JP2017184642A - Dementia marker, evaluation method of dementia using same, evaluation reagent, and evaluation kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel dementia marker capable of evaluating the possibility of dementia.SOLUTION: A dementia marker is at least one miRNA selected from the group consisting of: hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let-7b-5p, hsa-miR-126-3p, hsa-miR-25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101-3p, hsa-miR-222-3p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR-27b-3p, hsa-miR-484, hsa-miR-125a-5p, hsa-miR-93-5p, etc.SELECTED DRAWING: None

Description

  The present invention relates to a dementia marker, a method for evaluating dementia using the marker, an evaluation reagent, and an evaluation kit.

  With the aging of society, the number of patients with dementia is increasing, and miRNA is used as the marker (Non-patent Document 1). For the appropriate treatment of dementia, new markers are required in the diagnosis of dementia.

“MicroRNA-Seq Data Analysis Pipeline to Identify Blood Biomarkers for Alzheimer's Disease from Public Data.” Satoh J, Kino Y, Niida S. Biomark Insights. 2015 Apr 15; 10: 21-31.

  Then, an object of this invention is to provide the novel dementia marker which can judge the morbidity of dementia.

  The dementia markers of the present invention are hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let-7b-5p, hsa-miR- 126-3p, hsa-miR-25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101-3p, hsa-miR-222- 3p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR-27b-3p, hsa- miR-484, hsa-miR-125a-5p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa-miR-16-5p, hsa-miR-20a- 5p, hsa-miR-199a-5p, hsa-miR-17-5p, hsa-miR-486-3p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let-7c, hsa- miR-340-5p, hsa-miR-224-5p, hsa-miR-1180, hsa-miR-152, hsa-let-7e-5p, hsa-miR-223-3p, hsa-miR-183-5p, hsa-miR-126-5p, hsa-miR-652-3p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-30e-3p, hsa-miR-92b-3p, hsa- miR-424-3p, hsa-miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR-379-5p, hsa-miR-98, hsa-miR-339-5p, hsa-miR-654-3p, hsa-miR-150-3p, hsa-miR-130b-5p, hsa-miR-664-5p, hsa-miR-20b-5p, hsa- miR-194-5p, h sa-miR-142-5p, hsa-miR-361-5p, hsa-miR-423-5p, hsa-miR-483-5p, hsa-miR-148a-5p, hsa-miR-92b-5p and hsa- It is at least one miRNA selected from the group consisting of miR-4669.

  The evaluation method for evaluating the possibility of dementia according to the present invention comprises the steps of detecting the expression level of a dementia marker in a biological sample, and the expression level of the dementia marker detected in the detection step and the evaluation criteria. Including a step of determining the possibility of dementia, wherein the dementia marker is the dementia marker of the present invention.

  The evaluation reagents for evaluating the possibility of dementia of the present invention are hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let. -7b-5p, hsa-miR-126-3p, hsa-miR-25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101 -3p, hsa-miR-222-3p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa -miR-27b-3p, hsa-miR-484, hsa-miR-125a-5p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa-miR-16 -5p, hsa-miR-20a-5p, hsa-miR-199a-5p, hsa-miR-17-5p, hsa-miR-486-3p, hsa-miR-199a-3p, hsa-miR-199b-3p , Hsa-let-7c, hsa-miR-340-5p, hsa-miR-224-5p, hsa-miR-1180, hsa-miR-152, hsa-let-7e-5p, hsa-miR-223-3p , Hsa-miR-183-5p, hsa-miR-126-5p, hsa-miR-652-3p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-30e-3p, hsa -miR-92b-3p, hsa-miR-424-3p, hsa-miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR-379 -5p, hsa-miR-98, hsa-miR-339-5p, hsa-miR-654-3p, hsa-miR-150-3p, hsa-miR-130b-5p, hsa-miR-664-5p hsa-miR-20b-5p, hsa-miR-194-5p, hsa-miR-142-5p, hsa-miR-361-5p, hsa-miR-423-5p, hsa-miR-483-5p, hsa- a miRNA detection reagent that detects at least one miRNA selected from the group consisting of miR-148a-5p, hsa-miR-92b-5p and hsa-miR-4669, and used for the evaluation method of the present invention. Features.

  The evaluation kit for evaluating the possibility of dementia of the present invention comprises the evaluation reagent of the present invention and is used for the evaluation method of the present invention.

  Since the expression level of the dementia marker of the present invention varies depending on the onset of dementia, for example, the possibility of suffering from dementia can be determined by measuring the expression level.

  In the dementia marker of the present invention, for example, the miRNA is miRNA in serum.

  The dementia marker of the present invention includes, for example, the above-mentioned dementia: Alzheimer's disease, mild cognitive impairment (MCI), frontotemporal dementia, Lewy body dementia, cerebrovascular dementia, juvenile dementia, pick At least one dementia selected from the group consisting of Alzheimer's disease, Parkinson's disease, and alcoholic dementia. In the dementia marker of the present invention, for example, the dementia is mild cognitive impairment (MCI).

