JP7436004B2 - Diagnostic aid method and diagnostic kit for depression - Google Patents

Description

The present invention relates to a method and diagnostic kit for assisting in the diagnosis of depression.

In Japan, 1 million patients with depression visit medical institutions. However, in this diagnosis, a doctor evaluates the presence or absence of specific symptoms based on the results of interviews with the patient and family members. Diagnostic criteria for depression are based on how long specific symptoms have lasted, and because they partially include subjective evaluations by patients and others, they cannot be called objective evaluation methods.
Various methods have been proposed to objectively evaluate depression, such as genomic information and brain imaging information, but all have low sensitivity and specificity and have not been applied in actual clinical practice.

In recent years, the use of microRNA (miRNA) as a biomarker has been actively researched. Regarding depression, there are some reports suggesting that specific miRNA in blood can be used as a diagnostic marker (Non-Patent Documents 1-3). However, to date, there have been no reports investigating the relationship between miRNA contained in exosomes and depression.

In addition, five miRNAs, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-15b-5p, hsa-miR-204-3p, and hsa-miR-4497, are diagnostic markers for depression. Until now, it was completely unknown what could happen. On the other hand, it has been reported that serum or plasma levels of hsa-miR-106a-5p and hsa-miR-26b-5p are increased in depressed patients and mouse models (Non-Patent Documents 1-3).

Journal of Affective Disorders, 2014, 163:133-139 Acta Psychiatrica Scandinavica, 2017, 136(6):594-606 Journal of Cellular and Molecular Medicine, 2019, 23(10):7021-7028

An object of the present invention is to identify a novel depression diagnostic marker with high accuracy and provide a means for objectively diagnosing depression using the marker as an index.

Through comprehensive analysis using microarrays, the present inventors identified seven species that are detected in blood-derived exosomes of healthy individuals, but not detected or at low levels in blood-derived exosomes of depressed patients. discovered miRNA, and completed the present invention.

That is, the present invention is as follows.
[1] A method for assisting in the diagnosis of depression, comprising measuring the level of a specific miRNA contained in exosomes in a biological sample collected from a subject, the specific miRNA being hsa-miR- From 106a-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-26b-5p, hsa-miR-15b-5p, hsa-miR-204-3p and hsa-miR-4497 at least one method selected from the group consisting of:
[2] The method described in [1], wherein the biological sample is a body fluid.
[3] The method according to [2], wherein the body fluid is blood, serum, or plasma.
[4] A kit for diagnosing depression, comprising a nucleic acid probe and/or a nucleic acid primer capable of specifically detecting hsa-miR-106a-5p, and a nucleic acid capable of specifically detecting hsa-miR-15a-5p. Probe and/or nucleic acid primer, nucleic acid probe and/or nucleic acid primer capable of specifically detecting hsa-miR-20a-5p, nucleic acid probe and/or nucleic acid primer capable of specifically detecting hsa-miR-26b-5p , a nucleic acid probe and/or a nucleic acid primer capable of specifically detecting hsa-miR-15b-5p, a nucleic acid probe and/or a nucleic acid primer capable of specifically detecting hsa-miR-204-3p, and hsa-miR- A kit comprising at least one nucleic acid probe and/or nucleic acid primer selected from the group consisting of nucleic acid probes and/or nucleic acid primers capable of specifically detecting 4497.
[5] A method for diagnosing and treating depression, comprising:
(1) Measuring the level of specific miRNA contained in exosomes in biological samples collected from subjects,
(2) If the level of the specific miRNA is below the detection limit or is low compared to the control value, the subject is suffering from depression or will suffer from depression in the future. (3) administering antidepressant therapy to a subject who is determined to be suffering from depression or likely to suffer from depression in the future;
The specific miRNAs include hsa-miR-106a-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-26b-5p, hsa-miR-15b-5p, hsa - at least one selected from the group consisting of miR-204-3p and hsa-miR-4497.

According to the present invention, a testing method that enables objective diagnosis of depression, and a kit for the same are provided.

