A tsRNA biomarker, a kit and its application for diagnosing depression

Abstract

The present invention relates to the field of biotechnology, and specifically discloses a tsRNA biomarker, a kit and an application thereof for diagnosing depression. The marker includes a tsRNA numbered tiRNA-Gly-GCC-001; the number is the number of RNA in the MINTbase database, and the nucleotide sequence of the tiRNA-Gly-GCC-001 is shown in SQEIDNO.1 in the sequence table. The present invention uses tsRNA as a biomarker for diagnosing depression and a biomarker for predicting the efficacy of antidepressant drugs, which has high stability, good specificity and sensitivity, and high diagnostic reference value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a tsRNA biomarker, kit, and application for the diagnosis of depression. Background Technology

[0002] The core symptoms of depression are depressed mood, loss of interest, and lack of energy. Globally, over 350 million people suffer from depression. It is estimated that by 2030, depression will account for the largest share of disability-related lost healthy life years worldwide. Depression is characterized by high prevalence and high relapse rates, and can even lead to serious consequences such as suicide and self-harm. Depression places enormous economic and psychological burdens on patients, families, and society. Currently, the diagnosis of depression relies primarily on the patient's subjective description. Clinicians' subjective assessments of symptoms and diagnoses based on questionnaires are inherently subjective, and the lack of objective and effective clinical biomarkers easily leads to misdiagnosis and missed diagnosis, resulting in prolonged illness and unnecessary economic and psychological burdens on patients. Therefore, clinicians need to identify biomarkers to help them make early diagnoses of depression.

[0003] Non-coding RNA (ncRNA) is RNA that does not encode proteins. Based on length, it is divided into long non-coding RNA (>200 bp) and short non-coding RNA (<200 bp). Broadly speaking, it includes mRNA, tRNA, rRNA, small interfering RNA, miRNA, and long non-coding RNAs and circular RNAs with unclear functions. In recent years, small RNAs derived from tRNA (tRNA-derived small RNAs, tsRNAs) have attracted widespread attention. They mainly fall into two types: tRNA-derived stress-induced RNA (tiRNA) and tRNA-derived fragments (tRFs). They are generated based on their cleavage site in the precursor or mature tRNA transcript. These tRF and tiRNA molecules exhibit heterogeneity in nucleotide composition, biogenesis, and function. Recent studies have found that tsRNAs are not merely fragments of tRNA degradation but also play regulatory roles in many specific physiological and pathological processes. Many scholars have studied the differential expression of miRNAs in patients with depression, suggesting that miRNAs may participate in the development and progression of depression through various mechanisms, such as neuroplasticity, neuroendocrine factors, psychosocial factors, and antidepressants. Furthermore, miRNA-26b, miRNA-1972, miRNA-4485, miRNA-4498, and miRNA-4743 are considered potential biomarkers for depression. Studies using lncRNA microarrays have revealed aberrant expression of multiple lncRNAs in patients with depression, and the expression of six lncRNAs in peripheral blood could serve as potential biomarkers for the diagnosis and treatment response of clinical MDD. Subsequent research found that the circular RNA hsa_circRNA_103636 is downregulated in patients with depression, and its expression increases after 8 weeks of antidepressant treatment, suggesting that hsa_circRNA_103636 could serve as an important diagnostic and therapeutic biomarker for depression. Further investigation revealed upregulation of hsa_circRNA_003251, hsa_circRNA_015115, hsa_circRNA_100918, and hsa_circRNA_001520 in patients with type 2 diabetes and depression. These circRNAs may be involved in the signaling pathways of thyroid hormones, Wnt, ErbB, and mitogen-activated protein kinase, thus contributing to the occurrence and development of depression. This study elucidates the pathogenesis of depression in type 2 diabetes and suggests potential new targets for clinical diagnosis and treatment. However, the aforementioned biomarkers for depression all suffer from low stability, limiting their clinical application.

[0004] tsRNAs play a crucial role in human mental illnesses by regulating gene expression and silencing, modulating cell proliferation and apoptosis, and regulating translation. The tissue-specific, highly abundant, stable, and widespread nature of tsRNAs makes them a promising biomarker for the diagnosis of depression with high sensitivity, specificity, and stability. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the current situation where the stability of biomarkers used for the diagnosis of depression is not high, and to provide a tsRNA with high stability, specificity and sensitivity as a biomarker for the diagnosis of depression.

