Use of a dancre inhibitor in the preparation of a medicament for the treatment of depression

Abstract

This invention relates to the application of lncRNA-DANCR inhibitors in the preparation of drugs for treating depression. Extensive research has revealed that lncRNA DANCR is a key factor contributing to and promoting the development of depression. Inhibiting DANCR expression levels with inhibitors effectively promotes nerve cell proliferation and inhibits nerve cell apoptosis. Simultaneously, DANCR plays a crucial role in inflammation, especially neuroinflammatory inflammation and its related inflammatory factors. Results show that inhibiting DANCR expression significantly suppresses the levels of inflammatory factors such as TNF-α, IL-6, IL-2, IL-1β, IFN-γ, and G-CSF in vivo, thereby effectively treating neuroinflammatory inflammation and preventing further progression of depression. Mechanistic studies have revealed a significant interaction between lncRNA DANCR and DDX3X. LncRNA DANCR protects DDX3X protein, slowing its degradation, promoting DDX3X stability, and increasing DDX3X protein abundance, thereby mediating DDX3X protein-mediated inflammasome activation.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of LncRNA-DANCR inhibitors in the preparation of drugs for treating depression. Background Technology

[0002] Depression is a chronic, relapsing illness, a type of depressive disorder. Its main symptoms include low mood, loss of interest, slowed thinking, impaired attention and memory, negative self-esteem, decreased appetite, and reduced activity. The prevalence of depression is on the rise; it is currently the fourth leading cause of disease worldwide. Conservative estimates suggest there are 350 million people suffering from depression globally, and 10-15% of those with depression eventually die by suicide.

[0003] To date, the etiology of depression remains unclear. Research indicates that numerous biological, psychological, and social environmental factors may contribute to its pathogenesis. Biological factors primarily involve genetics, neurobiochemistry, neuroendocrinology, and neuroregeneration. A psychological predisposition closely related to depression is premorbid personality traits, such as depressive temperament. Stressful life events in adulthood are significant triggers for clinically significant depressive episodes. However, these factors do not act in isolation; the interaction between genetics and environment or stress factors, and the timing of this interaction, plays a crucial role in the development of depression. The complex and difficult-to-identify causes of depression make its treatment extremely challenging. Therefore, identifying biomarkers closely related to depression for targeted treatment has become a critical and urgent problem to be solved. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the application of lncRNA-DANCR inhibitors in the preparation of drugs for treating depression. This invention, through the construction of an in vitro model of depression, demonstrates that lncRNA DANCR is one of the key factors leading to and promoting the development of depression. By inhibiting DANCR expression, it is possible to effectively promote nerve cell proliferation and inhibit nerve cell apoptosis. Simultaneously, it can significantly inhibit the levels of inflammatory factors such as TNF-α, IL-6, IL-2, IL-1β, IFN-γ, and G-CSF in vivo, thereby effectively treating neuroinflammatory conditions and preventing the further development of depression.

[0005] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0006] The first aspect of this invention provides the use of LncRNA DANCR inhibitors in the preparation of medicaments for the prevention and / or treatment of depression.

[0007] It should be understood that, unless otherwise specified, in the context of this invention, the LncRNAs and DANCRs listed are LncRNA types known in the art, and those skilled in the art are capable of obtaining molecular biological information related to LncRNAs and DANCRs, such as sequences, from conventional databases. The terms "LncRNA DANCR," "LncRNA-DANCR," and "DANCR" all refer to the same thing, and are simplified expressions for long non-coding RNA-DANCR. The LncRNA DANCR inhibitor refers to a substance that can specifically downregulate and / or inhibit the expression level of LncRNA DANCRs and / or the activity of their mature LncRNAs in cells and / or tissues, such as siRNA, sgRNA, shRNA, etc., as long as it can achieve the knockdown and / or knockout of LncRNA DANCRs. The LncRNA DANCR inhibitor can be purchased or synthesized commercially available according to experimental needs, or it can be designed and synthesized based on the LncRNA DANCR sequence.

