Circular RNA circ-0004801 and application thereof in treatment of Alzheimer disease

Abstract

The invention provides a circular RNA circ0004801 and an application of the circular RNA circ0004801 in treatment of an Alzheimer's disease. The nucleotide sequence of the circular RNA circ0004801 is as shown in SEQ ID NO: 1 (Sequence Identity Number 1). The expression of the circular RNA circ0004801 in the hippocampal tissue of the AD mouse is obviously up-regulated, and the circular RNA circ0004801 is used as a'sponge 'of miR-7688-5p and plays a role by adjusting the TTBK1 expression and tau phosphorylation level. By knocking down the circular RNA circ0004801, the spatial learning and memory of a 3 * Tg mouse can be obviously improved, and the tau protein phosphorylation level of a hippocampus region and the NFTs density of a hippocampus DG region can be obviously reduced. Therefore, the circular RNA circ0004801 and the miR-7688-5p can be used as new targets for treating the AD (Alzheimer's disease). According to the invention, it is found for the first time that circular RNA circ0004801 regulates TTBK1 through a ceRNA mechanism, so that excessive phosphorylation of tau is driven, and a new molecular target is provided for AD early intervention treatment based on circRNA and miRNA.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to circular RNA circ_0004801 and its application in the treatment of Alzheimer's disease. Background Art

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

[0003] Tau-tubulin kinase 1 (TTBK1) is a major regulator of tau phosphorylation and drives the pathogenesis of AD through multiple mechanisms: (1) phosphorylation at specific sites (Ser199 / Ser202 / Ser396) directly catalyzes the formation of NFTs; (2) activates downstream kinases such as GSK-3β, establishing a phosphorylation cascade; and (3) regulates the pathological modification of proteins associated with neurodegeneration (such as TDP-43). However, the mechanisms of TTBK1 dysregulation and its upstream regulatory network remain unclear, significantly hindering the development of tau-targeted therapeutics.

[0004] Current approaches to regulating TTBK1 primarily focus on small molecule inhibitors, but these have limitations such as low blood-brain barrier penetration and significant off-target effects. In recent years, non-coding RNAs (such as circRNAs and miRNAs) have emerged as emerging therapeutic targets for neurodegenerative diseases due to their brain tissue enrichment and high targeting potential. However, studies have yet to demonstrate 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 the present 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 solutions.

[0007] The first aspect of the present invention provides a circular RNA circ_0004801, wherein the nucleotide sequence of the circular RNA circ_0004801 is as shown in SEQ ID NO: 1, or 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 present invention provides use of an agent for inhibiting or silencing the expression of the circular RNA circ_0004801 as described above in the preparation of a drug for treating Alzheimer's disease.

[0009] The third aspect of the present invention provides the use of an agent for inhibiting or silencing the expression of circular RNAhsa_ANKRD32_0001200 in the preparation of a drug for treating Alzheimer's disease.

[0010] In some embodiments, the agent comprises at least one of siRNA, shRNA, and a recombinant expression vector comprising the shRNA.

[0011] In some embodiments, the siRNA is selected from at least one siRNA having a nucleotide sequence 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 is shown in SEQ ID NO: 10, and the nucleotide sequence of the antisense strand is 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 present invention provides use of miR-7688-5p or a miR-7688-5p overexpression vector in the preparation of a drug for treating Alzheimer's disease.

[0015] A fifth aspect of the present invention provides a drug for treating Alzheimer's disease, wherein the active ingredient of the drug comprises at least one of the following (1) to (4): (1) an agent that inhibits or silences the expression of the circular RNA circ_0004801 as described above; (2) an agent that inhibits or silences the expression of the circular RNA hsa_ANKRD32_0001200; (3) miR-7688-5p; and (4) a 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 siRNA having a nucleotide sequence as shown in SEQ ID NO: 3 to SEQ ID NO: 5; and / or,

[0018] The shRNA includes a sense strand and an antisense strand. The nucleotide sequence of the sense strand is shown in SEQ ID NO: 10, and the nucleotide sequence of the antisense strand 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] The present invention obtained a circular RNA circ_0004801 through research and screening, and found its homologous circular RNA hsa_ANKRD32_0001200 in humans through search and analysis. The present invention found that the expression of the circular RNA circ_0004801 in the hippocampus of AD mice was significantly upregulated, and it can 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) significantly reduced tau phosphorylation levels in neuronal cells and reduced cell apoptosis, accompanied by enhanced cell viability; (2) improved spatial learning and memory in 3×Tg mice, and significantly reduced tau protein phosphorylation levels in the hippocampus and NFTs density in the DG region of the hippocampus. Therefore, the circular RNA circ_0004801 and its homologous circular RNA hsa_ANKRD32_0001200 can serve as new targets for the treatment of AD.

