Application of Circ-sappa and sappa-199aa as a detection marker for alzheimer's disease

Abstract

This invention belongs to the fields of biotechnology and medical technology, and specifically relates to a... Circ-sAPPα And sAPPα-199aa as a biomarker for Alzheimer's disease and its application. circ-sAPPα The application of sAPPα-199aa as a drug target for early diagnosis, treatment, mitigation or prevention of AD metastasis, and / or improvement of AD prognosis. The tissue specificity and stability of the circRNA described in this invention have made circRNA a promising biomarker, as confirmed by experiments. circ-sAPPα Nucleic acid fragments and peptides sAPPα-199aa play a significant and stable role in AD susceptibility, AD pathogenesis, prognostic assessment and treatment.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medical technology, and specifically relates to a... Circ-sAPPα And sAPPα-199aa as a biomarker for Alzheimer's disease and its application. Background Technology

[0002] Circular RNAs (circRNAs) can serve as potential biomarkers for drugs or clinical diagnostics. Studies have shown that in addition to linear RNA molecules, a large number of closed circular RNA molecules exist in living organisms, exhibiting specific expression in individual development, different tissues, and diseases. Sequencing analysis has identified these circRNA molecules as typically formed by the circularization of 2-4 exons of a protein-coding gene through complementary binding between the 5' end of the preceding exon and the 3' end of the following exon. Due to their circular and closed nature, circRNAs can evade the action of exonucleases. Therefore, circRNAs can remain stable in cells for extended periods, making them a recent research hotspot in the RNA field. This stability readily leads us to consider whether circRNAs can serve as potential biomarkers for drugs or clinical diagnostics.

[0003] Circular RNAs (circRNAs) have important physiological functions. Based on research on the structure and function of circRNAs, it has been found that they play a very important role in the development and progression of various diseases, such as nervous system disorders, atherosclerosis, diabetes, and neurological diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). Their main functions include: 1) circRNAs can act as a "sponge" to adsorb miRNAs and inhibit their function; 2) circRNAs can directly regulate the levels of other RNAs through base pairing; 3) circRNAs can bind to proteins, inhibiting protein activity, recruiting components of protein complexes, and / or regulating protein activity; 4) circRNAs can also serve as templates for translation, guiding protein synthesis and directly translating into small peptides and proteins.

[0004] Although its specific functions are not yet fully understood, research on circular RNA may have the following important implications: 1) The unique competitive endogenous (ceRNA) characteristics of circRNA can provide new ideas for drug development; 2) The tissue specificity and stability of circRNA have made it a good biomarker; 3) The proteins or small peptides / polypeptides translated from circRNA should have good physiological functions.

[0005] Circular RNA (cRNA) has become an emerging research hotspot, with recent studies on its functions primarily focused on tumors, accounting for one-third to one-half of the total. Compared to other biomarkers, research on cRNA and its translated proteins in the growth, development, and disease occurrence and progression of the nervous system is relatively limited.

[0006] Currently, there are few reports, both domestically and internationally, on using circular RNA and its translated proteins as markers for Alzheimer's disease (AD) or for their prognostic correlation and therapeutic applications. There is an urgent need in this field to identify important target molecules that can be effectively used for AD diagnosis and treatment selection, and to utilize them for these purposes.

[0007] In 2018, the Sanford Burnham Prebys Medical Research Institute discovered gene recombination in brain neurons, particularly the amyloid precursor protein (APP) gene, which they focused on and is associated with Alzheimer's disease (AD). APP gene expression of amyloid precursor protein is hydrolyzed by secretases, producing neurotoxic β-amyloid peptides and protein precipitates—one of the pathological factors of Alzheimer's disease. Increased APP copy numbers are observed in patients with sporadic Alzheimer's disease (SAD) and Down syndrome, suggesting that the APP gene likely plays a role in the progression of the disease.

[0008] High-throughput sequencing of brain tissue from AD patients revealed that APP precursor RNA can generate dozens of circAPP molecules, but there are few reports on their related physiological functions and clinical applications. By searching the circular RNA database circBase, we discovered multiple circAPP molecules formed by APP gene splicing. circAPP s is specifically highly expressed in the brain. Compared with the circular RNA produced by APP in humans and mice, we focused on circular RNAs that are species homologous and abundant in humans and mice, namely orthologous circRNA molecules (overlapped orthologs, OO-type). Zhao Fangqing's research team has reported that these orthologous circRNA molecules may have important biological functions.

[0009] Based on this, we propose a... Circ-sAPPα The study explores sAPPα-199aa as a biomarker for Alzheimer's disease and its applications, aiming to address the shortcomings of existing technologies. Summary of the Invention

[0010] The purpose of this invention is to address the existing problems and provide a solution. Circ-sAPPα And sAPPα-199aa as a biomarker for Alzheimer's disease and its application.