  The dementia marker of the present invention is, for example, a marker for determining the onset, preclinical stage, clinical stage or prognosis of dementia.

  In the evaluation method of the present invention, for example, the evaluation criterion is an expression level of the dementia marker in a normal human biological sample, and in the determination step, the detected expression level of the dementia marker and the evaluation criterion If the difference is significant, the subject determines that the possibility of the dementia is high.

  In the evaluation method of the present invention, for example, the dementia marker is hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let-7b. -5p, hsa-miR-126-3p, hsa-miR-25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101-3p , Hsa-miR-222-3p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR -27b-3p, hsa-miR-484, hsa-miR-125a-5p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa-miR-16-5p , Hsa-miR-20a-5p, hsa-miR-199a-5p, hsa-miR-17-5p, hsa-miR-486-3p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa -let-7c, hsa-miR-340-5p, hsa-miR-224-5p, hsa-miR-1180, hsa-miR-152, hsa-let-7e-5p, hsa-miR-223-3p, hsa -miR-183-5p, hsa-miR-126-5p, hsa-miR-652-3p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-30e-3p, hsa-miR -92b-3p, hsa-miR-424-3p, hsa-miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR-379-5p , Hsa-miR-98, hsa-miR-339-5p, hsa-miR-654-3p, hsa-miR-150-3p, hsa-miR-130b-5p, hsa-miR-664-5p at least one selected from the group consisting of hsa-miR-20b-5p, hsa-miR-194-5p, hsa-miR-142-5p, and hsa-miR-361-5p, When the expression level of the detected dementia marker is significantly lower than the evaluation criteria, the subject determines that the possibility of the dementia is high.

  In the evaluation method of the present invention, for example, the dementia marker is hsa-miR-423-5p, hsa-miR-483-5p, hsa-miR-148a-5p, hsa-miR-92b-5p, and hsa- at least one selected from the group consisting of miR-4669, and in the determination step, when the detected expression level of the dementia marker is significantly higher than the evaluation criteria, the subject Determines that the dementia is likely.

  In the evaluation method of the present invention, for example, the evaluation standard is the expression level of the dementia marker in a biological sample of a dementia patient, and the expression level of the dementia marker detected in the determination step and the evaluation standard If the difference is not significant, the subject determines that the possibility of the dementia is high.

  In the evaluation method of the present invention, for example, the biological sample is a sample containing serum.

  In the evaluation method of the present invention, for example, the dementia is Alzheimer's disease, mild cognitive impairment (MCI), frontotemporal dementia, Lewy body dementia, cerebrovascular dementia, juvenile dementia, Pick's disease And at least one dementia selected from the group consisting of Parkinson's disease and alcoholic dementia. In the evaluation method of the present invention, for example, the dementia is mild cognitive impairment (MCI).

  In the evaluation method of the present invention, for example, the expression level of the dementia marker is represented by the expression level of the dementia marker in the biological sample.

<Dementia marker>
As described above, the dementia marker of the present invention is hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let-7b-5p. , Hsa-miR-126-3p, hsa-miR-25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101-3p, hsa -miR-222-3p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR-27b -3p, hsa-miR-484, hsa-miR-125a-5p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa-miR-16-5p, hsa -miR-20a-5p, hsa-miR-199a-5p, hsa-miR-17-5p, hsa-miR-486-3p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let -7c, hsa-miR-340-5p, hsa-miR-224-5p, hsa-miR-1180, hsa-miR-152, hsa-let-7e-5p, hsa-miR-223-3p, hsa-miR -183-5p, hsa-miR-126-5p, hsa-miR-652-3p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-30e-3p, hsa-miR-92b -3p, hsa-miR-424-3p, hsa-miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR-379-5p, hsa -miR-98, hsa-miR-339-5p, hsa-miR-654-3p, hsa-miR-150-3p, hsa-miR-130b-5p, hsa-miR-664-5p, hsa-miR-20b -5p , Hsa-miR-194-5p, hsa-miR-142-5p, hsa-miR-361-5p, hsa-miR-423-5p, hsa-miR-483-5p, hsa-miR-148a-5p, hsa -at least one miRNA selected from the group consisting of -miR-92b-5p and hsa-miR-4669.

  As a result of intensive studies, the present inventors have found that the expression level of these miRNAs varies specifically in the living body with the onset of dementia, and have reached the present invention. Specifically, it has been clarified that, with the onset of dementia, for example, the expression level decreases or increases in serum. For this reason, for example, evaluation of the possibility of dementia can be performed by detecting the expression level of these miRNAs. Further, since the onset of dementia and the variation in the expression of the dementia marker of the present invention are related in this way, for example, the onset of dementia can only be indirectly determined by detecting the expression level of the marker. In addition, it is possible to determine the possibility of developing dementia or to determine the degree of improvement of dementia even when no symptoms appear.

  In the present invention, “dementia” generally means a state in which cognitive function has been acquired.

