Cyclic rna circ-0004801 and application thereof in treatment of alzheimer's disease

Abstract

The application provides a circular RNA circ_0004801 and application thereof in Alzheimer's disease treatment; a nucleotide sequence of the circular RNA circ_0004801 is shown as SEQ ID NO:1. Expression of the circular RNA circ_0004801 in hippocampus tissue of AD mice is significantly up-regulated, the circular RNA circ_0004801 acts as a "sponge" of miR-7688-5p, and plays a role by regulating TTBK1 expression and tau phosphorylation level. Knocking down the circular RNA circ_0004801 can obviously improve spatial learning and memory of 3xTg mice, significantly reduce tau protein phosphorylation level in hippocampus and NFTs density in hippocampus DG region. Therefore, the circular RNA circ_0004801 and miR-7688-5p can be used as a new target for treating AD. The application first finds that the circular RNA circ_0004801 regulates TTBK1 through a ceRNA mechanism, thereby driving tau hyperphosphorylation, and provides a new molecular target for early intervention treatment of AD based on circRNA and miRNA.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to circular RNA circ_0004801 and its application in the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, currently affecting more than 55 million people worldwide, a number projected to rise to 139 million by 2050, placing a tremendous burden on the socioeconomic system. Although β-amyloid (Aβ) plaque deposition and neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau protein remain hallmarks of AD, repeated failures in Aβ-targeting clinical trials have prompted a reassessment of the central role of tau pathology in AD. Emerging evidence suggests that aberrant phosphorylation of tau protein at key residues (Ser199 / Ser202 / Ser396) not only disrupts microtubule dynamics but also directly induces synaptic dysfunction by impairing glutamate receptor transport, highlighting tau as an important molecular target for early intervention.

[0003] Tau-tubulin kinase 1 (TTBK1) is a major regulator of tau phosphorylation, driving the pathogenesis of Alzheimer's disease (AD) through multiple mechanisms: (1) phosphorylation at specific sites (Ser199 / Ser202 / Ser396) directly catalyzes the formation of non-fibroblastic proteins (NFTs); (2) it activates downstream kinases such as GSK-3β, establishing a phosphorylation cascade amplification; and (3) it regulates the pathological modification of proteins associated with neurodegenerative diseases (such as TDP-43). However, the mechanisms of TTBK1 dysregulation and its upstream regulatory network remain unclear, which significantly hinders the development of tau-targeted therapies.

[0004] Currently, the main regulatory approaches to TTBK1 focus on small molecule inhibitors, but these suffer from problems such as low blood-brain barrier penetration and significant off-target effects. In recent years, non-coding RNAs (such as circRNAs and miRNAs) have become an emerging direction for the treatment of neurodegenerative diseases due to their brain tissue enrichment and high targeting properties. However, no studies have yet revealed the feasibility of circRNAs regulating the TTBK1-tau phosphorylation pathway through competitive miRNA sponging. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a circular RNA circ_0004801 and its application in the treatment of Alzheimer's disease.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] The first aspect of the present invention provides a circular RNA circ_0004801, the nucleotide sequence of which is shown in SEQ ID NO: 1, or is a functionally equivalent variant having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

[0008] A second aspect of the invention provides the use of agents that inhibit or silence the expression of circular RNA circ_0004801 as described above in the preparation of medicaments for treating Alzheimer's disease.

[0009] A third aspect of the invention provides the use of reagents for inhibiting or silencing the expression of circular RNA hsa_ANKRD32_0001200 in the preparation of medicaments for treating Alzheimer's disease.

[0010] In some embodiments, the reagent includes at least one of siRNA, shRNA, and a recombinant expression vector containing the shRNA.

[0011] In some embodiments, the siRNA is selected from at least one of the siRNAs with nucleotide sequences as shown in SEQ ID NO: 3 to SEQ ID NO: 5; and / or,

[0012] The shRNA comprises a sense strand and an antisense strand, the nucleotide sequence of the sense strand being shown in SEQ ID NO: 10, and the nucleotide sequence of the antisense strand being shown in SEQ ID NO: 11; and / or,

[0013] The recombinant expression vector is a recombinant lentiviral expression vector.

[0014] A fourth aspect of the invention provides the use of miR-7688-5p or miR-7688-5p overexpression vector in the preparation of medicaments for treating Alzheimer's disease.

[0015] A fifth aspect of the present invention provides a medicament for treating Alzheimer's disease, the active ingredient of which comprises at least one of the following (1) to (4): (1) a reagent that inhibits or silences the expression of circular RNA circ_0004801 as described above; (2) a reagent that inhibits or silences the expression of circular RNA hsa_ANKRD32_0001200; (3) miR-7688-5p; (4) miR-7688-5p overexpression vector.

