Application of circRNA-103546 as diagnostic marker in preparation of depression detection product

Abstract

The invention discloses an application of circRNA-103546 as a diagnostic marker in preparation of a depression detection product, and relates to the technical field of biomedicine, and the key points of the technical scheme are as follows: the circRNA-103546 detection kit comprises a determination assembly capable of detecting the level of circRNA-103546 in plasma, according to the assembly, differential expression between a depression patient and a healthy individual can be confirmed through a quantitative reverse transcription polymerase chain reaction method, and the nucleotide sequence of circRNA-103546 is as shown in SEQ ID NO. 1. The circRNA-103546 in the plasma provided by the invention is applied to the depression detection product and an antidepressant drug in the preparation of the depression detection product, the depression is detected and treated based on the circRNA-103546 in the plasma, and the depression detection product can effectively improve the accuracy of depression detection, is high in detection efficiency and is easy to popularize and use in a large scale.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically, to the use of circRNA-103546 as a diagnostic marker in the preparation of depression detection products. Background Art

[0002] Depression is a mental illness with persistent low mood as its main symptom. Its etiology is complex and its clinical manifestations are diverse, which has a serious impact on social economy and people's quality of life. According to the World Health Organization, by 2030, depression will become the disease with the highest disability rate and disease burden in the world. At present, the diagnosis of depression mainly relies on clinical manifestations and scales, and lacks objective experimental diagnostic indicators. In the human genome, a large number of DNA sequences are transcribed into RNA, most of which are non-coding RNA (ncRNA), including circRNA. CircRNA is a special type of ncRNA. They exist in a circular structure and do not encode proteins, but play an important role in regulating gene expression and miRNA sponge effect. Studies have shown that specific circRNAs may play a key role in diseases such as depression. Their circular structure allows them to exist stably in cells and peripheral blood. There are obvious differences in expression profiles between patients with depression and healthy people. This differential expression helps to distinguish patients with depression from healthy individuals. In addition, the detection of circRNA is convenient because they can be detected in peripheral samples such as blood, avoiding complex and invasive sampling methods.

[0003] At present, the diagnosis of depression mainly relies on clinical manifestations and scale assessments, and lacks objective experimental diagnostic indicators. This diagnostic method is difficult to meet the precise requirements of clinical diagnosis and classification of depression due to large individual differences, complex etiologies, and diverse symptoms. Therefore, it is particularly important to explore the pathogenesis of depression and find effective new therapeutic targets. The development of next-generation sequencing technology has made it possible to study circRNAs, revealing their regulatory roles in a variety of diseases and providing a new perspective for the diagnosis and treatment of depression. Although the study of circRNAs has provided a new direction for the diagnosis and treatment of depression, existing technologies still have defects in the clinical diagnosis of depression. First, the symptoms of depression are similar to other emotional states and mental illnesses and are difficult to distinguish. Second, depression is often comorbid with other diseases, which increases the complexity of diagnosis. In addition, differences in patients' subjective expressions also affect the accurate description of symptoms. These problems limit the accuracy and reliability of existing diagnostic methods, and new biomarkers are urgently needed to improve the accuracy of depression diagnosis.

[0004] In summary, the present invention provides the potential and importance of circRNA-103546 as a diagnostic marker in the preparation of depression detection products. Summary of the invention

[0005] The purpose of the present invention is to provide the use of circRNA-103546 as a diagnostic marker in the preparation of depression detection products, so as to solve the problems of insufficient diagnostic accuracy in the prior art and the need for new biomarkers to improve diagnostic accuracy.

[0006] The above technical purpose of the present invention is achieved through the following technical scheme: the use of circRNA-103546 as a diagnostic marker in the preparation of depression detection products, comprising a measurement component capable of detecting the level of circRNA-103546 in plasma, the component being capable of confirming the differential expression between depression patients and healthy individuals by a quantitative reverse transcription polymerase chain reaction method, the nucleotide sequence of the circRNA-103546 being as shown in SEQ ID NO.1.

[0007] The present invention is further configured as follows: the assay component includes a reagent for extracting total RNA from a plasma sample, and specific primers for reverse transcription and amplification of circRNA-103546.