  The dementia that is a target of the dementia marker of the present invention is not particularly limited, and examples thereof include Alzheimer's disease, mild cognitive impairment (MCI), frontotemporal dementia, Lewy body dementia, cerebrovascular dementia, Examples include juvenile dementia, Pick's disease, Parkinson's disease, and alcoholic dementia.

  In the present invention, the miRNA may be, for example, a single strand (monomer) or a double strand (dimer). In the present invention, the miRNA is preferably a mature miRNA cleaved by a ribonuclease such as Dicer.

As described above, the expression level of the dementia marker of the present invention decreases or increases with the onset of dementia, for example. For example, a marker whose expression level decreases with the onset of dementia is hereinafter referred to as a decrease marker. Examples of the decrease marker include miRNA shown below. For example, the sequence of the miRNA is registered under the following accession number.
hsa-miR-486-5p (Accession number: MIMAT0002177)
hsa-miR-21-5p (Accession number: MIMAT0000076)
hsa-miR-92a-3p (Accession number: MIMAT00000092)
hsa-miR-451a (Accession number: MIMAT0001631)
hsa-let-7b-5p (Accession number: MIMAT00000063)
hsa-miR-126-3p (Accession number: MIMAT000045)
hsa-miR-25-3p (Accession number: MIMAT00000081)
hsa-miR-10b-5p (Accession number: MIMAT0000254)
hsa-miR-30e-5p (Accession number: MIMAT0000692)
hsa-miR-140-3p (Accession number: MIMAT0004597)
hsa-miR-101-3p (Accession number: MIMAT000009)
hsa-miR-222-3p (Accession number: MIMAT00000200)
hsa-miR-10a-5p (Accession number: MIMAT000003)
hsa-miR-221-3p (Accession number: MIMAT0000278)
hsa-miR-375 (Accession No .: MIMAT000028)
hsa-miR-30a-5p (Accession number: MIMAT0000087)
hsa-miR-584-5p (Accession number: MIMAT0003249)
hsa-miR-27b-3p (Accession number: MIMAT000019)
hsa-miR-484 (Accession number: MIMAT0002174)
hsa-miR-125a-5p (Accession number: MIMAT000043)
hsa-miR-93-5p (Accession number: MIMAT00000093)
hsa-miR-182-5p (Accession number: MIMAT0000259)
hsa-miR-223-5p (Accession number: MIMAT0004570)
hsa-miR-16-5p (Accession number: MIMAT00000069)
hsa-miR-20a-5p (Accession number: MIMAT000075)
hsa-miR-199a-5p (Accession number: MIMAT000001)
hsa-miR-17-5p (Accession number: MIMAT00000070)
hsa-miR-486-3p (Accession number: MIMAT0004762)
hsa-miR-199a-3p (Accession number: MIMAT0000232)
hsa-miR-199b-3p (Accession number: MIMAT0004563)
hsa-let-7c (Accession number: MIMAT000063)
hsa-miR-340-5p (Accession number: MIMAT0004692)
hsa-miR-224-5p (Accession number: MIMAT000000281)
hsa-miR-1180 (Accession number: MIMAT0026735)
hsa-miR-152 (Accession number: MIMAT00026479)
hsa-let-7e-5p (Accession number: MIMAT000063)
hsa-miR-223-3p (Accession number: MIMAT0000280)
hsa-miR-183-5p (accession number: MIMAT000001)
hsa-miR-126-5p (Accession number: MIMAT0000444)
hsa-miR-652-3p (Accession number: MIMAT0003322)
hsa-miR-197-3p (Accession number: MIMAT0000227)
hsa-miR-432-5p (Accession number: MIMAT0002814)
hsa-miR-30e-3p (Accession number: MIMAT000053)
hsa-miR-92b-3p (Accession number: MIMAT0003218)
hsa-miR-424-3p (Accession number: MIMAT0004749)
hsa-miR-338-5p (accession number: MIMAT0004701)
hsa-miR-140-5p (Accession number: MIMAT000031)
hsa-miR-543 (Accession number: MIMAT0004954)
hsa-miR-625-3p (Accession number: MIMAT0004808)
hsa-miR-379-5p (Accession number: MIMAT000033)
hsa-miR-98 (Accession number: MIMAT00000096)
hsa-miR-339-5p (Accession number: MIMAT000064)
hsa-miR-654-3p (Accession number: MIMAT0004814)
hsa-miR-150-3p (accession number: MIMAT0004610)
hsa-miR-130b-5p (Accession number: MIMAT0004680)
hsa-miR-664-5p (Accession number: MIMAT0005948)
hsa-miR-20b-5p (Accession number: MIMAT0001413)
hsa-miR-194-5p (Accession number: MIMAT0000460)
hsa-miR-142-5p (Accession number: MIMAT000033)
hsa-miR-361-5p (Accession number: MIMAT0000703)

For example, a marker whose expression level increases with the onset of dementia is hereinafter referred to as an elevation marker. Examples of the elevation marker include miRNA shown below. For example, the sequence of the miRNA is registered under the following accession number.
hsa-miR-423-5p (Accession number: MIMAT0004748)
hsa-miR-483-5p (Accession number: MIMAT0004761)
hsa-miR-148a-5p (Accession number: MIMAT0004549)
hsa-miR-92b-5p (Accession number: MIMAT0004792)
hsa-miR-4669 (Accession number: MIMAT0019749)

  The miRNA in the present invention further includes, for example, a variant in which several bases are different from the aforementioned mature sequence.

