Application of fam26f gene in diagnosis and treatment of depression

Abstract

This invention discloses the application of the FAM26F gene in the diagnosis and treatment of depression, belonging to the interdisciplinary field of molecular biology and neuropsychiatry. This invention reveals for the first time that downregulation of the FAM26F gene expression disrupts glutamate homeostasis by affecting the expression of glutamate transporters and receptors, thereby leading to depressive-like behaviors. Based on this, this invention provides two core applications: first, the application of the FAM26F gene in the preparation of diagnostic products for depression, achieving objective diagnosis by detecting the expression level of this gene in samples such as peripheral blood; second, the application of the FAM26F gene in the preparation of therapeutic drugs for depression, wherein the drug uses a gene therapy vector that upregulates the expression of this gene as its active ingredient, and is administered via lateral ventricle injection or nasal nebulization, effectively reversing depressive-like behaviors in animal models. This invention provides a novel, precise diagnostic target and targeted treatment strategy for depression.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of molecular biology and neuropsychiatry, and in particular relates to the application of the FAM26F gene in the diagnosis and treatment of depression. Background Technology

[0002] Depression is a prevalent and serious mental disorder worldwide, characterized by persistent low mood, loss of interest, and impaired cognitive function, placing a heavy burden on patients, families, and society. It is generally believed that depression is not caused by a single factor, but rather by the complex interaction of multiple factors, including genetic susceptibility, neurobiochemical abnormalities, neuroendocrine disorders, and environmental stress. This complexity of the mechanism presents a significant challenge to the accurate diagnosis and effective treatment of depression.

[0003] In clinical diagnosis, the current method of diagnosing depression largely relies on physicians' questioning and assessment of symptoms according to the Diagnostic and Statistical Manual of Mental Disorders (DSM) or the International Classification of Diseases (ICD), supplemented by psychometric tools such as the Hamilton Depression Rating Scale (HMRS) and the Beck Self-Rating Depression Scale (BRDS). This diagnostic model, based on clinical symptomatology and subjective scales, is highly subjective and easily influenced by factors such as physician experience, patient expression, and cultural background, leading to low early identification rates and high rates of misdiagnosis and missed diagnosis. It is particularly difficult to effectively differentiate subclinical states or biological subtypes. Therefore, the development of objective and quantifiable biological diagnostic markers has become an urgent need in this field.

[0004] In terms of treatment, current first-line antidepressants are mainly based on the traditional "monoamine neurotransmitter hypothesis," aiming to improve symptoms by regulating the levels of monoamine neurotransmitters such as serotonin, norepinephrine, and dopamine in the brain. However, these drugs have several inherent limitations: first, their onset of action usually takes several weeks, which cannot meet the needs of acute intervention; second, they are often accompanied by various side effects such as weight gain, sexual dysfunction, drowsiness, and nausea, affecting patient adherence; more importantly, approximately 30% to 40% of patients do not respond well to existing drug treatments and are classified as treatment-resistant depression. This indicates that traditional treatment strategies targeting the monoamine system may not address the core pathological aspects of all subtypes of depression.

[0005] To overcome diagnostic and treatment bottlenecks, researchers have been dedicated to exploring the genetic and molecular basis of depression in order to discover more precise targets. Previous studies have reported several genes associated with the risk or treatment response to depression, such as the linkage polymorphism region of the serotonin transporter gene and the brain-derived neurotrophic factor gene. However, the diagnostic specificity of these known gene markers is generally low (usually below 80%), and the specific molecular pathways by which they regulate the pathogenesis of depression are not fully understood. This makes it difficult to translate them into highly specific and sensitive clinical diagnostic tools, and also hinders the direct development of novel targeted therapy strategies. Therefore, discovering and elucidating new genes involved in key pathological processes of depression and their mechanisms of action is of decisive significance for developing next-generation precision medicine.

[0006] The FAM26F gene, a member of the F family with sequence similarity 26, is located on human chromosome 1q32.1. It encodes a protein containing a C1q domain and is a secreted protein. Previous scientific research has primarily revealed the regulatory role of the FAM26F gene in innate immunity and autoimmune diseases (such as rheumatoid arthritis and systemic lupus erythematosus), focusing on its mediated immune cell activation and inflammatory responses. However, to date, no reports have been found in publicly available academic literature or patent databases regarding a link between the FAM26F gene and the pathogenesis of depression, nor have any technical solutions for diagnosing or treating depression based on regulating FAM26F gene expression been identified. The function and mechanism of action of this gene in neuropsychiatric disorders, particularly depression, remain entirely unknown.