[0008] Another object of the present invention is to provide a method for assisting the detection of depression by detecting the level of circRNA-103546 in plasma, the method comprising the following steps:

[0009] a) extracting total RNA from plasma samples of test subjects;

[0010] b) Detect the expression level of circRNA-103546 by quantitative reverse transcription polymerase chain reaction method using specific primers;

[0011] c) Compare the detected circRNA-103546 expression level with the reference standard of healthy individuals to assist in determining the risk of depression in the subject.

[0012] The present invention is further configured as follows: the normal standard is based on the expression level of circRNA-103546 in plasma samples of healthy individuals.

[0013] Another object of the present invention is to provide a kit for detecting the expression level of circRNA-103546, the kit comprising:

[0014] Component a: Reagents for extracting total RNA from plasma samples;

[0015] Component b: primers and enzymes required for reverse transcription-polymerase chain reaction;

[0016] Component c: standard control samples used to detect the expression level of circRNA-103546;

[0017] Component d: software for analyzing the expression level of circRNA-103546.

[0018] Another object of the present invention is to provide a depression detection product based on circRNA-103546 in plasma, which includes the method and kit described above.

[0019] By adopting the above technical scheme, the present invention provides a new depression detection product. The product includes a determination component capable of detecting the level of circRNA-103546 in plasma, which confirms the differential expression between depression patients and healthy individuals by quantitative reverse transcription polymerase chain reaction method. The detection of this differential expression provides a high-precision and high-efficiency method for the diagnosis of depression, which is easy to promote and use on a large scale; the determination component of the present invention includes a reagent for extracting total RNA from a plasma sample and a specific primer for reverse transcription and amplification of circRNA-103546. The accuracy and specificity of these components ensure the reliability of the test results, making the diagnosis of depression more accurate. Through this method, the present invention provides a new tool for assisting the detection of depression, which includes extracting total RNA from a plasma sample of a subject, using specific primers to detect the expression level of circRNA-103546 by RT-qPCR method, and comparing the test results with the reference standard of a healthy individual to assist in judging the risk of depression in the subject.

[0020] The present invention also provides a depression detection product based on circRNA-103546 in plasma, which includes the method and kit described above, wherein the kit comprises component a: a reagent for extracting total RNA from a plasma sample; component b: primers and enzymes required for reverse transcription-polymerase chain reaction; component c: a standard control sample for detecting the expression level of circRNA-103546; and component d: software for analyzing the expression level of circRNA-103546, which makes the detection process more convenient and standardized, and helps to improve the consistency and repeatability of the detection.

[0021] The product provided by the present invention has high stability, expression specificity and detection convenience. The circular structure of circRNA-103546 makes it highly resistant to exonucleases, and it can stably exist for a long time in cells and peripheral blood, and is easy to be accurately detected; in addition, the expression spectrum of circRNA-103546 in samples of patients with depression and healthy people is significantly different, and this differential expression helps to accurately distinguish patients with depression from healthy individuals. Finally, since circRNA can be detected in peripheral samples such as blood, this makes the detection process relatively simple and safe, avoiding complex and invasive sampling methods such as brain tissue biopsy. Therefore, the depression detection product provided by the present invention not only improves the accuracy and efficiency of depression detection, but also has broad application prospects and practical application value.

[0022] In summary, the present invention has the following beneficial effects:

[0023] 1. The present invention detects the level of circRNA-103546 in plasma by quantitative reverse transcription polymerase chain reaction (RT-qPCR) method, providing a detection product that can accurately distinguish patients with depression from healthy individuals. The high accuracy and efficiency of this product make it easy to promote and use on a large scale, greatly improving the diagnostic accuracy of depression.

[0024] 2. The detection product of the present invention includes reagents for extracting total RNA from plasma samples, specific primers, standard control samples and analysis software, which standardizes and facilitates the detection process. The stability of circRNA-103546 and its detectability in peripheral blood avoid invasive sampling, such as brain tissue biopsy, making the detection simple and safe, and has broad application prospects and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a chart used to analyze the difference in circRNA expression between depression patients and a healthy control group in Example 2 of the present invention;

[0026] in, Figure 1 A is a hierarchical clustering diagram of different circRNA expression patterns among samples; Figure 1 B is a scatter plot of statistically significant differentially expressed circRNAs between the two groups; Figure 1 C is the volcano plot of significantly differentially expressed circRNAs; Figure 1 D is the circRNA expression profile obtained by confirming the microarray data using RT-qPCR;