  The miRNA in the present invention is, for example, a polynucleotide composed of a base sequence having the same identity as the base sequence of the miRNA, or a polynucleotide composed of a base sequence complementary thereto, and has the function of the miRNA in the present invention. Including polynucleotides. The “identity” is the same degree when sequences to be compared are appropriately aligned, and means the appearance rate (%) of an exact match of amino acids between the sequences. The alignment can be performed by using an arbitrary algorithm such as BLAST, for example. The identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99%. The polynucleotide comprising a base sequence having the identity may have, for example, a point mutation, a deletion and / or an addition in the base sequence of the miRNA. The number of bases such as the point mutation is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1. The polynucleotide comprising the complementary base sequence is, for example, a polynucleotide that hybridizes with the polynucleotide comprising the base sequence of the miRNA under stringent conditions, and has the function of the miRNA in the present invention. including. The stringent conditions are not particularly limited. For example, in a solution containing 6 × SSC, 0.5% SDS, 5 × Denhardt, 0.01% denatured salmon sperm nucleic acid, a temperature of “Tm−25 ° C.” For example, the conditions for keeping the temperature overnight.

  The dementia marker of the present invention is, for example, miRNA present in a biological sample. The biological sample is, for example, a sample collected from a subject's living body. The kind of the biological sample is not particularly limited, and examples thereof include blood, saliva, urine, and diluted solutions thereof. Among these, a blood sample is preferable. The type of the blood sample is not particularly limited, and examples thereof include a whole blood sample, a plasma sample, a serum sample, a whole blood or hemolyzed hemolyzed sample, and diluted samples thereof.

<Evaluation method>
As described above, the evaluation method of the present invention is capable of dementia from the step of detecting the expression level of a dementia marker in a biological sample, and the expression level of the dementia marker detected in the detection step and the evaluation criteria. A step of determining sex, wherein the dementia marker is the dementia marker of the present invention.

  According to such an evaluation method, for example, the possibility of dementia can be determined with high reliability. For this reason, for example, in the prevention or treatment of dementia, important information for determining a policy such as medication can be obtained with high reliability.

  The evaluation method of the present invention is characterized by detecting the expression level of the dementia marker of the present invention in a biological sample, and other conditions and processes are not particularly limited. Moreover, the detection method of the expression level of miRNA which is a dementia marker of this invention is not restrict | limited at all, For example, a conventionally well-known method is employable. In the present invention, the dementia marker to be detected may be, for example, any one of the dementia markers of the present invention, or a combination of two or more.

  In the present invention, the dementia to be evaluated is not particularly limited, and examples thereof include the aforementioned dementia.

  In the present invention, the possibility of dementia includes, for example, the possibility of developing dementia, whether or not dementia has developed, the degree of progression of dementia, or prognosis. In the present invention, the “normal person” is, for example, a person who has not been determined to develop the dementia to be evaluated, or a person who has not been determined to have the possibility of developing the dementia. Means.

  As described above, the detection step in the present invention is a step of detecting the expression level of the dementia marker in the biological sample. The expression level of the dementia marker includes, for example, the expression level of the dementia marker in the biological sample. The expression level may be, for example, the actual miRNA amount or a value correlated with the actual miRNA amount. Examples of the latter include a signal value obtained upon detection of the miRNA. This signal value can be appropriately determined according to, for example, the miRNA detection method and the type of signal value detection device. For example, when the detection method is a PCR method such as a real-time RT-PCR (real-time reverse transcription-polymerase chain reaction) method, it can also be represented by, for example, copies / μL.

  In the present invention, the biological sample is not particularly limited, and examples thereof include blood, saliva, urine, etc. Among them, a blood sample is preferable, and a serum sample is more preferable.

  The serum sample can be collected by a known method. As a specific example, for example, it can be prepared by collecting whole blood from a subject and removing the blood cell fraction by centrifugation.

  The method for detecting miRNA in a biological sample is not particularly limited as long as it can detect the expression of miRNA as described above. Detection of miRNA in a biological sample can be performed, for example, by extracting total RNA from the biological sample and detecting the target miRNA from the total RNA. The method for extracting total RNA is not particularly limited, and a known method can be adopted. Examples of the extraction method include a guanidine-cesium chloride ultracentrifugation method, an AGPC (Acid Guanidinium-Phenol-Chloroform) method, and the like.

  Specific examples of the miRNA detection method include Northern blot analysis, real-time RT-PCR detection method, microarray analysis method and the like.

  When measuring the expression level of the dementia marker of the present invention, for example, an internal standard such as a correction marker may be measured, and the expression level corrected with the internal standard may be calculated for the miRNA to be detected.

  In the present invention, the origin of the biological sample, that is, the subject is not particularly limited, and examples thereof include non-human animals such as humans, dogs and cats.