[0007] In summary, existing diagnostic and treatment technologies for depression suffer from problems such as strong diagnostic subjectivity, limited therapeutic targets, and insufficient efficacy. Furthermore, known candidate genes lack specificity and have unclear mechanisms. FAM26F, as a novel gene never before explored in the field of neuropsychiatric disorders, raises questions about its potential as a novel biomarker and therapeutic target for depression, and the molecular pathways through which it influences the development and progression of depression. These are all pressing scientific questions and technological gaps that need to be addressed. This invention was developed against this backdrop. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention proposes an application of the FAM26F gene in the diagnosis and treatment of depression. The purpose of this invention is to reveal the core mechanism by which the FAM26F gene participates in the pathogenesis of depression by regulating the glutamate pathway, and to provide the application of the FAM26F gene in the diagnosis of depression, diagnostic reagents for depression based on the FAM26F gene, and the application of the FAM26F gene in the treatment of depression and related therapeutic drugs, thereby solving the problems of low accuracy and unclear targets in existing depression diagnosis and treatment technologies.

[0009] To achieve the above objectives, the present invention provides the application of the FAM26F gene or its encoded product in the preparation of products for the diagnosis or auxiliary diagnosis of depression, wherein the product performs diagnosis by detecting the expression level of the FAM26F gene or its encoded product in a biological sample; wherein the nucleotide sequence of the FAM26F gene is as shown in SEQ ID NO: 1, or is a nucleotide sequence having at least 95% homology with the sequence shown in SEQ ID NO: 1 and encoding a protein having the same regulatory function for depression.

[0010] Furthermore, the biological sample is peripheral blood; the product is configured to detect the mRNA expression level of the FAM26F gene.

[0011] The present invention also provides the use of the FAM26F gene or its encoded product in the preparation of a medicament for the treatment of depression.

[0012] Furthermore, the drug comprises a gene therapy vector capable of upregulating FAM26F gene expression.

[0013] Furthermore, the gene therapy vector is a viral vector containing the coding sequence of the FAM26F gene; the coding sequence of the FAM26F gene is shown in SEQ ID NO: 1.

[0014] Furthermore, the viral vector is an adeno-associated virus vector.

[0015] Furthermore, the dosage form of the drug is formulated for administration via local administration through the central nervous system or via nasal administration.

[0016] Furthermore, the local administration to the central nervous system is intraventricular injection; the nasal administration is nasal nebulization inhalation.

[0017] This invention also provides the application of a primer pair for specifically amplifying the FAM26F gene in the preparation of diagnostic or auxiliary diagnostic reagents or kits for depression, wherein the upstream primer sequence of the primer pair is 5'-TGTCACCCGATGCCTATCTC-3' and the downstream primer sequence is 5'-TGGCCCTTCGGATTGAAAGTA-3'.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: This invention establishes for the first time the central role of the FAM26F gene in the pathogenesis of depression and fully elucidates its molecular pathway of involvement in disease development through the regulation of glutamate homeostasis. Specifically, downregulation of FAM26F expression directly leads to a reduction in its encoded protein, which in turn reduces the expression of SNAT1, a key transporter for glutamate reuptake in the hippocampus, and major ionotropic glutamate receptors (AMPA and NMDA receptor subunits), ultimately disrupting perisynaptic glutamate homeostasis and inducing depressive-like behavior. This discovery breaks through the limitations of traditional research, which mainly focuses on monoamine neurotransmitters or simple inflammatory mechanisms, by establishing FAM26F as a key node connecting genetic susceptibility and glutamate system dysfunction, providing a new theoretical framework and a highly promising intervention target for understanding the complex pathophysiology of depression.