1. Method for assisting diagnosis of depression The present invention provides a method for assisting diagnosis of depression (hereinafter sometimes referred to as "diagnosis aid method of the present invention"). Specifically, the method includes measuring the level of a specific miRNA contained in exosomes in a biological sample collected from a subject. The specific miRNAs are hsa-miR-106a-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-26b-5p, hsa-miR-15b-5p, hsa-miR- At least one selected from the group consisting of 204-3p and hsa-miR-4497. These seven miRNAs (hereinafter also referred to as "marker miRNAs of the present invention") are detected in exosomes collected from healthy individuals, but are not substantially detected in exosomes collected from depressed patients. Or the value is low compared to normal value. Here, "substantially not detected" means that it is below the detection limit at least at the microarray analysis level.

In the diagnostic assistance method of the present invention, "assisting the diagnosis of depression" refers to providing information that serves as an index for determining whether or not the subject is suffering from depression or is likely to suffer from depression in the future. It does not include the process itself of determining whether a person is suffering from depression or is likely to suffer from depression in the future, which is a medical procedure.

In the present invention, depression is a type of mood disorder, and is a mental disorder characterized by depressed mood, decreased motivation, interest, and mental activity, irritability, decreased appetite, insomnia, and persistent sadness and anxiety. In the American Psychiatric Association (APA)'s "Diagnostic and Statistical Manual of Mental Disorders," 5th edition (DSM-5), any depressive disorder that is classified as a "depressive disorder group" is considered "depression" in the present invention. included in.

In the diagnostic aid method of the present invention, the subject is not particularly limited as long as it is a human. Examples of subjects include humans who are suspected of suffering from depression.

In the diagnostic assistance method of the present invention, the biological sample is not particularly limited as long as it is obtained from a subject and contains exosomes, and examples thereof include body fluids, tissues, or cell culture supernatants. Examples of body fluids include blood, plasma, serum, cerebrospinal fluid, lymph fluid, urine, and saliva. Preferably, blood, plasma, serum, etc. are used. Examples of the tissue include brain tissue, and examples of the cells include cell populations obtained from the tissue. The biological sample is preferably a body fluid, since it is less invasive to the subject. When using tissue or cell culture supernatants, these tissues or cells can be cultured under appropriate conditions known per se. In a preferred embodiment, the tissue or cells are cultured in a medium containing serum (e.g., fetal bovine serum (FBS), etc.), and since the serum contains exosomes derived from the source animal, the tissues or cells are cultured in a medium containing serum (eg, fetal bovine serum (FBS), etc.). serum should be used. Serum from which exosomes have been removed can be prepared, for example, by passing serum through a filter having a pore size smaller than the particle size of exosomes and obtaining a passed liquid.

In the diagnostic aid method of the present invention, miRNA contained within exosomes is measured after isolation of exosomes from a biological sample. Exosomes can be obtained from biological samples by known methods. Examples of such methods include centrifugation, density gradient centrifugation, ultracentrifugation, polymer precipitation, separation using size exclusion chromatography, and separation using affinity. Alternatively, exosomes may be obtained from a biological sample by using a commercially available kit according to the attached protocol. Commercially available kits include, for example, ExoQuick (System Biosciences), EVSecond (GL Sciences), Total Exosome Isolation Kit (Invitrogen), and CD9 Exo-Flow capture kit (System Biosciences).

Alternatively, as shown in Examples below, exosomes can also be easily isolated by passing a biological sample through a filter with a pore size smaller than the particle size of exosomes (for example, a filter with a pore size of 50 nm). Since exosomes are captured without being able to pass through a filter whose pore size is smaller than the particle size of exosomes, exosomes captured by the filter can be obtained by backflowing a buffer solution or the like. In another preferred embodiment, exosomes can be obtained from a biological sample using two or more types of filters with different pore sizes. That is, biological samples are sequentially passed through a filter with a pore size larger than the exosome particle size (e.g., a filter with a pore size of 220 nm) and a filter with a pore size smaller than the exosome particle size (e.g., a filter with a pore size of 50 nm). Exosomes captured by a filter with a pore size smaller than the particle size of the exosomes can be obtained by backflowing a buffer solution or the like through the filter.

