206-3p level, and panel B is a statistical analysis diagram.
FIG. 7 is a graph showing the result of detection of 3' -UTR of miR-206-3p in combination with BDNF. FIG. 7A is a schematic diagram of binding sites, FIG. 7B is a diagram of sequencing peaks of the binding sites and mutation sites, and FIG. 7C is a diagram of detection results of a dual-luciferase experiment.
FIG. 8 is a graph of the expression level of miR-206-3p in plasma of APP/PS1 mice.
FIG. 9 is a graph comparing the results of measurements of miR-206-3p expression levels in the plasma of AD patients and other psychiatric patients.
Detailed description of the preferred embodiments
The specific implementation of the present invention is explained by way of examples, and except that the technology used for detection does not limit the present invention in any way, some solutions in the described examples belong to some examples of the present invention, and those skilled in the art can obtain examples without inventive efforts, and all of them belong to the protection scope of the present invention.
Example 1 detection of high expression of miR-206-3p in astrocyte exosomes in AD pathological states by small RNA sequencing technology
Using the IIIumina HiseqTMThe 2500 system detects miRNAs in astrocyte exosomes, fig. 1A is a miRNAs difference analysis result (the left side is a control group and the right side is a treatment group), 10 miRNAs with significant differences in astrocyte exosomes under AD pathology and normal state are shown in the figure, and the fold difference of miR-206-3p is at the top among the 10 different miRNAs. Fig. 1B shows the enrichment of the signaling pathway in which these differential miRNA target genes participate. As can be seen, the first 2 th position is the pathway for regulating axon growth, a neurotrophic factor, and signaling, respectively. And the significant miRNAs participating in the regulation of 2 ways comprise miR-206-3p, which shows that miR-206-3p possibly plays an important role in the pathological process of AD and prompts that miR-206-3p probably has potential value in the diagnosis of AD.
The procedure of example 1 was as follows:
collecting exosomes secreted by the AD model astrocytes, extracting total RNA by adopting a Trizol method, carrying out reverse transcription on the total RNA by connecting joints at the 3 'end and the 5' end to generate cDNA, and carrying out in-vitro polymerase chain amplification reaction (PCR) amplification on the generated cDNA. And then tapping to recover target fragments, and starting sequencing after the quality inspection is qualified. And comparing the sequenced library information with a known database to determine the type and level of miRNA in the sample, and finally, further performing expression clustering analysis on the identified miRNAs, and performing function annotation and KEGG signal channel enrichment analysis on miRNAs target genes.
Example 2 differential validation of miR-206-3p in exosomes
Differential miRNAs in exosomes are verified by a real-time fluorescent quantitative PCR method, and the result is shown in FIG. 2, and miR-206-3p in exosomes secreted by astrocytes of AD model cells is significantly higher than that in a control group (n is 5, and p is less than 0.0001) and has the highest level.
The procedure of example 2 was as follows:
(1) starvation of astrocytes with a purity of 98% or more for 24 hours, followed by addition of oligo A β containing 4 micromoles1-42Treating for 72 hours, and setting a normal control group;
(2) the medium after 72 hours of cell processing was collected, centrifuged at 500 Xg for 15 minutes, 2000 Xg for 15 minutes, 10,000 Xg for 30 minutes in a 4 ℃ centrifuge, and then passed through a 0.22 micron filter to remove cell debris;
(3) passing through a 30kD cutoff column, and centrifuging at 6,000rpm for 50 minutes in a 4 ℃ centrifuge to concentrate the collected supernatant;
(4) concentrating the supernatant and an exosome extraction reagent according to the ratio of 2: 1 proportion, mixing evenly, and incubating overnight at 4 ℃;
(5) the next day, centrifuging at 12,000rpm for 1 hr in a 4 deg.C centrifuge, discarding the supernatant, and collecting the precipitate as exosome;
(6) adding 800 microliters of Trizol to the collected exosomes, and vigorously shaking and cracking for 5 minutes;
(7) adding 200 microliters of chloroform, oscillating and standing for 5 minutes;
(8) centrifuge at 1,2000rpm for 15 minutes at 4 ℃ and transfer the supernatant to a clean Ep tube;
(9) adding isopropanol with the same volume, gently mixing uniformly, and standing for 10 minutes;
(10)1,2000rpm, centrifuging for 10 minutes, and collecting the precipitate;
(11) adding 70% ethanol solution, washing for 2 times, air drying at room temperature, adding 20 microliter of RNase-free water for dissolving;
(12) quantitatively measuring the concentration of the extracted RNA, and performing reverse transcription on 2000 micrograms of RNA to generate cDNA;
(13) using cDNA diluted by 5 times as a template, and performing denaturation at 95 ℃ for 10 seconds; annealing at 60 ℃ for 20 seconds; and (3) carrying out extension for 10 seconds at 72 ℃, carrying out amplification cycle for 40 times, collecting fluorescence signals, and analyzing the level of miR-206-3 p.
