Salivary biomarkers for depression

Abstract

To provide depression biomarkers in saliva.SOLUTION: The present invention is based on finding that metabolites and micro RNAs in saliva can be used as biomarkers for depression, and finding that they can be applied to a method of determining and assessing the condition and severity of depression using the biomarkers, a program for determining depression, a test kit for determining depression, and a method of screening and evaluating therapeutic agents for depression.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a salivary biomarker for depression, a method for determining and evaluating the condition and severity of depression using this biomarker, a program for determining depression, a test kit for determining depression, and a method for screening and evaluating therapeutic drugs for depression. [Background technology]

[0002] To live a healthy life, it is necessary to maintain both a healthy body and a healthy mind. However, in recent years, factors such as increased stress have led to a decline in mental health and an increase in the number of people suffering from depression, which has become a social problem.

[0003] To date, there have been searches for candidate biomarkers that indicate the severity of depression through metabolomic analysis (Patent Document 1), searches for microRNAs that are candidate biomarkers for depression (Patent Document 2), and searches for candidate blood biomarkers for diagnosing depression (Patent Document 3), and it has been disclosed that tryptophan or its degradation products are candidate biomarkers for depression (Patent Document 4). However, all of these disclosures concern biomarkers in blood or cerebrospinal fluid.

[0004] It has been reported that the search for biomarkers using saliva samples through metabolome analysis, miRNAseq (microRNA sequencing analysis), flora analysis, etc. may lead to the diagnosis of oral diseases, various cancers, Alzheimer's disease, and lifestyle-related diseases, but there have been no reports to date of saliva-based biomarkers for depression. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Re-tabled publication 2017 / 082103 [Patent Document 2] Re-tabled publication 2016 / 117582 [Patent Document 3] Re-tabled publication No. 2011 / 019072 [Patent Document 4] Special Publication No. 2008-537111 [Non-patent literature]

[0006] [Non-Patent Document 1] Behav Brain Res., 2014;270:339-48 [Non-patent document 2] J Vis Exp., 2015;105:52973 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a salivary biomarker for depression, a method for determining and evaluating the condition and severity of depression using this biomarker, a program for determining depression, a test kit for determining depression, and a method for screening and evaluating therapeutic drugs for depression. [Means for solving the problem]

[0008] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that certain metabolites and certain microRNAs are increased or decreased in saliva samples from subjects with depression compared to saliva samples from control subjects, and thus came up with the present invention.

[0009] That is, the present invention is as follows. [1] Use of one or more metabolites selected from the group consisting of the metabolites listed in Tables 1 and 2, and / or one or more microRNAs selected from the group consisting of the microRNAs listed in Tables 3 and 4, or human microRNAs corresponding to these microRNAs, in a saliva sample collected from a test subject as biomarkers for diagnosing depression. [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2] [2] A method for determining and / or assessing the condition or severity of depression in a subject, comprising: Measuring the amount of the biomarker defined in [1] in a saliva sample collected from the subject; comparing the amount of biomarker from the test subject with the amount of biomarker in a saliva sample taken from a control subject; and determining and / or assessing that the subject has depression and / or is severely depressed when the amount of the biomarker derived from the subject is significantly different from the amount of the biomarker derived from the control subject; A method comprising: [3] When the biomarker is one listed in Table 1, by CE-FTMS analysis The method according to [2], wherein the nucleotides are measured by LC-TOFMS analysis when they are listed in Table 2, and / or by next-generation sequencing analysis when they are listed in Tables 3 to 4. [4] A program for determining and / or assessing the condition or severity of depression in a subject, A program for causing a computer to execute the following steps (1) and (2): (1) A procedure for calculating whether there is a significant difference between the measurement data derived from the test subject and the measurement data derived from the control subject, based on input measurement data of the amount of the biomarker defined in [1] in the saliva sample collected from the test subject and input measurement data of the amount of the biomarker in the saliva sample collected from the control subject; (2) A procedure for displaying a judgment and / or evaluation that the subject has depression and / or is severely depressed when there is a significant difference between the two measurement data as a result of the calculation. [5] A kit for determining and / or assessing the condition or severity of depression in a subject, comprising a reagent for measuring the amount of the biomarker defined in [1] in a saliva sample collected from the subject; The reagent is a nucleic acid probe and / or nucleic acid primer that specifically detects at least one microRNA listed in Tables 3 to 4 or a human microRNA corresponding to such a microRNA. Includes a kit. [6] A method for screening and / or evaluating a therapeutic agent for depression, comprising: Measuring the amount of the biomarker defined in [1] in a saliva sample collected from a subject with depression who has been administered a candidate therapeutic agent; comparing the amount of the biomarker in a subject with depression after administration of the candidate therapeutic agent with the amount of the biomarker in a subject with depression who is not administered the candidate therapeutic agent, or comparing the amount of the biomarker in a subject with depression after administration of the candidate therapeutic agent with the amount of the biomarker in the subject with depression before administration of the candidate therapeutic agent; and a step of evaluating the candidate substance as effective as a therapeutic agent for depression when the amount of the biomarker in a subject with depression after administration of the candidate substance is significantly different from the amount of the biomarker in a subject with depression who has not been administered the therapeutic agent candidate substance, or when the amount of the biomarker in a subject with depression after administration of the candidate substance is significantly different from the amount of the biomarker in the subject with depression before administration of the therapeutic agent candidate substance; A screening method and / or evaluation method comprising: [Effects of the Invention]