[0008] Preferably, the LncRNA DANCR inhibitor is selected from siRNA (siDANCR) designed based on the LncRNA DANCR gene.

[0009] Preferably, the siRNA sequence designed based on the LncRNA DANCR gene is as shown in SEQ ID NO: 1, which is 5'-GGTGAAGTAGTCGCACATTTATT-3'(sense); correspondingly, its antisense sequence is as shown in SEQ ID NO: 2, which is 5'-TAAATGTGCGACTACTTCACCTT-3'(antisense).

[0010] The second aspect of this invention provides the use of LncRNA DANCR inhibitors in the preparation of medicaments for treating neuroinflammatory diseases and / or improving physiological indicators related to neuroinflammatory diseases.

[0011] Preferably, the LncRNA DANCR inhibitor is selected from siRNA (siDANCR) designed based on the LncRNA DANCR gene.

[0012] Preferably, the siRNA sequence designed based on the LncRNA DANCR gene is as shown in SEQ ID NO: 1, which is 5'-GGTGAAGTAGTCGCACATTTATT-3'(sense); correspondingly, its antisense sequence is as shown in SEQ ID NO: 2, which is 5'-TAAATGTGCGACTACTTCACCTT-3'(antisense).

[0013] Preferably, the physiological indicators related to neuroinflammatory disorders are selected from one or more of TNF-α, IL-6, IL-2, IL-1β, IFN-γ, G-CSF, and DDX3X.

[0014] A third aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of depression, comprising an LncRNA DANCR inhibitor and pharmaceutically acceptable excipients.

[0015] Preferably, the LncRNA DANCR inhibitor is selected from siRNA (siDANCR) designed based on the LncRNA DANCR gene.

[0016] Preferably, the siRNA sequence designed based on the LncRNA DANCR gene is as shown in SEQ ID NO: 1, which is 5'-GGTGAAGTAGTCGCACATTTATT-3'(sense); correspondingly, its antisense sequence is as shown in SEQ ID NO: 2, which is 5'-TAAATGTGCGACTACTTCACCTT-3'(antisense).

[0017] Preferably, the pharmaceutically acceptable excipient is selected from one or more of solvents, solubilizers, wetting agents, antioxidants, antibacterial agents, chelating agents, and surfactants.

[0018] Inflammation is a defensive response of tissues to biological, physical, and chemical damaging factors. The immune response in the brain is known as neuroinflammation. Neuroinflammation acts as a double-edged sword in the central nervous system; on the one hand, it can maintain homeostasis; on the other hand, an overreaction can damage normal tissues and organs. There are generally two pathways that trigger neuroinflammation. First, some peripheral cytokines can cross the blood-brain barrier and enter the brain, causing central nervous system inflammation. Second, the central nervous system's own immune system can also cause an inflammatory response. Microglia in the central nervous system function similarly to peripheral macrophages. When the brain is stimulated by external factors such as trauma or infection, microglia are activated, secreting large amounts of pro-inflammatory cytokines, chemokines, and reactive oxygen species. Excessive activation of inflammation can cause pathological damage. In the classical inflammasome pathway, when the formation of the NLRP3 inflammasome activates Caspase-1, it transforms chronic stress into an inflammatory response, causing inflammatory damage to the brain and leading to neurological diseases.

[0019] DDX3 is a member of the DEAD (Asp-Glu-Ala-Asp)-box helicase family and is involved in almost all aspects of eukaryotic RNA metabolism. The human genome encodes two types of DDX3 genes: DDX3X and its homolog DDX3Y. DEAD-box RNAhelicase 3 (DDX3X), as a highly conserved member of the DEAD-box protein family, is a key RNA-binding protein and transcriptional cofactor with multiple functions, including immune regulation, viral manipulation, and tumor progression. Studies have found that the activation of the NLRP3 inflammasome is strongly inhibited in the absence of DDX3X; therefore, reducing DDX3X expression is an important pathway to inhibiting NLRP3 activation.