Example 3 high expression of miR-206-3p in astrocytes under in vitro pathological conditions
Starvation of astrocytes for 24 hours, followed by the use of oligomeric A β at a final concentration of 4 micromolar1-42Astrocytes were treated for 72 hours. Oligomeric Abeta1-42Is formed by A beta1-42The monomer is polymerized into a small molecule which has the strongest toxic effect on cells. Addition of oligomeric Abeta1-42After treatment, the cells were harvested and the expression level of miR-206-3p in astrocytes was detected using the method of qPCR (FIG. 3). FIG. 3 shows oligomeric Abeta1-42Remarkably up-regulates the expression level of miR-206-3p in astrocytes (n-4, p)<0.05)。
The procedure of example 3 was as follows:
(1) collecting astrocytes with the purity of more than 98 percent to be respectively inoculated into 2 cell culture bottles, wherein the cell culture bottles are respectively numbered as (i) and (ii), and culturing for 36 hours at 37 ℃ to ensure that the cells are recovered to be in a normal state;
(2) rinsing the cells with PBS for 2 times, adding serum-free DMEM-F12 culture medium, and culturing for 24 hours;
(3) add oligo Abeta to a final concentration of 4 micromolar1-42In the bottle II, the bottle I is compared with a bottle II added with an equal volume of solvent and cultured for 72 hours;
(4) digesting cells in the next bottle (I) and the next bottle (II), and collecting the cells in 2 clean Ep tubes;
(5) the subsequent experimental procedures were the same as those in (6) to (12) of example 2.
Example 4 high expression of miR-206-3p in cortex and Hippocampus of 9-month-old APP/PS1 mice
The in vitro detection finds oligomeric Abeta1-42After 72 hours of treatment, the level of miR-206-3p in the cells was significantly upregulated. Given that the main pathological features of APP/PS1 mice appear 7 months later and worsen with age, the present inventionIn the invention, 9-month-old mice are selected for detection, and oligomeric Abeta already appears in brain1-42Presumably, the level of miR-206-3p on the in vivo AD model would also be similar to the results in the cell model. To confirm the speculation, the present invention sacrificed 9-month old APP/PS1 mice and wild-type control mice, the hippocampal and prefrontal cortex sites were removed from the mice, and the miR-206-3p levels in the hippocampal and prefrontal cortex were examined by qPCR (fig. 5). The results show that miR-206-3p in hippocampus and prefrontal cortex of 9-month-old APP/PS1 mice is higher than that of normal control mice (n-4, p)<0.05), which indicates that miR-206-3p has relevance to AD.
The procedure of example 4 was as follows:
the prefrontal cortex and hippocampus of APP/PS1 mice and wild-type mice were taken, Trizol reagent was added to the tissue blocks, and the subsequent steps were the same as those in (7) - (12) of example 2.
Example 5 in situ hybridization detection of expression level of miR-206-3p in APP/PS1 mouse brain
Analysis and determination of the results of the in situ hybridization experiments in this example: the same area of different animals is observed under a low power lens, shooting is carried out by using the same excitation light exposure parameters, then the expression level of miR-206-3p is analyzed according to the intensity of a fluorescence signal, and the interpretation result can be determined according to the following description: observing the corresponding area, and if the fluorescence is brighter, the area spread by the fluorescence is larger, which indicates that the expression level of miR-206-3p is higher. As shown in fig. 6, miR-206-3p was significantly higher in APP/PS1 mouse brain than wild-type mice (n ═ 4, p < 0.05).