[0016] In some embodiments, the reagents in (1) and (2) include siRNA, shRNA, and a recombinant expression vector containing the shRNA.

[0017] In some embodiments, the siRNA is selected from at least one of the siRNAs with nucleotide sequences as shown in SEQ ID NO: 3 to SEQ ID NO: 5; and / or,

[0018] The shRNA comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO: 10 and the nucleotide sequence of which is shown in SEQ ID NO: 11.

[0019] In some embodiments, the recombinant expression vector is a recombinant lentiviral expression vector.

[0020] Compared with the prior art, the present invention has the following beneficial effects.

[0021] This invention obtained a circular RNA circ_0004801 through research and screening, and found its homologous circular RNA hsa_ANKRD32_0001200 in humans through search analysis. This invention found that the expression of the circular RNA circ_0004801 was significantly upregulated in the hippocampus of AD mice, and that it could act as a "sponge" for miR-7688-5p, regulating the progression of AD by regulating TTBK1 expression and tau phosphorylation levels. Knockdown of the circular RNA circ_0004801 has the following effects: (1) it significantly reduces the level of tau phosphorylation in neuronal cells and reduces apoptosis, accompanied by enhanced cell viability; (2) it improves spatial learning and memory in 3×Tg mice, and significantly reduces the phosphorylation level of tau protein in the hippocampus and the density of NFTs in the hippocampal DG region. Therefore, the circular RNA circ_0004801 and its homologous circular RNA hsa_ANKRD32_0001200 can serve as novel targets for the treatment of AD.

[0022] Furthermore, this invention confirms the regulatory role of the circular RNA circ_0004801 / miR-7688-5p / TTBK1 regulatory axis in AD progression. Circular RNA circ_0004801 adsorbs miR-7688-5p through a sponge-like mechanism, relieving its inhibitory effect on TTBK1, driving the formation of abnormal tau protein phosphorylation, ultimately leading to cognitive impairment and promoting AD progression. Silencing or inhibiting the expression of circular RNA circ_0004801 in HT22 cells, or transducing miR-7688-5p, can enhance the inhibitory effect on TTBK1, inhibit abnormal tau protein phosphorylation, alleviate cognitive impairment, and thus exert a therapeutic effect on AD. Therefore, both miR-7688-5p and circular RNA circ_0004801 can serve as novel therapeutic targets for AD.

[0023] This invention is the first to discover that the circular RNA circ_0004801 targets miR-7688-5p through the ceRNA mechanism to regulate TTBK1, thereby driving the hyperphosphorylation of tau, providing a new molecular target for early intervention therapy of AD based on circRNA and miRNA. Attached Figure Description

[0024] Figure 1 The results of immunoblotting of tau and TTBK1 in hippocampal tissue and primary neurons of 3×Tg-AD mice.

[0025] Figure 2 The results of screening for differentially expressed circRNAs and miRNAs.

[0026] Figure 3 The results of qRT-PCR validation of the important circRNAs and miRNAs screened out were obtained.

[0027] Figure 4 The effect of inhibiting circ_0004801 expression on the proliferation and apoptosis of HT22 cells was detected.

[0028] Figure 5 To investigate the effects of transfection with miR-7688-5p and silencing circ_0004801 on TTBK1 expression and tau phosphorylation in HT22 cells.

[0029] Figure 6 The results show the interaction detection between circ_0004801 and miR-7688-5p.

[0030] Figure 7 To investigate the effect of inhibiting circ_0004801 expression on improving spatial learning and memory in 3×Tg mice.

[0031] Figure 8 The results show the effect of inhibiting circ_0004801 expression on NFTs in 3×Tg mice. Detailed Implementation

[0032] Experimental methods in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0034] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0035] The term "and / or" as used in this invention describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0036] The following description is based on specific embodiments.

[0037] 3×Tg transgenic AD mice and matched WT mice were purchased from the Laboratory Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). Mice were provided with food and water at will under controlled temperature and humidity conditions with a 12-hour light / dark cycle. All procedures were approved by the Laboratory Animal Ethics Committee of Guangzhou Medical University, China (Approval No.: GD 2019-129).

[0038] HT22 cell culture: In Dulbecco modified Eagle medium (DMEM; Invitrogen, Camario, California, USA) containing 10% FBS (Invitrogen) and 1% penicillin / streptomycin (Invitrogen), the cells were cultured at 37°C in a CO2-free environment.

