Use of circrna molecules in the preparation of a medicament for treating depression

Abstract

The application discloses application of a circRNA molecule in preparation of a medicine for treating depression. The exogenous circHERC1 is introduced into a mouse body, so that the memory, learning ability and muscle strength of the mouse can be effectively improved, and the depression degree of the mouse can be weakened. The application provides a new strategy for developing a gene therapy for resisting depression, improving cognitive ability and memory ability of the mouse, and expands the selectable range of the gene therapy and the drug therapy.

Description

Technical Field

[0001] This invention discloses a novel pharmaceutical use for circRNA molecules, belonging to the field of pharmaceutical applications. Background Technology

[0002] Depression is a prevalent mental illness worldwide, affecting over 300 million people according to World Health Organization (WHO) data. It is often accompanied by cognitive impairments, such as decreased attention span and impaired learning and memory. Research indicates that the hippocampus, a key brain region responsible for learning and memory, shows a close correlation between reduced neuroplasticity and decreased neurogenesis, and depressive behavior and cognitive deficits. While traditional antidepressants (such as SSRIs) can alleviate some mood symptoms, their effects on improving cognitive function are limited, and they suffer from delayed onset of action and significant side effects. Therefore, developing multi-target intervention strategies that combine antidepressant and cognitive-enhancing effects has become a current research hotspot.

[0003] In neuroscience research, mouse models are widely used to explore the mechanisms of depressive-like behaviors and cognitive functions due to their strong genetic manipulation and mature behavioral evaluation systems. Paradigms such as chronic unpredictable mild stress (CUMS) or social defeat can effectively simulate core symptoms of human depression. Combined with behavioral experiments such as the Morris water maze and novel object recognition, learning and memory abilities in mice can be systematically assessed. Recent studies have found that natural products (such as flavonoids) or novel synthetic molecules exhibit potential antidepressant and cognitive-enhancing synergistic effects by regulating pathways such as oxidative stress and inflammatory factors (such as IL-6 and TNF-α), providing a theoretical basis for the development of novel therapeutic drugs.

[0004] circRNAs are a class of non-coding RNA molecules present in organisms that lack a 5' cap and a 3' poly(A) tail and form a circular structure covalently linked to each other. With the development of high-throughput sequencing and bioinformatics analysis techniques, research on circRNAs has become quite extensive. Circular RNAs are covalently closed endogenous biomolecules in eukaryotes, exhibiting tissue- and cell-specific expression patterns. Their biogenesis is controlled by different cis-acting elements and trans-acting factors. circRNAs interact with specific RNAs and proteins, acting as microRNA (miRNA) sponge decoys and protein scaffolds. Some circRNAs containing internal ribosome entry sites (IRES) and AUG sites can be translated under specific conditions, resulting in unique peptides.

[0005] The purpose of this invention is to provide a circRNA that can combat depression, relieve anxiety, and improve cognitive function. Summary of the Invention

[0006] Based on the above objectives, this invention provides an application of a circRNA molecule in the preparation of a drug for treating depression, wherein the sequence of the circRNA molecule is shown at bases 21-1163 of SEQ ID NO. 1. In this invention, the circRNA molecule is named "circHERC1" or "circ_0035796". "circHERC1" or "circ_0035796" is a circular RNA disclosed in CN115948548A associated with the development and progression of non-small cell lung cancer. The application described in this invention refers to the direct preparation of the circRNA molecule into a pharmaceutical formulation, or the preparation of a pharmaceutical formulation from a substance capable of producing and / or releasing the circRNA molecule. The depression described in this invention, also known as depressive disorder, is a mental disorder with a high incidence, high clinical cure rate but low treatment acceptance rate and high relapse rate. Its main characteristics are significant and persistent low mood, which may even be accompanied by psychotic symptoms such as delusions and hallucinations, severe cognitive impairment, and some patients may exhibit self-harm or suicidal behavior. Clinically, depressive disorders can be classified into mild, moderate, and severe based on the number, type, and severity of symptoms. These classifications are tailored to different groups, such as the elderly, children, and postpartum women, and are termed geriatric depression, childhood depression, postpartum depression, etc. Depression is primarily characterized by low mood, loss of interest, and lack of energy; it may also present with slowed reactions, sluggish thinking, and memory loss.

[0007] In a preferred embodiment, the application is to prepare a drug for treating depression by overexpressing the circRNA molecule circHERC1. The overexpression of circHERC1 in this invention refers to a substance that provides sufficient expression of circHERC1 through genetic engineering techniques.