The procedure of example 5 was as follows:
(1) taking out the slices, fixing the slices with 4% paraformaldehyde at room temperature for 15 minutes, and rinsing the slices with PBS for 5 minutes;
(2) treating with 0.2mol/L hydrochloric acid for 12 minutes;
(3) after drying hydrochloric acid, the tissue was treated with a solution containing 0.5% triton for 12 minutes;
(4) the solution containing 0.5% triton was aspirated off and rinsed with PBS (DEPC water) for 5 minutes;
(5) 20 micrograms/ml of proteinase K is dripped on the sheet, and the sheet is kept stand for 12 minutes at room temperature and then washed for 5 minutes by PBS (DEPC water preparation);
(6)3％H2O2incubate sections for 15 minutes at room temperature, followed by washing sections with 0.1% DEPC water for 5 minutes;
(7) after throwing away the residual DEPC water, dripping paraformaldehyde on the tissue, and fixing for 10 minutes;
(8) sucking away paraformaldehyde, and soaking the slices in a PBS solution for 5 minutes;
(9) pre-hybridization: covering the tissue with 40 microliters of miRNA Hybridization Buffer, covering the tissue with a cover slip treated with DEPC water, and incubating for 1.5 hours at 55 ℃ in a thermostat;
(10) preparing a probe: 10 minutes before the end of prehybridization, the miR-206-3p probe labeled with digoxigenin: mixing miRNA Hybridization Buffer at 1:100, denaturing at 85 deg.C for 3 min, and balancing at 37 deg.C for 5 min;
(11) and (3) hybridization: dropwise adding a probe diluent, gently placing the probe diluent into a wet box, and finally closing a cover and transferring the wet box into a thermostat at 41 ℃ for incubation for 48 hours;
(12) after completion of hybridization, the cells were washed 3 times for 5 minutes each with a washing solution (10 × washing buffer: DEPC water: 1: 9);
(13) spin-drying the washing solution, dripping Blockingbuffer, and incubating at 37 ℃ for 1 hour;
(14) throwing away a Blocking buffer, adding dropwise Digoxin-resistant HRP (anti-Digoxin HRP Conjugate: Blocking buffer 1: 100), and incubating at 37 ℃ for 1 hour;
(15) PBS (DEPC water) for 3 times, 5 minutes each;
(16) dropwise adding a solution containing TSA-488: TSA amplification Buffer: 0.15% H2O 2-1: 100: 1; incubating for 15 minutes at 37 ℃ in the dark;
(17) washing with PBS (DEPC water) for 3 times, each for 5 min, in dark conditions;
(18) spin-drying PBS, dripping DAPI-antipode solution, covering a cover glass, standing at room temperature for 20 minutes, and storing in the dark;
(19) and (5) taking a picture.
Example 6 binding of miR-206-3p to BDNF
The online prediction software MiRanda and Targetscan are used to find that miR-206-3p has a binding site with BDNF, as shown in FIG. 7A, which indicates that miR-206-3p can potentially regulate BDNF. Therefore, the invention selects a section of sequence possibly combined by miR-206-3p and 3' -UTR-BDNF to carry out PCR amplification, and simultaneously, the sequence of the site-directed mutation binding site is used as a contrast. Then, the obtained fragment was inserted into a vector for detecting dual luciferase activity, thereby obtaining a constructed wild-type recombinant vector (wt-3 'UTR-BDNF-psiCHECK 2) and a mutant recombinant vector (mut-3' UTR-BDNF-psiCHECK2), and the sequencing result showed that the constructed vector was error-free (FIG. 7B). As shown in FIG. 7C, the constructed wild-type vector and mutant vector and miR-206-3p mimic are co-transfected into HEK293 cells, after culturing for 48h, luciferase activity in the cells is detected by using a dual-luciferase detection kit, and the relative activity of renilla luciferase is calculated by using firefly luciferase activity calibration. The results in fig. 7 show that miR-206-3p mimetics significantly reduced luciferase activity after cotransformation with wt-3 ' UTR-BDNF-psiCHECK2 (n is 3, p <0.05), and that after cotransformation with mut-3 ' UTR-BDNF-psiCHECK2, the relative activity of intracellular luciferase was not inhibited by miR-206-3p mimetics, indicating that miR-206-3p can bind to sites in the 3 ' UTR region of BDNF. BDNF is taken as a pathological index of AD, the content of the BDNF in blood plasma is low, and the BDNF is difficult to detect, but miR-206-3p and BDNF have negative correlation, which indicates that miR-206-3p has potential feasibility as a marker of gene level.