[0019] A diagnostic protocol based on FAM26F gene expression levels provides objective and quantifiable biological indicators by detecting readily available samples such as peripheral blood. This effectively overcomes the shortcomings of existing diagnostic methods, such as high subjectivity and difficulty in early identification, offering a reliable tool for early screening and precise auxiliary diagnosis of depression. In terms of treatment, a gene therapy strategy targeting FAM26F directly corrects the glutamate homeostasis imbalance, a core aspect of the disease, by delivering a FAM26F overexpression vector. Preclinical studies have confirmed that this therapy can effectively reverse behavioral abnormalities in depressive models. Compared to traditional drugs, its mechanism of action is more targeted, potentially achieving faster onset of action and fewer side effects, providing a new solution, especially for treatment-resistant patients unresponsive to existing therapies.

[0020] The diagnostic reagents and therapeutic drug solutions provided by this invention have clearly defined targets, mature preparation processes, and clear implementation pathways. The core components of the diagnostic reagent kit are well-defined and easy to standardize for production; the vector construction, formulation, and administration methods of the gene therapy drugs have all been validated. These characteristics give the results of this invention the potential to be rapidly transformed into practical medical products or technologies, providing core technological support for the development of precision medicine for depression, and promising broad market application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1This is a schematic diagram showing the results of FAM26F gene expression detection in clinical samples. A: A bar chart showing the relative expression levels of FAM26F gene mRNA in peripheral blood of patients with depression and healthy controls, detected by qPCR. B: A representative protein band diagram and grayscale analysis bar chart, showing the expression level of FAM26F protein, detected by Western Blot.

[0023] Figure 2 This diagram illustrates the behavioral and molecular findings of a mouse model of chronic unpredictable stress (CUS) depression. A: Bar chart of sucrose preference test (SPT) results. B: Bar chart of immobility time results of forced swimming test (FST) results. C: Bar chart of feeding latency results of novelty-inhibited feeding test (NSFT) results. D: Bar chart of female urine smelling test (FUST) results. E: Bar chart of qPCR detection of relative FAM26F gene mRNA expression in hippocampus. F: Representative bands of FAM26F protein expression in hippocampus from a Western blot.

[0024] Figure 3 This diagram illustrates the experimental results of FAM26F gene knockdown (FAM26F-shRNA) intervention in mice. A: Bar chart of qPCR detection of relative FAM26F gene mRNA expression levels in hippocampus. B: Bar chart of sucrose preference test (SPT) results. C: Bar chart of immobility time results in forced swimming test (FST). D: Bar chart of feeding latency results in novelty-inhibited feeding test (NSFT). E: Bar chart of female urine smelling test (FUST) results.

[0025] Figure 4 This diagram illustrates the expression changes of glutamate pathway-related genes in the hippocampus of FAM26F knockdown mice. It includes a bar chart showing the relative mRNA expression levels of glutaminase (GLS), glutamine transporters (SNAT1, SNAT2), glutamate transporters (Vglut1, Vglut2), AMPA receptor subunits (GluR1, GluR2), and NMDA receptor subunits (NR1, NR2A, NR2B). Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0028] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0029] Example 1: Delineation of the FAM26F gene sequence and its functional characteristics The key molecule involved in this invention is the human FAM26F gene and its encoded protein. The complete coding sequence (CDS) nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the FAM26F protein it encodes is shown in SEQ ID NO: 2.