[0020] The present invention has the following advantages over the prior art:

[0021] (1) This invention has demonstrated through extensive research that lncRNA DANCR plays an important role in depression and neuroinflammatory disorders. LncRNA DANCR can inhibit the proliferation of nerve cells and promote their apoptosis. Furthermore, inhibiting the expression of lncRNA DANCR using inhibitors can significantly promote nerve cell proliferation and inhibit apoptosis. This provides a new therapeutic target for the treatment of depression and offers novel ideas and strategies for the development of drugs for the diagnosis and treatment of depression.

[0022] (2) This invention further investigates the mechanism of action of LncRNA DANCR, finding that LncRNA DANCR is mainly located in the cell nucleus and has a significant interaction with DDX3X. It can protect DDX3X protein, slow its degradation, promote DDX3X stability, and increase the abundance of DDX3X protein, thereby mediating DDX3X protein to promote inflammasome activation. Inhibiting the expression level of LncRNA DANCR helps to reduce the expression level of DDX3X protein in vivo, inhibit the levels of inflammatory factors such as TNF-α, IL-6, IL-2, IL-1β, IFN-γ, and G-CSF, thereby inhibiting the inflammatory response. This plays an important role in alleviating and treating depression and preventing its further development. Attached Figure Description

[0023] Figure 1 This is a schematic diagram showing the expression levels of LncRNA DANCR in rats in the depression model group and the normal group.

[0024] Figure 2 This is a schematic diagram showing the effect of different concentrations of CORT on PC12 cell proliferation.

[0025] Figure 3 This is a schematic diagram showing the effect of siDANCR on PC12 cell proliferation.

[0026] Figure 4 This is a schematic diagram showing the effect of siDANCR on apoptosis in PC12 cells.

[0027] Figure 5 This is a schematic diagram showing the effect of siDANCR on the expression of intracellular inflammatory factors such as TNF-α, IL-6, IL-2, IL-1β, IFN-γ, and G-CSF.

[0028] Figure 6 This is a schematic diagram of the results of the cell nucleus-cytoplasm separation experiment.

[0029] Figure 7 This is a schematic diagram of the results of a fluorescence in situ hybridization experiment.

[0030] Figure 8 This is a schematic diagram of the Pull-down experiment results.

[0031] Figure 9 This is a schematic diagram of the results of an RNA-binding protein immunoprecipitation experiment.

[0032] Figure 10 This is a schematic diagram of the results from a Western blot experiment. Detailed Implementation

[0033] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] Unless otherwise specified, all cell lines listed in this invention, including PC12, were cultured using conventional methods. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan) and their presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). They were also cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available. The experimental methods used in this invention, such as cell culture, nucleic acid extraction, genome sequencing, primer design, PCR, cell transfection, fluorescence staining, animal experiments, cell proliferation experiments, and apoptosis experiments, are all conventional methods and techniques in the field. For animal experiments, the experimental protocols were approved by the Animal Experiment Ethics Committee of Jinan University and followed its guidelines.

[0035] Representative results from biological experiments were selected from replicates and presented in the contextual figures. Data were displayed as mean ± SD and mean ± SEM as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 8.0 or SPSS 22.0 software. Standard medical statistical methods such as t-tests, chi-square tests, and ANOVA were used to compare differences in means between two or more groups. p < 0.05 was considered statistically significant.