[0022] Furthermore, the present invention demonstrates the regulatory role of the circular RNA circ_0004801 / miR-7688-5p / TTBK1 axis in the progression of AD. Circular RNA circ_0004801 acts as a sponge to absorb miR-7688-5p, relieving its inhibitory effect on TTBK1. This drives the formation of abnormal tau phosphorylation, ultimately leading to cognitive impairment and promoting AD disease progression. Silencing or inhibiting the expression of circular RNA circ_0004801 in HT22 cells, or transfecting miR-7688-5p, enhances the inhibitory effect on TTBK1, inhibits abnormal tau phosphorylation, alleviates cognitive impairment, and thus plays a therapeutic role in AD. Therefore, both miR-7688-5p and circular RNA circ_0004801 may serve as new targets for the treatment of AD.

[0023] This study first discovered that circular RNA circ_0004801 regulates TTBK1 by targeting miR-7688-5p through the ceRNA mechanism, thereby driving the hyperphosphorylation of tau, providing a new molecular target for early intervention therapy of AD based on circRNA and miRNA. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 Screening results for differentially expressed circRNA and miRNA.

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

[0027] Figure 4 The results of the test show that the inhibition of circ_0004801 expression affects the proliferation and apoptosis of HT22 cells.

[0028] Figure 5 Effects of miR-7688-5p transfection and circ_0004801 silencing on TTBK1 expression and tau phosphorylation in HT22 cells.

[0029] Figure 6 This is the interaction detection result between circ_0004801 and miR-7688-5p.

[0030] Figure 7 To inhibit the expression of circ_0004801 and improve the spatial learning and memory of 3×Tg mice.

[0031] Figure 8 These are the test results of the effect of inhibiting circ_0004801 expression on NFTs in 3×Tg mice. DETAILED DESCRIPTION

[0032] Experimental procedures in the following examples, unless otherwise specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0033] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

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

[0035] The "and / or" mentioned in the present invention describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0036] The following describes the method in conjunction with 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 housed under temperature- and humidity-controlled conditions with a 12-h light / dark cycle with ad libitum access to food and water. All procedures were approved by the Laboratory Animal Ethics Committee of Guangzhou Medical University, China (Approval No. GD 2019-129).

[0038] HT22 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Camarillo, CA, USA) containing 10% FBS (Invitrogen) and 1% penicillin / streptomycin (Invitrogen) at 37°C in a CO2-free environment.

[0039] qRT-PCR assay: Total RNA was extracted from samples using Trizol reagent (Invitrogen), and genomic DNA was subsequently digested with DNase I (Promega). RNA was reverse transcribed using M-MLV (Promega) according to the instructions after purity and integrity testing. SYBR Green qPCR SuperMix Kit (Invitrogen) was used at ABI qPCR was performed on a 7500 Sequence Detection System with the following parameters: 50°C for 2 minutes, 95°C for 30 seconds, and then 40 cycles of 95°C for 5 seconds and 60°C for 34 seconds. -ΔΔCt For calculations, β-actin or U6 was used as an internal reference. The primers used are listed in Table 1.

[0040] Table 1

[0041]

[0042]

[0043]

[0044] Western blot analysis: Western blot analysis was performed according to previous research methods (Q. Li et al., 2022). Protein was extracted from the tissues or cells to be tested. Total protein content was determined using a bicoumaric acid (BCA) kit (Beyotime Biotechnology, Jiangsu, China). The antibodies used in the present 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 horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, Sigma-Aldrich). The membrane was incubated with the primary antibodies overnight at 4°C, followed by incubation with the secondary antibodies at 20-25°C for 2 hours. GAPDH was used as an internal control. The optical density of the target band was estimated using ImageJ software (National Institutes of Health; http: / / rsb.info.nih.gov / ij / ).