[0010] The present invention has discovered that metabolites and microRNAs in saliva can be used as biomarkers for depression, and has also made it possible to provide a method for determining and evaluating the condition and severity of depression using these biomarkers, a program for determining depression, a test kit for determining depression, and a method for screening and evaluating therapeutic drugs for depression. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Biomarkers for diagnosing depression> One embodiment of the present invention is the use of one or more metabolites selected from the group consisting of the metabolites listed in Tables 1 and 2, and / or one or more microRNAs selected from the group consisting of the microRNAs listed in Tables 3 and 4, or human microRNAs corresponding to these microRNAs, in a saliva sample collected from a test subject as biomarkers for diagnosing depression.

[0012] The biomarkers of the present invention are contained in saliva samples. Saliva samples can be collected non-invasively from test subjects, which has the advantage of reducing the physical and / or mental burden on the test subjects. The saliva sample may be collected at any time of day. The method for collecting saliva is not particularly limited and can be any method known to those skilled in the art. If the biomarker fluctuates throughout the day, collecting the saliva sample at a specific time period can enable a more accurate diagnosis.

[0013] After collection from the subject, the saliva sample may be optionally pretreated, frozen, or freshly prepared. Pretreatment may include, for example, preparation so that the concentration of an internal standard in the saliva sample is constant among individual samples, or ultrafiltration. When the biomarker is a microRNA, the microRNA can be isolated from a sample by a method known to those skilled in the art. For example, the microRNA can be isolated using a commercially available reagent or kit (e.g., miRNeasy Serum / Plasma Kit (Qiagen)) according to the protocol provided with the kit.

[0014] The method for measuring each biomarker is not particularly limited as long as it can measure the biomarker, but when the biomarker is a metabolite, it can be measured by, for example, metabolome analysis. When the biomarker is a metabolite listed in Table 1, it can be measured by CE-FTMS (capillary electrophoresis-Fourier transform mass spectrometry) analysis, and when it is a metabolite listed in Table 2, it can be measured by LC-TOFMS (liquid chromatography-time-of-flight mass spectrometry) analysis. Furthermore, when the biomarker is a microRNA, it may be measured by, for example, next-generation sequencing, microarray, or quantitative PCR. Any of these measurement methods can be performed by methods known to those skilled in the art. For example, when the biomarker is a microRNA listed in Tables 3 and 4, it can be measured by next-generation sequencing.