[0036] Example 1

[0037] Female rats weighing approximately 250g were selected and provided with free access to water and pelleted feed. The rats were kept in an acclimatization room with alternating 12h / 12h light and humidity maintained at 60-75% and a temperature of 22-25℃ for one week. Subsequently, the rats were randomly divided into two groups of 10 each, designated as Group 1 and Group 2. Group 1 was kept normally without any treatment; Group 2 was used to establish a Chronic Unpredictable Mild Stress (CUMS) depression model, specifically as follows: Various stressors were randomly administered over a weekly cycle for 6 weeks: 16h water deprivation, 16h fasting, 1h empty bottle, 8h cage tilting (45 degrees), 16h overcrowding (8-10 rats per cage), 24h continuous lighting, 8h wet bedding, 5min heat stimulation (45℃), and 5min cold stimulation (4℃). Each day, the rats received a different stressor, with no repetition of the same stressor for two consecutive days to minimize the predictability of each stressor. Subsequently, whole-transcriptome sequencing was used to analyze the expression profiles of the cerebral cortex of rats in groups 1 and 2, and differentially expressed lncRNAs were screened. The results showed that the expression of 20 lncRNAs differed significantly between the depression model group and the normal group, of which 12 lncRNAs were significantly upregulated and 8 lncRNAs were downregulated. The expression difference of lncRNA DANCR was the most significant (see [link to original text]). Figure 1 ).

[0038] Subsequently, corticosterone was used to construct an in vitro model of depression. The specific steps are as follows:

[0039] (1) Rat adrenal medullary pheochromocytoma differentiated cell line (PC12 cells) were cultured in RPMI-1640 (Hyclone) medium containing 10% fetal bovine serum for 24 h under the following conditions: 5% CO2, 37℃.

[0040] (2) Dissolve corticosterone (CORT, MCE) in DMSO, filter to remove bacteria, and add serum-free RPMI-1640 medium to make the CORT concentrations 0 μM, 20 μM, 50 μM, 100 μM and 200 μM respectively.

[0041] (3) After the cells have filled the bottom of the well, the upper cell solution is removed, and serum-free RPMI-1640 medium containing different concentrations of CORT prepared in step (2) is added. The cells are then cultured in a 5% CO2, 37℃ cell culture incubator for 24 hours. CCK8 solution is then added, and the cells are incubated for another 2 hours. The absorbance is measured at 450 nm using an ELISA reader to observe the effect of CORT on the proliferation of PC12 cells.

[0042] The results are as follows Figure 2As shown in the figure. The results showed that adding different concentrations of CORT to PC12 cells reduced cell viability. Compared with the untreated control group, treatment with CORT at concentrations of 20 μM, 50 μM, 100 μM, and 200 μM significantly inhibited cell proliferation, with statistically significant differences. The viability of PC12 cells decreased to the lowest level at a CORT concentration of 200 μM, and this concentration will be used for subsequent cell treatment. CORT can significantly inhibit the proliferation of PC12 cells, and this inhibitory ability is positively correlated with the CORT concentration, thus confirming the successful establishment of an in vitro depression model using CORT.

[0043] Furthermore, the role of lncRNA DANCR in depression was clarified by inhibiting its expression, and the optimal treatment time for CORT was determined. The specific steps are as follows:

[0044] (1) PC12 cells were placed in 6-well plates and cultured overnight under the following conditions: 5% CO2, 37°C, and RPMI-1640 medium containing 10% fetal bovine serum.

[0045] (2) After discarding the cell culture medium, add 1 mL of Opti-MEM medium, and then add siDANCR (sense sequence as shown in SEQ ID NO: 1, 5'-GGTGAAGTAGTCGCACATTTATT-3', antisense sequence as shown in SEQ ID NO: 2, 5'-TAAATGTGCGACTACTTCACCTT-3', the same below) into the cells and incubate in a 5% CO2, 37℃ cell culture incubator for 6 h.

[0046] (3) Discard the Opti-MEM medium, add RPMI-1640 medium containing 2 mL of 10% fetal bovine serum, and incubate in a 5% CO2, 37℃ cell culture incubator for 24 h.

[0047] (4) After digesting the cells with trypsin, centrifuging and resuspending them to a cell concentration of 80,000 cells / mL, the cells were then seeded into 96-well plates (100 μL / well).

[0048] (5) Add CORT (200 μM) to the cells for treatment. After 24 h and 48 h, add 10 μL of CCK8 solution to each well and continue incubation for 2 h. Detect the absorbance at 450 nm using an ELISA reader.