[0045] Statistical Analysis: Data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS 26.0 software (IBM, USA), and graphs were generated using GraphPad 9.0 software (GraphPad Software, USA). Differences between two groups were analyzed using the Student's t-test (two-tailed). Variance between multiple groups was analyzed using one-way analysis of variance (ANOVA) with Tukey's post hoc test. P < 0.05 was considered statistically significant.

[0046] The nucleotide sequence of the circular RNA circ_0004801 of the present invention is shown in SEQ ID NO: 1. The nucleotide sequence is the mature sequence after the coding gene is transcribed and spliced. The database annotates the mature circRNA in DNA sequence format, using U instead of T.

[0047]

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

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

[0050] This is the only alignment result clearly marked as a conserved species (conserved = TRUE), indicating that this circRNA has conservation support in cross-species evolution, significantly improving its credibility as a homologous candidate.

[0051] 2. Excellent performance of full sequence alignment (blastWhole)

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

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

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

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

[0056] The phastCons scores were 0.825|0.891|0.907, and all three scores were high (close to or above 0.8), indicating that the sequence was significantly evolutionarily conserved in multiple species.

[0057] After retrieval, the mean Expression of hsa_ANKRD32_0001200 in human lung 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] Immunoblotting detection of tau and TTBK1 in hippocampal tissues and primary neurons of 3×Tg-AD mice.

[0063] Hippocampal tissues and primary neurons were obtained from WT mice and 3×Tg-AD mice, and proteins were extracted and immunoblotted for tau and TTBK1.

[0064] The results are as follows Figure 1 As shown in Figure 2, the phosphorylation levels of tau(199), tau(202), and tau(396) sites in the hippocampus of 3×Tg-AD mice were significantly upregulated compared with those of matched WT mice ( Figure 1 A~ Figure 1 B); The phosphorylation levels of 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] In addition, compared with matched WT mice, TTBK1 protein ( Figure 1 E~ Figure 1 F) and mRNA ( Figure 1 G) expression levels were significantly increased.

[0066] Figure 1 In the figure, A and B are representative images and quantitative analysis of tau immunoblotting in hippocampal tissues of 3×Tg-AD mice (animal model); C and D are representative images and quantitative analysis of tau immunoblotting in primary hippocampal neurons of 3×Tg-AD mice (cell model); E and F are representative images and quantitative analysis of TTBK1 immunoblotting in hippocampal tissues and primary hippocampal neurons of 3×Tg-AD mice; G is the expression of TTBK1 mRNA in hippocampal tissues 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 hippocampal tissues of 3×Tg AD mice.

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

[0070] Sequencing results were analyzed using bioinformatics pipelines for quality control (FastQC), removal of low-quality sequences (Trimmomatic), and alignment to the reference genome (STAR). CircRNAs were identified using CIRCexplorer2 to identify backsplicing sites, and miRNAs were annotated using miRDeep2 based on the miRBase database (v22). Differential expression analysis was performed using the DESeq2 algorithm (|log2FC| > 1, FDR < 0.05), and Graphpad Prism 10.1.2 was used to create volcano plots to display significantly differentially expressed molecules. The genomic distribution and co-expression network of circRNAs were visualized using Cytoscape (v3.8) and Circos (v0.69).

[0071] After analysis, a total of 3399 circRNAs were identified in the hippocampus tissues of 3×Tg AD mice and WT mice, of which 1681 circRNAs were common in different samples ( Figure 2 A). The volcano plot showed that 273 circRNAs were upregulated and 301 circRNAs were downregulated in the hippocampus of 3×Tg AD mice compared with WT mice ( Figure 2 B). The volcano plot shows the expression of miRNAs. There are 71 up-regulated miRNAs and 83 down-regulated miRNAs ( Figure 2 C). Data are expressed as mean ± SEM.

[0072] We further screened circRNA and miRNA molecules based on the regulatory network, performed circRNA-miRNA interaction prediction, differential miRNA screening, and miRNA-AD key gene targeted sorting, and screened out 10 important circRNAs and 12 miRNAs for further testing.

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

[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 targeting prediction was performed, and the results showed that circ-0004801 and miR-7688-5p had a targeting relationship ( Figure 3 E), and the downstream target gene of miR-7688-5p was predicted to be TTBK1 ( Figure 3 F).