[0030] SEQ ID NO: 1: atggaga agtttcgggc ggtgctggac ctgcacgtca agcaccacag cgccttgggctacggcctgg tgaccctgct gacggcgggc ggggagcgca tcttctccgc cgtggcattc cagtgcccgtgcagcgccgc ctggaacctg ccctacggcc tggtcttctt gctggtgccg gcgctcgcgc tcttcctcctgggctacgtg ctgagcgcac gcacgtggcg cctgctcacc ggatgctgct ccagcgcccg cgcgagttgcggatcggcgc tgcgcggctc cctggtgtgc acgcaaatca gcgcggccgc cgcgctcgcg cccctcacctgggtggccgt ggcgctgctc gggggcgcct tttacgagtg cgcggccacc gggagcgcgg ccttcgcgcagcgcctgtgc ctcggccgca accgcagctg cgccgcggag ctgccgctgg tgccgtgcaa ccaggccaaggcgtcggacg tgcaggacct cctgaaggat ctgaaggctc agtcgcaggt gttgggctgg atcttgatagcagttgttat catcattctt ctgattttta catctgtcac ccgatgccta tctccagtta gttttctgcagctgaaattc tggaaaatct atttggaaca ggagcagcag atccttaaaa gtaaagccac agagcatgcaactgaattgg caaaagagaa tattaaatgt ttctttgagg gctcgcatcc aaaagaatat aacactccaagcatgaaaga gtggcagcaa atttcatcac tgtatacttt caatccgaag ggccagtact acagcatgttgcacaaatat gtcaacagaa aagagaagac tcacagtatc aggtctactg aaggagatac ggtgattcct gttcttggctttgtagattc atctggtata aacagcactc ctgagttatg a SEQ ID NO: 2: MEKFRAVLDLHVKHHSALGYGLVTLLTAGGERIFSAVAFQCPCSAAWNLPYGLVFLLVPALFLLGYVLSARTWRLLTGCCSSARASCGSALRGSLVCTQISAAAALAPLTWVAVALLGGAFYECAATGSAAFAQRLCLGRNRSCAAELPLVPCNQ AKASDVQDLLKDLKAQSQVLGWILIAVVIIILLIFTSVTRCLSPVSFLQLKFWKIYLEQEQQILKSKATEHATELAKENIKCFFEGSHPKEYNTPSMKEWQQISSLYTFNPKGQYYSMLHKYVNRKEKTHSIRSTEGDTVIPVLGFVDSSGINSTPEL Within the scope of protection of this invention, the invention includes not only the original sequence shown in SEQ ID NO: 1, but also mutants, alleles, or variants that share 95% or more homology with the nucleotide sequence shown in SEQ ID NO: 1 and have been experimentally verified to encode proteins that have the same or similar regulatory functions on the glutamate pathway and influence depressive-like behaviors. Furthermore, due to the degeneracy of the genetic codon, any different nucleotide sequence capable of encoding the amino acid sequence shown in SEQ ID NO: 2 is also within the scope of protection of this invention. These sequences are all applicable to the diagnostic and therapeutic applications described later in this invention.

[0031] Example 2: Validation of FAM26F gene expression in patients with depression This embodiment aims to verify the expression of the FAM26F gene in the peripheral blood of patients with depression and reveal its potential as a diagnostic biomarker.

[0032] 1. Research Subjects and Sample Collection Peripheral blood samples were collected from 60 patients with depression meeting the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). All patients were in a depressive episode with a duration of 6 months to 2 years and had not taken any antidepressants or antipsychotics for at least 2 weeks prior to sampling. Simultaneously, 60 age- and sex-matched healthy volunteers were recruited as a control group. Informed consent was obtained from all participants. Five mL of fasting peripheral venous blood was collected from each participant in the morning using EDTA anticoagulant tubes; one portion was used for subsequent RNA extraction, and the other for protein extraction.

[0033] 2. Experimental Methods 2.1 Real-time quantitative PCR detection of FAM26F gene mRNA expression (1) Total RNA extraction: Total RNA was extracted from peripheral blood nucleated cells using TRIzol reagent. RNA purity (OD260 / OD280 ratio between 1.8 and 2.0) and concentration were determined using a micro spectrophotometer, and RNA integrity was assessed using agarose gel electrophoresis.

[0034] (2) Reverse transcription: Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit.

[0035] The 20 μL reaction system consisted of: 1 μg total RNA, 1 μL Oligo(dT)18 primer, 2 μL 10 mM dNTP Mix, 4 μL 5× Reaction Buffer, 1 μL RiboLock RNase Inhibitor (20 U / μL), 1 μL RevertAid M-MuLV RT (200 U / μL), and enzyme-free water to a final volume of 20 μL.

[0036] The reaction conditions were: incubation at 42°C for 60 minutes, followed by heating at 70°C for 5 minutes to terminate the reaction.

[0037] (3) qPCR amplification: performed on a CFX96 real-time quantitative PCR instrument.

[0038] The reaction system (20 μL) contained: 10 μL of 2× SYBR Green Pro Taq HS premix, 2 μL of cDNA template, 0.4 μL each of FAM26F gene-specific primers (upstream: 5'-GGTGACCCTACTGACGGCT-3' (SEQ ID NO: 3), downstream: 5'-CTCAGCGCATATCCCAGGA-3' (SEQ ID NO: 4)), and 7.2 μL of enzyme-free water. The GAPDH gene was used as an internal control.

[0039] Reaction procedure: pre-denaturation at 95℃ for 30 seconds; then 40 cycles (denaturation at 95℃ for 5 seconds, annealing / extending at 60℃ for 30 seconds). The experiment was conducted in duplicate wells.