  As described above, the determination step in the present invention is a step of determining the possibility of dementia from the expression level of the dementia marker detected in the detection step and the evaluation criteria. As described above, the expression level in the living body of the dementia marker of the present invention varies specifically with the onset of dementia. For this reason, for example, the possibility of dementia can be determined by comparing the expression level of the dementia marker detected from a biological sample derived from a living body with an evaluation standard. The evaluation criterion is not particularly limited, and may be, for example, the expression level of the dementia marker in a normal biological sample or the expression level of the dementia marker in a biological sample of a dementia patient.

  When the evaluation standard is the expression level of the dementia marker in a normal human biological sample, the evaluation method of the present invention includes, for example, in the determination step, the detected expression level of the dementia marker and the evaluation standard. If the difference is significant, the subject determines that the possibility of the dementia is high.

  Among the dementia markers of the present invention, the expression level of the decrease marker decreases in the biological sample with the onset of dementia, for example, as described above. Therefore, when the expression level of the lowering marker is detected as the dementia marker, in the determination step, for example, when the detected expression level of the dementia marker is significantly lower than the evaluation criterion. The subject determines that the possibility of the dementia is high. In addition, among the dementia markers of the present invention, the expression level of the elevation marker increases in the biological sample with the onset of dementia, for example, as described above. Therefore, when the expression level of the elevated marker is detected as the dementia marker, in the determination step, for example, when the detected expression level of the dementia marker is significantly higher than the evaluation criterion. The subject determines that the possibility of the dementia is high.

  When the evaluation criterion is the expression level of the dementia marker in the biological sample of the dementia patient, the evaluation method of the present invention includes, for example, in the determination step, the detected expression level of the dementia marker and the When the difference from the evaluation standard is not significant, the subject determines that the possibility of the dementia is high.

  When the evaluation standard is the expression level of the dementia marker in the biological sample of the dementia patient, specifically, it can be determined whether or not the dementia is the same level as the dementia patient used as the evaluation standard. . When the dementia marker is the decrease marker or the elevation marker, and the dementia patient of the evaluation criterion is mild (or severe), if the difference from the evaluation criterion is not significant, the subject Similarly, it can be judged as mild (or severe). When the dementia marker is the decrease marker and the dementia patient of the evaluation criterion is mild, when the detected expression level is significantly lower than the evaluation criterion, the subject is It can be judged to be more severe than the dementia patient. When the cognitive amount marker is the elevation marker and the dementia patient of the evaluation criterion is mild, when the detected expression level is significantly higher than the evaluation criterion, the subject is: It can be judged to be more severe than the dementia patient.

  It is preferable that the dementia marker detected in the detection step and the evaluation criterion marker are the same dementia marker. The evaluation criterion is preferably determined in advance, and it is not necessary to determine the evaluation criterion for each evaluation. That is, it is preferable to set the expression level of the dementia marker of the present invention in a normal person or a dementia patient that has been detected in advance as the evaluation criterion. The evaluation criterion may be, for example, a value obtained from the expression level of one normal person or a patient with dementia, or may be statistically determined from the expression levels of a plurality of normal persons or patients with dementia. It may be a calculated value.

  When the expression level of the subject is compared with the expression level of a normal person or a dementia patient, the significant difference is, for example, statistical such as T-test (T test), F test, chi-square test, etc. It can be judged by the method.

  The expression level of a dementia marker of a normal person can be determined using, for example, a biological sample collected from a normal person in advance. Moreover, the expression level of the dementia marker of a dementia patient can also be determined using the biological sample extract | collected from the patient previously, for example. The normal specimen and the biological specimen of the patient are, for example, the same kind of biological specimen as the biological specimen of the subject. Moreover, the normal sample and the biological sample of the patient are samples prepared by the same method and conditions as the biological sample of the subject, for example.

<Evaluation reagent>
As described above, the evaluation reagent of the present invention is an evaluation reagent for use in the evaluation method of the present invention, the detection reagent for the dementia marker of the present invention, that is, the dementia marker of the present invention. It includes a miRNA detection reagent for detection. According to such an evaluation reagent, the evaluation method of the present invention can be easily carried out.

  As described above, the present invention is characterized by detecting at least one of the dementia markers of the present invention as a dementia marker, and the detection method of these miRNAs is not limited at all. A conventionally known method can be employed. The miRNA detection reagent only needs to be able to detect any of the dementia markers of the present invention. For example, the type and composition of the reagent are not limited. Moreover, those skilled in the art can set detection reagents for these miRNAs based on common technical knowledge.

  The evaluation reagent of the present invention may further contain, for example, various enzymes, buffers, washings, solutions, dispersions, dilutions, etc., depending on various detection methods. The form of the evaluation reagent of the present invention is not particularly limited, and may be, for example, a liquid wet system or a dry dry system.