[0027] Figure 2 is the expression profile of 15 differentially expressed circRNAs in Example 2 of the present invention;

[0028] Figure 3 is the correlation between circRNA levels and depressive symptoms in Example 2 of the present invention;

[0029] Figure 4 is a flow chart of the animal experiment procedure in Example 2 of the present invention;

[0030] Figure 5 is a chart of behavioral test results in Example 2 of the present invention;

[0031] in, Figure 5 A is a graph showing the changes in mouse body weight; Figure 5 B is the result of the tail suspension experiment; Figure 5 C is a graph showing the results of the sucrose preference test; Figure 5 D is the open field result image; Figure 5 E is a graph showing the total number of times mice entered the open arms in the elevated plus maze test; Figure 5 F is a graph showing the percentage of time mice spent in the open arms in the elevated plus maze test;

[0032] Figure 6 This is a diagram showing the results of quantitative reverse transcription-PCR analysis of circRNA expression in mice in Example 2 of the present invention. DETAILED DESCRIPTION

[0033] The specific implementation of the present application is described below in conjunction with the accompanying drawings.

[0034] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operating procedures used herein are reagents, materials and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0035] Example 1: Application of circRNA-103546 as a diagnostic marker in the preparation of depression detection products

[0036] This embodiment provides an application of a depression detection product based on the level of circRNA-103546 in plasma, which uses circRNA-103546 as a diagnostic marker, and the nucleotide sequence of the circRNA-103546 is shown in SEQ ID NO.1, and the differential expression between depression patients and healthy individuals is confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) method. The assay component further includes a reagent for extracting total RNA from a plasma sample, and a specific primer for reverse transcription and amplification of circRNA-103546, and these components work together to ensure efficient extraction and specific detection of circRNA-103546 from a plasma sample. Specifically, the detection product uses the content of circRNA-103546 in plasma as a detection indicator. When the content of circRNA-103546 is higher than the normal level of a healthy individual, it indicates that the subject may be at risk of depression. Preferably, the higher than normal level is higher than 2 times or more.

[0037] The depression detection product includes a kit for detecting the expression level of circRNA-103546, the kit includes a reagent for detecting the expression level of circRNA-103546, and the reagent includes a primer and / or a probe for the circRNA-103546; the kit includes:

[0038] Component a: Reagents for extracting total RNA from plasma samples;

[0039] Component b: primers and enzymes required for reverse transcription-polymerase chain reaction;

[0040] Component c: standard control samples used to detect the expression level of circRNA-103546;

[0041] Component d: software for analyzing the expression level of circRNA-103546.

[0042] This embodiment innovatively applies circRNA-103546 to depression detection products. Detection of depression based on specific circRNA-103546 in plasma can effectively improve the accuracy and reliability of depression detection. This embodiment also provides a depression detection product based on circRNA-103546 in plasma, which includes a kit for detecting the expression level of circRNA-103546 and a corresponding detection method. This method has high stability, expression specificity and detection convenience, provides a new and effective tool for the diagnosis of depression, and has broad application prospects and practical application value.

[0043] Example 2: Study on the expression changes of circular RNA in the blood of patients with depression

[0044] Participant selection: Twenty-seven patients with first-episode depression from Xiamen Xianyue Hospital between January and December 2019 were selected. All diagnoses were made according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition (DSM-IV) criteria using the Structured Clinical Interview for DSM-IV (SCID).

[0045] Inclusion criteria were (1) first diagnosis of depression and no history of clinical treatment for depression within 3 months before blood collection; (2) Han nationality; (3) no other diseases, drug abuse or blood transfusion history within 1 month before enrollment, and age between 16 and 65 years. Exclusion criteria included (1) severe organic disease, affective disorder or other mental disorder; (2) personality disorder caused by continuous medication; (3) alcohol or other drug abuse; (4) modified electroconvulsive therapy or long-term use of antidepressants within 6 months; (5) need for long-term combined use of mood-altering or central nervous system-acting drugs.