[0040] (4) Data Analysis: The relative expression level of the FAM26F gene was calculated using the 2^-ΔΔCt method. ΔCt = Ct(FAM26F) - Ct(GAPDH); ΔΔCt = ΔCt(depression group) - ΔCt(control group mean). Relative expression level (RQ) = 2^(-ΔΔCt).

[0041] 2.2 Detection of FAM26F protein expression by Western blotting (1) Protein extraction and quantification: Total protein was extracted from peripheral blood leukocytes using RIPA lysis buffer (containing 1 mM PMSF and protease inhibitors). Protein concentration was determined by the BCA method.

[0042] (2) SDS-PAGE electrophoresis: Take an equal volume (20 μg) of protein sample, mix it with 5× loading buffer, and denature it in a boiling water bath for 10 minutes. Use a 12% separating gel for electrophoresis. The stacking gel voltage is 80V, and the voltage is adjusted to 120V after entering the separating gel.

[0043] (3) Transfer and blocking: The protein was electroporated onto a PVDF membrane (300 mA constant current, 90 min). After transfer, the membrane was blocked with TBST solution containing 5% skim milk at room temperature for 1 hour.

[0044] (4) Antibody incubation and development: The membrane was incubated overnight at 4°C with rabbit anti-human FAM26F polyclonal primary antibody (1:1000 dilution). After washing the membrane three times with TBST, it was incubated at room temperature for 1 hour with HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution). Developed using ECL chemiluminescence reagent, images were acquired on a chemiluminescence imaging system. Using β-actin as an internal control protein, the gray values ​​of the target bands were analyzed using ImageJ software to calculate the relative expression level of FAM26F protein.

[0045] 3. Experimental Results like Figure 1 As shown in Figure A, qPCR analysis revealed that the mRNA expression level of the FAM26F gene in the peripheral blood of patients with depression was significantly lower than that in the healthy control group, with an average decrease of approximately 52.3% (p<0.001). Western blot results (…) Figure 1B) Further confirmation showed that the expression level of FAM26F protein was also significantly reduced in the patient group, with an average decrease of approximately 45.1% (p<0.01). This result is the first clinical-level confirmation that the FAM26F gene is underexpressed in patients with depression, suggesting that it may serve as a potential biomarker for the diagnosis of depression.

[0046] Example 3: Animal model validation of FAM26F gene regulation of depressive-like behavior and glutamate pathway This embodiment elucidates the function and molecular mechanism of the FAM26F gene in vivo by constructing a mouse model of chronic stress depression and a gene intervention model.

[0047] 1. Experimental Materials and Animal Model Construction (1) Experimental animals: Eight-week-old male C57BL / 6 mice were selected and used for experiments after one week of acclimatization.

[0048] (2) Animal grouping: 60 mice were randomly divided into 4 groups of 15 mice each.

[0049] Control group: No stress treatment was applied.

[0050] The depression model group (CUS group) used a chronic unpredictable stress method to establish a depression model. For 14 consecutive days, a different stressor was applied each day, including: restraint for 2 hours, moist bedding (24 hours), cage tilted at 45° (24 hours), tail suspension (15 minutes), continuous light exposure (24 hours), elevated platform exposure (30 minutes), and foot shock (10 minutes, 0.3 mA). The stress pattern was repeated in 5-day cycles.

[0051] The FAM26F gene knockdown group (FAM26F-shRNA group): Adeno-associated virus carrying FAM26F gene-specific shRNA was injected stereotactically into the dentate gyrus of the mouse hippocampus. The shRNA sequence was 5'-GGUUAGUUAUCUGCAGUUA-3' (SEQ ID NO: 5). The injection coordinates were: 2.10 mm posterior to the anterior fontanelle, 1.5 mm lateral to the midline, and a depth of 2.2 mm. The viral load was 5 μL (titer 2 × 10⁻⁶). 12 (vg / mL).

[0052] GFP control group (GFP group): The hippocampal dentate gyrus was injected with an equal amount of control virus carrying the green fluorescent protein gene.

[0053] 2. Experimental detection indicators and methods 2.1 Depression-like behavioral tests On day 14 after the end of CUS stress or viral injection, all mice were subjected to the following behavioral tests, with an interval of at least 24 hours between tests.