[0015] The subject is a subject who may develop depression, and is not particularly limited as long as the biomarker of the present invention can be used to diagnose depression. The subject is typically a human, but may be any mammal, such as pet animals such as dogs and cats, livestock animals such as cows, horses, and pigs, or laboratory animals such as mice and rats. When the biomarker is a microRNA, the microRNA in each mammal corresponding to the microRNA listed in Tables 3 and 4 can be used.

[0016] All metabolites in the present invention are disclosed in databases such as PubChem and HMDB (The Human Metabolome Database), and all microRNAs are disclosed in databases such as miRBase and GenBank.

[0017] In the present invention, "depression" refers to, for example, major depressive disorder Major depressive disorder is a type of mood and anxiety disorder characterized by depressed and anxious behavior, and decreased motivation. It is distinct from bipolar disorder and schizophrenia.

[0018] Depression may be diagnosed by comparing the amount of a biomarker in a saliva sample collected from a test subject with the amount of a biomarker in a saliva sample collected from a control subject, and determining and / or assessing that the test subject has depression and / or is severely depressed if there is a significant difference between the amount of the biomarker derived from the test subject and the amount of the biomarker derived from the control subject. Whether the amount of a biomarker in a sample is significantly increased or significantly decreased depends on the biomarker being measured. Control subjects are subjects without depression. The significance test can be performed using a test known to those skilled in the art, for example, Welch's t-test or Student's t-test. There is no particular limitation on the significance of a difference, and it may be, for example, a p-value of less than 0.05, a p-value of less than 0.01, or a p-value of less than 0.001. Although depression can be diagnosed based on the measurement results of only one biomarker, a more accurate diagnosis and / or evaluation of depression can be achieved by combining the measurement results of multiple biomarkers. The multiple biomarkers may be two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, twenty or more, thirty or more, thirty or more, for example, fifty or less, forty or less, thirty or less, or twenty or less. When multiple biomarkers are used, a risk score may be calculated based on the measurement results of the multiple biomarkers, and a diagnosis may be made based on the risk score.

[0019] Furthermore, depression can be diagnosed by setting the values ​​of these biomarkers in a saliva sample from a healthy mammal (e.g., a healthy individual) of the same species as the subject as reference values ​​in advance, and by determining whether the amount of the biomarkers in the saliva sample collected from the subject exceeds or falls below the reference value.

[0020] <Method for determining and / or assessing the condition or severity of depression> Another embodiment of the present invention is a method for determining and / or assessing the state or severity of depression in a subject, comprising: measuring the amount of any one or more biomarkers described in the above section <Biomarkers for diagnosing depression> in a saliva sample collected from the subject; comparing the amount of biomarker from the test subject with the amount of biomarker in a saliva sample taken from a control subject; and determining and / or assessing that the subject has depression and / or is severely depressed when the amount of the biomarker derived from the subject is significantly different from the amount of the biomarker derived from the control subject; The method includes:

[0021] The descriptions in the above section <Biomarkers for diagnosing depression> can be used to refer to each description in the method for determining and / or assessing the condition or severity of depression.

[0022] <Program for determining and / or assessing the condition or severity of depression> Another embodiment of the present invention is a program for determining and / or assessing the condition or severity of depression in a subject, comprising: A program for causing a computer to execute the following steps (1) and (2): (1) A step of calculating whether there is a significant difference between the measurement data derived from the test subject and the measurement data derived from the control subject, based on input measurement data of the amount of any one or more biomarkers described in the above section <Biomarkers for diagnosing depression> in a saliva sample collected from the test subject and input measurement data of the amount of biomarkers in a saliva sample collected from a control subject; (2) A procedure for displaying a judgment and / or evaluation that the subject has depression and / or is severely depressed when there is a significant difference between the two measurement data as a result of the calculation.

[0023] The program may further include a procedure for inputting measurement data.

[0024] This program can be implemented in a computer by a method known to those skilled in the art, for example, via a computer-readable recording medium on which the above-described program is recorded.

[0025] The descriptions in the above section <Biomarkers for diagnosing depression> can be used as reference for each description in the program for determining and / or assessing the condition or severity of depression.