[0008] In a more preferred embodiment, the substance overexpressing circHERC1 is a gene expression vector overexpressing circHERC1, the gene expression vector containing a DNA molecule with the sequence shown in positions 9-1175 of SEQ ID NO. 1.

[0009] In one specific embodiment of the present invention, the vector overexpressing circHERC1 is an adeno-associated virus vector.

[0010] In another preferred embodiment of the present invention, the substance overexpressing circHERC1 is an exosome containing the circRNA molecule. The exosomes described in this invention refer to small membrane vesicles (30-150 nm) containing complex RNA and proteins, particularly disc-shaped vesicles with a diameter of 40-100 nm. Various cell types can secrete exosomes under normal and pathological conditions, primarily originating from multivesicular bodies formed by the invagination of intracellular lysosomal microparticles. After the outer membrane of the multivesicular body fuses with the cell membrane, the exosomes are released into the extracellular matrix. All cultured cell types can secrete exosomes, and exosomes are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk.

[0011] In a more preferred embodiment, the substance overexpressing circHERC1 is a cell that secretes exosomes containing the circRNA molecule.

[0012] In one specific embodiment of the present invention, the cells that secrete exosomes containing circHERC1 are 293FT cells.

[0013] In another preferred embodiment of the present invention, the drug for treating depression refers to a drug for relieving anxiety. Anxiety, as described in this invention, is a major clinical symptom of depression. The circHERC1 drug alleviates or reduces anxious behavior in experimental mice by enhancing their interest and exploration abilities.

[0014] In a preferred embodiment of the present invention, the therapeutic drug for depression refers to a drug that improves cognitive impairment and enhances learning ability and / or memory. Cognitive impairment, learning ability, and / or memory, as described in this invention, are also major clinical symptoms of depression. The circHERC1 achieves an antidepressant therapeutic effect by enhancing learning ability and / or memory.

[0015] This invention, through the administration of adeno-associated virus carrying circHERC1 and exosomes carrying circHERC1 to mice, demonstrated that exogenous circHERC1 expression in mice effectively reduced depression levels, enhanced interest, energy, memory, learning ability, and exploratory ability, improved cognitive function, and reduced anxiety behavior. This suggests that circHERC1 is closely related to improved anxiety behavior and cognitive enhancement in the brain. Furthermore, circHERC1 expression improved memory, learning ability, and muscle strength in mice, reduced depression levels, and improved metabolism. circHERC1 holds promise as an effective molecular target for enhancing cognitive function.

[0016] The antidepressant effect described in this invention can be evaluated using conventional methods in the field, such as relevant behavioral experiments on mice: open field test, tail suspension test, elevated cruciate maze test, and forced swimming test, to detect the degree of depression and anxiety in mice; novel object recognition test to detect the curiosity and exploration ability of mice; and Morris water maze test to detect the memory and learning ability of mice. The improvement in cognitive ability described herein can not only restore, maintain, and enhance the physiological functions of the brain, but can also be used for drug evaluation and screening to establish animal models of depression and cognition. Attached Figure Description