[0006] To achieve the objectives of this invention, the technical solution is as follows: a tsRNA biomarker for the diagnosis of depression, the biomarker comprising tsRNA numbered tiRNA-Gly-GCC-001; wherein tiRNA-Gly-GCC-001 is the RNA number in the MINTbase database, and the nucleotide sequence of tiRNA-Gly-GCC-001 is shown in SEQ ID NO.1 of the sequence listing.

[0007] Compared with existing technologies, this invention uses tsRNA designated tiRNA-Gly-GCC-001 as a biomarker for diagnosing depression and predicting the efficacy of antidepressants. It exhibits high stability, good specificity and sensitivity, and high diagnostic reference value. The diagnosis of depression will no longer rely on subjective experience but can be confirmed through objective indicators such as tsRNA expression levels, improving diagnostic accuracy, simplifying the diagnostic process, and facilitating early prediction. By detecting the expression level of the specific tsRNA designated tiRNA-Gly-GCC-001, the efficacy of antidepressants and the prognosis of patients with depression can be predicted.

[0008] A kit containing primers and probes for detecting tsRNA designated tiRNA-Gly-GCC-001, wherein the primer sequences for tiRNA-Gly-GCC-001 are shown in SEQ ID NO.2 and SEQ ID NO.3, and the kit is capable of determining the expression level of tsRNA designated tiRNA-Gly-GCC-001 in a test sample. The kit can be used to detect the expression level of tsRNA designated tiRNA-Gly-GCC-001 in patient blood, plasma, or serum.

[0009] Furthermore, the test sample is selected from at least one of human blood, plasma, or serum. Since tsRNA is widely present in human blood, plasma, and serum, using human blood, plasma, and serum as test samples can effectively detect the expression level of tiRNA-Gly-GCC-001.

[0010] Furthermore, the kit contains a detection reagent, which is coupled with or carries a detectable marker. By coupling or carrying a detection marker in the detection reagent, it is convenient to observe the detection results through changes in the detection marker.

[0011] Furthermore, the detectable marker is selected from one of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes. Using one of these markers makes the detection results easier to observe.

[0012] Furthermore, the application of the tsRNA biomarker in the diagnosis of depression. Using this tsRNA biomarker in the diagnosis of depression can improve the stability of diagnostic results.

[0013] This invention also provides the application of the kit in the diagnosis of depression or the evaluation of the efficacy of antidepressants. When applied to the diagnosis of depression, the expression level of tsRNA designated tiRNA-Gly-GCC-001 can be used to determine whether a patient has depression; when applied to the evaluation of the efficacy of antidepressants, the expression level of tsRNA designated tiRNA-Gly-GCC-001 can be used to assess the efficacy of the antidepressant.

[0014] Furthermore, the application of the kit in evaluating the efficacy of antidepressants specifically includes the following steps:

[0015] S1. The concentration of tsRNA with the code tiRNA-Gly-GCC-001 in the blood, plasma or serum of normal subjects was determined using the kit described above, and recorded as the control reference value C0.

[0016] S2. The concentration of tsRNA with the code tiRNA-Gly-GCC-001 in the blood, plasma or serum of the test subject is determined using the kit described above, and recorded as the marker expression level C1.

[0017] S3. Determine whether the subject suffers from depression based on the C1 / C0 ratio. When the expression level of the marker C1 is close to the control reference value C0, it indicates that the antidepressant drug has a significant therapeutic effect.

[0018] The above methods can be used to assess whether antidepressants are effective for patients and whether their depression has been cured.

[0019] In summary, the tiRNA-Gly-GCC-001 provided by this invention, as a standard substance, reagent kit, and application for the diagnosis of depression, will facilitate the early detection of depression in clinical practice, reduce misdiagnosis and missed diagnosis, increase patient treatment compliance, and reduce the economic burden and mental stress on patients and society. Attached Figure Description

[0020] Figure 1 Heatmap of gene expression screening between depression case group and healthy control group;

[0021] Figure 2 A scatter plot showing gene expression screening between a case group of depression and a healthy control group;

[0022] Figure 3 Volcano plot for gene expression screening between depression case group and healthy control group;

[0023] Figure 4 The graph shows the differential expression of tiRNA-Gly-GCC-001 between the depression case group and the healthy control group, as confirmed by RT-PCR analysis.