[0026] <Kit for determining and / or assessing the condition or severity of depression> Another embodiment of the present invention is a kit for determining and / or assessing the condition or severity of depression in a subject, comprising a reagent for measuring the amount of any one or more biomarkers described above in the section <Biomarkers for diagnosing depression> in a saliva sample collected from the subject, The reagent is a nucleic acid probe and / or nucleic acid primer that specifically detects at least one microRNA listed in Tables 3 to 6 or a human microRNA corresponding to such a microRNA. The kit includes:

[0027] Examples of nucleic acid probes capable of specifically detecting microRNA or its complementary nucleic acid include polynucleotides that contain a contiguous nucleotide sequence of 10 or more bases (e.g., 10 to 24 bases, preferably 15 to 24 bases) that is complementary to part or all of the nucleotide sequence of the microRNA, or its complementary sequence, and that can hybridize to the microRNA or its complementary nucleic acid. Such nucleic acid probes can be appropriately designed by known methods based on the microRNA information described herein, or commercially available nucleic acid probes can be used. The nucleic acid primer for specifically detecting a microRNA may have any sequence, as long as it is designed to specifically amplify a partial or entire region of the nucleotide sequence of the microRNA or its complementary nucleic acid.

[0028] The nucleic acid probe and / or nucleic acid primer may be labeled with any labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, biotin, or the like.

[0029] The kit of this embodiment may further include, as appropriate, reagents for extracting RNA, internal standard reagents, quantitative PCR reagents, microarray membranes, and the like, depending on the method for measuring microRNA.

[0030] The descriptions in the above section <Biomarkers for diagnosing depression> can be used to describe each aspect of the kit for determining and / or assessing the condition or severity of depression.

[0031] <Method for screening and / or evaluating therapeutic drugs for depression> Another embodiment of the present invention is a method for screening and / or evaluating a therapeutic agent for depression, comprising the steps of: Measuring the amount of any one or more biomarkers described in the above section <Biomarkers for diagnosing depression> in a saliva sample collected from a subject with depression who has been administered a candidate therapeutic drug; comparing the amount of the biomarker in a subject with depression after administration of the candidate therapeutic agent with the amount of the biomarker in a subject with depression who has not been administered the candidate therapeutic agent, or comparing the amount of the biomarker in a subject with depression after administration of the candidate therapeutic agent with the amount of the biomarker in the subject with depression before administration of the candidate therapeutic agent. , and a step of evaluating the candidate substance as effective as a therapeutic agent for depression when the amount of the biomarker in a subject with depression after administration of the candidate substance is significantly different from the amount of the biomarker in a subject with depression who has not been administered the therapeutic agent candidate, or when the amount of the biomarker in a subject with depression after administration of the candidate substance is significantly different from the amount of the biomarker in the subject with depression before administration of the therapeutic agent candidate. The screening method and / or evaluation method includes:

[0032] The specific procedure is as follows, for example. Subjects with depression are divided into two groups, one of which is administered a candidate therapeutic substance, and the other of which is not. Saliva samples are collected from subjects in the group administered with the candidate therapeutic substance and from the group not administered with the candidate therapeutic substance, and the amounts of the biomarkers related to depression are compared between the two groups. If the results of the comparison show that the measured values ​​of the group administered with the candidate therapeutic substance are significantly different from the amounts of the biomarkers in subjects with depression who are not administered with the candidate therapeutic substance, but are not significantly different from the measured values ​​of healthy subjects without depression, the candidate therapeutic substance used can be selected as a candidate therapeutic substance for depression. Furthermore, saliva samples are collected from subjects with depression before and after administration of a candidate therapeutic agent, and the amount of the depression-related biomarker in the saliva sample after administration of the candidate therapeutic agent is compared with the amount of the biomarker in the saliva sample before administration of the candidate therapeutic agent. If the comparison results in a significant difference in the amount of the biomarker in the saliva sample after administration of the candidate therapeutic agent compared with the amount of the biomarker in the saliva sample before administration of the candidate therapeutic agent, but no significant difference compared with the measured value in a healthy group without depression, the candidate therapeutic agent used can be selected as a candidate therapeutic agent for depression. The test substance can be administered to the subject orally, intravenously, or topically, but it is preferable to add the test substance to the feed or drinking water.