[0039] qRT-PCR assay: Total RNA was extracted from the samples using Trizol reagent (Invitrogen), followed by digestion of genomic DNA using DNase I (Promega). RNA reverse transcription was performed using M-MLV (Promega), following the instructions after purity and integrity testing. The SYBR Green qPCR SuperMix kit (Invitrogen) was used at ABI. qPCR was performed on a 7500 sequencing system with the parameters set as follows: 50℃ for 2 minutes, 95℃ for 30 seconds, followed by 40 cycles, each cycle consisting of a reaction at 95℃ for 5 seconds and 60℃ for 34 seconds. Results were obtained via 2... -ΔΔCt Calculations were performed, with β-actin or U6 set as an internal reference. Primers used are shown in Table 1.

[0040] Table 1

[0041]

[0042]

[0043]

[0044] Western Blot (Protein Immunoblot Detection): Western blot was performed according to previous research methods (Q. Li et al., 2022). Proteins were extracted from the tissues or cells to be tested. Total protein content was determined using a dicumarol (BCA) kit (Beyotime Biotechnology, Jiangsu, China). The antibodies used in this invention are as follows: TTBK1 (1:1000, Sigma-Aldrich), Tau (1:1000, Sigma-Aldrich), Tau-Ser198 (1:1000, Sigma-Aldrich), Tau-Ser199 (1:1000, Cell Signaling Technology, Boston, USA), Tau-Ser202 (1:1000, Cell Signaling Technology), Tau-Ser422 (1:1000, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, Sigma-Aldrich), and the corresponding secondary antibody bound to horseradish peroxidase (HRP) (1:2000, Sigma-Aldrich). The membrane was incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody at 20-25°C for 2 hours. GAPDH was used as an internal control. The optical density values ​​of the target bands were estimated using ImageJ software (National Institutes of Health, http: / / rsb.info.nih.gov / ij / ).

[0045] Statistical analysis: Data are expressed as mean ± standard error (SEM). Statistical analysis was performed using SPSS 26.0 software (IBM, USA), and graphs were generated using GraphPad 9.0 software (GraphPad Software, USA). Student's t-test (two-tailed) was used to analyze differences between two groups. One-way ANOVA was used to analyze variances among multiple groups, with Tukey's follow-up test. P < 0.05 was considered statistically significant.

[0046] The nucleotide sequence of the circular RNA circ_0004801 described in this invention is shown in SEQ ID NO: 1. The nucleotide sequence is the mature sequence of the coding gene after transcription and splicing. The database uses DNA sequence format to label mature circRNAs, and uses U instead of T for labeling.

[0047]

[0048] Homology analysis of circ_0004801 revealed one homologous circRNA in humans: hsa_ANKRD32_0001200. The homology analysis results are as follows:

[0049] 1. Conservative marker (conserved = TRUE)

[0050] This is the only alignment result explicitly labeled as a conserved species (conserved=TRUE), indicating that the circRNA is conserved in cross-species evolution, significantly enhancing its credibility as a homology candidate.

[0051] 2. Excellent performance in whole-sequence alignment (blastWhole)

[0052] High sequence similarity: ident = 85.4 (identity score 0.85) and pident = 84.554% (fragment similarity percentage 84.55%), both higher than most other entries.

[0053] Excellent coverage: The alignment length (length = 1994) almost covers the entire query sequence (qlen = 1975) and target sequence (slen = 1966), indicating that the two sequences are highly matched across the entire length range.

[0054] The alignment regions are complete: qregion=1-1975 and sregion=1-1966 show that the alignment covers most of the sequence regions, further supporting their homology.

[0055] 3. The evolutionary conservation score (phastCons) is reliable.

[0056] The phastCons scores were 0.825, 0.891, and 0.907, all three scores being relatively high (close to or exceeding 0.8), indicating that the sequence has significant evolutionary conservation across multiple species.

[0057] According to the search, the mean expression of hsa_ANKRD32_0001200 in human lungs is 0.026.

[0058] This indicates that circ_0004801 has a homologous circRNA in humans: hsa_ANKRD32_0001200.

[0059] The nucleotide sequence of hsa_ANKRD32_0001200 is shown in SEQ ID NO: 2:

[0060]

[0061] Example 1

[0062] Immunoblot detection of tau and TTBKl in hippocampal tissue and primary neurons of 3×Tg-AD mice.

[0063] Hippocampal tissues and primary neurons were collected from WT mice and 3×Tg-AD mice, respectively. Proteins were extracted and tau and TTBK1 were detected by immunoblotting.

[0064] The results are as follows Figure 1 As shown, compared with matched WT mice, the phosphorylation levels of tau(199), tau(202), and tau(396) sites in the hippocampus of 3×Tg-AD mice were significantly upregulated. Figure 1 A~ Figure 1 B); phosphorylation levels at tau(199), tau(202), and tau(396) sites were also significantly upregulated in primary hippocampal neurons of 3×Tg-AD mice. Figure 1 C~ Figure 1 D).