[0024] Figure 5 The expression of tiRNA-Gly-GCC-001 is shown in the figure for case groups with different degrees of depression.

[0025] Figure 6 The expression diagram of tiRNA-Gly-GCC-001 after 8 weeks of antidepressant treatment;

[0026] Figure 7 The graph shows the changes in the expression of tiRNA-Gly-GCC-001 in the samples after being placed at room temperature for 4h, 8h, and 24h.

[0027] Figure 8 The graph shows the changes in the expression of tiRNA-Gly-GCC-001 after 2, 4, and 6 freeze-thaw cycles.

[0028] Figure 9 ROC curve for the diagnosis of depression using tiRNA-Gly-GCC-001. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Mullis KB et al., Polymerase Chain Reaction (Nature. 1986 Nov 13-19; 324(6093): 163-6.), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0030] Example 1

[0031] This embodiment provides a tsRNA biomarker for the diagnosis of depression. The biomarker includes tsRNA numbered tiRNA-Gly-GCC-001; the RNA number is from the MINTbase database, and the nucleotide sequence of tiRNA-Gly-GCC-001 is shown in SEQ ID NO.1 of the sequence listing.

[0032] Compared with existing technologies, this invention uses tsRNA (tiRNA-Gly-GCC-001) as a biomarker for diagnosing depression and predicting the efficacy of antidepressants, exhibiting high stability, specificity, and sensitivity, and possessing significant diagnostic value. The diagnosis of depression will no longer rely on subjective experience but can be confirmed through objective indicators such as tsRNA expression levels, improving diagnostic accuracy, simplifying the diagnostic process, and facilitating early prediction. Detecting the expression level of a specific tsRNA (tiRNA-Gly-GCC-001) can predict the efficacy of antidepressants and the prognosis of patients with depression. Furthermore, altering the expression level of a specific tsRNA through tsRNA mimics and antagonists (modified oligonucleotides) can normalize abnormal gene regulatory networks and signaling pathways, thereby enabling targeted treatment of specific symptoms of depression.

[0033] Example 2

[0034] This embodiment provides a diagnostic kit for depression. The kit can detect tsRNA numbered tiRNA-Gly-GCC-001 in blood, plasma, or serum. Any one of the blood, plasma, or serum samples can be used as the test sample. The tsRNA numbered tiRNA-Gly-GCC-001 is a risk standard, and its number is the RNA number from the MiNTbase database. The blood, plasma, or serum is derived from human samples. The diagnostic reagent in the kit is coupled with or carries a detectable marker, which is selected from one of the following: chromophore, chemiluminescent group, fluorophore, isotope, or enzyme. The diagnostic kit contains primers and probes for detecting tsRNA numbered tiRNA-Gly-GCC-001. The primer sequence for tiRNA-Gly-GCC-001 is shown in SEQ ID NO. 2 and SEQ ID NO. 2. As shown in NO.3, the diagnostic kit also includes routine PCR amplification detection components, which include Taq enzyme, reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, standards, and controls. The diagnostic kit also includes auxiliary PCR amplification detection components, which include negative controls, positive controls, a real-time PCR reaction plate, and a PCR reaction plate sealing film.

[0035] Example 3

[0036] This embodiment provides a kit for evaluating the efficacy of antidepressant drugs. The kit can detect tsRNA numbered tiRNA-Gly-GCC-001 in blood, plasma, or serum; the RNA number is the same as the one in the MiNTbase database; the evaluation includes early diagnosis, auxiliary diagnosis, and their combination; the kit for evaluating the efficacy of antidepressant drugs contains primers and probes for detecting tsRNA numbered tiRNA-Gly-GCC-001, the forward primer of tiRNA-Gly-GCC-001 is shown in SEQ ID NO.2, and the reverse primer of tiRNA-Gly-GCC-001 is shown in SEQ ID NO.3; the evaluation kit also includes routine PCR amplification detection components, including Taq enzyme, reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, standards, and controls; the evaluation kit also includes auxiliary PCR amplification detection components, including negative controls, positive controls, a real-time PCR reaction plate, and a PCR reaction plate sealing film.