[0076] These results indicate that the circ_0004801-miR-7688-5p-TTBK1 regulatory axis may play a regulatory role in AD.

[0077] Example 3

[0078] 1. siRNA inhibits the expression of circ_0004801

[0079] siRNAs were designed to specifically silence circ_0004801 against the nucleotide sequence of circ_0004801. The following three siRNAs were designed and screened, with the following sense 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); and NC sequence: 5'-TTCTCCGAACGTGTCACGTT-3' (SEQ ID NO: 6). siRNAs 1 to 3 and the NC sequence were provided by BlueLife Biotechnology Co., Ltd. (Guangzhou, China).

[0080] Using Lipofectamine TM siRNAs 1–3 and NC sequences were transfected into HT22 and 293T cells using RNAiMAX (Invitrogen) 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 performed strictly according to the manufacturer's instructions. Interference efficiency was assessed by qRT-PCR 24 hours later.

[0081] like Figure 4As shown in A, transfection of siRNAs 1 to 3 can effectively inhibit the expression of circ_0004801. Among them, siRNA 2 has the best effect of interfering with and inhibiting the expression of circ_0004801 at a concentration of 100 nm / ml. Therefore, siRNA 2 was selected to continue the experiment at a concentration of 100 nm / ml.

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

[0083] Cell proliferation was detected by MTS assay in strict accordance with the manufacturer's instructions. HT22 cells transfected with the siRNA2 and NC sequences were seeded in 96-well plates (100 μl per well, 1×10 4 Cells were collected after 0 and 24 hours of incubation and mixed with cellTiter96AQ Single Solution Cell Proliferation Reagent (Promega, Cat. No. G3582) at a ratio of 1:10. After 1.5 hours, the absorbance was measured at 490 nm using a microplate reader.

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

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

[0086] Cell apoptosis was detected by Annexin V-FITC / PI double staining combined with flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Keygen, Cat. No. KGA106) in strict accordance with the manufacturer's instructions. HT22 cells transfected with the siRNA2 and NC sequences were seeded in 6-well plates and harvested after 48 hours of incubation for detection.

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

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

[0089] The above results show that specific inhibition of circ_0004801 expression by siRNA can promote the proliferation of HT22 cells and inhibit their apoptosis. Since a large number of neurons undergo apoptosis in the pathological process of AD, the results preliminarily suggest that circ_0004801 can be used as a therapeutic target for AD.

[0090] Example 4

[0091] 1. Effect 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), and the NC sequence shown in SEQ ID NO: 6 was used as a control. The cells were divided into the following groups: 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) and an empty vector plasmid (2 μg / ml); and miR-7688-5p+TTBK1 group: co-transfected with miR-7688-5p (50 nM) and a TTBK1 overexpression plasmid (2 μg / ml). After 48 hours of treatment, TTBK1 expression and tau phosphorylation were analyzed by Western blot. TTBK1 overexpression plasmid and empty vector plasmid were constructed and provided by Guangzhou Aisuoda Biopharmaceutical Technology Co., Ltd.

[0093] like Figure 5 A and Figure 5 As shown in Figure 2, transfection of miR-7688-5p significantly reduced the expression of TTBK1 and inhibited the phosphorylation of tau at ser199 and ser202, while total tau was not significantly changed.

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

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

[0096] like Figure 5 C and Figure 5As shown in 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 did not change significantly.

[0097] like Figure 5 E and Figure 5 As shown in F, immunofluorescence further verified that silencing circ-0004801 expression reduced tau phosphorylation at Ser199.

[0098] Figure 5 Data are expressed as mean + SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

[0099] These results indicate that circ_0004801 and miR-7688-5p play an important role 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 above results indicate that there may be an interaction between circ_0004801 and miR-7688-5p. To further verify the interaction between the two, RNAhybrid and miranda software were used to predict the binding sites of circ_0004801 and miR-7688-5p. Figure 6 As shown in A, circ-0004801 (WT) was mutated to obtain circ-0004801 (Mut), and the mutation site sequence of the circ-0004801 (Mut) nucleotide is as follows: 5′-GCUAAAGAUAC-3′ (SEQ ID NO: 8).