[0046] Twenty-seven age- and sex-matched healthy volunteers, all of Han nationality, were recruited as controls. Healthy controls (HCs) were assessed using the Structured Clinical Interview for DSM-IV Non-Patient Version (SCID-I / NP). None of the first-degree relatives in the healthy controls had Axis I psychiatric disorders. The Medical Ethics Committee of Xiamen Xianyue Hospital approved this study, and written informed consent was obtained from all participants.

[0047] Blood collection, RNA extraction, and reverse transcription: Whole blood (3 ml) was collected from each subject using EDTA anticoagulation tubes and processed within 2 h. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation, and then they were transferred into 2 ml fresh RNase / DNase-free tubes and stored at -80 °C until further use. TM Total RNA was extracted from PBMCs using miRNA isolation reagent (Applied Biosystems, product number AM1560) according to the manufacturer's protocol and quantified on a NanoDrop ND-1000 (Thermo Scientific, ME, USA). RNA integrity was assessed after agarose gel electrophoresis. For reverse transcription, the reaction system contained 2 μg of total RNA, 4 μl of 5× RT buffer, 1 μl of RT enzyme mix, 1 μl of primer mix, and nuclease-free water according to the instructions of the ReverTraAceqPCRRT kit (Toyobo).

[0048] Labeling and hybridization: Blood RNA samples from four patients with depression and samples from three normal control subjects were selected for microarray studies. Sample labeling and array hybridization were performed according to the manufacturer's protocol (Arraystar Inc.). Briefly, total RNA was digested by RNaseR (Epicentre, Inc.) to remove linear RNA and enrich circular RNA. The enriched circular RNA was then amplified and transcribed into fluorescent cRNA using a random primer method (Arraystar Super RNA Labelling Kit; Arraystar). The labeled cRNA was purified using RNeasy Mini Kit (Qiagen). The concentration and specific activity (pmol Cy3 / μg cRNA) of the labeled cRNA were measured using NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 5 μl of 10× blocker and 1 μl of 25× fragmentation buffer, and then the mixture was heated at 60°C for 30 min, and finally 25 μl of 2× hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was added to the sealed slide and mounted on the circRNA expression microarray slide. The slide was incubated at 65 °C for 17 h in an Agilent hybridization oven. The hybridized array was washed, fixed, and scanned using an Agilent G2505C scanner.

[0049] Microarray data analysis: The raw data were extracted by importing images scanned by the Agilent G2565C microarray scanner into the Agilent Feature Extraction software (version 11.0.1.1; Agilent Technologies, Inc.). A series of data processing including quantile normalization of the raw data was performed using the R software package limma (http: / / bioconductor.riken.jp / packages / 3.0 / bioc / html / limma.html). The differentially expressed circRNAs between the depression and normal groups were then evaluated using the t-test, and the P values ​​were false discovery rate (FDR) corrected by the Benjamini-Hochberg (BH) procedure. CircRNAs considered to be significantly differentially expressed had a fold change of ≥1.5 and an FDRP value of <0.05. Hierarchical clustering was used to display the different circRNA expression patterns between samples, as shown in the attached figure. Figure 1 A. The scatter plot was used to evaluate the statistically significant differentially expressed circRNAs between the two groups, as shown in Supplementary Figure 2. Figure 1 B. Volcano plot filtering was used to visualize the significantly differentially expressed circRNAs between each pair of comparisons, as shown in the attached Figure 1C. Hierarchical clustering, scatter plots, and volcano plot filtering were performed using Agilent Feature Extraction software (version 11.0.1.1; Agilent Technologies, Inc.).

[0050] qPCR validation of candidate circRNAs: RT-qPCR was performed to confirm the circRNA expression profiles obtained in the microarray data. Considering the expression fold changes detected in the microarray analysis and the target circRNAs predicted to be associated with the progression of depression in previous studies, 15 differentially expressed circRNAs were selected for further study. CircRNAs that showed a fold change (FC) ≥ 2 (P ≤ 0.05) between the two groups were classified as significantly differentially expressed, as shown in the attached Figure 2 As shown. We selected the 4 most upregulated circRNAs and all 11 downregulated circRNAs for subsequent validation. Briefly, total RNA was extracted from 27 pairs of serum samples from depression patients and controls using TRIzol reagent. cDNA was synthesized from 500 ng of total RNA using the Prime-Script RT kit (Takara Biotechnology). qPCR was performed on a Bio-Rad PCR instrument using the SYBR Green (Takara Bio Inc., Dalian, China) detection method and the divergent primers listed in Table 1 below. GAPDH was used as an internal control. Relative gene expression levels were analyzed using the 2-ΔΔCq method, as shown in the attached Figure 3 shown.