  The miRNA detection reagent is not particularly limited, and examples thereof include reagents used for Northern blot analysis and real-time RT-PCR detection method as described above. Specific examples of the miRNA detection reagent used for Northern blot analysis include, for example, a labeled probe that can hybridize to any of the dementia markers of the present invention. Specific examples of reagents for real-time RT-PCR detection include primers for amplifying cDNA from total RNA, primers for amplifying any of these miRNAs, and specifically binding to double-stranded nucleic acids Or the fluorescent reagent which intercalates, the various reagents which can be used for nucleic acid amplification, etc. are mention | raise | lifted. Examples of the various reagents include enzymes such as DNA polymerase, nucleotide triphosphate (dNTP), and the like.

<Evaluation kit>
As described above, the evaluation kit of the present invention is an evaluation kit for use in the evaluation method of the present invention, and the miRNA detection reagent for detecting the dementia marker of the present invention (for example, the evaluation reagent of the present invention) is used. It is characterized by including. According to such an evaluation kit, the evaluation method of the present invention can be easily performed. In the evaluation kit of the present invention, the evaluation reagent of the present invention is, for example, as described above.

  The form of the evaluation kit of the present invention is not particularly limited, and may be a liquid wet system or a dry system. The various reagents in the evaluation kit of the present invention are, for example, separate and may be used together at the time of use, or may be mixed before use.

  The evaluation kit of the present invention may be, for example, a test tool in which the above-described evaluation reagent of the present invention and other various reagents are arranged. The form of the inspection tool is not particularly limited, and examples thereof include a reactor such as a microreactor, a chip such as a microchip, a plate such as a microtiter plate, and an array such as a microarray. If it is such a test | inspection tool, the evaluation method of this invention can be easily performed by detecting a required signal with existing various detection apparatuses, such as a real-time PCR apparatus, for example. Further, for example, digitization of detected signals by computer processing, creation of data files for each subject, storage of data files in a predetermined directory, statistical analysis of measurement results of subjects, normal subjects, and patients, etc. It may be accompanied by a system for performing. Those skilled in the art can design such a data processing system from existing techniques, methods, and procedures based on common general knowledge.

  The evaluation kit of the present invention may further include, for example, various devices that can be used for executing the evaluation method of the present invention. Moreover, the evaluation kit of the present invention further includes, for example, instructions for use.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

[Example 1]
As a dementia marker, using the dementia marker of the present invention (No. 1 to 60 in Table 2 and No. 61 to 65 in Table 3), the expression level of the dementia marker in normal persons and patients with dementia is determined. It was measured.

  Dementia patients (mild dementia (MCI), 4 men, 6 women) and normal subjects (3 men, 7 women) are subjects and the symptoms of dementia are alleviated as shown in Table 1 below A supplement of the composition (manufactured by Healthy Pass Co., Ltd.) was taken twice a day for 5 weeks. As for the ingestion method, the powdered supplement was dissolved in hot water or the like and ingested after meals. When the symptoms were diagnosed for a dementia patient before and after taking the supplement, it was confirmed that the symptoms were alleviated by the intake.

  Thus, serum was collected before and after ingestion for each patient with dementia and normal, and the expression level of the dementia marker of the present invention was confirmed. If there is a difference in the expression level between a dementia patient and a normal person, the miRNA can be said to be a dementia marker. Moreover, as described above, since the symptoms of dementia are alleviated by taking the supplement, the expression level of miRNA before ingestion is different from that of normal subjects in patients with dementia, and the level of normal subjects after ingestion is If approached, it can be said that the miRNA is a dementia marker.

  Hereinafter, a method for measuring the expression level of each miRNA will be described. First, blood was collected from the subject before ingestion and after ingestion for 5 weeks. The collected blood was centrifuged at 3000 rpm and 15 ° C. for 15 minutes, and the separated serum fraction was collected and used as a serum sample. And about the said serum sample of a total of 8 groups (before ingestion of a dementia patient male group, a dementia patient female group, a normal male group, a normal female group, and after five weeks intake), it is equivalent so that it may become 200 microliters each. A total of 8 pooled sera corresponding to each group were prepared.

  Using each pooled serum, miRNA was prepared by miRNeasy Serum / Plasma kit (Qiagen) and dissolved in 14 μl of water to prepare a miRNA sample. For each miRNA sample, the molecular weight distribution and concentration were confirmed by a bioanalyzer (manufactured by Agilent Technologies).

  For the miRNA sample, a library was prepared using a Next Small RNA Library Prep Set for Illumina kit (manufactured by NEB). The number of PCR cycles was 15 cycles, except that 20 cycles were taken for the pre-intake samples of the normal female group. And miRNA fraction (19-22nt) was cut out from the reaction product. Next, the next-generation sequencer HiSeq 1500 manufactured by Illumina was used, and the SR50 was run to cut out 20 bp from the read. This sequence was mapped to hg19 using Strand NGS (manufactured by Agilent Technologies). In addition, a heat map and a dendrogram were created with all the expression data, and it was confirmed that a specific sample was not greatly different from others. Quantile correction was performed on the obtained data, and this was used as the expression level of each miRNA.