The procedure of example 6 was as follows:
(1) predicting a 3' -UTR sequence of the seed region of miR-206-3p and the combination of the target gene BDNF by targetscan/mirDB/microRNA software;
(2) designing a sequence containing a mutation site, and synthesizing the mutation sequence (the end contains XholI and NotI enzyme cutting sites) by Shanghai;
(3) enriching target fragments, cutting and recovering;
(4) carrying out enzyme digestion on the psiCHECK-2 vector by using XholI and NotI restriction enzymes, and then carrying out gel cutting recovery on the product;
(5) connecting the target gene fragment with the sequence of the enzyme digestion vector psiCHECK-2, transforming, selecting a monoclonal colony, extracting a plasmid and sequencing.
(6) Recovering HEK293T cells, and inoculating 4000 cells per well on a 96-well white board after passage once;
(7) adding 2.25 microliters of lipofectamine 3000 transfection reagent into 500 microliters of Opti-MEM culture medium, uniformly mixing, and standing for 5 minutes;
(8) adding 2 microliters of miR-206-3p micic/miR-206-3 p Inhibitor (the final concentration is 40nM) with the concentration of 20 micromoles into 250 microliters of Opti-MEM, uniformly mixing and standing for 5 minutes;
(9) (0.9. mu.g) vector (psiCHECK-2, psiCHECK-2-BDNF-wt-3 '-UTR, psiCHECK-2-BDNF mut-3' -UTR) was added to 250. mu.LOpti-MEM, mixed well and left to stand for 5 minutes;
(10) and adding the carrier and the mixed solution of miR-206-3p imic/miR-206-3p Inhibitor into the mixed solution of the transfection reagent, uniformly mixing, and standing for 20 minutes. When the cell density is 60%, washing the cells for 3 times by PBS, adding the reagent into the cells, culturing for 6 hours in an incubator at 37 ℃, then replacing the complete culture medium, and continuing culturing for 48 hours;
(11) discarding the culture medium, washing with PBS 2 times, adding Dual-Glo Luciferase Reagent for lysis, transferring to detection plate for detecting firefly Luciferase luminescence signal, adding Dual-Glo Stop & Glo Reagent, and detecting Renilla Luciferase luminescence signal.
Example 7 high expression of miR-206-3p in APP/PS1 mouse plasma
Examples 3 and 4 have shown whether miR-206-3p is highly expressed in APP/PS1 brain, and has such a phenomenon in peripheral blood, and if it exists, it can avoid obtaining detection of pathological changes in AD in a manner that affects the brain. Firstly, the method collects the blood plasma of 9-month-old APP/PS1 mice and wild mice by means of heart blood sampling, extracts RNA by means of example 2, and detects the level of miR-206-3p in the blood plasma by means of a qPCR method. The results show that the level of miR-206-3p in plasma was also higher in APP/PS1 mice than in wild-type control mice (FIG. 8, p <0.0001), consistent with the results for miR-206-3p in hippocampus and prefrontal cortex.
The procedure for example 7 was as follows:
(1) collecting blood collected by the heart by using an EDTA anticoagulation tube;
(2) turning upside down and mixing evenly, and standing for 10 minutes at room temperature;
(3) centrifuging at 3,000rpm for 10 minutes at room temperature;
(4) transfer supernatant to enzyme-free Ep tube;
(5) the plasma levels of miR-206-3p were analyzed in the same manner as in steps (6) to (12) of example 2.