[0017] Figure 1 : A schematic diagram of a transgenic mouse that conditionally overexpresses circHERC1 at the Rosa26 site using gene editing technology; Figure 2 : Identification of transgenic mice with site-directed integration induced by circHERC1 expression; Figure 3 Expression of circHERC1 in different organs of male and female mice; Figure 4 Expression of circHERC1 in male control mice and transgenic circHERC1 mice; Figure 5 Expression of circHERC1 in female control mice and transgenic circHERC1 mice; Figure 6 The distance the mouse traveled in the open field experiment; Figure 7 The time a mouse spends in the designated area during the elevated cross maze experiment; Figure 8 The duration of swimming struggle in mice during the forced swimming experiment; Figure 9 The time the mouse remained still during the forced swimming experiment; Figure 10 The climbing time of mice in the tail suspension experiment; Figure 11 In a new object recognition experiment, the recognition index of mice for new objects; Figure 12 The number of times a mouse traverses the escape platforms in the Morris water maze; Figure 13 The path a mouse takes through the escape platforms in the Morris water maze; Figure 14 The time it takes for mice to reach the platform in the Morris water maze varies over time. Figure 15Expression of circHERC1 in various organs of aged mice after injection of AVVcircHERC1; Figure 16 In the open field experiment, mice were injected with AVV. circHERC1 The percentage of the total distance traveled by the mouse in the central region; Figure 17 In the open field experiment, mice were injected with AVV. circHERC1 The resting time of the mice afterward; Figure 18 In the elevated cross maze experiment, mice were injected with AVV. circHERC1 The time mice spent in the open area afterward; Figure 19 In the elevated cross maze experiment, mice were injected with AVV. circHERC1 The distance the mouse traveled in the open area afterward; Figure 20 In the tail suspension experiment, mice were injected with AVV. circHERC1 The climbing time of the mice after the climbing; Figure 21 In the tail suspension experiment, mice were injected with AVV. circHERC1 The resting time of the mice afterward; Figure 22 In a new object recognition experiment, the recognition index of mice for new objects; Figure 23 The number of times a mouse comes into contact with a new object in a new object recognition experiment; Figure 24 In the new object recognition experiment, the contact distance of the mouse to the new object; Figure 25 In the Morris water maze, mice were injected with AVV. circHERC1 The distance the mouse traveled across the escape platform; Figure 26 In the Morris water maze, male mice were injected with AVV. circHERC1 The time it takes for mice to reach the platform varies over time; Figure 27 In the Morris water maze, female mice were injected with AVV. circHERC1 The time it takes for mice to reach the platform varies over time; Figure 28 Transmission electron microscopy (TEM) images of exosomes from circHERC1-overexpressing 293FT cells and vector controls; Figure 29 Western blot analysis of exosome-labeled proteins in 293FT cells, cell supernatant, and exosomes; Figure 30Automated nanoparticle tracking analysis (NTA) of exosomes from circHERC1-overexpressing 293FT cells and vector controls; Figure 31 qRT-PCR analysis of circHERC1 expression in exosomes from circHERC1-overexpressing 293FT cells and vector control cells; Figure 32 In the open field experiment, mice were injected with EVs. circHERC1 The percentage of the total distance traveled by the mouse in the central region; Figure 33 In the open field experiment, mice were injected with EVs. circHERC1 The resting time of the mice afterward; Figure 34 In the elevated cross maze experiment, mice were injected with EVs. circHERC1 The percentage of the distance traveled by mice in the open area; Figure 35 In the elevated cross maze experiment, mice were injected with EVs. circHERC1 The number of times the mouse entered the open area; Figure 36 In the elevated cross maze experiment, mice were injected with EVs. circHERC1 The time it took for the mouse to enter the open area; Figure 37 In the tail suspension experiment, mice were injected with EVs. circHERC1 The climbing time of the mice after the climbing; Figure 38 In the Morris water maze, mice were injected with EVs. circHERC1 The distance the mouse traveled across the escape platform; Figure 39 In the Morris water maze, male mice were injected with EVs. circHERC1 The time it takes for mice to reach the platform varies over time; Figure 40 In the Morris water maze, female mice were injected with EVs. circHERC1 The time it takes for mice to reach the platform varies over time. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0021] Example 1. Construction of a transgenic mouse model The circular RNA circHERC1 is derived from the circular RNA circ_0035796 disclosed in CN115948548A. In this invention, the circRNA is named "circHERC1". The nucleotide sequence of the circular RNA circHERC1 is bases 21-1163 of the sequence shown in SEQ ID NO.1.

[0022] 1. Construction and identification of circHERC1 transgenic mice Gene editing technology was used to construct transgenic mice that conditionally overexpressed circHERC1 at the Rosa26 locus. A vector flanked by loxP sites was inserted between the promoter and the sequence encoding circHERC1, and then integrated into the mouse ROSA26 gene locus using CRISPR / Cas9-mediated homologous recombination. (See...) Figure 1 The tails of mice were cut off and the genome was extracted. Multiple primer pairs were designed for PCR verification.

[0023] Table 1. Primer sequence information used in PCR reactions

[0024] According to the primer synthesis instructions, add an appropriate amount of RNase-free water to prepare a 10 μM primer solution, and prepare a 2' reverse transcription mixture (Table 2). Keep it on ice until needed.

[0025] Table 2. Reverse transcription reaction system

[0026] After preparing the reverse transcription mixture, add it to each of the eight-tube strips, then add 1 μL of mouse tail DNA sample to each strip, making a total reaction volume of 20 μL. Gently pipette to mix, seal tightly, and briefly centrifuge; place in a PCR instrument and set the reaction conditions as follows (Table 3): Table 3. PCR conditions

[0027] The results showed corresponding bands in the nucleic acid electrophoresis image, indicating that the conditionally overexpressing circHERC1 site-directed integration transgenic mouse (Rosa26-LSL-circHERC1) was successfully constructed.