[0049] The results are as follows Figure 3As shown in the figure. Results showed that after 24 hours, cell viability was significantly increased in both the Control group and the si-DANCR+200μM CORT group compared to the 200μM CORT group. However, after 48 hours, cell viability was significantly increased in the Control group compared to the Control+200μM CORT group, but not in the si-DANCR+200μM CORT group. Subsequent treatments will use 200μM for 24 hours (compared to the control group, *p<0.05, **p<0.01, ***p<0.001; compared to the si-DANCR group, #p<0.05, ##p<0.01, ###p<0.001).

[0050] Furthermore, the effect of lncRNA DANCR on CORT-induced apoptosis in PC12 cells was investigated, and the specific steps are as follows:

[0051] (1) PC12 cells were divided into three groups, designated as Group 1 to Group 3. Group 1 was the blank control group, transfected with the blank vector siNC and without CORT treatment; Group 2 was the negative control group, transfected with the blank vector siNC and treated with 200 μM CORT in the same manner as above; Group 3 was the experimental group, transfected with siDANCR and treated with 200 μM CORT in the same manner as above. After the cells reached the logarithmic growth phase, they were digested with trypsin, counted, and seeded into 96-well plates.

[0052] (2) Cells were cultured in a 5% CO2 incubator at 37°C for 24 hours. Cells were then collected in 10mL centrifuge tubes, with each sample containing 1×10⁻⁶ cells. 5 -1×10 6 Collect samples per mL, centrifuge at 1000 rpm for 5 min, and discard the culture medium.

[0053] (3) Wash once with PBS buffer and centrifuge at 1000 rpm for 5 min.

[0054] (4) Resuspend the cells in 100 μL of labeling buffer and incubate at room temperature in the dark for 15 min.

[0055] (5) Centrifuge at 1000 rpm for 5 min, and rinse the precipitated cells once with PBS.

[0056] (6) Add 5 μL Annexin V-APC and 10 μL 7AAD, gently vortex to mix, and incubate at room temperature in the dark for 15 min.

[0057] (7) Add 485 μL of pre-cooled 1× Binding Buffer to each tube for resuspending and mixing, and perform detection using a flow cytometer. Excitation peak at 488 nm, emission peak at 575 nm. Flow cytometry was used to detect cell fluorescence, and NovoExpress was used to analyze the data (x-axis: Annexin V-APC; y-axis: 7AAD).

[0058] The results are as follows Figure 4 As shown, flow cytometry was used to detect the apoptosis rate in the CORT group and the control group, and the proportion of apoptotic cells, including early and late apoptotic cells, was statistically analyzed. The results showed that the apoptosis rate in the Control-si-NC group was lower than that in the CORT-si-NC group, and the apoptosis rate in the CORT-si-DANCR group was also lower than that in the CORT-si-NC group, with statistically significant differences. These results suggest that DANCR induces neuronal apoptosis, and inhibiting DANCR expression can significantly reduce neuronal apoptosis.

[0059] Example 2

[0060] The foregoing examples demonstrated that inhibiting the expression of LncRNA-DANCR helps promote CORT-induced PC12 cell proliferation while simultaneously inhibiting cell apoptosis. Numerous studies have shown that inflammation, especially neuroinflammatory inflammation, is a key factor contributing to and exacerbating depression. Therefore, this example further investigates the anti-inflammatory effect of LncRNA-DANCR, with the specific steps as follows:

[0061] (1) Coating process: Dilute the antigen to an appropriate concentration with coating diluent (generally the required amount of antigen to be coated is 20-200 μg per well), add 100 μL of antigen to each well, place at 37℃ for 4 hours and discard the liquid in the well.

[0062] (2) Blocking enzyme-labeled reaction wells: 5% fetal bovine serum was placed at 37°C for 40 min to block; during blocking, the blocking solution was filled into each reaction well and the air bubbles in each well were removed. After blocking, the wells were washed with washing solution for 3 times, each time for 3 min; washing method: aspirate the reaction solution in the well, fill the well with washing solution, let it stand for 2 min and shake it slightly, aspirate the liquid in the well, pour out the liquid and pat dry on absorbent paper.