[0033] The descriptions in the above section <Biomarkers for diagnosing depression> can be used to describe the methods for screening and / or evaluating therapeutic agents for depression. [Example]

[0034] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0035] <Creation of social defeat stress model mice (depression model mice)> C57BL / 6J (B6) strain mice (6-week-old, male, CLEA Japan, Inc.) were housed under a 12-hour light / 12-hour dark cycle (lights on at 7:00 AM, lights off at 7:00 PM) for 2 weeks (acclimation). AIN-93G (Oriental Yeast Co., Ltd.) diet was used, and the mice were allowed to feed ad libitum throughout the experiment. After acclimation, B6 mice were subjected to social defeat stress for 10 days (Day 1 to Day 10) using ICR strain (ICR) mice (retired, male, Japan SLC Co., Ltd.) as previously reported (Non-Patent Documents 1 and 2), to obtain social defeat stress model mice. This social defeat stress model mouse is a representative animal model of depression used worldwide.

[0036] <Metabolomic analysis of saliva using depression model mice> Salivary metabolome analysis was performed using the depression model mice (6 mice) prepared by the above method. On the day after the stress exposure period (Day 11), a social behavior test was conducted to evaluate the social behavior of B6 mice in relation to ICR. On day 12, pilocarpine hydrochloride (15 mg / kg, Fujifilm Wako Pure Chemical Corporation) was administered intraperitoneally, and saliva overflowing from the oral cavity was collected. The collected saliva samples were frozen and stored at -80°C until metabolomic analysis. Metabolomic analysis was performed at Human Metabolome Technologies, Inc. (HMT) using CE-FTMS (capillary electrophoresis-Fourier transform mass spectrometry) and LC-TOFMS (liquid chromatography-time-of-flight mass spectrometry).

[0037] [CE-FTMS analysis] <Measurement> Saliva samples from six depression model mice and six control mice were each subjected to measurement using CE-FTMS in cation mode and anion mode, a method known to those skilled in the art. To prepare the saliva samples, 40 μL of saliva sample was mixed with 10 μL of an aqueous solution containing the internal standard at a concentration of 100 μM, stirred, and transferred to an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5 kDa), centrifuged (9,100 × g, 4°C, 60 minutes), ultrafiltered, and then used for measurement. For the measurement in the cation mode, the saliva sample was diluted two-fold, and for the measurement in the anion mode, the saliva sample was diluted five-fold. For saliva samples from each mouse, measurements were performed in cation mode and anion mode under the conditions shown below, and the obtained data were integrated. As a result, a total of 401 peaks (242 cations and 159 anions) were detected. Cationic metabolites (cation mode) Device CE: Agilent CE system MS: Q Exactive Plus No. 2 Capillary: Fused silica capillary id 50 μm × 80 cm Measurement conditions Run buffer: Cation Buffer Solution (p / n : H3301-1001) Rinse buffer: Cation Buffer Solution (p / n : H3301-1001) Sample injection: Pressure injection 50 mbar, 10 seconds CE voltage: Positive, 30 kV MS ionization: ESI Positive MS capillary voltage: 4,000 V MS scan range: m / z 60-900 Sheath liquid: HMT Sheath Liquid (p / n : I3301-1040) Anionic metabolites (anion mode) Device CE: Agilent CE system MS: Q Exactive Plus No. 2 Capillary: Fused silica capillary id 50 μm × 80 cm Measurement conditions Run buffer: Anion Buffer Solution (p / n : H3302-1023) Rinse buffer: Anion Buffer Solution (p / n : H3302-1023) Sample injection: Pressure injection 50 mbar, 22 seconds CE voltage: Positive, 30 kV MS ionization: ESI Negative MS capillary voltage: 3,500 V MS scan range: m / z 70-1,050 Sheath liquid: HMT Sheath Liquid (p / n : I3301-1040)