[0051] Table 1 Divergent primers

[0052]

[0053] Animal experimentation: These experiments were performed on male BALB / c mice (18–22 g, 8 weeks old) provided by the Experimental Animal Center of Xiamen University (Laboratory Animal Production License: GB / T35892-2018). The animals had free access to food and water throughout the experiment. The room was maintained at a temperature of 25 ± 1 °C and a relative humidity of approximately 65%, and a 12:12 h light-dark cycle (lights on at 8 a.m.). All experiments were performed in accordance with the guidelines of the National Institutes of Health (NIH) and were approved by the Ethics Committee of Xiamen Xianyue Hospital and the Laboratory Animal Ethics Committee.

[0054] The experimental procedure is as follows Figure 4As shown. Before the CUMS (chronic unpredictable mild stress) procedure on day 0, the mice were randomly divided into four groups: control group (n=10, Con), control + fluoxetine group (n=10, Con+F), CUMS group (n=10) and CUMS + fluoxetine group (n=10, CUMS+F). After 28 days of fluoxetine treatment, the mice were subjected to behavioral tests from day 57 to day 60. On day 60, all mice were euthanized and their brains were removed for analysis. The changes in brain weight of the mice are shown in the attached figure. Figure 5 As shown in A.

[0055] Chronic mild unpredictable stress depression model (CUMS): Mice were housed in single cages and given any two of the six stimulation methods (fixed in a plastic tube, tilted cage, no nesting, crowded environment, light and dark reversal, white noise) every day. The two stimulation methods were randomly selected every day, and the same stimulation method did not appear at the same time. Each stimulation method was randomly distributed in each stress cycle. The mice were given the above stimulation for 4 weeks to establish the CUMS depression model. The behavioral experiment was used to evaluate whether the model was successful. The CUMS procedure was performed as described above, but with slight modifications, such as: confining mice in plastic tubes, tilting cages, placing them in empty cages without nesting materials, placing them in crowded cages, turning on the lights for a short time during the dark period, and white noise. In order to prevent habituation and maintain the unpredictability of stress factors, two stress factors were used at different times within a week and repeated in the four-week experiment. The control group animals were placed in a separate room and did not come into contact with the stress group. Stress presentation did not include deprivation stress factors of food or water.

[0056] Drug treatment: To explore whether the antidepressant fluoxetine can reverse the changes caused by CUMS, mice were randomly divided into four groups: control group (Con), control + fluoxetine group (Con+F), CUMS group, and CUMS + fluoxetine group (CUMS+F). After 4 weeks of CUMS exposure, the animals received 4 weeks of intraperitoneal injection of fluoxetine at a dose of 10 mg / kg.

[0057] Behavioral tests: Behavioral tests included outdoor motor activity test, elevated plus maze test, tail suspension test and sucrose preference test.

[0058] Tail suspension test: On day 59, mice underwent the tail suspension test (TST) to assess depression levels. In this test, mice are placed in an unpleasant environment from which they cannot escape, and they alternate between active periods (foraging behavior) and resting periods (waiting behavior, reflecting "behavioral despair"). Mice are suspended 58 cm above the table from the edge of the rack by tape. The experiment lasts for 6 minutes, and the last 4 minutes of immobility are recorded, reflecting the animal's depressive state (Yan et al. 2007). The experimental results are as follows Figure 5 As shown in B.

[0059] Experimental results: The immobility time of the Con group and the Con+F group was similar, indicating that fluoxetine had no significant effect on the immobility behavior of mice in the absence of stress conditions. This may mean that under normal conditions, fluoxetine does not change the basic behavioral patterns of mice. The immobility time of the Con group and the Con+F group was significantly lower than that of the CUMS group, indicating that the CUMS procedure successfully induced depressive-like behavior and increased the immobility time of mice, reflecting a more obvious state of "behavioral despair". The immobility time of the CUMS+F group was slightly lower than that of the Con group and the Con+F group, but higher than that of the CUMS group, indicating that fluoxetine treatment reversed the depressive-like behavior caused by CUMS to a certain extent and reduced the immobility time, indicating that fluoxetine has a certain antidepressant effect.