[0063] (3) Add the cell sample to be tested: Collect PC12 cells in a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm), then collect the supernatant. Dilute the cell suspension with PBS (pH 7.2-7.4) to a cell concentration of 1×10⁻⁶. 6Approximately 100 μL / mL. After repeated freeze-thaw cycles, centrifuge for about 20 minutes (2000-3000 rpm), and add the supernatant to the enzyme-labeled reaction wells. Add at least two wells per sample, 100 μL per well, and incubate at 37°C for 40-60 minutes. Wash the wells three times with washing buffer, 3 minutes each time.

[0064] (4) Add enzyme-labeled antibody: Add enzyme-labeled antibody according to the reference working dilution provided by the enzyme conjugate provider, incubate at 37°C for 30-60 min, add 100 μL to each well; wash as before.

[0065] (5) Adding substrate solution: TMB-hydrogen peroxide urea solution is preferred, followed by OPD-hydrogen peroxide substrate solution system; Substrate addition amount: 100 μL per well, place at 37℃ in the dark for 3-5 minutes, add 50 μL of stop solution to each well for color development, and measure the experimental results within 20 minutes.

[0066] Test results as follows Figure 5 As shown in the figure, the results indicated that compared with the control group, the CORT group showed significantly increased expression levels of inflammatory factors such as TNF-α, IL-6, IL-2, IL-1β, IFN-γ, and G-CSF. However, inhibiting the expression of intracellular LncRNA-DANCR using siDANCR significantly reduced the expression of inflammatory factors such as TNF-α, IL-6, IL-2, IL-1β, IFN-γ, and G-CSF (compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the CORT group, #p<0.05, ##p<0.01, ###p<0.001). Therefore, inhibiting the expression of LncRNA-DANCR can significantly suppress the levels of inflammatory factors in vivo, effectively treating neuroinflammatory diseases and preventing the further development of depression.

[0067] To clarify the mechanism of action of LncRNA DANCR in depression and inflammation, we investigated its intracellular location using nucleocytoplasmic separation and fluorescence in situ hybridization (FISH) experiments.

[0068] The steps for the cell nucleus-cytoplasm separation experiment are as follows: Using the Paris™ Kit cell nucleus-cytoplasm separation kit (Invitrogen), the cell lysate was separated into two parts: the nucleus and the cytoplasm. RNA was extracted using the TRAZOL method, and 1 μg of RNA was reverse transcribed into cDNA. The expression of related genes was detected by qRT-PCR (U6 RNA was used as the internal reference gene for nuclear RNA qRT-PCR, and GAPDH was used as the internal reference gene for cytoplasmic RNA qRT-PCR).

[0069] The steps for fluorescence in situ hybridization are as follows:

[0070] (1) PC12 cells were digested with 0.25% trypsin solution to prepare cell crawling slides, fixed with 4% paraformaldehyde for 20 min, and 0.3% Triton X-100 solution was prepared. 2 mL / well was incubated for 20 min, and prehybridization solution was prepared at 95℃ for 5 min. The cells were then rapidly cooled on ice, and prehybridization solution was added dropwise. The cells were then prehybridized at 42℃ for 2 h.

[0071] (2) Preparation of probe working solution: The probe concentration is 1 μg / μL. 1 μL probe + 99 μL probe dilution solution, incubate at 95℃ for 5 min, cool on ice, add probe working solution, and hybridize overnight at 42℃; preheat 2×SSC at 37℃, wash on a shaker for 2×5 min; preheat 0.5×SSC at 37℃, wash on a shaker for 15 min; preheat 0.2×SSC at 37℃, wash on a shaker for 15 min; add 100 μl DAPI working solution, incubate at room temperature in the dark for 4 min, rinse with PBS 3 times, 3 min each time; after slightly drying the sections, mount them with anti-fluorescence quenching mounting medium; observe the cell luminescence under a fluorescence microscope and take pictures.