[0038] <Data processing> Peaks detected by CE-FTMS were automatically extracted using the automatic integration software MasterHands ver. 2.18.0.1 (developed by Keio University) for peaks with a signal-to-noise (S / N) ratio of 3 or greater, and the mass-to-charge ratio (m / z), peak area, and migration time (MT) were obtained. The peak area values ​​obtained were converted to relative area values ​​using the following formula:

number

[0039] <Search for candidate metabolites> The detected peaks were compared and searched against all substances registered in the HMT's metabolic library based on their m / z and MT values. The search tolerance was ±0.5 min for MT and ±5 ppm for m / z. Based on the m / z and MT values ​​of the substances registered in the library, a candidate compound was assigned to peak 401 (cation 242, anion 159).

number

[0040] <Quantitation of target metabolic compounds> Some of the target metabolic compounds were analyzed. The calibration curve was created using peak areas corrected for the internal standard, and the concentration of each compound was calculated using a 10 μM calibration point (internal standard 20 μM). The quantitative results are shown in Table 5. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] NA: Not Available. Although the item is subject to calculation, calculation was not possible due to insufficient data. ¶ The ratio of the detection mean values ​​between the two groups was calculated using the latter as the denominator. || Welch's t-test p-value and its range (*<0.05, **<0.01, ***<0.001)

[0041] <Intergroup comparison> For the 401 peaks from which candidate compounds were narrowed down, the relative area ratios of each group were calculated and a Welch t-test was performed. The results showed that the concentrations of five metabolites (CE-FTMS) were significantly altered by depression (Table 6). The p-values ​​of the Welch t-test are shown (*<0.05, **<0.01, ***<0.001). [Table 6]

[0042] [LC-TOFMS analysis] <Measurement> Saliva samples from six depression model mice and six control mice were each subjected to measurement in positive and negative modes by LC-TOFMS, a method known to those skilled in the art. To prepare the saliva samples, 80 μL of the sample was mixed with 240 μL of methanol solution containing the internal standard at a concentration of 4 μM. The mixture was then centrifuged at 2,300 × g, 4°C, for 5 minutes, and the supernatant was collected. The supernatant was then dried and dissolved in 160 μL of 50% isopropanol (v / v) for further analysis. For saliva samples from each mouse, measurements were performed in both positive and negative modes under the conditions shown below, and the data obtained were integrated. As a result, a total of 66 peaks (40 positive, 26 negative) were detected. Cationic metabolites (positive mode) Device LC system: Agilent 1200 series RRLC system SL (Agilent Technologies) Column: ODS column, 2×50 mm, 2 μm MS system: Agilent LC / MSD TOF (Agilent Technologies) No. 10 Measurement conditions Column temperature: 40℃ Mobile phase A: H2O / 0.1% HCOOH Mobile phase B: Isopropanol: Acetonitrile: H2O (65:30:5) / 0.1% HCOOH, 2 mM HCOONH4 Flow rate: 0.3 mL / min Run time: 20 min Post time: 7.5 min Gradient condition: 0-0.5 min: B 1%, 0.5-13.5 min: B 1-100%, 13.5-20 min: B 100% MS ionization mode: ESI Positive MS Nebulizer pressure: 40 psi MS dry gas flow: 10 L / min MS dry gas temperature: 350℃ MS capillary voltage: 4,000 V MS scan range: m / z 100-1,700 Sample injection: 1 μL Anionic metabolites (negative mode) Device LC system: Agilent 1200 series RRLC system SL (Agilent Technologies) Column: ODS column, 2×50 mm, 2 μm MS system: Agilent LC / MSD TOF (Agilent Technologies) Model 10 Measurement conditions Column temp.: 40°C Mobile phase A: H2O / 0.1% HCOOH Mobile phase B: Isopropanol: Acetonitrile: H2O (65:30:5) / 0.1% HCOOH, 2 mM HCOONH4 Flow rate: 0.3 mL / min Run time: 20 min Post time: 7.5 min Gradient condition: 0 - 0.5 min: B 1%, 0.5 - 13.5 min: B 1 - 100%, 13.5 - 20 min: B 100% MS ionization mode: ESI Negative MS Nebulizer pressure: 40 psi MS dry gas flow: 10 L / min MS dry gas temp: 350°C MS capillary voltage: 3,500 V MS scan range: m / z 100 - 1,700 Sample injection: 1 μL