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

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

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

[0106] Figure 6 Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

[0107] The above results showed 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) was used to assess the spatial learning and memory abilities of experimental animals. The testing apparatus consisted of a circular pool with a diameter of 120 cm and a height of 60 cm. Four spatial orientation points (N, S, E, and W) were marked on the pool wall, dividing the pool into four virtual quadrants. A platform (10 cm in diameter) was fixed at the center of the target quadrant (NW), with the top of the platform 1 cm above the water surface. The pool water temperature was maintained at 23 ± 1°C, and the water was rendered opaque by the addition of food-grade titanium dioxide.

[0111] The experimental procedure was divided into two phases: (1) Hidden platform training (days 1-5): 4 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, the experimenter gently guided it to the platform and kept it there for 15 seconds. The EthoVision XT 14.0 video tracking system (Noldus, Netherlands) was used to record the escape latency (EL), which was defined as the time interval from the animal entering the water to the forelimb contacting the platform. The starting position was rotated in a pseudo-random order (N / S / E / W) to avoid directional preference. (2) Spatial exploration test (day 6): After the platform was removed, the animal was allowed to explore freely for 60 seconds. The target quadrant residence time (quadrant time, QT) and the number of crossings to the original platform site (platform crossings) were recorded. The ambient noise level was kept below 50 dB and the light intensity was kept at 20 lux throughout the experiment. All animal experiments were carried out in accordance with the NIH Guide 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 with an intraperitoneal injection of 1.2g / kg 20% urethane. No response was observed by gently pinching the tail or toes, indicating deep anesthesia. The mice were fixed in a stereotaxic apparatus, with the head aligned horizontally and horizontally. The hair on the top of the skull was trimmed, the skin disinfected, and the skin and subcutaneous tissue were cut open to expose the skull. Using the bregma as the reference zero point, the mouse head was adjusted so that it was at the same level, both left and right, and front and back, from zero. The bregma was repositioned, returning the coordinates to zero. Locate the lateral ventricle (0.25 mm behind the anterior fontanelle, 1 mm beside the raphe, 1.9 mm below the skull), use a dental drill to drill a hole (about 2 mm in diameter) at the site where the drug delivery device is to be inserted, keeping the dura mater intact, and use a 5 μL Hamilton injection needle with a 33-gauge needle to inject the drug into the bilateral lateral ventricles (0.25 mm behind the anterior fontanelle, 1 mm beside the raphe, 2.7 mm below the skull, and gradually retreat to 2.4 mm). After the injection is completed, keep the injection needle in place for 5 minutes and slowly withdraw the needle to avoid reflux. The experimental groups and drug treatments were as follows: sh-NC group: 2 μL of recombinant lentivirus containing random shRNA control was injected into the lateral ventricle; sh-circ group: 2 μL of shRNA recombinant lentivirus targeting circ_0004801 was injected into the lateral ventricle. The nucleotide sequence of the shRNA was as follows: positive 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 was injected into the lateral ventricle; sh-circ+TTB K1 group: 1 μL of sh-circ lentivirus + 1 μL of TTBK1 overexpression lentivirus was injected into the lateral ventricle. All virus titers were 1×10 9 TU / mL, behavioral experiments were performed 15 days after viral transfection. The shRNA recombinant lentivirus targeting circ_0004801, TTBK1 overexpression lentivirus, and empty vector control lentivirus were constructed and provided by Guangzhou Aisuoda Biopharmaceutical Technology Co., Ltd.

[0113] Figure 7 A is the change of escape time (ET) of each group in the water maze hidden platform test; Figure 7B shows the trajectory of each group of mice in the water maze spatial exploration experiment and the proportion of time spent in the quadrant where the platform was located (QT). The results showed that in the hidden platform experiment, the escape time (ET) of all mouse groups gradually shortened 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 time spent in the platform quadrant of the shRNA-circ group was significantly increased, while that of the TTBK1 group was significantly reduced. These results indicate that knockdown of circ_0004801 significantly improved the spatial learning and memory ability of mice, while overexpression of TTBK1 significantly weakened the spatial learning and memory ability of mice.