[0060] Experimental conclusion: The results of the tail suspension test showed that the CUMS procedure could successfully induce depressive-like behavior in mice, and fluoxetine treatment could partially reverse this depressive-like behavior and reduce the immobility time of mice.

[0061] Sucrose preference test: On day 60, a sucrose preference test (SPT) was performed to assess the degree of depression. Starting at 08:00 on day 57, two bottles of 1% (w / v) sucrose solution were placed in each cage, and all mice were adapted to sucrose for 24 hours. Then one bottle of sucrose solution was replaced with tap water, and sucrose adaptation was performed for another 24 hours. Starting at 20:00 on day 59, each mouse was given two pre-weighed bottles, one containing sucrose solution and the other containing tap water, and the positions of the two bottles were exchanged once at 02:00 on day 60 to avoid position preference. The weight of each bottle was measured 12 hours after the start of sucrose intake, and the intake of sucrose solution and tap water was calculated at 08:00 on day 60. Sucrose preference (%) is defined as the ratio of the volume of sucrose consumed to the total amount of liquid consumed (sucrose + water). The experimental results are shown in the attached figure. Figure 5 As shown in C.

[0062] Experimental results: The sucrose preference of the Con group (control group) and the Con+F group (control group + fluoxetine) was similar, indicating that there was no significant difference in the preference for sucrose between the two groups of mice. The sucrose preference of the CumS group (chronic unpredictable mild stress group) was significantly lower than that of the Con group and the Con+F group, indicating that the CUMS procedure may have successfully induced depressive-like behavior and reduced the mice's preference for sweetness. The sucrose preference of the CumS+F group (CUMS+fluoxetine) was slightly lower than that of the Con group and the Con+F group, but higher than that of the CUMS group, which may indicate that fluoxetine has a certain improvement effect on the depressive-like behavior induced by CUMS.

[0063] Experimental conclusion: The similar results of the Con group and the Con+F group indicate that fluoxetine has no significant effect on the sucrose preference of mice in the absence of stress. The low sucrose preference of the CumS group was associated with depressive behavior, which is consistent with the reduced sucrose preference in depression models in the literature. The results of the CumS+F group indicate that fluoxetine may have a certain improvement effect on the depressive-like behavior induced by CUMS, although this improvement is not as good as the baseline level of the Con group and the Con+F group.

[0064] Open field test: On the 57th day, an open field test was conducted in a motor activity box. The box is a square area (90 cm × 90 cm × 30 cm high) surrounded by gray walls. The bottom of the box is divided into 36 identical squares (20 outer squares against the wall and 16 inner squares). The movement of the mice was measured by counting the number of times the mice moved from one square to another. The locomotor activity test of the mice lasted for 5 minutes and was recorded by a camera installed on the top of the locomotor activity box. The results are shown in the figure. Figure 5 As shown in D.

[0065] Experimental results: The outdoor exercise activity levels of mice in the Con group and the Con+F group were similar, indicating that fluoxetine had no significant effect on the exercise activity levels of mice in the absence of stress conditions. This may mean that fluoxetine does not change the motor behavior of mice under normal conditions. The outdoor exercise activity levels of mice in the Con group and the Con+F group were significantly higher than those in the CUMS group, indicating that the CUMS procedure successfully reduced the exercise activity levels of mice, which may be an indicator of depressive-like behavior, reflecting the decreased activity of mice. The outdoor exercise activity levels of mice in the CUMS+F group were slightly lower than those in the Con group and the Con+F group, but higher than those in the CUMS group, indicating that fluoxetine treatment reversed the decrease in exercise activity levels caused by CUMS to a certain extent and increased the exercise activity of mice, indicating that fluoxetine has a certain antidepressant effect.

[0066] Experimental conclusion: The results of the open field test showed that the CUMS procedure could successfully induce depressive-like behavior in mice, characterized by reduced locomotor activity levels, while fluoxetine treatment could partially reverse this depressive-like behavior and increase the locomotor activity levels of mice.