[0043] <Data processing> Peaks detected by LC-TOFMS were automatically extracted using the automatic integration software MasterHands ver. 2.18.0.1 (developed by Keio University) for peaks with a signal-to-noise (S / N) ratio of 3 or greater, and mass-to-charge ratios (m / z), peak areas, and retention times (RT) were obtained. The peak areas were converted to relative area values ​​using the following formula. Furthermore, the concentration of each substance could be calculated using these relative area values.

number

[0044] <Search for candidate metabolites> The detected peaks were compared and searched against all substances registered in HMT's metabolic library based on their m / z and RT values. The search tolerances were ±0.3 min for RT and ±25 ppm for m / z. Based on the m / z and RT values ​​of the substances registered in the library, 66 peaks (40 positive, 26 negative) were assigned candidate compounds.

number

[0045] <Intergroup comparison> For the 66 peaks narrowed down to candidate compounds, the relative area ratios of each group were calculated and Welch's t-test was performed. The results showed that the concentrations of four metabolites (LC-TOFMS) were significantly altered by depression (Table 7). The p-values ​​of the Welch's t-test are shown (*<0.05, **<0.01, ***<0.001). [Table 7]

[0046] <Saliva microRNA analysis using depression model mice> Saliva microRNA analysis was performed on each of the depression model mice (6 mice) created by the above method. On the day after the stress exposure period (Day 11), a social behavior test was conducted to evaluate B6's social behavior in response to the ICR, and depressive-like behavior was observed. On Day 12, pilocarpine hydrochloride (15 mg / kg, Fujifilm Wako Pure Chemical Corporation) was administered intraperitoneally, and saliva overflowing from the oral cavity was collected. The collected saliva samples were frozen at -80°C until metabolomic analysis. RNA extraction was performed using the miRNeasy Serum / Plasma Kit (Qiagen) according to the protocol attached to the kit. MicroRNA sequence analysis was performed by DNA Chip Research Institute, Inc. using methods known to those skilled in the art. Saliva samples from each mouse were analyzed separately, and the resulting data were combined.

[0047] The results of the microRNA analysis showed that depression increases some microRNAs in saliva, while others decrease in saliva. These are shown in Tables 8 and 9 below.

[0048] Table 8 shows the microRNAs that increase in saliva. [Table 8-1] [Table 8-2] [Table 8-3]

[0049] Table 9 shows the microRNAs that are decreased in saliva.

Table 9-1

Table 9-2

Claims

1. In a saliva sample collected from a test subject, (1) miRNA-208b-3p (Accession ID: MIMAT0004939), (2) A human microRNA corresponding to the microRNA miRNA-208b-3p (Accession ID: MIMAT0004939), (3) miRNA-7092-5p (Accession ID: MIMAT0028090), and (4) miRNA-6985-3p (Accession ID: MIMAT0027873) of one or more microRNAs selected from the group consisting of: Use as a biomarker to aid in the diagnosis of depression, when the (1) miRNA-208b-3p (Accession ID: MIMAT0004939), the (3) miRNA-7092-5p (Accession ID: MIMAT0028090), or the (4) miRNA-6985-3p (Accession ID: MIMAT0027873) is used as a biomarker, the subject is a mouse; (2) Use in which, when a human microRNA corresponding to the microRNA miRNA-208b-3p (Accession ID: MIMAT0004939) is used as a biomarker, the subject is a human.