[0037] This embodiment also provides a method for evaluating the efficacy of antidepressants using the kit provided in this embodiment, including the following steps:

[0038] S1. The concentration of tsRNA with the code tiRNA-Gly-GCC-001 in the blood, plasma or serum of normal subjects was determined using a kit and recorded as the control reference value C0.

[0039] S2. The concentration of tsRNA with the code tiRNA-Gly-GCC-001 in the blood, plasma or serum of the test subjects was determined using a kit and recorded as the marker expression level C1.

[0040] S3. Determine whether the subject suffers from depression based on the C1 / C0 ratio. When the expression level of the marker C1 is close to the control reference value C0, it indicates that the antidepressant drug has a significant therapeutic effect.

[0041] Application examples

[0042] I. Research Subjects

[0043] This study was approved by the Medical Ethics Committee of Ningbo Kangning Hospital, and all subjects or their family members (guardians) signed informed consent forms.

[0044] Case group: The case group consisted of 90 patients who met the diagnostic criteria for depression and were treated at the outpatient and inpatient departments of the Department of Psychiatry at Ningbo Kangning Hospital from January 2019 to December 2021. All patients met the following inclusion criteria: 1. Meeting the diagnostic criteria for depressive episode in the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-V); 2. A Hamilton Scale (24-item version) score of ≥7 by two attending physicians; 3. Age 18-60 years; 4. Right-handed; 5. No use of any antidepressants or antipsychotics in the past 3 months. Exclusion criteria were: 1. History of severe organic disease or drug-induced affective, mental or personality disorders; history of alcohol or other substance abuse; 2. Pregnant or lactating women; 3. Those who have undergone nonconvulsive electroconvulsive therapy (MECT) within the past 6 months or have used antipsychotic drugs for a long time; those who need to use drugs that may affect mood for a long time; those who need to use drugs with central nervous system effects for a long time in the near future; and those who are allergic to the investigational drugs used (mirtazapine, sertraline, escitalopram).

[0045] Control group: The healthy control group consisted of 60 healthy volunteers who underwent physical examinations during the same period. Volunteers were aged between 20 and 60 years and matched for age and sex with the case group. They underwent a structured clinical interview for DSM-V, nonpatient edition (SCID-I / NP) to confirm they did not have depression or other mental illnesses, and had no history of mental illness in themselves or their extended families. They also had no history of serious neurological diseases or other serious physical illnesses, severe head injuries, or severe stress events within the past 3 months. They were right-handed and had not been ill or taking any medications within one month prior to blood collection.

[0046] Before conducting the research, the participating psychiatrists and graduate students were trained. The Hamilton Depression Rating Scale (HAMD), Gross General Rating Scale (GAS), and Clinical Overall Evaluation Scale (CGI) were used for assessment before case enrollment, 4 weeks after drug treatment, and 8 weeks after drug treatment. Two attending psychiatrists made diagnoses based on the DSM-IV-TR; in case of discrepancies, a chief psychiatrist was consulted to finalize the diagnosis.

[0047] General information (name, gender, age, education level, occupation, income level, marital status, history of drug addiction, and family history of mental illness, etc.) was recorded upon enrollment. Similarly, general information of the normal control group was recorded.

[0048] II. Research Methods

[0049] ① Use of scales

[0050] The Hamilton Depression Rating Scale (HAMD), developed by psychologist Hamilton in 1960, is currently the most widely used scale in clinical assessment of depressive states. This study used the 24-item Chinese version, divided into 7 factors: anxiety / somatization, weight, cognitive symptoms, diurnal variation, retardation, sleep disturbance, and helplessness. Its reliability and validity have been validated.

[0051] The General Rating Scale (GAS) is a scale used to evaluate symptoms of various mental illnesses and is one of the most widely used scales of its kind. This study used the 1976 Spitzer version from the National Institute of Mental Health (NIMH), which has a 10-point scale (1-10) and is assessed based on the patient's clinical symptoms. A higher total score indicates a milder condition. This study used the Chinese version of the GAS scale for evaluation, and this version has been tested for reliability and validity and meets psychometric standards.