[0065] Furthermore, compared to matched WT mice, 3×Tg-AD mice showed higher levels of TTBK1 protein in the hippocampus and primary neurons. Figure 1 E~ Figure 1 F) and mRNA ( Figure 1 The expression levels of G were significantly increased.

[0066] Figure 1 In the table, A and B are representative images and quantitative analyses of tau immunoblotting in the hippocampus of 3×Tg-AD mice (animal model); C and D are representative images and quantitative analyses of tau immunoblotting in primary hippocampal neurons of 3×Tg-AD mice (cell model); E and F are representative images and quantitative analyses of TTBK1 immunoblotting in the hippocampus and primary hippocampal neurons of 3×Tg-AD mice; G shows the expression of TTBK1 mRNA in the hippocampus and primary hippocampal neurons of 3×Tg-AD mice; *P<0.05, **P<0.01, ***P<0.001.

[0067] Example 2

[0068] Differentially expressed circRNAs and miRNAs were screened from the hippocampus of 3×Tg AD mice.

[0069] Hippocampal tissues from 3×Tg AD mice and matched WT mice were collected, and total RNA was extracted using TRIzol reagent (Invitrogen). RNA sequencing was then performed by Beijing BioMed Biotechnology Co., Ltd.

[0070] Sequencing results were analyzed using a bioinformatics workflow, including FastQC, trimmomatic removal, and STAR alignment of the raw sequencing data. circRNA identification utilized CIRCexplorer2 to identify backsplicing sites, and miRNA analysis was performed using miRDeep2 based on the miRBase database (v22). Differential expression analysis employed the DESeq2 algorithm (|log2FC|>1, FDR<0.05), and volcano plots were generated using Graphpad Prism 10.1.2 to visualize significantly differentially expressed molecules. Genomic distribution and co-expression networks of circRNAs were visualized using Cytoscape (v3.8) and Circos (v0.69).

[0071] Analysis revealed 3399 circRNAs in the hippocampus of 3×Tg AD mice and WT mice, of which 1681 circRNAs were co-occurring in different samples. Figure 2 A). Volcano plots showed that, compared to WT mice, 273 circRNAs were upregulated and 301 circRNAs were downregulated in the hippocampus of 3×Tg AD mice. Figure 2 B). The volcano plot shows the expression of miRNAs; 71 miRNAs were upregulated, and 83 were downregulated. Figure 2 C). Data are expressed as mean ± SEM.

[0072] Further screening of circRNA and miRNA molecules based on regulatory networks, including circRNA-miRNA interaction prediction, differential miRNA screening, and miRNA-AD key gene targeting sequencing, identified 10 important circRNAs and 12 miRNAs for further detection.

[0073] The expression levels of the 10 circRNAs and the 12 miRNAs in the hippocampus of 6 3×Tg-AD mice and 6 WT mice were detected by qRT-PCR.

[0074] The results are as follows Figure 3 As shown, among the 10 circRNAs detected, circ_0004801 was significantly upregulated, and circ_0011110 was significantly downregulated. Figure 3 A~ Figure 3 B). Among the 12 miRNAs detected, miR-7688-5p and miR-128-2-5p were significantly downregulated, while miR-6948-5p was significantly upregulated. Figure 3 C~ Figure 3 D).

[0075] Further target prediction revealed a target relationship between circ-0004801 and miR-7688-5p. Figure 3 E), and the downstream target gene of miR-7688-5p was predicted to be TTBK1 ( Figure 3 F).

[0076] The above results suggest that the circ_0004801-miR-7688-5p-TTBK1 control axis may play a regulatory role in AD.

[0077] Example 3

[0078] 1. siRNA inhibits the expression of circ_0004801.

[0079] siRNAs specifically designed to silence the circ_0004801 nucleotide sequence were developed. Three siRNAs were designed and screened, with the following positive strands: siRNA1: 5'-CTTTCCTGTGATGATGGAA-3' (SEQ ID NO: 3); siRNA2: 5'-TCCTGTGATGATGGAAGAT-3' (SEQ ID NO: 4); siRNA3: 5'-GTGATGATGGAAGATAGTG-3' (SEQ ID NO: 5); NC sequence: 5'-TTCTCCGAACGTGTCACGTT-3' (SEQ ID NO: 6). siRNAs 1–3 and the NC sequence were provided by BlueLife Biotechnology Co., Ltd. (Guangzhou, China).

[0080] Use Lipofectamine TM RNAiMAX (Invitrogen) was used to transfect siRNA1–3 and NC sequences into HT22 and 293T cells, respectively, at the following transfection concentrations: siRNA1 50 nm / ml, siRNA1 100 nm / ml, siRNA2 50 nm / ml, siRNA2 100 nm / ml, siRNA3 100 nm / ml, and NC 100 nm / ml. Experimental procedures were strictly performed according to the manufacturer's instructions. Interference efficiency was assessed using qRT-PCR after 24 hours.