Example 8 high expression of miR-206-3p in plasma of AD patients
Plasma was collected from clinically confirmed AD patients and used as a control with plasma of an age corresponding to the subjects identified as healthy. The level of miR-206-3p in the plasma of AD patients was detected using the qPCR technique. The results show (fig. 9A) that the fluorescence signal collected in the qPCR assay for the AD patient plasma samples was significantly earlier than for the normal subjects, and that the levels of exogenous cel-miR-39-3p were nearly identical in the normal and AD patient plasma samples (fig. 9D), with better primer specificity (fig. 9B-9C, fig. 9E-9F), indicating that the level of miR-206-3p in the AD patient plasma was significantly higher than in the normal subjects (fig. 9G). In this embodiment, qPCR may be used for detection, and specifically, one of the following primers may be used for amplification detection:
(upstream primer sequence) 5'-GGAATGTAAGGAAGTGTGTGGAAA-3', (downstream primer sequence) 5'-GTCCAGTTTTTTTTTTTTTTTCTCG-3'
Or (upstream primer sequence) 5'-CACGCATGGAATGTAAGGAAGT-3', (downstream primer sequence) 5'-CCAGTGCAGGGTCCGAGGT-3'
Or (upstream primer sequence) 5'-GGCCACATGCTTCTTTATATCCT-3', (downstream primer sequence) 5'-CCAAAACCACACACTTCCTTAC-3'
Or (upstream primer sequence) 5'-AGGCCACATGCTTCTTTATATCC-3', (downstream primer sequence) 5'-CCAAAACCACACACTTCCTTACAT-3'
Or (upstream primer sequence) 5'-CAGGCCACATGCTTCTTTATATC-3', (downstream primer sequence) 5'-CAAAACCACACACTTCCTTACA-3'; or the nucleic acid hybridization technology can be adopted, and the probe is utilized to detect the abundance of miR-206-3 p; specifically, the probe can be one of the following sequences: 5 '-Digoxin-CCACACACUUCCUUACAUUCCA-Digoxin-3' or 5 '-Digoxin-ACACACUUCCUUACAUUCCA-Digoxin-3' or 5 '-Digoxin-CACACUUCCUUACAUUCCA-Digoxin-3'.
The procedure for example 8 was as follows:
(1) collecting plasma of patients with confirmed diagnosis of AD, MCI, mental disorder, schizophrenia, depression, anxiety, and collecting plasma of healthy subjects with corresponding age;
(2) RNA was extracted from plasma and the level of miR-206-3p in plasma was measured according to the procedure in example 7.
Example 9 the level of miR-206-3p in the plasma of AD subjects is higher than the level of miR-206-3p in the plasma of other psychiatric patients
miR-206-3p, although it has a significant difference in plasma in normal subjects and AD patients, has not yet demonstrated that miR-206-3p is a specific marker for AD. For this purpose, the invention collects the blood plasma of a subject with symptoms of anxiety, depression, schizophrenia, Parkinson and mental disorder, and detects the level of miR-206-3p in the blood plasma by a qPCR method. Although schizophrenia, psychotic disorder, etc. are most commonly found in a young population, considering that the age of onset of AD is generally 55 years old or later, subjects with anxiety, depression, schizophrenia, psychotic disorder, age 55 years old were selected for comparison by the present invention (fig. 9H). The results show that the level of miR-206-3p in AD is significantly higher than in normal subjects (fig. 9G), and that the level of miR-206-3p in the plasma of AD and MCI subjects is significantly higher than in the plasma of anxiety, depression, parkinson, schizophrenia and psychotic disorders subjects (normal n ═ 7, alzheimer's disease n ═ 6, mild cognitive impairment n ═ 4, anxiety n ═ 12, depression n ═ 6, parkinson's n ═ 4, schizophrenia n ═ 6 and psychotic disorder n ═ 5, × <0.05, × <0.01, × < 0.001). According to the results, the invention sets the reference (base line, B line) of the highest miR-206-3p in the plasma of the subjects with anxiety, depression, Parkinson, schizophrenia and mental disorder, the A line is taken as mild cognitive disorder, and the C line is taken as AD. For subjects over 55 years of age, miR-206-3p levels are considered to be as advanced as possible as MCI if they approach the age-corresponding healthy group by a factor of 4.23; if the blood pressure is 4.23 times higher and 5.7 times lower than that of the normal healthy group, the MCI is judged to be in the AD stage. As the abundance of miR-206-3p is related to the development of AD, whether the medicament has a treatment effect on AD can be detected by detecting the abundance of miR-206-3 p.