[0011] This invention is achieved through the following technical solution: In a first aspect of the invention, a biomarker for predicting and assessing Alzheimer's disease is provided, said biomarker being a circ-sAPPα nucleic acid fragment, or a nucleic acid sequence comprising the nucleic acid fragment, or a nucleic acid sequence encoding an amino acid sequence equivalent to sAPPα-199aa, or a sequence comprising the nucleic acid sequence, or sAPPα-199aa, or a polypeptide modified with sAPPα-199aa, or a protein comprising the polypeptide or the modified polypeptide, or a biomarker for overexpression. circ-sAPPα and substances expressed by sAPPα-199aa; The sequence of the circ-sAPPα nucleic acid fragment is shown in SEQ ID NO.1, and the sequence of the sAPPα-199aa is shown in SEQ ID NO.2.

[0012] In a second aspect of the invention, it is provided that circ-sAPPα The application of sAPPα-199aa as a drug target for early diagnosis, treatment, relief or prevention of AD progression.

[0013] In a third aspect of the invention, a method for detecting circ-sAPPα The use of substances at sAPPα-199aa levels in products for predicting AD susceptibility, AD onset or onset, diagnosis or treatment of AD.

[0014] Furthermore, the product testing is performed on mammals that have or are suspected of having AD or are at risk of having AD, or on samples obtained from said mammals.

[0015] Furthermore, the mammals have been identified as having AD or at high risk of having AD using methods known in the art.

[0016] Furthermore, the sample is selected from the mammalian: tissue or cell samples, such as brain tissue, cerebrospinal fluid, serum, or / and samples from AD-like mice or AD-like cell models.

[0017] Furthermore, the method for detection circ-sAPPα Substances that measure sAPPα-199aa expression levels are used to detect at the gene and / or protein levels. circ-sAPPα And substances containing sAPPα-199aa.

[0018] Furthermore, the method for detection circ-sAPPα The substance used to measure sAPPα-199aa expression level is a substance selected from one or more detection techniques or methods from the group consisting of: immunohistochemistry (such as immunofluorescence assay, ELISA, immunogold assay), Western blotting, Northern blotting, PCR, and microarray.

[0019] Furthermore, the method for detection circ-sAPPα Substances expressing sAPPα-199aa were selected from the study of... circ- sAPPα And substances specific to sAPPα-199aa, such as anti-sAPPα-199aa antibodies or their antigen-binding fragments, preferably monoclonal antibodies; circ-sAPPα Specific probes, gene chips, PCR primers, etc.

[0020] Furthermore, the method for detection circ-sAPPα Substances expressing sAPPα-199aa are accompanied by detectable markers, such as those selected from the group consisting of: radioisotopes, fluorophores, chemiluminescent components, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, or ligands (e.g., biotin or haptens).

[0021] Furthermore, the aforementioned circ-sAPPα Decreased sAPPα-199aa levels indicate that the subject is susceptible to or has developed AD, or that the subject has a poor prognosis, or that the subject already has AD.

[0022] Furthermore, the kit used for the detection is an immunohistochemical method based on immunoenzyme labeling for detecting sAPPα-199aa expression in biological samples, comprising: blocking buffer (e.g., 10% goat serum), anti-sAPPα-199aa antibody (e.g., rabbit anti-human or mouse sAPPα-199aa monoclonal or polyclonal antibody), secondary antibody (e.g., anti-rabbit biotinylated secondary antibody), labeled conjugate (e.g., HRP-labeled streptavidin), substrate buffer (e.g., DAB substrate buffer), chromogenic solution (e.g., DAB chromogenic solution), and / or substrate solution.

[0023] Furthermore, the aforementioned detection kit is based on fluorescently labeled in situ hybridization (FISH) technology to detect [the substance] in biological samples. circ-sAPPα The expression kit contains: fluorescent molecular probes.

[0024] Furthermore, compared to a normal control, the object or the sample obtained from the object... circ-sAPPα Decreased sAPPα-199aa levels indicate that the subject is susceptible to or has already developed AD, or that the subject has a poor prognosis for AD, or that the subject already has AD.

[0025] In a fourth aspect of the invention, it is provided that circ-sAPPα Application of the sequence, or the sAPPα-199aa sequence encoded therein, in the preparation of drugs for treating AD, alleviating or preventing the onset of AD and / or improving the prognosis of AD. Furthermore, the aforementioned circ-sAPPα The sAPPα-199aa can be obtained through plasmid expression vectors, chemical synthesis, and recombinant expression.

[0026] Furthermore, the drug can be administered orally, parenterally (e.g., intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, or subcutaneously), subcutaneously, intraperitoneally, intranasally, intracerebrally, and locally.

[0027] In a fifth aspect of the invention, a method for detecting Circ-sAPPα Application of substances at the sAPPα-199aa level in predicting and assessing products related to aggregated amyloid levels.

[0028] In a sixth aspect of the invention, a method for screening candidate drugs for treating, alleviating, and / or improving Alzheimer's disease (AD), characterized in that the method includes testing the candidate drug object or a sample obtained from the object for... circ-sAPPα and the effect on sAPPα-199aa levels, wherein, after using the candidate drug, circ-sAPPα The increased levels of sAPPα-199aa indicate that the candidate drug has therapeutic, alleviating, and / or improving effects on AD.