[0114] The brains of mice in each group were frozen and sectioned (7 μm), and the total Tau protein and P-tau (199) protein in the hippocampus of mice in each group were detected by immunofluorescence. Figure 7 C is a representative image of immunofluorescence of P-tau (199) protein in the hippocampus and quantitative analysis. The results showed that the phosphorylation level of tau protein in the hippocampus was significantly reduced after circ_0004801 knockdown; Figure 7 D is a representative image of total Tau protein immunofluorescence and quantitative analysis in the hippocampus. The results showed that there was no significant change in the total amount of tau protein in the hippocampus after circ_0004801 knockdown.

[0115] Brain tissue paraffin sections (7 μm) from each group were stained with modified Bielschowsky silver stain to assess NFts (kit catalog number: G1038, Servicebio; version: 2023.01). The procedure included tissue dewaxing and hydration, dark-proof impregnation with silver nitrate, ammoniacal silver development, dark-proof incubation, and fixation. Specific steps were performed strictly according to the kit instructions. After mounting with neutral gum, NFTs were assessed under a light microscope.

[0116] Figure 8 A is a comparison of silver-stained NFTs in the DG region of the mouse hippocampus; Figure 8 B shows quantitative analysis of silver-stained NFTs in the DG region of the mouse hippocampus. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Knockdown of circ_0004801 decreased the density of NFTs in the DG region of the hippocampus, while overexpression of TTBK1 reversed this effect.

[0117] These results indicate that knockdown of circ_0004801 significantly improves spatial learning and memory in mice and effectively slows the pathological progression of Alzheimer's disease by reducing tau phosphorylation in the hippocampus and decreasing the accumulation of NFTs. Therefore, circ_0004801 may serve as a new target for the treatment of Alzheimer's disease, and agents that specifically inhibit circ_0004801 may be used to develop drugs for the treatment of Alzheimer's disease.

[0118] Furthermore, because circRNA circ-0004801 absorbs miR-7688-5p through a sponging effect, it relieves its inhibitory effect on TTBK1, driving the formation of abnormal tau protein phosphorylation, ultimately leading to cognitive dysfunction and promoting the progression of AD. Silencing or inhibiting the expression of circRNA circ_0004801 in HT22 cells, or transfecting miR-7688-5p, can enhance the inhibitory effect on TTBK1, inhibit abnormal tau protein phosphorylation, alleviate cognitive dysfunction, and thus play a therapeutic role in AD. Therefore, circRNA circ_0004801 and its homologous circRNA hsa_ANKRD32_0001200 and miR-7688-5p can serve as new targets for the treatment of AD.

[0119] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, 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 above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A circular RNA circ 0004801, characterized in that The nucleotide sequence of the circular RNA circ 0004801 is shown in SEQ ID NO: 1, or a functionally equivalent variant having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:

1.

2. Use of an agent for inhibiting or silencing the expression of the circular RNA circ_0004801 as claimed in claim 1 in the preparation of a drug for treating Alzheimer's disease.

3. Use of an agent for inhibiting or silencing the expression of circular RNA hsa_ANKRD320001200 in the preparation of a drug for treating Alzheimer's disease.

4. The use according to claim 2 or 3, characterized in that The reagent includes at least one of siRNA, shRNA and a recombinant expression vector containing the shRNA.

5. The use according to any one of claims 2 to 4, characterized in that: The siRNA is selected from at least one siRNA having a nucleotide sequence 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 the sense strand is shown in SEQ ID NO: 10, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 11; and / or, The recombinant expression vector is a recombinant lentiviral expression vector.

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

7. A drug for treating Alzheimer's disease, characterized in that: The active ingredients of the drug include at least one of the following (1) to (4): (1) an agent that inhibits or silences the expression of the circular RNA circ_0004801 as described in claim 1; (2) an agent that inhibits or silences the expression of the circular RNA hsa_ANKRD320001200; (3) miR-7688-5p; (4) a miR-7688-5p overexpression vector.

8. The drug according to claim 7, wherein The reagents in (1) and (2) include siRNA, shRNA, and a recombinant expression vector containing the shRNA.

9. The drug according to claim 8, wherein The siRNA is selected from at least one of the siRNAs whose nucleotide sequences are shown in SEQ ID NO: 3 to SEQ ID NO: 5; and / or, The shRNA includes a sense strand and an antisense strand. The nucleotide sequence of the sense strand is shown in SEQ ID NO: 10, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

11.

10. The drug according to claim 8, wherein The recombinant expression vector is a recombinant lentiviral expression vector.

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