[0081] like Figure 4As shown in Figure A, transfection with siRNA1–3 can effectively inhibit the expression of circ_0004801. Among them, siRNA2 at a concentration of 100 nm / ml has the best effect in inhibiting the expression of circ_0004801. Therefore, siRNA2 at a concentration of 100 nm / ml was selected for further experiments.

[0082] 2. Effect of inhibiting circ_0004801 expression on HT22 cell proliferation

[0083] Cell proliferation was detected using the MTS assay, strictly following the manufacturer's instructions. HT22 cells transfected with the stated siRNA2 and NC sequences were seeded in 96-well plates (100 μl per well, 1 × 10⁻⁶ cells / well). 4 Cells / well). Cells incubated for 0 and 24 hours were collected and mixed with CellTiter 96AQ single-solution cell proliferation reagent (Promega, Cat. No. G3582) at a 1:10 ratio. After 1.5 hours, absorbance was measured at 490 nm using a microplate reader.

[0084] like Figure 4 As shown in Figure B, MTS assay results indicate that silencing circ_0004801 promotes the proliferation of HT22 cells.

[0085] 3. Effect of inhibiting circ_0004801 expression on apoptosis in HT22 cells

[0086] Apoptosis was detected by Annexin V-FITC / PI double staining combined with flow cytometry using the Annexin V-FITC apoptosis detection kit (Keygen, catalog number KGA106), strictly following the manufacturer's instructions. HT22 cells transfected with the stated siRNA2 and NC sequences were seeded in 6-well plates and incubated for 48 hours before cell collection for analysis.

[0087] like Figure 4 C~ Figure 4 As shown in Figure D, silencing the expression of circ_0004801 inhibited apoptosis in HT22 cells.

[0088] Figure 4 Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

[0089] The above results indicate that siRNA-specific inhibition of circ_0004801 expression can promote the proliferation of HT22 cells and inhibit their apoptosis. Since apoptosis of neurons occurs frequently in the pathological process of AD, the results preliminarily suggest that circ_0004801 can serve as a therapeutic target for AD.

[0090] Example 4

[0091] 1. Effects of miR-7688-5p transfection on TTBK1 expression and tau phosphorylation.

[0092] HT22 cells were transfected with miR-7688-5p (5′-UAGCUGGGCAUGAUCUGAUGAGC-3′, SEQ ID NO: 7), with the NC sequence shown in SEQ ID NO: 6 serving as a control. The groups were as follows: NC group: transfected with the negative control NC sequence at a final concentration of 50 nM; miR-7688-5p group: transfected with miR-7688-5p at a final concentration of 50 nM; miR-7688-5p+vector group: co-transfected with miR-7688-5p (50 nM) + empty vector plasmid (2 μg / ml); miR-7688-5p+TTBK1 group: co-transfected with miR-7688-5p (50 nM) + TTBK1 overexpression plasmid (2 μg / ml). After 48 hours of treatment, TTBK1 expression and tau phosphorylation were detected by Western blot. The TTBK1 overexpression plasmid and empty vector plasmid were constructed and provided by Guangzhou Asoda Biomedical Technology Co., Ltd.

[0093] like Figure 5 A and Figure 5 As shown in Figure B, transfection with miR-7688-5p significantly reduced TTBK1 expression and inhibited tau phosphorylation at ser199 and ser202, while total tau remained largely unchanged.

[0094] 2. Effects of silencing circ_0004801 on TTBK1 expression and tau phosphorylation

[0095] The groups were as follows: NC group: transfected with the negative control siRNA as shown in SEQ ID NO: 6, final concentration 100 nM; si-circ group: transfected with the siRNA2 (si-circ) specifically silencing circ_0004801 expression, final concentration 100 nM; si-circ+vector group: co-transfected with si-circ (100 nM) + empty vector plasmid (2 μg / ml); si-circ+TTBK1 group: co-transfected with si-circ (100 nM) + TTBK1 overexpression plasmid (2 μg / ml). After 48 hours of treatment in each group, TTBK1 expression and tau phosphorylation levels were detected by Western blot and immunofluorescence, respectively.

[0096] like Figure 5 C and Figure 5As shown in Figure D, silencing the expression of circ_0004801 significantly reduced the expression of TTBK1 and inhibited the phosphorylation of tau at ser199, ser202, and ser396, while total tau remained unchanged.

[0097] like Figure 5 E and Figure 5 As shown in F, immunofluorescence further confirmed that silencing the expression of circ-0004801 reduced the phosphorylation of tau in ser199.