[0067] Elevated plus maze test: The elevated plus maze test was performed on day 58. The elevated plus maze consists of two open arms and two closed arms (50 × 10 × 10 cm). The closed arms have 45 cm high walls on both sides and ends. The entire maze is 45 cm from the ground. Each mouse is placed in the arm facing the wall of the central square (10 × 10 cm) and allowed to explore the maze for 5 min. The total number of times the mouse entered the open arm and the time spent in the open arm were recorded. The results of the total number of times the mouse entered the open arm are shown in Figure 5E shows the percentage of time spent in the open arms position. Figure 5 As shown in F.

[0068] Experimental results: The performance of the Con group and the Con+F group in the elevated plus maze test was similar, indicating that fluoxetine had no significant effect on the exploratory behavior and anxiety level of mice in the absence of stress conditions. This may mean that fluoxetine does not change the behavioral pattern of mice under normal conditions. The exploratory behavior of the Con group and the Con+F group was significantly higher than that of the CUMS group, indicating that the CUMS procedure successfully induced anxiety and depression-like behaviors and reduced the exploratory activities of mice in the elevated plus maze, especially the exploration of the open arms. The exploratory behavior of the CUMS+F group was slightly lower than that of the Con group and the Con+F group, but higher than that of the CUMS group, indicating that fluoxetine treatment reversed the anxiety and depression-like behaviors caused by CUMS to a certain extent and increased the exploratory activities of mice in the elevated plus maze, especially the exploration of the open arms.

[0069] Experimental conclusion: The results of the elevated plus maze test showed that the CUMS procedure could successfully induce anxiety- and depression-like behaviors in mice, while fluoxetine treatment could partially reverse such anxiety- and depression-like behaviors and increase the exploratory activities of mice.

[0070] Mouse RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0071] After completing all behavioral tests, mice were deeply anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection) and perfused with 30 ml of 0.1 M phosphate buffered saline (PBS, pH 7.4). Total RNA was extracted from mouse samples of the hippocampus and cortex using the TIANGEN reagent (TIANGEN, Beijing, China). The concentration and purity of all RNA samples were then measured using a NanoDrop2000 spectrophotometer (ThermoScientific, Wilmington, DE, USA). cDNA was reverse transcribed from total RNA (TOYOBO, Osaka, Japan). Relative quantification of circRNA and messenger RNA (mRNA) expression was performed by the 2-ΔΔCt method, with β-actin as an internal control. All primer pairs were designed and synthesized by Fuzhou Shangya Biotechnology Co., Ltd. (Fuzhou, China), and the sequences of forward and reverse primers are in Table 2 below.

[0072] Table 2 Forward and reverse primers

[0073]

[0074] Statistical analysis: Data analysis was performed using GenEx qPCR and SPSS 20.0 software. The significance of qRT-PCR validation between each group was tested by two-way analysis of variance (ANOVA), and P < 0.05 was considered statistically significant.

[0075] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. The application of circRNA-103546 as a diagnostic marker in the preparation of depression detection products is characterized by: The method comprises a determination component capable of detecting the level of circRNA-103546 in plasma, wherein the component can confirm the differential expression between depression patients and healthy individuals by a quantitative reverse transcription polymerase chain reaction method, and the nucleotide sequence of the circRNA-103546 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The assay components include reagents for extracting total RNA from plasma samples and specific primers for reverse transcription and amplification of circRNA-103546.

3. A method for assisting the detection of depression by detecting the level of circRNA-103546 in plasma, characterized by: The method comprises the following steps: a) extracting total RNA from plasma samples of test subjects; b) Detect the expression level of circRNA-103546 by quantitative reverse transcription polymerase chain reaction method using specific primers; c) Compare the detected circRNA-103546 expression level with the reference standard of healthy individuals to assist in determining the risk of depression in the subject.

4. The method according to claim 3, characterized in that: The normal standard is based on the expression level of circRNA-103546 in plasma samples of healthy individuals.

5. A kit for detecting the expression level of circRNA-103546, characterized in that: The kit comprises: Component a: Reagents for extracting total RNA from plasma samples; Component b: primers and enzymes required for reverse transcription-polymerase chain reaction; Component c: standard control samples used to detect the expression level of circRNA-103546; Component d: software for analyzing the expression level of circRNA-103546.

6. A depression detection product based on circRNA-103546 in plasma, characterized by: The product comprises the method described in claim 3 and the kit described in claim 5.

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