[0072] Experimental results are as follows Figure 6-7 As shown. The relative expression levels of DANCR in the nucleus and cytoplasm of cells in the control group and CORT group were detected by the nucleus-cytoplasm separation experiment. U6 was used as the nuclear internal reference gene, and GAPDH was used as the cytoplasmic internal reference gene. The results showed that DANCR was mainly present in the nucleus in both the control group and the CORT group (*p<0.05, **p<0.01, ***p<0.001).

[0073] Similarly, fluorescence in situ hybridization was used to further confirm the localization of DANCR. The results showed that the DANCR fluorescent probe was distributed in both the nucleus and cytoplasm (scale bar = 20 μM). However, semi-quantitative analysis revealed that the fluorescence of DANCR and the nuclear marker DAPI overlapped significantly, suggesting that the subcellular localization of DANCR was mainly in the nucleus, consistent with the results of the nucleocytoplasmic separation experiment (*p < 0.05, **p < 0.01, ***p < 0.001).

[0074] LncRNAs in eukaryotes are diverse in type and function. Most lncRNAs are distributed in the nucleus and cytoplasm, while a few are found in organelles such as mitochondria and endoplasmic reticulum. These lncRNAs with different subcellular locations are endowed with different functions, but it remains unclear how cells sort different lncRNAs to specific subcellular compartments to exert their effects. LncRNAs located in the nucleus or chromosomes often regulate gene expression by influencing epigenetic modifications and transcriptional processes, and can also activate or inhibit target gene expression by directly binding to target genes. However, cytoplasmic lncRNAs are more likely to participate in translational regulation or function as ceRNAs by attaching to miRNAs. Tissue-specific lncRNAs located in specific organelles can participate in the functional and metabolic regulation of organelles. This invention uses FISH and nucleocytoma separation to detect the expression differences of DANCR in the cytoplasm and nucleus, finding that DANCR is mainly distributed in the nucleus, indicating that post-transcriptional or post-translational regulation is the main mechanism of epigenetic regulation by lncRNA DANCR.

[0075] Existing research has clearly established that stress granule proteins DDX3X and NLRP3 can interact to activate inflammasomes. Stress granule assembly leads to DDX3X retention, thereby inhibiting NLRP3 inflammasome activation. Stress granules and NLRP3 inflammasomes compete for DDX3X molecules to coordinate the activation of intrinsic responses under stress and subsequent cell fate determination. Macrophages utilize DDX3X availability to process stress signals, selecting between pro-survival stress granules and pyroptotic ASCs. To clarify the interaction between lncRNA-DANCR and DDX3X and NLRP3, pull-down assays, RNA-binding protein immunoprecipitation (RIP) assays, and Western blot experiments were performed. The specific steps of the pull-down and Western blot experiments are as follows:

[0076] (1) RNA pull-down assays were performed using the Magnetic RNA-Protein Pull-Down Kit (Axl-Bio) according to the manufacturer's instructions. The probe against DANCR was synthesized by Axl-Bio.

[0077] (2) The mixture of cell lysate and biotinylated RNA probe was hybridized at 4°C for 37 hours, and then the binding complex was recovered using streptavidin-coupled magnetic beads.

[0078] (3) Elute and purify the protein from the magnetic beads, and load it onto a 12% SDS-PAGE gel for electrophoretic separation.

[0079] (3) After electrophoresis, the membrane was transferred to a PVDF membrane and blocked with 5% skim milk powder for 2 hours. The membrane was then placed in a solution containing DDX3X primary antibody (Proteintech, 1:2000), NLRP3 primary antibody (Bioss, 1:2000), ASC primary antibody (Proteintech, 1:2000), Caspase-1 primary antibody (Bioss, 1:2000), and GAPDH primary antibody (Proteintech, 1:5000) and incubated overnight at 4°C.