  And about each miRNA of the dementia marker of this invention, the expression level of a dementia patient when the expression level of a normal person was made into the expression level 1 was computed (dementia / normal person).

  These results are shown in Tables 2 and 3 below. Table 2 shows the results of measurement of the expression level in normal subjects and patients with dementia for the decrease marker among the dementia markers of the present invention, and Table 3 shows the normal subject for the increase marker among the dementia markers of the present invention. It is the result of measuring the expression level in patients with dementia. In Tables 2 and 3, “dementia marker” indicates the type of miRNA detected, “dementia / normal person (before ingestion)” indicates the value of dementia / normal person before supplement intake, “Dementia / normal person (after ingestion)” indicates the value of dementia / normal person after taking the supplement for 5 weeks.

  As shown in Table 2, among the dementia markers of the present invention, the expression level of the decrease marker in the patient with dementia was lower than that of the normal person before taking the supplement. From this, it turned out that a depressive marker can determine dementia as a dementia marker among the dementia markers of this invention. Among them, hsa-miR-92a-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR-27b-3p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa- miR-16-5p, hsa-miR-20a-5p, hsa-miR-199a-5p, hsa-miR-17-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let- 7c, hsa-miR-224-5p, hsa-let-7e-5p, hsa-miR-223-3p, hsa-miR-183-5p, hsa-miR-126-5p, hsa-miR-197-3p, hsa-miR-30e-3p, hsa-miR-424-3p, hsa-miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR- 98, hsa-miR-654-3p, hsa-miR-130b-5p, hsa-miR-20b-5p, hsa-miR-194-5p, and hsa-miR-361-5p compared to normal The expression level in patients with dementia was 0.5 or less for both men and women and was greatly reduced. That is, it was found that these markers can determine dementia with higher sensitivity. In addition, after taking the supplement for 5 weeks, the dementia marker expression level was all recovered in correlation with the alleviation of the symptoms of dementia in the patient.

  Moreover, as shown in Table 3, as for the elevation marker among the dementia markers of this invention, the expression level in a dementia patient was rising compared with a normal person before supplement intake. From this, it was found that the elevation marker among the dementia markers of the present invention can determine dementia as a dementia marker. Among them, hsa-miR-423-5p, hsa-miR-483-5p, hsa-miR-92b-5p, and hsa-miR-4669 have an expression level in patients with dementia compared to normal subjects. Both males and females were 2 or more, and they had risen significantly. That is, it was found that these markers can determine dementia with higher sensitivity. In addition, after taking the supplement for 5 weeks, the dementia marker expression level was all recovered in correlation with the alleviation of the symptoms of dementia in the patient.

  From the above results, it was found that the possibility of suffering from dementia can be determined by the dementia marker of the present invention.

  As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  According to the present invention, by detecting the expression level of the dementia marker of the present invention in a biological sample, for example, the possibility of dementia can be evaluated. For this reason, the present invention can be said to be extremely useful in the field of medicine and the like.

Claims (16)

hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let-7b-5p, hsa-miR-126-3p, hsa-miR- 25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101-3p, hsa-miR-222-3p, hsa-miR-10a- 5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR-27b-3p, hsa-miR-484, hsa-miR- 125a-5p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa-miR-16-5p, hsa-miR-20a-5p, hsa-miR-199a- 5p, hsa-miR-17-5p, hsa-miR-486-3p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let-7c, hsa-miR-340-5p, hsa- miR-224-5p, hsa-miR-1180, hsa-miR-152, hsa-let-7e-5p, hsa-miR-223-3p, hsa-miR-183-5p, hsa-miR-126-5p, hsa-miR-652-3p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-30e-3p, hsa-miR-92b-3p, hsa-miR-424-3p, hsa- miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR-379-5p, hsa-miR-98, hsa-miR-339-5p, hsa-miR-654-3p, hsa-miR-150-3p, hsa-miR-130b-5p, hsa-miR-664-5p, hsa-miR-20b-5p, hsa-miR-194-5p, hsa- miR-142-5p, hsa-miR-361-5p, hsa-mi It is at least one miRNA selected from the group consisting of R-423-5p, hsa-miR-483-5p, hsa-miR-148a-5p, hsa-miR-92b-5p and hsa-miR-4669 Characteristic dementia marker. The dementia marker according to claim 1, wherein the miRNA is a miRNA in serum. The dementia is from Alzheimer's disease, mild cognitive impairment (MCI), frontotemporal dementia, Lewy body dementia, cerebrovascular dementia, juvenile dementia, Pick's disease, Parkinson's disease, alcoholic dementia The dementia marker according to claim 1 or 2, which is at least one dementia selected from the group consisting of: The dementia marker according to claim 1 or 2, wherein the dementia is mild cognitive impairment (MCI). The dementia marker according to any one of claims 1 to 4, wherein the dementia marker is a marker for determining the onset, preclinical phase, clinical phase, or prognosis of dementia. Detecting the expression level of a dementia marker in a biological sample, and determining the possibility of dementia from the expression level of the dementia marker detected in the detection step and the evaluation criteria,
The evaluation method for evaluating the possibility of dementia, wherein the dementia marker is the dementia marker according to any one of claims 1 to 5. The evaluation criterion is the expression level of the dementia marker in a biological sample of a normal person,
In the determination step, when the difference between the detected expression level of the dementia marker and the evaluation criterion is significant, the subject determines that the possibility of the dementia is high. The evaluation method described. The dementia marker is hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let-7b-5p, hsa-miR-126- 3p, hsa-miR-25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101-3p, hsa-miR-222-3p, hsa-miR-10a-5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR-27b-3p, hsa-miR- 484, hsa-miR-125a-5p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa-miR-16-5p, hsa-miR-20a-5p, hsa-miR-199a-5p, hsa-miR-17-5p, hsa-miR-486-3p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let-7c, hsa-miR- 340-5p, hsa-miR-224-5p, hsa-miR-1180, hsa-miR-152, hsa-let-7e-5p, hsa-miR-223-3p, hsa-miR-183-5p, hsa- miR-126-5p, hsa-miR-652-3p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-30e-3p, hsa-miR-92b-3p, hsa-miR- 424-3p, hsa-miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR-379-5p, hsa-miR-98, hsa- miR-339-5p, hsa-miR-654-3p, hsa-miR-150-3p, hsa-miR-130b-5p, hsa-miR-664-5p, hsa-miR-20b-5p, hsa-miR- 194-5p, hsa-miR -142-5p, and at least one selected from the group consisting of hsa-miR-361-5p,
In the determination step, when the expression level of the detected dementia marker is significantly lower than the evaluation criterion, the subject determines that the possibility of the dementia is high. 7. The evaluation method according to 7. The dementia marker is selected from the group consisting of hsa-miR-423-5p, hsa-miR-483-5p, hsa-miR-148a-5p, hsa-miR-92b-5p, and hsa-miR-4669 At least one,
In the determination step, when the expression level of the detected dementia marker is significantly higher than the evaluation criterion, the subject determines that the possibility of the dementia is high. 7. The evaluation method according to 7. The evaluation criterion is the expression level of the dementia marker in a biological sample of a dementia patient,
The said determination process WHEREIN: The said subject determines that the possibility of the said dementia is high when the difference of the expression level of the said detected dementia marker and the said evaluation criteria is not significant. Evaluation method. The evaluation method according to any one of claims 6 to 10, wherein the biological sample is a sample containing serum. The dementia is from Alzheimer's disease, mild cognitive impairment (MCI), frontotemporal dementia, Lewy body dementia, cerebrovascular dementia, juvenile dementia, Pick's disease, Parkinson's disease, alcoholic dementia The evaluation method according to any one of claims 6 to 11, wherein the evaluation method is at least one dementia selected from the group consisting of: The evaluation method according to any one of claims 6 to 11, wherein the dementia is mild cognitive impairment (MCI). The evaluation method according to any one of claims 6 to 13, wherein the expression level of the dementia marker is represented by an expression level of the dementia marker in the biological sample. hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-451a, hsa-let-7b-5p, hsa-miR-126-3p, hsa-miR- 25-3p, hsa-miR-10b-5p, hsa-miR-30e-5p, hsa-miR-140-3p, hsa-miR-101-3p, hsa-miR-222-3p, hsa-miR-10a- 5p, hsa-miR-221-3p, hsa-miR-375, hsa-miR-30a-5p, hsa-miR-584-5p, hsa-miR-27b-3p, hsa-miR-484, hsa-miR- 125a-5p, hsa-miR-93-5p, hsa-miR-182-5p, hsa-miR-223-5p, hsa-miR-16-5p, hsa-miR-20a-5p, hsa-miR-199a- 5p, hsa-miR-17-5p, hsa-miR-486-3p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let-7c, hsa-miR-340-5p, hsa- miR-224-5p, hsa-miR-1180, hsa-miR-152, hsa-let-7e-5p, hsa-miR-223-3p, hsa-miR-183-5p, hsa-miR-126-5p, hsa-miR-652-3p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-30e-3p, hsa-miR-92b-3p, hsa-miR-424-3p, hsa- miR-338-5p, hsa-miR-140-5p, hsa-miR-543, hsa-miR-625-3p, hsa-miR-379-5p, hsa-miR-98, hsa-miR-339-5p, hsa-miR-654-3p, hsa-miR-150-3p, hsa-miR-130b-5p, hsa-miR-664-5p, hsa-miR-20b-5p, hsa-miR-194-5p, hsa- miR-142-5p, hsa-miR-361-5p, hsa-mi MiRNA that detects at least one miRNA selected from the group consisting of R-423-5p, hsa-miR-483-5p, hsa-miR-148a-5p, hsa-miR-92b-5p and hsa-miR-4669 Including a detection reagent,
An evaluation reagent for evaluating the possibility of dementia, characterized by being used in the evaluation method according to any one of claims 6 to 14. An evaluation reagent according to claim 15,
An evaluation kit for evaluating the possibility of dementia, characterized by being used in the evaluation method according to any one of claims 6 to 14.