[0098] Figure 5 In this study, data are expressed as mean + SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

[0099] The above results indicate that circ_0004801 and miR-7688-5p play important roles in the pathological process of Alzheimer's disease, especially in TTBK1 regulation and tau protein phosphorylation.

[0100] Example 5

[0101] circ_0004801 interacts with miR-7688-5p.

[0102] The aforementioned results suggest a potential interaction between circ_0004801 and miR-7688-5p. To further verify this interaction, the binding sites of circ_0004801 and miR-7688-5p were predicted using RNAhybrid and Miranda software. The predicted binding sites of circ-0004801 (WT) and miR-7688-5p are shown below. Figure 6 As shown in A, circ-0004801(WT) was mutated to obtain circ-0004801(Mut). The mutation site sequence of circ-0004801(Mut) nucleotides is as follows: 5′-GCUAAAGAUAC-3′ (SEQ ID NO: 8).

[0103] Next, the effect of silencing circ_0004801 on miR-7688-5p was investigated. The expression of circ_0004801 in HT22 cells was silenced using the siRNA2 described in Example 3. HT22 cells transfected with the siRNA2 were seeded in 96-well plates (100 μl per well, 1 × 10⁻⁶ cells / well). 4 Cells / well). After 48 hours of incubation, the level of miR-7688-5p was detected by qRT-PCR. Figure 6 As shown in Figure B, the level of miR-7688-5p in HT22 cells did not change significantly after silencing circ-0004801.

[0104] After co-transfecting 293T cells with miR-7688-5p or a miR-7688-5p inhibitor, the fluorescence intensity of wild-type circ_0004801 (circ_0004801WT) and mutant (circ_0004801Mut) fragments was analyzed. Normalization was performed using Renilla luciferase. Wild-type (WT) or mutant (Mut) circ-0004801 fragments were inserted downstream of the luciferase reporter gene in psiCHECK-2. Then, miR-7688-5p or a miR-7688-5p inhibitor and the reporter gene were co-transfected into 293T cells using Lipofectamine 2000 (Invitrogen). The groups are as follows: Blank group: transfected only with 0.5 μg wild-type (WT) or mutant (Mut) circ_0004801-psiCHECK2 plasmid (without microRNA or inhibitor); NC group: co-transfected with 0.5 μg WT / Mut circ_0004801 plasmid + 50 nM negative control microRNA (NC); miR-7688-5p group: co-transfected with 0.5 μg WT / Mut circ_0004801 plasmid ± 50 nM miR-7688-5p mimics; NC inhibitor group: co-transfected with 0.5 μg WT / Mut circ_0004801 plasmid + 100 nM negative control inhibitor (NC inhibitor); miR-7688-5p inhibitor group: co-transfected with 0.5 μg WT / Mut circ_0004801 plasmid + 100 nM miR-7688-5p Inhibitors. The NC inhibitor was a negative control inhibitor (SEQ ID NO: 9: 5′-CAGUACUUUUGUGUAGUACAA-3′, non-target sequence), and the miR-7688-5p inhibitor was an antisense oligonucleotide that specifically inhibits miR-7688-5p. After 48 hours, the luciferase activity of fireflies and jellyfish was detected using an automated luminescence detector. Figure 6 As shown in Figure C, the results of the dual-luciferase reporter system assay indicated that co-transfection with miR-7688-5p significantly inhibited the luciferase activity of the circ_0004801WT vector compared to the negative control group, but had no significant effect on the Mut vector. This verified the binding sites of the two vectors, suggesting that circ_0004801 can directly bind to miR-7688-5p via the ceRNA mechanism. All plasmids used in the experiment were provided by Guangzhou Blue Dolphin Biotechnology Co., Ltd.

[0105] Further analysis revealed the enrichment of circ_0004801 and miR-7688-5p, as well as miR-7688-5p and TTBK1, in the Ago2 protein. The results showed that circ_0004801 and miR-7688-5p were enriched in the Ago2 protein. Figure 6 D), miR-7688-5p and TTBK1 are enriched in Ago2 protein ( Figure 6 E). Figure 6 F is a positive control for RNA immunoprecipitation (RIP) test.

[0106] Figure 6 In this study, data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

[0107] The above results indicate that circ_0004801, miR-7688-5p, and TTBK1 were significantly enriched by Ag02 protein, verifying the existence of the circ_0004801-Ag02-miR-7688-5p complex.