2. 1. A method for assisting in determining and / or assessing the condition or severity of depression in a subject, comprising: Measuring the amount of a biomarker as defined in claim 1 in a saliva sample taken from the subject; comparing the amount of biomarker from the test subject with the amount of biomarker in a saliva sample taken from a control subject; and determining and / or assessing that the subject has depression and / or is severely depressed when the amount of the biomarker derived from the subject is significantly increased compared to the amount of the biomarker derived from the control subject; Including, when the (1) miRNA-208b-3p (Accession ID: MIMAT0004939), the (3) miRNA-7092-5p (Accession ID: MIMAT0028090), or the (4) miRNA-6985-3p (Accession ID: MIMAT0027873) is used as a biomarker, the subject is a mouse; The method (2) above, wherein the subject is a human when a human microRNA corresponding to the microRNA miRNA-208b-3p (Accession ID: MIMAT0004939) is used as a biomarker.

3. The method of claim 2, wherein the biomarker is measured by next-generation sequencing analysis.

4. A program for determining and / or assessing the condition or severity of depression in a subject, comprising: Follow the steps (1) and (2): (1) A procedure for calculating whether there is a significant difference between the measurement data derived from the test subject and the measurement data derived from the control subject, based on input measurement data of the amount of the biomarker defined in claim 1 in a saliva sample collected from the test subject and input measurement data of the amount of the biomarker in a saliva sample collected from a control subject; and (2) A procedure for displaying a judgment and / or evaluation that the subject has depression and / or is severely depressed when the measurement data derived from the subject significantly increases compared to the measurement data derived from the control subject as a result of the calculation. It is a program for making a computer execute the when the (1) miRNA-208b-3p (Accession ID: MIMAT0004939), the (3) miRNA-7092-5p (Accession ID: MIMAT0028090), or the (4) miRNA-6985-3p (Accession ID: MIMAT0027873) is used as a biomarker, the subject is a mouse; The program (2) wherein the subject is a human when a human microRNA corresponding to the microRNA miRNA-208b-3p (Accession ID: MIMAT0004939) is used as a biomarker.

5. A kit for determining and / or assessing the condition or severity of depression in a subject, comprising a reagent for measuring the amount of a biomarker defined in claim 1 in a saliva sample collected from the subject, The reagent is a nucleic acid probe and / or a nucleic acid primer that specifically detects the biomarker defined in claim 1. Including, when the (1) miRNA-208b-3p (Accession ID: MIMAT0004939), the (3) miRNA-7092-5p (Accession ID: MIMAT0028090), or the (4) miRNA-6985-3p (Accession ID: MIMAT0027873) is used as a biomarker, the subject is a mouse; (2) The kit, wherein when a human microRNA corresponding to the microRNA miRNA-208b-3p (Accession ID: MIMAT0004939) is used as a biomarker, the subject is a human.

6. A method for screening and / or evaluating therapeutic agents for depression, comprising: Measuring the amount of a biomarker defined in claim 1 in a saliva sample collected from a subject with depression who has been administered a candidate therapeutic agent; comparing the amount of the biomarker in a subject with depression after administration of the candidate therapeutic agent with the amount of the biomarker in a subject with depression who has not been administered the candidate therapeutic agent, or comparing the amount of the biomarker in a subject with depression after administration of the candidate therapeutic agent with the amount of the biomarker in the subject with depression before administration of the candidate therapeutic agent. , and a step of evaluating the candidate substance as effective as a therapeutic agent for depression when the amount of the biomarker in a subject with depression after administration of the candidate substance is significantly reduced compared to the amount of the biomarker in a subject with depression not administered the therapeutic agent candidate substance, or when the amount of the biomarker in a subject with depression after administration of the candidate substance is significantly reduced compared to the amount of the biomarker in the subject with depression before administration of the therapeutic agent candidate substance; Including, when the (1) miRNA-208b-3p (Accession ID: MIMAT0004939), the (3) miRNA-7092-5p (Accession ID: MIMAT0028090), or the (4) miRNA-6985-3p (Accession ID: MIMAT0027873) is used as a biomarker, the subject is a mouse; (2) The screening method and / or evaluation method, wherein the subject is a human, when a human microRNA corresponding to the microRNA miRNA-208b-3p (Accession ID: MIMAT0004939) is used as a biomarker.

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