[0028] Rosa26-LSL-circHERC1 transgenic mice were mated with Dppa3-Cre transgenic mice (transgenic mice expressing Cre recombinase in the reproductive system) to obtain circHERC1 site-directed integration transgenic mice and circHERC1-induced site-directed integration transgenic mice, respectively. Both types of mice were circHERC1 transgenic mice, and the corresponding bands were verified by nucleic acid surge after PCR. Figure 2 ).

[0029] To study the systemic effects of circHERC1, the expression levels of circHERC1 in various organs of circHERC1 transgenic mice were detected using real-time PCR technology.

[0030] RNA was extracted from various organs of the Control and circHERC1 transgenic mice to be tested, and cDNA was obtained by reverse transcription. The primers shown in Table 4 were prepared, and qRT-PCR was performed using the following system and procedure.

[0031] Table 4. Primer sequence information used in PCR reactions

[0032] According to the primer synthesis instructions, add an appropriate amount of RNase-free-water to prepare a 10 μM primer solution, and prepare a 2' reverse transcription mixture (Table 5). Keep it on ice until needed.

[0033] Table 5. Reverse transcription reaction system

[0034] After preparing the reverse transcription mixture, add it to each of the eight-tube strips, then add 15 μL of RNA sample to each strip, making a total reaction volume of 30 μL. Gently pipette to mix, seal tightly, and briefly centrifuge; place in a 96-well PCR instrument and set the reaction conditions as follows (Table 6): Table 6. Reverse Transcription PCR Conditions

[0035] The system was added to a 96-well PCR plate. 18 μL of the qRT-PCR reaction mixture shown in Table 5 was added to each well, followed by 2 μL of cDNA to prepare a 20 μL reaction mixture. The plate was then placed in a real-time quantitative PCR instrument for the reaction. The PCR reaction conditions are as follows (Table 7): Table 7. qRT-PCR reaction conditions

[0036] Each sample was configured with three replicates, using U6 as the internal control gene. After the qPCR reaction, the average Ct value of the target gene in the three replicates for each sample was taken as the final result. The relative expression level of each target gene was calculated using the formula 2^-ΔCt, where ΔCt = Ct(target gene) - Ct(internal control gene).

[0037] qPCR showed that circHERC1 was strongly expressed in different organs of both male and female mice. Figure 3 ), while the baseline circHERC1 level in mice showed no significant change in control mice and male and female circHERC1 transgenic mice. Figures 4-5 ).

[0038] Example 2: Assessment of Depressive State in circHERC1 Transgenic Mice To assess the emotional state and cognitive abilities of mice, we used six classic behavioral tests: Morris water maze (MWM), novel object recognition test, suspended tail test (TST), elevated cross maze (EPM), forced swimming test (FST), and open field test.

[0039] 1. Depressive symptoms were reduced in circHERC1 transgenic mice. The emotional and anxiety states of the circHERC1 transgenic mice constructed in Example 1 were verified using the open field test, elevated cruciate maze test, and tail suspension test. We then assessed the effects of circHERC1 overexpression on behavioral and cognitive performance.

[0040] Open Field Test (OFT): Mice are placed in a large, neutral, open field for 5 minutes. Mouse behavior, total distance traveled, time spent in the center and periphery, movement, stillness, and grooming are recorded. These behaviors assess exploratory activity, anxiety (preference for the periphery indicates anxiety), and motor skills. Elevated Cross Maze (EPM): Mice are placed in the center of a maze and allowed free exploration for 5 minutes. Behaviors such as time spent in open and closed areas, and the tendency to enter open areas are observed. More time spent in closed areas indicates higher anxiety levels. Tail Suspension Test (TST): Mice are suspended by their tails in a controlled environment for 6 minutes. Their behavior is observed; immobility indicates a depressive-like state, while active movements such as struggling indicate adaptive coping mechanisms.