[0108] Example 6

[0109] Morris Water Maze Test

[0110] The Morris Water Maze (MWM) behavioral testing system (model: XR-XM101, Shanghai Xinruan) is used to assess the spatial learning and memory abilities of laboratory animals. The testing device consists of a circular pool with a diameter of 120 cm and a height of 60 cm. Four spatial orientation points (N, S, E, W) are marked on the pool walls, dividing the pool into four virtual quadrants. A platform (10 cm in diameter) is fixedly placed at the center of the target quadrant (NW), with its top 1 cm above the water surface. The water temperature is maintained at 23±1℃, and the water is made opaque by adding food-grade titanium dioxide.

[0111] The experimental procedure was divided into two stages: (1) Hidden platform training (days 1-5): Four training sessions were conducted daily, with a maximum duration of 60 seconds per session. If the animal failed to locate the platform within the time limit, it was gently guided to the platform by the experimenter and remained there for 15 seconds. The escape latency (EL) was recorded using the EthoVision XT 14.0 video tracking system (Noldus, Netherlands), defined as the time interval from when the animal entered the water to when its forelimbs touched the platform. The starting positions were rotated in a pseudo-random order (N / S / E / W) to avoid orientation bias. (2) Spatial exploration test (day 6): After the platform was removed, the animal was given 60 seconds of free exploration. The target quadrant time (QT) and the number of times the animal crossed the original platform site (Platform Crossings) were recorded. The ambient noise was kept <50dB and the light intensity was 20 lux throughout the experiment. All animal experiments followed the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 8023).

[0112] Experiments were conducted using 3×Tg transgenic AD mice. Mice were anesthetized by intraperitoneal injection of 1.2 g / kg 20% ​​urethane; no response was observed when the tail and toes were gently pinched, indicating deep anesthesia. The mice were fixed to a stereotactic apparatus, ensuring their heads were centered and horizontally aligned. The hair on the top of the skull was shaved, the skin was disinfected, and the skin and subcutaneous tissue were incised to expose the skull. Using the anterior fontanelle as the reference zero point, the mouse's head position was adjusted so that its left, right, front, and back were all equidistant from the zero point and aligned horizontally. The anterior fontanelle was then repositioned, and the coordinates were reset to 0. Locate the lateral ventricles (0.25mm posterior to the anterior fontanelle, 1mm lateral to the midline, 1.9mm subcranially). Use a dental drill to make a hole (approximately 2mm in diameter) at the site where the drug delivery device will be inserted, keeping the dura mater intact. Using a 5μL Hamilton needle with a 33-gauge needle, inject the drug into both lateral ventricles (0.25mm posterior to the anterior fontanelle, 1mm lateral to the midline, 2.7mm subcranially, gradually withdrawing to 2.4mm). After injection, hold the needle in place for 5 minutes, then slowly withdraw the needle to avoid backflow. The experimental groups and drug treatments were as follows: sh-NC group: 2 μL of recombinant lentivirus containing a random shRNA control was injected into the lateral ventricle; sh-circ group: 2 μL of recombinant lentivirus targeting circ_0004801 was injected into the lateral ventricle. The nucleotide sequence of the shRNA was as follows: Sensitive strand: 5'-GATCCGTCCTGTGATGATGGAAGATTTCAAGAGAATCTTCCATCATCACAGGATTTTTG-3' (SEQ ID NO: 10); Antisense strand: 5'-AATTCAAAAATCCTGTGATGATGGAAGATTCTCTTGAAATCTTCCATCATCACAGGACG-3' (SEQ ID NO: 11); sh-circ+vector group: 1 μL of sh-circ lentivirus + 1 μL of empty vector control lentivirus were injected; sh-circ+TTB K1 group: 1 μL of sh-circ lentivirus + 1 μL of TTBK1 overexpressing lentivirus were injected. All viral titers were 1 × 10⁻⁶. 9 TU / mL, behavioral experiments were initiated 15 days after viral transfection. The shRNA recombinant lentivirus targeting circ_0004801, the TTBK1 overexpressing lentivirus, and the empty vector control lentivirus were constructed and provided by Guangzhou Asoda Biomedical Technology Co., Ltd.

[0113] Figure 7 A represents the change in escape time (ET) for each group in the water maze hidden platform experiment; Figure 7B represents the trajectory of each group of mice in the water maze spatial exploration experiment and the percentage of time spent in the platform quadrant (QT). The results showed that in the hidden platform experiment, the escape time (ET) of all mouse groups gradually decreased over time. Compared with the control group, knockdown of circ_0004801 significantly reduced the escape time of mice, while overexpression of TTBK1 led to a prolonged escape time. In the spatial exploration experiment, the shRNA-circ group showed a significant increase in the time spent in the platform quadrant, while the TTBK1 group showed a significant decrease. These results indicate that knockdown of circ_0004801 significantly improved the spatial learning and memory abilities of mice, while overexpression of TTBK1 significantly weakened these abilities.