[0052] The Clinical Evaluation and Treatment Scale (CGI): Designed by the WHO for the International Pilot Survey of Mental Illness (IPSS), the CGI is now used to assess clinical efficacy and is applicable to any psychiatric treatment and research subjects. The scale consists of three subscales: Severity Ofillness (SI), Global Improvement (GI), and Efficacy Index (EI). This study used the Chinese version of the CGI scale for assessment; this version has undergone reliability and validity testing and meets psychometric standards. Statistical analysis in this study used the Severity Ofillness (SI) and Global Improvement (GI) scores.

[0053] ② Data collection

[0054] General information (name, gender, age, education level, occupation, income level, marital status, history of drug addiction, and family history of mental illness) was recorded upon enrollment. Twenty-three attending psychiatrists or physicians assessed the case group participants using the Hamilton Depression Rating Scale (HAMD) and the Clinical Overall Improvement Scale (CGI) before and at least six weeks after medication administration. Prior to assessment, all participating researchers received standardized training and adhered to standardized methodologies to ensure the standardized administration process. HAMD scores were calculated using total and factor scores, while CGI scores were calculated using the global improvement (GI) score.

[0055] ③ Drug intervention

[0056] Ninety patients underwent clinical drug intervention and follow-up observation. Among them, 25 patients received mirtazapine (dose range 7.5-45 mg, starting dose 7.5 mg, mean dose 30 mg) in combination with escitalopram (dose range 20-40 mg, starting dose 20 mg, mean dose 30 mg), 20 patients received sertraline (dose range 50-150 mg, starting dose 50 mg, mean dose 100 mg) daily in combination with mirtazapine, 22 patients received fluvoxamine (dose range 50-150 mg, starting dose 50 mg, mean dose 100 mg) in combination with mirtazapine, 12 patients received venlafaxine (dose range 75-150 mg, starting dose 75 mg, mean dose 125 mg), and 11 patients received fluoxetine (dose range 20-40 mg, starting dose 20 mg, mean dose 30 mg), as shown in Table 2.

[0057] Table 2. Types and dosages (mg) of antidepressants in 90 patients.

[0058] Antidepressants Starting dose Dosage range Average dose mirtazapine 7.5 7.5～45 30 Escitrapram 20 20～40 30 Sertraline 50 50～150 100 Fluvoxamine 50 50～150 100 Venlafaxine 75 75-150 125 Fluoxetine 20 20-40 30

[0059] ④ Main reagents and consumables

[0060] LowInputQuick-AmpLabelingKit, one-color (24*) (Agilent, catalog number: 5190-2305); GeneExpressionWashPack (Agilent, 5188-5327); GeneExpression HybridizationKit (5188-4242); RNASpikeInKit, one-color (5188-5327); Real-time PCR primers: CustomPlusTQMNRNAAssays (ABI, USA); Real-time PCR reagents: TaqMan Universal Mixed Kit II (ABI, USA, catalog number: 4440047); Reverse transcription kit: QuantiTectRev.Transcription Kit (QIAGEN, catalog number: 205311).

[0061] ⑤ Main instruments

[0062] Scanner (Affymetrix, Scanner 3000); Hybridization Oven (Affymetrix, Hybridization Oven 640); Washing Workstation (Affymetrix, Fluidics Station 450); 2100 (Agilent, G2939A); 2100 Shaker (Agilent, 9600); NanoDrop (Thermo, 2000); PCR Instrument (ABI, 9700); Centrifuge (Eppendorf, 5418); Concentrator (Eppendorf, 5301); Centrifuge (Chelline Bell, LX-200); Centrifuge-1 (Chelline Bell, LX-300); Shaker-1 (Chelline Bell, GL-88B); Magnetic Stirrer (Chelline Bell, GL-3250B); Metal Bath-2 (Borgward, HB-100); Metal Bath-3 (Bori CHB-100); Electric thermostatic incubator (Jinghong Experimental Equipment, XMTD-8222); Refrigerator (Rongshida, BCD-265F); ABI 9700 PCR instrument (ABI Corporation, USA); Tianmei CT14RD benchtop high-speed refrigerated centrifuge (Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.); NanoDrop1000 ultra-micro UV spectrophotometer (with computer, Thermo Scientific, USA); ABI 7900HT FastRead-Time PCR System (ABI Corporation, USA); WH-2 miniature vortex mixer (Shanghai Huxi Analytical Instrument Factory Co., Ltd.); JS-400A thermostatic metal bath (Shanghai Peiqing Technology Co., Ltd.); DK-8D digital display thermostatic water bath (Jintan Medical Instrument Factory); SW-CJ-IFD single-person single-sided clean bench (Sujing Purification Equipment Co., Ltd.); -80℃ ultra-low temperature freezer (Haier Corporation).