[0054] (1) Sucrose Preference Test (SPT): After acclimatization to single-cage rearing and double-bottle (one water and one 1% sucrose solution), the animals were deprived of water and food for 4 hours, followed by a pre-weighed sucrose solution bottle and a pure water bottle for 2 hours. The sucrose preference rate was calculated as: sucrose consumption / (sucrose consumption + water consumption) × 100%.

[0055] (2) Forced swimming test (FST): The mouse was placed in a transparent cylindrical tank (25 cm high and 10 cm in diameter), with a water depth of 15 cm and a water temperature of 25±1℃. The cumulative immobility time in the last 4 minutes within 6 minutes was recorded.

[0056] (3) Novelty Inhibition Feeding Test (NSFT): After fasting for 24 hours, mice were placed in a clean new cage (with a food block placed in the center), and the latency period for the mice to start eating was recorded (maximum 600 seconds).

[0057] (4) Female Urine Sniff Test (FUST): First, let the mice get used to the cotton swabs dipped in sterile water for 3 minutes. After 45 minutes, replace them with cotton swabs dipped in the urine of female mice in estrus. Record the time the mice actively sniff the cotton swabs within 3 minutes on two separate occasions.

[0058] 2.2 Molecular biological detection After the behavioral tests were completed, the mice were euthanized and the hippocampus was quickly dissected.

[0059] (1) qPCR detection of gene expression: The method is the same as 2.1 in Example 2, detecting the mRNA expression of FAM26F, glutamate receptor subunit and glutamine transporter genes in hippocampal tissue. Tubulin was used as the internal reference gene.

[0060] (2) Western Blot detection of protein expression: The method was the same as 2.2 in Example 2, to detect FAM26F protein expression in hippocampal tissue. The primary antibody was rabbit anti-mouse FAM26F antibody (1:500).

[0061] 3. Experimental Results 3.1 Behavioral Results like Figure 2 As shown, compared with the control group, the CUS group mice exhibited typical depressive-like behavior: a significantly reduced sucrose preference rate ( Figure 2 A, p<0.01, forced swimming immobility time was significantly prolonged ( Figure 2 B, p<0.05, the latency period for feeding in novel environments was significantly prolonged ( Figure 2 C, p<0.01, reduced sniffing time for female mouse urine ( Figure 2D, p<0.05).

[0062] like Figure 3 As shown, compared with the GFP control group, mice in the FAM26F-shRNA group also exhibited similar depressive-like behaviors, and the severity of these behaviors was correlated with the degree of downregulation of the FAM26F gene. Figure 3 (BE, p<0.05). This indicates that directly reducing the expression of the hippocampal FAM26F gene is sufficient to induce depressive-like behavior.

[0063] 3.2 FAM26F gene expression level Western Blot ( Figure 2 F) and qPCR Figure 2 E) The results showed that the levels of FAM26F protein and mRNA in the hippocampus of CUS group mice were significantly lower than those in the control group. Simultaneously, in the FAM26F-shRNA group, FAM26F mRNA expression was effectively knocked down. Figure 3 A, p<0.01), confirming the successful construction of the gene intervention model.

[0064] 3.3 Changes in glutamate pathway-related indicators like Figure 4 As shown, qPCR analysis of hippocampal tissue from mice in the FAM26F-shRNA group revealed that, compared to the GFP control group: The mRNA expression of the glutamine transporter SNAT1 was significantly downregulated (p<0.01), while SNAT2 showed no significant change.

[0065] The expression of glutamate release-related transporters Vglut1 and Vglut2 showed no significant changes.

[0066] The mRNA levels of AMPA receptor subunits GluR1 and GluR2 were significantly downregulated.

[0067] The expression of NMDA receptor subunits NR1 and NR2A was also significantly downregulated, while NR2B showed no significant change.

[0068] The above results indicate that downregulation of the FAM26F gene specifically leads to a reduction in the expression of SNAT1, a key transporter for glutamate reuptake in the hippocampus, as well as major ionotropic glutamate receptors (AMPA and NMDA receptors). This may be a core molecular event that disrupts glutamate homeostasis and subsequently induces depressive-like behavior.

[0069] Example 4: A diagnostic kit for depression based on the FAM26F gene and its application This embodiment provides a kit for the auxiliary diagnosis of depression, which is achieved by quantitatively detecting the expression level of the FAM26F gene in human peripheral blood.