In the diagnostic aid method of the present invention, miRNAs to be measured include hsa-miR-106a-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-26b-5p, hsa-miR -15b-5p, hsa-miR-204-3p, and hsa-miR-4497.
The miRNA is already a known molecule, and its sequence information is available from databases such as miRBase (http://www.mirbase.org/). hsa-miR-106a-5p is Accession No. MIMAT0000103, hsa-miR-15a-5p is Accession No. MIMAT0000068, hsa-miR-20a-5p is Accession No. MIMAT0000075, hsa-miR-26b-5p is Registered in miRBase as Accession No. MIMAT0000083, hsa-miR-15b-5p as Accession No. MIMAT0000417, hsa-miR-204-3p as Accession No. MIMAT0022693, and hsa-miR-4497 as Accession No. MIMAT0019032. has been done. In this specification, the nucleotide sequence of hsa-miR-106a-5p is SEQ ID NO: 1, the nucleotide sequence of hsa-miR-15a-5p is SEQ ID NO: 2, the nucleotide sequence of hsa-miR-20a-5p is SEQ ID NO: 3, The nucleotide sequence of hsa-miR-26b-5p is SEQ ID NO: 4, the nucleotide sequence of hsa-miR-15b-5p is SEQ ID NO: 5, the nucleotide sequence of hsa-miR-204-3p is SEQ ID NO: 6, hsa-miR-4497 The nucleotide sequence of is represented by SEQ ID NO:7.

hsa-miR-106a-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-26b-5p, hsa-miR-15b-5p, hsa-miR-204-3p and hsa- Each miRNA of miR-4497 may contain mutations (nucleotide substitutions, deletions, insertions, or additions) due to naturally occurring polymorphisms, mutations, etc. Therefore, in the diagnostic aid method of the present invention, such The expression level of the miRNA containing the mutation may be measured.

The expression level of miRNA is determined by methods known per se such as hybridization and polymerase chain reaction (PCR) using nucleic acid probes or nucleic acid primers that can specifically detect each miRNA or its complementary nucleic acid (cRNA or cDNA). It can be measured. Examples of such measurement methods include miRNA arrays, Northern blotting, quantitative PCR (real-time PCR, etc.), and the like.

The expression level of miRNA is measured using miRNA derived from exosomes in the biological sample obtained from the subject. The method for extracting miRNA is not particularly limited as long as it is a method for extracting RNA containing miRNA (e.g., total RNA) from exosomes in a biological sample obtained from a subject. (Nippongene), QIAZOL (Qiagen)) and kits (e.g., NucleoSpin miRNA Plasma Kit (Macherey-Nagel), phenol-based reagent Tripure Isolation Reagent (Roche Applied Science), phenol-based reagent ISOGEN-LS (Nippon Gene) for liquid samples, miRNeasy RNA including miRNA can be extracted using a Mini Kit (Qiagen), RNeasy MinElute Cleanup Kit (Qiagen), etc. according to the attached protocol. Alternatively, total RNA including miRNA contained in exosomes may be extracted from a biological sample obtained from a subject. For example, total RNA including miRNA in exosomes can be extracted using commercially available reagents or kits such as Plasma/Serum Exosome Purification and RNA Isolation (Norgen Biotek Corp.).

Furthermore, after extracting total RNA including miRNA in exosomes from a biological sample obtained from a subject, miRNA may be further isolated. Isolation of miRNA can be performed by a method known per se, for example, using commercially available reagents or kits (e.g., RNeasy Mini Column (Qiagen), High Pure miRNA Isolation Kit (Roche Applied Science), etc.) according to the attached protocol. This can be implemented by using Here, "isolation of miRNA" means that an operation is performed to remove components other than miRNA.

When measuring the expression level of miRNA, it is preferable to correct the expression level of miRNA using an appropriate internal standard in order to correct for variations between samples due to the extraction and isolation of the miRNA. The internal standard is not particularly limited as long as it is naturally included in the sample derived from the subject. For example, U6 etc. can be used as an internal standard.

As used herein, the expression that a nucleic acid probe is "capable of specifically detecting miRNA or its complementary nucleic acid" means that the nucleic acid probe is capable of specifically detecting miRNA or its complementary nucleic acid; By having an affinity higher than , it is meant that under appropriate hybridization conditions it will hybridize to the miRNA or its complementary nucleic acid, but not to other nucleic acids.