[0063] ⑥ tsRNA sequencing screening

[0064] Using the Kangcheng Biotechnology tRF & tiRNA seq platform, ten samples from five patients with severe depression and five healthy controls were analyzed. Total RNA was quantified using a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was checked using an Agilent Bioanalyzer 2100 (Agilent Technologies). Sequencing analysis was performed after the RNA passed quality control.

[0065] ⑦ Total RNA extraction and quantitative PCR

[0066] Ten differentially expressed tsRNAs from the sequencing screening results were subsequently validated by qRT-PCR. All subjects had 5 ml of venous blood collected in EDTA anticoagulant tubes. After collection, the tubes were gently shaken to mix the anticoagulant with the blood. RNA extraction was performed on all blood samples within 2 hours of collection. Total RNA (including tsRNA) was extracted from serum using the TRIzol (USA) method, following the kit instructions. Reverse transcription was performed according to the instructions of the RNA reverse transcription kit (TaqMan RNA Reverse Transcription Kit, ABI, USA). The total reaction volume was 15 μL (5 μL total RNA, 3 μL TaqMan MicroRNA Assay, 4.16 μL nuclease-free water, 0.19 μL RNase inhibitor, 1.5 μL buffer, 1 μL Multiscribe reverse transcriptase, and 0.15 μL dNTPs). Reactions were performed at different temperatures (16℃, 42℃, 85℃, 4℃) for different durations (30 mins, 30 mins, 5 mins, 10 mins). Real-time quantitative PCR was performed according to the TaqMan kit (TaqMan Universal Mix Kit II, ABI, USA). The total volume of the PCR amplification system was 10 μL, and the reaction was repeated for 40 cycles. The Ct value of the PCR reaction was measured using a 7900 real-time quantitative PCR instrument (ABI, USA), and each reaction was repeated three times. Data were read and analyzed using SDS 2.4 and DataAssist v3.0 software, and β-Actin was selected as an internal control for data standardization.

[0067] ⑧ Statistical processing

[0068] All data were statistically analyzed using SPSS v19.0 software. Independent samples t-tests and Fisher's exact probabilities in a 2×2 table were used to detect significant differences in age and sex between the two groups. Two-class difference genetic screening (2DF) was performed after correction using a random variance model (RVM) to identify differentially expressed tsRNAs. The ΔCT value was calculated as the difference in threshold cycle (Ct) between tsRNAs and the internal control β-Actin. The Wilcoxon rank sum test was used to compare the expression differences of tsRNAs between the depression group and the control group. Nonparametric tests were used to analyze the expression changes of the screened tiRNA-Gly-GCC-001 before treatment, at 4 weeks of treatment, and at 8 weeks of treatment, as well as the differences between the depression group and the control group in terms of sex and age.

[0069] III. Research Results

[0070] 1. Sequencing results analysis revealed that both the 5 patients in the depression group and the 5 healthy controls showed two major trends in tsRNA expression after sequencing analysis, indicating a possible differential expression of tsRNAs between the depression group and the control group. A p-value < 0.05 was considered statistically significant, and the false negative rate (FDR) was used as the corrected p-value. RVMt-test analysis identified 37 differentially expressed tsRNAs, of which 34 were upregulated and 3 were downregulated. Figure 1 As shown, log2(fold change) is plotted on the x-axis, representing a 2-fold increase or decrease in gene regulation; the negative logarithm of the p-value in the t-test is used to assess significance. 10 The p-value is plotted on the ordinate; therefore, the black area represents gene expression with statistically significant differences. Results are shown below. Figure 1 , Figure 2 and Figure 3 .