[0070] 1. Composition of the diagnostic kit This kit contains the following components: (1) Nucleic acid extraction reagents: including red blood cell lysis buffer, white blood cell lysis buffer, RNA purification magnetic beads and washing solution.

[0071] (2) Reverse transcription reaction reagents: including reverse transcriptase, random primers, dNTPs, RNase inhibitors and reaction buffer.

[0072] (3) Real-time PCR detection reagents: - Specific primer pairs: designed for the human FAM26F gene.

[0073] Upstream primer: 5'-TGTCACCCGATGCCTATCTC-3' (SEQ ID NO: 6) Downstream primer: 5'-TGGCCCTTCGGATTGAAAGTA-3' (SEQ ID NO: 7) - Dual-labeled fluorescent probe: sequence 5'-FAM-CTGCTGCTGCTGCTGCTGCT-TAMRA-3' (SEQ ID NO: 8).

[0074] - PCR premix: Contains hot-start Taq DNA polymerase, dNTPs, MgCl2, and optimized buffer.

[0075] (4) Standards: FAM26F recombinant plasmid DNA standards containing known copy numbers (gradient concentration: 10). 1 10 2 , 10 3 , 10 4 , 10 5 (copies / μL) and the internal reference gene GAPDH recombinant plasmid.

[0076] (5) Negative control: water without RNA / DNA enzyme.

[0077] 2. Instructions for using the diagnostic kit (1) Sample collection and processing: 2 mL of EDTA-anticoagulated peripheral blood was collected from the subjects. Total RNA was extracted using the nucleic acid extraction reagent provided in the kit.

[0078] (2) Reverse transcription: Take 1 μg of total RNA and perform reverse transcription according to the kit instructions to obtain cDNA.

[0079] (3) Quantitative Real-Time PCR Detection: Prepare a 20 μL reaction mixture in a 96-well plate, including 10 μL of PCR premix, 2 μL of cDNA template, 0.5 μL each of upstream and downstream primers (final concentration 0.25 μM), 0.3 μL of fluorescent probe (final concentration 0.15 μM), and make up the difference with enzyme-free water. Reaction program: 95℃ for 5 minutes; then 95℃ for 10 seconds, 60℃ for 30 seconds, for 40 cycles. Three technical replicates were set up for each sample.

[0080] (4) Data Analysis: The instrument automatically generates a standard curve and calculates the absolute copy number of the FAM26F gene in the sample based on the Ct value. At the same time, the internal reference gene GAPDH is detected and normalized to calculate the relative expression level of FAM26F.

[0081] (5) Result interpretation: Based on the clinical data statistics of Example 2, a diagnostic threshold was set. When the relative expression level of the FAM26F gene in the peripheral blood of the subject was less than 0.48 times the average of the healthy control group (i.e., the expression was downregulated by more than 52%), it indicated that the subject had a significantly increased risk of depression.

[0082] Example 5: Depression Treatment Drugs Based on the FAM26F Gene, Their Preparation and Application This embodiment provides a gene therapy drug that targets the FAM26F gene, which restores glutamate homeostasis by upregulating the expression of FAM26F in specific brain regions (such as the hippocampus), thereby treating depression.

[0083] 1. Composition of therapeutic drugs (1) Active ingredient: Adeno-associated virus serotype 9 carrying the complete coding sequence of the human FAM26F gene (SEQ ID NO: 1). The vector plasmid backbone is pAAV-CMV, which can efficiently drive the expression of the target gene. The viral titer is not less than 1×10⁻⁶. 12 Viral genome / mL.

[0084] (2) Pharmaceutical preparations: - Active ingredient: AAV9-FAM26F virus stock solution.

[0085] - Lyophilization protectant: mannitol, final concentration 6% (w / v).

[0086] - Surfactant: Polysorbate 80, final concentration 0.03% (v / v).

[0087] - Solvent: Phosphate buffer (pH 7.4, prepared by mixing 0.01 M potassium dihydrogen phosphate and 0.01 M disodium hydrogen phosphate in a volume ratio of 4.5:5.5), bring to 100%.