[0080] (4) The PVDF membrane was washed with TBST, then bound with HRP secondary antibody (1:1000), incubated at room temperature for 1 h, and then developed with ECL luminescence kit and quantitatively analyzed with ImageJ software.

[0081] The RNA-binding protein immunoprecipitation assay was performed as follows: Following the manufacturer's instructions, the RIP assay was performed using the RNA-binding protein immunoprecipitation kit (Axl-Bio). Approximately 90% confluent cells were lysed using RIP lysis buffer containing RNase and protease inhibitors. Then, 100 μL of whole-cell extract was incubated with RIP buffer containing magnetic beads conjugated to specific antibodies. The negative control was normal rat anti-IgG antibody (Abcam), and the positive control was anti-DDX3X antibody (Proteintech).

[0082] Experimental results are as follows Figure 8-9 As shown in the figure. The results showed that by using the experimental group LncRNA DANCR probe and the control probe in the RNA pulldown experiment, the protein bound to DANCR was pulled out, eluted, and then silver stained. The RPD group protein silver staining pattern showed obvious bands, indicating that DANCR was bound to the protein. The RIP experiment, which used RT-qPCR to detect the expression level of DANCR in the RIP-RNA product, showed that the DANCR expression level in the IP group was significantly higher than that in the negative control IgG group, indicating that there is a binding between LncRNA DANCR and DDX3X (*p<0.05, **p<0.01, ***p<0.001). This proves that DDX3X is a key binding protein of LncRNA DANCR.

[0083] Western blot results showed that ( Figure 10When DANCR was overexpressed intracellularly (Control+oe-DANCR group), the levels of DDX3X and inflammasome proteins increased. In the CORT group, DANCR was knocked down using siDANCR (CORT+siDANCR group), resulting in a decrease in DDX3X and inflammasome protein levels (*p<0.05, **p<0.01, ***p<0.001). This indicates that lncRNA DANCR can protect DDX3X protein, slowing its degradation, promoting DDX3X stability, and increasing DDX3X protein abundance, thereby mediating DDX3X protein's promotion of inflammasome activation. Inhibiting the expression level of lncRNA DANCR helps reduce the expression level of DDX3X protein in vivo, thereby inhibiting the occurrence and further progression of the inflammatory response.

[0084] This invention, through extensive research, has discovered that lncRNA DANCR plays a crucial role in the development and progression of depression. Both in vivo and in vitro experiments have demonstrated that lncRNA DANCR is a key factor contributing to and promoting the development of depression. LncRNA DANCR not only inhibits nerve cell proliferation but also promotes nerve cell apoptosis. Furthermore, inhibiting the expression level of lncRNA DANCR using inhibitors effectively promotes nerve cell proliferation and inhibits nerve cell apoptosis. Simultaneously, lncRNA DANCR has a crucial influence on inflammation, especially neuroinflammatory inflammation and its related inflammatory factors. Experimental results show that inhibiting the expression of lncRNA-DANCR significantly suppresses the levels of inflammatory factors such as TNF-α, IL-6, IL-2, IL-1β, IFN-γ, and G-CSF in vivo, thereby effectively treating neuroinflammatory inflammation and preventing the further development of depression. In-depth research into its mechanism of action revealed that lncRNA DANCR is primarily located in the cell nucleus, and there is a significant interaction between lncRNA DANCR and DDX3X. LncRNA DANCR can protect DDX3X protein, slowing its degradation, promoting DDX3X stability, and increasing DDX3X protein abundance, thereby mediating DDX3X protein's promotion of inflammasome activation. Inhibiting the expression level of lncRNA DANCR helps reduce the expression level of DDX3X protein in vivo, thereby inhibiting the occurrence and further progression of the inflammatory response.

[0085] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. The use of LncRNA DANCR inhibitors in the preparation of drugs for the prevention and / or treatment of depression, characterized in that, The LncRNA DANCR inhibitor is selected from siRNAs designed based on the LncRNA DANCR gene, and the sequence of the siRNA designed based on the LncRNA DANCR gene is shown in SEQ ID NO: 1.

Images