Such hybridization conditions can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringency conditions. Low stringency conditions include, for example, 42°C, 5x SSC, 0.1% SDS, preferably 50°C, 2x SSC, 0.1% SDS in the washing after hybridization. More preferable hybridization conditions include highly stringent conditions. High stringency conditions are, for example, 65°C, 0.1×SSC, 0.1% SDS. However, there are multiple factors that affect the stringency of hybridization, such as temperature and salt concentration, and those skilled in the art can achieve similar stringency by appropriately selecting these factors. be.

Nucleic acid probes that can specifically detect miRNA or its complementary nucleic acid include, for example, about 10 or more bases (for example, 10 to 24 bases, preferably 15 to 24 bases) that are complementary to part or all of the nucleotide sequence of the miRNA. Examples include polynucleotides that include a continuous nucleotide sequence (24 bases) or its complementary sequence and are capable of hybridizing to the miRNA or its complementary nucleic acid. Such a nucleic acid probe can be appropriately designed by a method known per se based on the nucleotide sequence information described herein. Alternatively, commercially available nucleic acid probes can also be used. For example, miRNA consisting of each nucleotide sequence represented by SEQ ID NOs: 1 to 7 or its complementary nucleic acid are hsa-miR-106a-5p (Assay ID: 478225_mir) and hsa-miR- provided by ThermoFisher Scientific, respectively. 15a-5p (Assay ID: 477858_mir), hsa-miR-20a-5p (Assay ID: 000580), hsa-miR-26b-5p (Assay ID: 478418_mir), hsa-miR-15b-5p (Assay ID: 000390) ), hsa-miR-204-3p (Assay ID: 463101_mat) and hsa-miR-4497 (Assay ID: 479836_mir) can be detected using the nucleic acid probe included in the quantification kit.

As used herein, the phrase "a nucleic acid primer can specifically detect miRNA or its complementary nucleic acid" means that the nucleic acid primer is capable of detecting miRNA or its complementary nucleic acid using the miRNA or its complementary nucleic acid as a template under appropriate PCR reaction conditions. This means that all or part of the region is amplified by PCR, but all or part of the region is not PCR amplified using a nucleic acid other than the miRNA or its complementary nucleic acid as a template.

Nucleic acid primers that can specifically detect miRNA can be any primer designed to specifically amplify part or all of the nucleotide sequence of the miRNA or its complementary nucleic acid. Good too. Quantification of miRNA by PCR usually involves adding a poly(A) sequence to the 3' end using poly(A) polymerase, synthesizing cDNA by reverse transcription using an oligo(dT) primer, and then quantifying the cDNA. Do by doing. In this case, the polynucleotide ( forward primer) and a nucleotide sequence of about 10 bases or more (for example, 10 to 24 bases, preferably 15 to 24 bases) that hybridizes to a part of the complementary sequence of the cDNA sequence on the 3' side of this hybridization site. A part or all of the nucleotide sequence of each miRNA or its complementary nucleic acid can be specifically amplified by combination with a polynucleotide (reverse primer) containing the following. The forward primer and reverse primer can be appropriately designed by a method known per se based on information such as the nucleotide sequence of each miRNA or its complementary nucleic acid and the sequence of the primer used in the reverse transcription reaction. Alternatively, commercially available nucleic acid primers can also be used. For example, for specific amplification of miRNA complementary nucleic acids consisting of each nucleotide sequence represented by SEQ ID NOs: 1 to 7, hsa-miR-106a-5p (Assay ID: 478225_mir) provided by ThermoFisher Scientific is used. ), hsa-miR-15a-5p (Assay ID: 477858_mir), hsa-miR-20a-5p (Assay ID: 000580), hsa-miR-26b-5p (Assay ID: 478418_mir), hsa-miR-15b- Primer sets included in the 5p (Assay ID: 000390), hsa-miR-204-3p (Assay ID: 463101_mat), and hsa-miR-4497 (Assay ID: 479836_mir) quantification kits can be used.