[0114] After the brains of mice in each group were frozen and sectioned (7μm), the total Tau protein and P-tau(199) protein in the hippocampus of each group of mice were detected by immunofluorescence. Figure 7 C represents the immunofluorescence map and quantitative analysis of P-tau(199) protein in the hippocampus. The results show that the phosphorylation level of tau protein in the hippocampus was significantly reduced after circ_0004801 knockdown. Figure 7 D is a representative immunofluorescence map and quantitative analysis of total Tau protein in the hippocampus. The results showed that the total amount of tau protein in the hippocampus did not change significantly after circ_0004801 knockdown.

[0115] NFTs were assessed in paraffin sections (7 μm) of brain tissue from each group using a modified Bielschowsky silver staining kit (kit number: G1038, Servicebio; version: 2023.01). The simplified procedure included: tissue dewaxing and hydration, silver nitrate staining in the dark, ammoniacal silver imaging, incubation in the dark, and fixing. Specific steps were strictly performed according to the kit instructions. After mounting with neutral resin, NFTs were evaluated under an optical microscope.

[0116] Figure 8 A shows a comparison of silver-stained NFTs in the DG region of the mouse hippocampus; Figure 8 B represents the quantitative analysis of silver-stained NFTs in the DG region of the mouse hippocampus. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001. It can be seen that the density of NFTs in the DG region of the hippocampus decreased after circ_0004801 knockdown, and overexpression of TTBK1 reversed this effect.

[0117] The above results indicate that knockdown of circ_0004801 significantly improves spatial learning and memory function in mice, and effectively slows the pathological progression of Alzheimer's disease by reducing the phosphorylation level of tau protein in the hippocampus and decreasing the accumulation of non-freezing factors (NFTs). Therefore, circ_0004801 can serve as a novel therapeutic target for Alzheimer's disease, and agents targeting and inhibiting circ_0004801 can be used to prepare drugs for treating Alzheimer's disease.

[0118] Furthermore, because the circular RNA circ_0004801 adsorbs miR-7688-5p through a sponge-like mechanism, it relieves the inhibition of TTBK1, driving the formation of abnormal tau protein phosphorylation, ultimately leading to cognitive impairment and promoting the progression of Alzheimer's disease (AD). Silencing or inhibiting the expression of circular RNA circ_0004801 in HT22 cells, or transducing miR-7688-5p, can enhance the inhibition of TTBK1, inhibit abnormal tau protein phosphorylation, alleviate cognitive impairment, and thus exert a therapeutic effect on AD. Therefore, circular RNA circ_0004801 and its homologous circular RNAs hsa_ANKRD32_0001200 and miR-7688-5p can all serve as novel therapeutic targets for AD.

[0119] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0120] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of a reagent for inhibiting or silencing the expression of circular RNA circ_0004801 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The nucleotide sequence of the circular RNA circ_0004801 is shown in SEQ ID NO:

1.

2. The application of a reagent that inhibits or silences the expression of circular RNA hsa_ANKRD32_0001200 in the preparation of drugs for treating Alzheimer's disease, characterized in that, The nucleotide sequence of the circular RNA hsa_ANKRD32_0001200 is shown in SEQ ID NO:

2.

3. The application as described in claim 1 or 2, characterized in that, The reagents include at least one of siRNA, shRNA, and a recombinant expression vector containing the shRNA.

4. The application as described in claim 3, characterized in that, The siRNA is selected from at least one of the siRNAs with nucleotide sequences as shown in SEQ ID NO: 3 to SEQ ID NO: 5; and / or, The shRNA comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO: 10, and the nucleotide sequence of which is shown in SEQ ID NO: 11; and / or, The recombinant expression vector is a recombinant lentiviral expression vector.

5. Application of miR-7688-5p or miR-7688-5p overexpression vector in the preparation of drugs for treating Alzheimer's disease.

6. A drug for treating Alzheimer's disease, characterized in that, The active ingredient of the drug includes at least one of the following (1) to (2): (1) a reagent for inhibiting or silencing the expression of circular RNA circ_0004801, the nucleotide sequence of which is shown in SEQ ID NO: 1; (2) a reagent for inhibiting or silencing the expression of circular RNA hsa_ANKRD32_0001200, the nucleotide sequence of which is shown in SEQ ID NO: 2; The reagents in (1) and (2) include siRNA, shRNA, and recombinant expression vectors containing the shRNA; The siRNA is selected from at least one of the siRNAs with nucleotide sequences as shown in SEQ ID NO: 3 to SEQ ID NO: 5; The shRNA comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO: 10 and the nucleotide sequence of which is shown in SEQ ID NO:

11.

7. The drug as described in claim 6, characterized in that, The recombinant expression vector is a recombinant lentiviral expression vector.

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