[0041] In open field tests, the center distance of male control mice was approximately 10.7%, while that of circHERC1 transgenic mice was approximately 16.8%; the center distance of female control mice was approximately 16.1%, while that of circHERC1 transgenic mice was approximately 21.6% (P < 0.05), indicating that circHERC1 transgenic mice traveled a longer distance. Figure 6 This indicates that circHERC1 transgenic mice exhibited more exploratory behavior. These behavioral changes are consistent with enhanced exploratory behavior and are generally associated with better physical and emotional health. In the elevated cross maze, male control mice spent approximately 10.88% of their time in the open area, while circHERC1 transgenic mice spent approximately 22.43%; female control mice spent approximately 15.65% of their time in the open area, while circHERC1 transgenic mice spent approximately 26.45% (P < 0.05). Figure 7 Mice transgenic with circHERC1 exhibited increased exploratory behavior and spent more time in the released area, suggesting reduced anxiety-like behaviors. Forced swimming tests showed that male control mice swam for approximately 48.22 seconds, while circHERC1 transgenic mice swam for approximately 107.63 seconds; female control mice swam for approximately 53.58 seconds, while circHERC1 transgenic mice swam for approximately 72.63 seconds (P < 0.05). Figure 8 The circHERC1 transgenic mice exhibited longer swimming struggle times. In the resting time assay, the resting time of male control mice was approximately 157.29 seconds, while that of circHERC1 transgenic mice was approximately 84.12 seconds; the resting time of female control mice was approximately 160.37 seconds, while that of circHERC1 transgenic mice was approximately 82.43 seconds (P < 0.05), indicating a shorter resting time for circHERC1 transgenic mice. Figure 9 Tail suspension tests showed that the climbing time for male control mice was approximately 17.07 seconds, while the swimming time for circHERC1 transgenic mice was approximately 52.95 seconds; the climbing time for female control mice was approximately 23.24 seconds, while the climbing time for circHERC1 transgenic mice was approximately 62.61 seconds (P < 0.05). Transgenic mice exhibited significantly greater climbing ability. Figure 10 ).

[0042] 2. Enhanced cognitive function in transgenic mice New Object Recognition Test: Mice were initially acclimatized to the laboratory environment to minimize external influences. The test was conducted in a closed square or rectangular container (40×40×40 cm) with a transparent observation window and soft ambient lighting. Video recording equipment captured the animals' behavior. Mice explored the test chamber for 5 minutes to acclimatize. 24 hours later, two identical objects were placed 5 cm from the chamber wall. The mice were then allowed to explore the objects for 10 minutes or 20 seconds with their backs to the objects. The next day, one object was replaced with a new object, and exploration continued for 10 minutes. The analysis focused on the exploration time and frequency of each object; longer exploration time or a discrimination rate higher than 0.5 for the new object indicated normal memory and object recognition.

[0043] The novel object recognition test showed that the control group mice had approximately 9 encounters with novel objects, while the circHERC1 transgenic mice had approximately 12 encounters (P < 0.05), revealing that the treated mice had better memory and exploration abilities, such as a higher recognition index and more frequent encounters with novel objects. Figure 11 ).

[0044] Morris Water Maze (MWM): This apparatus consists of a large circular pool, 1.5-2 meters in diameter, filled with water to a depth of 25-30 centimeters, maintained at a temperature of 20-24 degrees Celsius. A hidden platform made of transparent acrylic is submerged in one of the four quadrants. External visual cues provide spatial clues to help the mice orient themselves. Pre-training is conducted to acclimate the mice to the task and minimize stress. During pre-training, the mice are briefly placed in the maze without the platform to familiarize themselves with the environment. The initial phase includes a hidden platform task, where the mice learn to locate the platform using spatial cues. The mice are placed at different starting positions and given a fixed time limit (60 seconds) to find the platform over five consecutive days, with four trials per day. Decreases in delay and path length indicate successful learning. A probing test assesses memory retention by removing the platform and allowing 60 seconds of free swimming. Key metrics, such as time spent in the target area and the number of times the platform's previous position is crossed, are recorded. Video tracking systems capture animal movements and perform statistical analysis, typically using repeated measures ANOVA, to assess differences between groups or changes in performance during an experiment.

[0045] The results of the Morris water maze test showed that the control group mice crossed the escape platform approximately twice, while the circHERC1 transgenic mice crossed the platform approximately four times (P < 0.05). The transgenic mice crossed the platform more frequently. Figure 12The platform crossing distance for male control mice was approximately 316.18 mm, while that for circHERC1 transgenic mice was approximately 669.132 mm; for female control mice, the platform crossing distance was approximately 219.495 mm, while that for circHERC1 transgenic mice was approximately 602.71 mm (P < 0.05). The platform crossing distance was significantly increased in transgenic mice. Figure 13 The time it took for control mice to reach the plateau was approximately twice that of circHERC1 transgenic mice (P < 0.05), while the time for transgenic mice to reach the plateau was significantly shorter. Figure 14 This suggests that transgenic mice have stronger memory and learning abilities.