Nucleic acid probes and nucleic acid primers may contain additional sequences (nucleotide sequences that are not complementary to the polynucleotide to be detected) as long as they do not interfere with specific detection.

In addition, the nucleic acid probe and the nucleic acid primer may contain appropriate labeling agents, such as radioisotopes (e.g., ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S, etc.), enzymes (e.g., β - Galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (e.g., fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (e.g., luminol, luminol derivatives, luciferin, lucigenin) etc.), may be labeled with biotin, etc. Alternatively, a quencher (quencher) that absorbs fluorescence energy emitted by the fluorescent substance may be further bonded near the fluorescent substance (eg, FAM, VIC, etc.). In such embodiments, the fluorescent substance and quencher are separated during the detection reaction and fluorescence is detected.

Nucleic acid probes and nucleic acid primers may be DNA, RNA, or a DNA/RNA chimera, but are preferably DNA. Nucleic acid probes and nucleic acid primers may be single-stranded or double-stranded, but are usually single-stranded. In the present invention, when the nucleic acid probe and the nucleic acid primer are RNA, thymine (T) in the base sequences of the nucleic acid probe and the nucleic acid primer may be replaced with uracil (U).

Nucleic acid probes and nucleic acid primers may be nucleic acid derivatives with artificial modifications. Examples of artificially modified nucleic acid derivatives include those modified with a phosphate backbone such as phosphorothioate type and boranophosphate type DNA/RNA; 2'-OMe modified RNA, 2'-F modified RNA, etc. 2' modified nucleotide; Modified nucleotide that bridges the sugar molecules of nucleotides such as LNA (Locked Nucleic Acid) and ENA (2'-O,4'-C-Ethylene-bridged nucleic acids); PNA (peptide nucleic acid), morpholino Examples include, but are not limited to, modified nucleotides having different basic skeletons such as nucleotides; base-modified nucleotides such as 5-fluorouridine and 5-propyluridine.

The above-mentioned nucleic acid probe and nucleic acid primer can be synthesized according to a conventional method using an automatic DNA/RNA synthesizer, for example, based on the nucleotide sequence information described in this specification.

Nucleic acid probes capable of specifically detecting miRNA to be measured or its complementary nucleic acid may be bound onto a suitable support to provide a nucleic acid array. The support is not particularly limited as long as it is a support commonly used in the field, and examples thereof include membranes (eg, nylon membranes), beads, glass, plastics, metals, and the like.

As described above, the marker miRNA of the present invention is detected in exosomes derived from healthy individuals, but is substantially not detected in exosomes derived from depressed patients, or has a lower value compared to normal values. Therefore, when using the above nucleic acid array as a means for measuring miRNA, if the marker miRNA of the present invention is not detected in exosomes in a biological sample collected from a subject, or if the level is lower than the normal value, the subject It can be determined that the person is suffering from depression or is likely to suffer from depression in the future. For example, when using microarray analysis as a means of measuring miRNA, exosomes derived from depressed patients contain hsa-miR-106a-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, and hsa-miR -26b-5p is usually not detected. Therefore, if these marker miRNAs are below the detection limit in exosomes in a biological sample collected from a subject, it can be determined that the subject is suffering from depression or is likely to suffer from depression in the future. Alternatively, in the exosomes in the biological sample collected from the subject, the level of the marker miRNA of the present invention is equal to or higher than a preset control value (cutoff value), for example, in a sufficient number (e.g., 5, 10, 20, 30 If the level of the miRNA in exosomes obtained from a group of healthy subjects (40 or 50 or more) is greater than or equal to the mean value + 3SD, preferably greater than or equal to the mean value + 5SD, the subject is suffering from or will be diagnosed with depression. It can be determined that there is a high possibility of contracting the disease. In a preferred embodiment, if the level of the marker miRNA of the present invention is less than 1/3 of the minimum level of the miRNA in exosomes obtained from the blood of a sufficient number of healthy subjects, the subject It can be determined that the person is suffering from depression or is likely to suffer from depression in the future. Even when using a method with high detection sensitivity such as quantitative real-time PCR as a means of measuring miRNA, the level of the marker miRNA of the present invention in exosomes in a biological sample collected from a subject is determined by a preset control value ( For example, the average level of the miRNA in exosomes obtained from a sufficient number (e.g., 5, 10, 20, 30, 40, or 50 or more) of healthy subjects, preferably 3SD or more. Subjects were diagnosed with depression if the miRNA level was greater than the mean value + 5 SD or less than 1/3 of the minimum level of the miRNA in exosomes obtained from the blood of a sufficient number of healthy subjects. It can be determined that the patient has the disease or is likely to develop the disease in the future.