The procedure for example 9 was as follows:
(1) comparing miR-206-3p levels in plasma of subjects over 55 years of age (4 years of age except psychotic disorder);
(2) comparing the age differences of the subjects in each group;
(3) setting thresholds of AD and MCI, and selecting the highest miR-206-3p level as a lower line according to changes of other mental diseases.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. Application of the marker miR-206-3p in preparation of medicines for diagnosing or treating Alzheimer's disease.
2. A kit for diagnosing alzheimer's disease, characterized in that: the kit comprises a detection reagent for detecting the content of miR-206-3 p.
3. The kit for diagnosing alzheimer's disease according to claim 2, wherein: the detection reagent comprises a primer for amplifying miR-206-3 p.
4. The kit for diagnosing alzheimer's disease according to claim 3, wherein: the sequence of the primer is as follows:
5’-GGAATGTAAGGAAGTGTGTGGAAA-3’，
5’-GTCCAGTTTTTTTTTTTTTTTCTCG-3’
or 5'-CACGCATGGAATGTAAGGAAGT-3', or a combination thereof,
5’-CCAGTGCAGGGTCCGAGGT-3’
or 5'-GGCCACATGCTTCTTTATATCCT-3', or a combination thereof,
5’-CCAAAACCACACACTTCCTTAC-3’
or 5'-AGGCCACATGCTTCTTTATATCC-3', or a combination thereof,
5’-CCAAAACCACACACTTCCTTACAT-3’
or 5'-CAGGCCACATGCTTCTTTATATC-3', or a combination thereof,
5'-CAAAACCACACACTTCCTTACA-3'.
5. The kit for diagnosing alzheimer's disease according to claim 2, wherein: the detection reagent comprises a probe for detecting the content of miR-206-3 p.
6. The kit for diagnosing alzheimer's disease according to claim 5, wherein: the sequence of the probe is at least one of 5 '-Digoxin-CCACACACUUCCUUACAUUC CA-Digoxin-3' or 5 '-Digoxin-ACACACUUCCUUACAUUCCA-Digoxin-3' or 5 '-Digoxin-CACACUUCCUUACAUUCCA-Digoxin-3'.
7. A method of screening for a drug for treating alzheimer's disease; the method is characterized in that:
step 1: treating an AD model animal or AD model cells with a candidate drug;
step 2: detecting the amount of miR-206-3p in the treated AD model animal or AD model cell;
and step 3: performing drug screening according to the content of the obtained miR-206-3 p; and if the content of the miR-206-3p in the candidate drug is reduced, the candidate drug is the target drug obtained by screening.
8. A storage medium having computer-executable program code stored thereon, wherein the program code, when executed by one or more processors of a computer system, causes the computer system to perform a method of diagnosing alzheimer's disease, the method comprising the steps of:
step 1: obtaining the content T of miR-206-3p in a detection sample of a subject and the content N of miR-206-3p in a detection sample of a normal person; the subject is older than 55 years;
step 2: calculating R ═ T/N;
and step 3: if R is less than or equal to 1, outputting that the subject is healthy, otherwise, outputting that the subject is unhealthy.
9. The storage medium of claim 8, wherein:
and step 3:
if R is less than or equal to 2.15, outputting that the subject is healthy;
if R is more than 2.15 and less than or equal to 4.23; outputting the subject as being in a stage of developing mild cognitive impairment; if R is more than 4.23 and less than or equal to 5.7; outputting the subject as being in mild cognitive impairment to progress toward the AD stage;
if 5.7< R; the subject is output as having AD.
10. An electronic device, comprising: one or more processors, and the storage medium of any one of claims 8-9; when executed by the one or more processors, cause the one or more processors to implement a method for Alzheimer's disease diagnosis.
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