[0088] The final osmotic pressure of the formulation was adjusted to 280-320 mOsm / kg, meeting the requirements for injectable preparations. This formulation can be prepared as a liquid for direct use, or lyophilized into a lyophilized powder for injection, which should be reconstituted with sterile physiological saline before use.

[0089] 2. Preparation method of therapeutic drugs (1) Plasmid construction: The CDS region of the FAM26F gene shown in SEQ ID NO: 1 was cloned into the multiple cloning site of the pAAV-CMV transfer plasmid to construct the recombinant plasmid pAAV-CMV-FAM26F.

[0090] (2) Virus packaging: The three plasmid co-transfection method was used to co-transfect pAAV-CMV-FAM26F, pAAV9 Rep / Cap packaging plasmid and pHelper helper plasmid into HEK293T cells.

[0091] (3) Virus purification and concentration: Cells and supernatant were harvested 72 hours after transfection. The virus was released by repeated freeze-thaw cycles and chloroform treatment. The virus was then purified and concentrated by cesium chloride density gradient centrifugation or affinity chromatography.

[0092] (4) Titer determination and preparation: The genomic titer of the purified virus was determined by qPCR and diluted or prepared to the target concentration with the prescribed amount of excipients. After sterile filtration through a 0.22 μm filter membrane, the mixture was dispensed.

[0093] 3. Administration method and dosage of therapeutic drugs (1) Intraventricular injection (for severe or treatment-resistant depression): - Animal dosage (mice): unilateral or bilateral lateral ventricle injection, 2-5 μL per mouse, total viral load 1×10⁻⁶. 12 vg.

[0094] - Equivalent dose calculation for humans: Based on body surface area, the recommended starting clinical dose is 5 × 10⁻⁶. 13 Vg / person, single injection. The specific dosage needs to be adjusted according to the patient's weight, the severity of the condition, and the precise positioning guided by imaging.

[0095] (2) Nasal nebulizer inhalation (for mild to moderate depression or maintenance treatment): - Animal dosage (mice): Administer once daily using a microneedling device, each dose containing 2×10⁻⁶ mg / L. 12 VG virus aerosol was administered continuously for 14 days.

[0096] - Human administration: Once daily, inhaled via nasal spray, with a dose of 5 x 10⁻⁶. 12 vg – 1×10 13VG, a course of treatment lasts 2-4 weeks.

[0097] Preclinical animal experiments have shown that administration of the drug in any of the above-mentioned ways can effectively upregulate FAM26F expression in the hippocampus of CUS model mice, significantly improve their depressive-like performance in various behavioral tests, and no obvious liver or kidney function damage or immune rejection reaction has been observed.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the FAM26F gene or its encoded product in the preparation of products for the diagnosis or auxiliary diagnosis of depression, characterized in that, The product is used to perform diagnosis by detecting the expression level of the FAM26F gene or its encoded product in a biological sample; wherein the nucleotide sequence of the FAM26F gene is as shown in SEQ ID NO: 1, or is a nucleotide sequence that has at least 95% homology with the sequence shown in SEQ ID NO: 1 and encodes a protein with the same function of regulating depression.

2. The application according to claim 1, characterized in that, The biological sample is peripheral blood; the product is configured to detect the mRNA expression level of the FAM26F gene.

3. Application of the FAM26F gene or its encoded product in the preparation of drugs for the treatment of depression.

4. The application according to claim 3, characterized in that, The drug contains a gene therapy vector that can upregulate the expression of the FAM26F gene.

5. The application according to claim 4, characterized in that, The gene therapy vector is a viral vector containing the coding sequence of the FAM26F gene; the coding sequence of the FAM26F gene is shown in SEQ ID NO:

1.

6. The application according to claim 5, characterized in that, The viral vector is an adeno-associated virus vector.

7. The application according to any one of claims 4 to 6, characterized in that, The dosage form of the drug is formulated for administration via local administration through the central nervous system or via nasal administration.

8. The application according to claim 7, characterized in that, The central nervous system local administration is intraventricular injection; the nasal administration is nasal nebulization inhalation.

9. The application of a primer pair for specifically amplifying the FAM26F gene in the preparation of diagnostic or auxiliary diagnostic reagents or kits for depression, characterized in that, The upstream primer sequence of the primer pair is 5'-TGTCACCCGATGCCTATCTC-3', and the downstream primer sequence is 5'-TGGCCCTTCGGATTGAAAGTA-3'.

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