[0046] Example 3: Intervention of mice using adeno-associated virus loaded with human circHERC1 Adeno-associated virus (AAV) was used to load circHERC1 and control vectors. The viral vectors were either ordinary AAV vectors or AAV-circHERC1. The AAV virus particles were diluted to the required concentration with phosphate-buffered saline (PBS). Fourteen-month-old mice (at this age, mice exhibit significant declines in memory, learning ability, and cognitive ability) were selected, with half being male and half female. After a single intravenous injection, the mice were fed normally for three months, and then their depression, anxiety levels, learning and memory abilities were assessed.

[0047] 1. Mice were treated with tail vein injection of AAVcircHERC1.

[0048] The expression of circHERC1 in various organs of mice was detected after injection. To study the systemic effects of circHERC1, AAV vectors carrying circHERC1 were administered to mice, and the AAV vectors were injected with the AAV vectors. circHERC1 and AAV control Mice were administered the drug via tail vein. Three months later, the expression levels of circHERC1 in various organs of the mice were detected using real-time PCR, following the same experimental procedures as above.

[0049] qPCR showed AAV control In the control group of mice, the expression levels were approximately 6.32 and 3.23 in the heart of male and female mice, approximately 7.01 and 3.21 in the lungs, approximately 19.54 and 6.69 in the liver, 5.41 and 1.98 in the spleen, 4.61 and 3.11 in the kidneys, 4.53 and 2.41 in the stomach, approximately 2.98 and 2.51 in the intestines, and approximately 5.11 and 1.23 in the reproductive organs, indicating that circHERC1 is strongly expressed in different organs. Figure 15circHERC1 is expressed at the highest levels in metabolically active organs such as the liver, intestines, and kidneys, while intermediate levels are detected in the heart and spleen.

[0050] 2. Tail vein injection of AAV circHERC1 Antidepressant effect of intervention in mice To assess the emotional state and cognitive abilities of mice, we used six classic behavioral tests: Morris water maze (MWM), novel object recognition test, suspended tail test (TST), elevated cross maze (EPM), forced swimming test (FST), and open field test.

[0051] (1) AAV circHERC1 The anxiety behavior of mice was reduced after injection. AAV was verified through open field experiments, elevated cross maze experiments, and tail suspension experiments. circHERC1 The effects of injection on mood and anxiety in mice were investigated. We then evaluated the effects of circHERC1 overexpression on behavior and cognitive performance.

[0052] In open field testing, AAV circHERC1 The treated mice exhibited more exploratory behavior, while the center distance of the control group mice was approximately 10.27%, and AAV... circHERC1 The central distance of the treated mice was approximately 16.07% (P < 0.05), which was characterized by AAV. circHERC1 The treated mice traveled a longer distance. Figure 16 The time spent at rest in the center of the arena was approximately 91.99 seconds for male control mice, according to AAV. circHERC1 The resting time of the treated mice was approximately 29.64 seconds; the resting time of the female control mice was approximately 70.17 seconds, AAV circHERC1 The resting time of the treated mice was approximately 40.46 seconds (P < 0.05), indicating that AAV... circHERC1 The treated mice spent less time in the center of the arena. Figure 17 These behavioral changes are consistent with enhanced exploratory behavior and are generally associated with better physical and emotional health. In the elevated cross maze, AAV... circHERC1 The treated mice exhibited increased exploratory behavior, while the control group mice spent approximately 28.52 seconds in open areas. circHERC1 The treated mice spent approximately 107.45 seconds in the open region (P < 0.05), AAV circHERC1 The treated mice spent more time in the open area. Figure 18 The control group mice had a distance of approximately 13.60% in the open area, and AAV... circHERC1 The treated mice spent approximately 32.64% of their time in the open region (P < 0.05), AAVcircHERC1 The treated mice also moved a greater distance in the open area. Figure 19 This indicates a reduction in anxiety-like behaviors. The tail suspension test showed that the climbing time for control mice was approximately 92.98 seconds, and AAV... circHERC1 The climbing time of the treated mice was approximately 144.10 seconds (P < 0.05), AAV circHERC1 The treated mice had the ability to climb for extended periods of time. Figure 20 The resting time of the control group mice was approximately 137.89 seconds, and the AAV... circHERC1 The resting time of the treated mice was approximately 91.46 seconds (P < 0.05), AAV circHERC1 The treated mice had a shorter resting time. Figure 21 ).