2. Methods for diagnosing and treating depression The present invention also provides methods for diagnosing and treating depression. As mentioned above, antidepressant therapy is administered to a subject who is determined to be suffering from depression or to be likely to suffer from depression in the future based on the information obtained by the diagnostic aid method of the present invention. Can be done. Examples of antidepressant therapy include rest, psychotherapy (eg, cognitive therapy, interpersonal therapy, etc.), drug therapy, and the like. Antidepressants are not particularly limited as long as they have therapeutic and/or preventive effects on depression, but examples include serotonin reuptake inhibitors (SSRIs) (e.g., fluvoxamine, paroxetine, sertraline, escitalopram, etc.); Serotonin/noradrenaline reuptake inhibitors (SNRIs) (e.g., duloxetine, milnacipran, etc.), noradrenergic/specific serotonergic antidepressants (NaSSAs) (e.g., mirtazapine, etc.), tricyclics/tetracyclics Antidepressants (eg, amoxapine, nortopriptyline, clomipramine, maprotiline, mianserin, etc.), etc. Two or more antidepressants can also be used together. The administration method and dosage of these antidepressants can follow those commonly used clinically.

3. Kit for diagnosis of depression The present invention provides hsa-miR-106a-5p, hsa-miR-15a-5p, hsa-miR-20a-5p, hsa-miR-26b-5p, hsa-miR-15b- A depression diagnostic kit comprising a nucleic acid probe and/or nucleic acid primer capable of specifically detecting at least one miRNA selected from the group consisting of 5p, hsa-miR-204-3p and hsa-miR-4497 provide. Hereinafter, it may be referred to as "the kit of the present invention". Using the kit of the present invention, it becomes possible to easily carry out the diagnostic aid method of the present invention.

In the kit of the present invention, depression is as described in "1. Method for assisting diagnosis of depression."

In the kit of the present invention, the nucleic acid probe and nucleic acid primer are as described in "1. Method for assisting diagnosis of depression." The nucleic acid probe and nucleic acid primer contained in the kit of the present invention are capable of specifically detecting hsa-miR-106a-5p, and the nucleic acid probe and/or nucleic acid primer are capable of specifically detecting hsa-miR-15a-5p. Nucleic acid probe and/or nucleic acid primer, nucleic acid probe and/or nucleic acid primer capable of specifically detecting hsa-miR-20a-5p, nucleic acid probe and/or nucleic acid capable of specifically detecting hsa-miR-26b-5p Primer, nucleic acid probe and/or nucleic acid primer capable of specifically detecting hsa-miR-15b-5p, nucleic acid probe and/or nucleic acid primer capable of specifically detecting hsa-miR-204-3p, and hsa-miR At least one nucleic acid probe and/or nucleic acid primer selected from the group consisting of nucleic acid probes and/or nucleic acid primers capable of specifically detecting -4497.

The kit of the present invention may be provided as a nucleic acid array by binding nucleic acid probes capable of specifically detecting each miRNA to be measured or its complementary nucleic acid onto a suitable support. The support is not particularly limited as long as it is a support commonly used in the field, and examples thereof include membranes (eg, nylon membranes), beads, glass, plastics, metals, and the like.

Each component included in the kit of the present invention is prepared separately (or in a mixed state if possible) at an appropriate concentration in water or an appropriate buffer (e.g. TE buffer, PBS, etc.). It is placed in a suitable container either in a dissolved or lyophilized state.