[0071] 2. RT-qPCR was used to validate the sequencing analysis results. Based on the 37 tsRNAs screened by sequencing, we validated the abnormally expressed tiRNA-Gly-GCC-001 in depression. Results are shown below. Figure 4 .

[0072] 3. A total of 90 patients in the depression group and 60 healthy controls were submitted for testing. Based on the HAMD-17 scale scores, the patients were divided into mild, moderate, and severe depression. The results are as follows: Figure 5 As shown, the expression level of tiRNA-Gly-GCC-001 increases with the severity of depression.

[0073] 4. After 8 weeks of antidepressant treatment, the expression of tiRNA-Gly-GCC-001 decreased in enrolled patients with depression, along with a decrease in HAMD scores and a reduction in depressive symptoms. The results are shown below. Figure 6 .

[0074] 5. After being incubated at room temperature for 4 hours, 8 hours, and 24 hours, the expression of tiRNA-Gly-GCC-001 showed no significant change. (See attached figures.) Figure 7 .

[0075] 6. The stability of tiRNA-Gly-GCC-001 showed no significant change after 2, 4, and 6 freeze-thaw cycles. (See results below.) Figure 8 .

[0076] 7. ROC curve analysis suggested the diagnostic value of differentially expressed tiRNA-Gly-GCC-001, validated by RT-PCR, in depression. The AUC of tiRNA-Gly-GCC-001 was 0.84 (95% CI: 0.65-0.96), with a sensitivity of 0.80 and a specificity of 0.93. (See attached data.) Figure 9 .

[0077] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention. sequence list <110> Ningbo University <120> A tsRNA biomarker, kit, and application for the diagnosis of depression. <130> P2024-0602 <140> 202210206281.0 <141> 2022-03-01 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 33 <212> RNA <213> Homo sapiens <400> 1 gcaugggugg uucaguggua gaauucucgc cug 33 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 gtcctgccca ctgggata 18 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <400> 3 tgtcctcttc cgatctatc 19

Claims

1. A use of a tsRNA biomarker or a detection reagent thereof for diagnosing depression, characterized in that: Used for preparing a kit for diagnosing depression or evaluating the efficacy of antidepressant drugs; Wherein, the marker includes a tsRNA numbered as tiRNA-Gly-GCC-001, and the nucleotide sequence of the tiRNA-Gly-GCC-001 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The kit contains primers and probes for detecting tsRNA numbered tiRNA-Gly-GCC-001, the primer sequences of the tiRNA-Gly-GCC-001 are shown in SEQ ID NO.2 and SEQ ID NO.3, and the kit can measure the expression level of tsRNA numbered tiRNA-Gly-GCC-001 in the sample to be tested.

3. The use according to claim 2, characterized in that The sample to be tested is selected from at least one of human blood, plasma or serum.

4. The use according to claim 1, characterized in that The kit contains a detection reagent, which is coupled with or carries a detection label.

5. The use according to claim 4, characterized in that The detection label is selected from a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

6. The use according to claim 1, characterized in that The kit is also used to distinguish between mild depression, moderate depression and severe depression.

7. The use according to claim 1, characterized in that The kit also includes conventional components for PCR amplification detection, which include Taq enzyme, reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, standard substances and control substances.

8. The use according to claim 1, characterized in that The kit includes the following steps for evaluating the efficacy of antidepressant drugs: S1. Using the kit to measure the concentration of tsRNA numbered tiRNA-Gly-GCC-001 in the blood, plasma or serum of normal subjects, recorded as a control reference value C0; S2. Using the kit to measure the concentration of tsRNA numbered tiRNA-Gly-GCC-001 in the blood, plasma or serum of the test subject, and recording it as the marker expression amount C1; S3. Determine whether the test subject suffers from depression according to the ratio of C1 / C0. When the marker expression amount C1 is close to the control reference value C0, it indicates that antidepressant drugs have a significant therapeutic effect.

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