[0053] (2) AAV circHERC1 The mice's cognitive abilities were enhanced after injection. The novel object recognition test revealed that the treated mice had better memory, as shown by the novel object recognition index, which was approximately 19.01% higher in the control group than in the AAV group. circHERC1 Approximately 47.44% of the treated mice (P < 0.05) had AAV. circHERC1 The treated mice showed a higher recognition index for new objects. Figure 22 ); The number of new object explorations for control mice was approximately 7 times, and for AAV... circHERC1 The mice were treated approximately 14 times (P < 0.05), AAV circHERC1 The treated mice explored more times ( Figure 23 The exploration distance for the control group mice was approximately 815.94 mm, and the AAV... circHERC1 The treated mice were approximately 2201.47 mm in length (P < 0.05), and AAV... circHERC1 The treated mice had a longer exploration distance for new objects. Figure 24 ).

[0054] The results of the Morris water maze test showed that the male control group mice traversed a distance of 121.53 mm across the platform, and the AAV... circHERC1 The treated mice traversed a platform a distance of 563.02 mm, while the female control group mice traversed a distance of 209.65 mm. (AAV) circHERC1 The distance the treated mice traveled across the platform was 431.64 mm (P < 0.05), while the control mice traveled approximately 13.60% of the distance in the open area. circHERC1 The treated mice spent approximately 32.64% of their time in the open region (P < 0.05), AAV circHERC1 The treated mice also moved a greater distance in the open area, and AAV circHERC1The treated mice showed a stronger ability to escape. Figure 25 Meanwhile, the recognition time for female and male mice was significantly shortened. Figures 26-27 ), prompt AAV circHERC1 The treated mice exhibited enhanced memory and learning abilities.

[0055] Example 4. Tail vein injection of EV circHERC1 Intervention in depressive states in mice Mice were injected with exosomes loaded with circherc1 and a control vector. Fourteen-month-old mice, half male and half female, were selected and injected with 1×10⁻⁶ oz. every other day. 11 Exosomes were continuously injected into mice for 3 months. After 3 months of normal feeding, the mice were tested for depression, anxiety, learning and memory abilities, etc.

[0056] 1. Tail vein injection of EV circHERC1 Intervention in mice.

[0057] Exosomes from 293FT cells overexpressing circHERC1 were validated. To investigate the therapeutic potential of extracellular vesicles (EVs) overexpressing circHERC1 in alleviating cognitive-related diseases, 293FT cells were transfected with the pLC5-circHERC1 overexpression vector. Culture supernatants from both 293FT cells and those overexpressing circHERC1 were collected. First, intact cells were removed by centrifugation at 300×g for 10 min, and the supernatant was transferred to new centrifuge tubes. Then, cell debris was removed by centrifugation at 2000×g for 10 min. The clarified supernatant was transferred to clean sample vials, and EVs were isolated using a high-performance exosome isolation system (EXODUS H-600, Huixin Life Science & Technology). EVs were recovered from the chip using 1 mL of phosphate-buffered saline (PBS). TEM imaging confirmed that the isolated EVs exhibited typical exosome morphology. Figure 28 Western blot (protein blotting) Figure 29 ) and NTA ( Figure 30 Further confirmation that EVs were rich in exogenous markers (ALIX, TSG101, CD9) and had an appropriate size distribution. Furthermore, as shown by quantitative PCR, circHERC1 was confirmed to be present in EVs. Figure 31 ).

[0058] 2. Tail vein injection of EV circHERC1 Intervene in the anxiety state and behavioral cognitive abilities of mice.