Depending on the method for measuring the miRNA expression level, the kit of the present invention may further contain other components necessary for carrying out the method. The kit of the present invention can assist in the diagnosis of depression by, for example, measuring miRNA expression levels using miRNA arrays, Northern blotting, quantitative PCR (real-time PCR, etc.), and the like. When using real-time PCR for measurement, the kit of the present invention further contains 10x PCR reaction buffer, 10x _MgCl2 aqueous solution, 10x dNTPs aqueous solution, Taq DNA polymerase (5 U/μL), reverse transcriptase, etc. can be included. When miRNA array or Northern blotting is used for measurement, the test reagent of the present invention can further contain a blotting buffer, a labeling reagent, a blotting membrane, and the like.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

(Materials and methods)
Blood was collected from 13 depressed patients (7 males, 6 females) and 13 healthy subjects (all males), separated into serum using a standard method, and 500 μL was filtered through a 50 nm syringe filter (SFPES013022N, Membrane Solutions Ltd, USA) to isolate exosomes. Next, physiological saline was passed through the syringe filter to wash the exosomes.

Exosomes captured with a 50 nm filter were eluted by 1 mL of ISOGEN (Nippongene, Tokyo, Japan). The eluate was mixed with 200 μL of chloroform and centrifuged (12,000×g, 15 min). Total RNA was purified by NucleoSpin miRNA Plasma Kit (Macherey-Nagel, Germany) according to the manufacturer's protocol. After eluting the entire amount of RNA with 100 μL of H ₂ O, RNA was precipitated with 10 μL of 3.0 M potassium acetate, 100 μL of isopropanol, and 1 μL of Pellet Paint. After washing and drying the pellet, the RNA was dissolved in 2 μL of H ₂ O for use in microarrays. MicroRNA purification was verified using Eukaryote Total RNA Nano Series II on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA).

Microarray analysis was performed using purified microRNA. Microarray analysis on Human miRNA Oligo chip-4 plex (TORAY, Tokyo, Japan) containing 2,565 probe sets was performed as follows by 3D-Gene array system (TORAY, Tokyo, Japan) according to the manufacturer's protocol. . For microRNA array, 2 μL of RNA solution was hybridized with Human miRNA Oligo chip. The obtained values (raw data) were subjected to global normalization (corrected so that the median value of the values obtained by subtracting the background from the raw data was 25).

(result)
A summary of the results of the microarray analysis is shown in Table 1.

(result)
(1) Four types of microRNAs (see below) were found that met the following conditions.
Condition: all healthy people have a value, all depressed patients have no value・hsa-miR-106a-5p
・hsa-miR-15a-5p
・hsa miR-20a-5p
・hsa-miR-26b-5p
(2) Three types of microRNAs (see below) that meet the following conditions were discovered.
Condition 1: Value exists for all healthy people (no condition for depressed patients)
Condition 2: "Average value for healthy people"/"Average value for depressed patients" is greater than 10
Condition 3: “Minimum value J for healthy people/maximum value J for depressed patients” is greater than 3
・hsa miR-15b-5p
・hsa-miR-204-3p
・hsa-miR-4497
(3) There was no microRNA that met the following conditions.
When "healthy person" and "depressed person" in (1) above are swapped. When "healthy person" and "depressed patient" in (2) above are swapped.

The above results showed that it is possible to diagnose depression by quantifying the above seven microRNAs in serum exosomes.

According to the present invention, by measuring the level of a specific miRNA in a biological sample containing exosomes obtained from a subject, it is possible to determine whether the subject is suffering from depression or is likely to suffer from depression in the future. This can be easily and objectively determined. In addition, it may be possible to diagnose patients with depression, who are difficult to diagnose using conventional methods, or pre-depressive patients at an early stage, which is extremely useful.

Claims (2)

A method for assisting in the diagnosis of depression, the method comprising measuring the level of a specific miRNA contained in exosomes in blood, serum or plasma collected from a subject, the specific miRNA being h sa-miR. -15a-5p and hsa-miR- 204-3p . A kit for diagnosing depression , comprising a nucleic acid probe and/or a nucleic acid primer capable of specifically detecting hsa -miR-15a-5p, and a nucleic acid probe capable of specifically detecting hsa-miR-204-3p. A kit comprising at least one nucleic acid probe and/or nucleic acid primer selected from the group consisting of: and/or nucleic acid primer.