[0059] EV injected via tail vein circHERC1The treated mice showed improved performance in several behavioral tests. In terms of walking distance, the central distance of the PBS-injected group was approximately 8.37%, the central distance of the exosome-injected group was approximately 11.94%, and the central distance of the EV-injected group was... circHERC1 The center distance in the mouse group was approximately 18.94% (P < 0.05), after injection of EVs. circHERC1 The mice traveled a longer distance. Figure 32 The time spent at the center of the arena was approximately 76.30 seconds for mice injected with PBS, approximately 55.48 seconds for mice injected with exosomes, and approximately 56.30 seconds for mice injected with EVs. circHERC1 The resting time in the mouse group was approximately 47.66 seconds (P < 0.05). After injection of EV... circHERC1 The mice spent less time in the center of the arena. Figure 33 In the open area movement distance, mice injected with PBS covered approximately 7.68% of the distance, mice injected with exosomes covered approximately 7.80%, and mice injected with EVs covered approximately 7.80%. circHERC1 The mice's distance in the open area was approximately 21.98% (P < 0.05), indicating that EV injection... circHERC1 Mice travel longer distances in open areas ( Figure 34 In terms of the number of times mice entered the open area, male and female mice injected with PBS entered the open area approximately twice each; male and female mice injected with exosomes entered the open area approximately three and two times respectively; and mice injected with EVs entered the open area approximately twice each. circHERC1 Male and female mice entered the open area approximately 5 and 3 times, respectively (P < 0.05), indicating that the injection of EVs was effective. circHERC1 Mice entered open areas more frequently. Figure 35 In terms of entry time into the open region, mice injected with PBS entered the open region in approximately 14.23 seconds, mice injected with exosomes entered the open region in approximately 13.52 seconds, and mice injected with EVs entered the open region in approximately 13.52 seconds. circHERC1 The time it took for mice to enter the open area was approximately 51.18 seconds (P < 0.05), indicating that the injection of EVs... circHERC1 Mice took longer to enter the open area ( Figure 36 Tail suspension tests showed that the climbing time of mice injected with PBS was approximately 85.62 seconds, while that of mice injected with exosomes was approximately 124.28 seconds. The climbing time of mice injected with EVs was... circHERC1 The climbing time for mice was approximately 134.79 seconds (P < 0.05). Injection of EVs... circHERC1 Mice have the ability to climb for extended periods of time. Figure 37 These results indicate that increased physical activity, reduced anxiety, and improved cognitive function.

[0060] In behavioral testing, EV circHERC1In the Morris water maze test, the distances from the PBS group to the platforms of male and female mice were approximately 69.27 mm and 95.53 mm, respectively. The distances from the platforms of exosome-injected mice were approximately 120.32 mm and 381.51 mm, respectively. The distances from the EV-injected mice were also approximately... circHERC1 Mice crossed the platform at distances of approximately 339.7 mm from males and 460.50 mm from females (P < 0.05), showing a significant improvement in performance. Figure 38 Compared with PBS and EV controls, males and females injected with EVs... circHERC1 Mice have a faster escape latency. Figures 39-40 ).

[0061] The results showed that circHERC1 significantly enhanced cognitive abilities in mice, reduced depression and anxiety levels, improved exploratory, memory, and learning abilities, and improved multiple aspects of mouse health, with better exosome delivery. In conclusion, our findings suggest that circHERC1 delivery represents a promising therapeutic strategy for improving cognitive abilities in mice, potentially providing a novel intervention for cognitive-related diseases.

Claims

1. The application of circRNA molecules in the preparation of antidepressant drugs, characterized in that, The sequence of the circRNA molecule is shown as bases 21-1163 of SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application is to prepare an antidepressant from a substance that overexpresses the circRNA molecule.

3. The application according to claim 2, characterized in that, The substance that overexpresses the circRNA molecule is a vector that overexpresses the circRNA molecule.

4. The application according to claim 3, characterized in that, The vector overexpressing the circRNA molecule is an adeno-associated virus vector.

5. The application according to claim 2, characterized in that, The substance that overexpresses the circRNA molecule is an exosome containing the circRNA molecule.

6. The application according to claim 2, characterized in that, The substance that overexpresses the circRNA molecule is a cell that secretes exosomes containing the circRNA molecule.

7. The application according to claim 1, characterized in that, The antidepressant mentioned refers to medication that relieves anxiety.

8. The application according to claim 1, characterized in that, The antidepressant mentioned refers to medication used to treat cognitive impairment.

9. The application of circRNA molecules as diagnostic biomarkers in diagnostic kits for depression, characterized in that, The sequence of the circRNA molecule is shown as bases 21-1163 of SEQ ID NO.1, and the kit contains reagents for detecting the content or expression level of the circRNA molecule in the sample to be tested.

10. The application according to claim 9, characterized in that, The depression diagnostic kit is a gene testing kit.

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