[0029] The present invention has the following advantages over the prior art: 1. In this invention, molecules that simultaneously meet this condition are circular RNAs in humans with the ID number (ID) [missing information]. hsa_ circ_0115728 The ID number in mice is mmu_circ_0000705 Its mature form in both humans and mice is 597 nt in length, and it is named... circ-sAPPα .

[0030] 2. The tissue specificity and stability of the circRNA described in this invention have made circRNA a good biomarker. circ-sAPPα The application of sAPPα-199aa as a drug target for early diagnosis, treatment, mitigation or prevention of AD metastasis, and / or improvement of AD prognosis. The tissue specificity and stability of the circRNA described in this invention have made circRNA a promising biomarker, as confirmed by experiments. circ-sAPPα Nucleic acid fragments and peptides sAPPα-199aa play a significant and stable role in AD susceptibility, AD pathogenesis, prognostic assessment and treatment. Attached Figure Description

[0031] Figure 1 This is a graph used to verify the effectiveness of the antibody. Figure 2 For AD patients and healthy individuals in blood circ-sAPPα Differences in expression; Figure 3 Differences in sAPPα-199aa expression in the blood of AD patients and healthy individuals; Figure 4 This is an electrophoresis image for RNA quality control. Figure 5 Gene sequence peak diagram; Figure 6 This is the relative expression for mmu_circ_0000705; Figure 7 Verification of circ-sAPPα expression in mouse brain tissue overexpressing circ-sAPPα; Figure 8 Expression of AD biomarkers in mouse brain tissue; Figure 9 Immunohistochemical staining of brain tissue from mice in the 3×Tg-AD group and the OV-circ-sAPPα group; Figure 10 For the 3×Tg-AD group and OV- circ-sAPPα Transcriptome results of mouse brain tissue; Figure 11 Electrophysiological measurements of brain tissue from mice in the 3×Tg-AD group and the OV-circ-sAPPα group; Figure 12 The expression difference of the immediate early gene in mice in the 3×Tg-AD group and the OV-circ-sAPPα group; Figure 13 Differential expression of HSP family in brain tissue of mice in the 3×Tg-AD group and the OV-circ-sAPPα group; Figure 14 The results of Western blot analysis of autophagy proteins in brain tissue. Detailed Implementation

[0032] To further explain the present invention, the following specific embodiments are described.

[0033] Example 1; circ-sAPPα (hsa_circ_0115728) and its translational proteins and MMU- circ-sAPPα (mmu_circ_0000705) and the sequence of its translated protein Focus on an ultra-abundant and species-conserved circular RNA circ-sAPPα And sAPPα-199aa, this invention cleaves the APP gene into circ-sAPPα(hsa_circ_0115728), circbase database http: / / www.circbase.org / . The sequence is shown in nucleic acid SEQ ID NO.1, and it was also found... circ-sAPPα A protein encoding sAPPα-199aa molecular weight, the protein sequence of which is shown in SEQ ID NO.2. Mouse-derived MMU- circ-sAPPα (mmu_circ_0000705), can be found in the circbase database http: / / www.circbase.org / , the nucleic acid sequence is shown in SEQ ID NO.3, and the translated protein "mmu-sAPPα-199aa" is shown in SEQ ID NO.4.

[0034] As used in this article, the term " circ-sAPPα " and "hsa_circ_0115728" can be used interchangeably, "mmu- circ- sAPPα " and "mmu_circ_0000705" are interchangeable. "sAPPα-199aa" means " circ-sAPPα "Translated human protein, "mmu-sAPPα-199aa" means "mmu- circ-sAPPα "Translated mouse-derived protein."

[0035] SEQ ID NO.1: TTCCTACAACAGCAGCCAGTACCCCTGATGCCGTTGACAAGTATCTCGAGACACCTGGGGATGAGAATGAACATGCCCATTTCCAGAAAGCCAAAGAGAGGCTTGAGGCCAAGCACCGAGAGAGAATGTCCCAGGTCATGAGAGAATGGGAAGAGGCAGAACGTCAAGCAAAGAACTTGCCTAAAGCTGATAAGAAGGCAGTTATCCAGCATTTCCAGGAGAAAGTGGAATCTTTGGAACAGGAAGCAGCCAACGAGAGACAGCAGCTGGTGGAGACACACATGGCCAGAGTGGAAGCCATGCTCAATGACCGCCGCCGCCTGGCCCTGGAGAACTACATCACCGCTCTGCAGGCTGTTCCTCCTCGGCCTCGTCACGTGTTCAATATGCTAAAGAAGTATGTCCGCGCAGAACAGAAGGACAGACAGCACACCCTAAAGCATTTCGAGCATGTGCGCATGGTGGATCCCAAGAAAGCCGCTCAGATCCGGTCCCAGGTTATGACACACCTCCGTGTGATTTATGAGCGCATGAATCAGTCTCTCTCCCTGCTCTACAACGTGCCTGCAGTGGCCGAGGAGATTCAGGATGAAGTTG SEQ ID NO.2: MSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRER SEQ ID NO.3: TTCCCACGACAGCAGCCAGCACCCCCGACGCCGTCGACAAGTACCTGGAGACACCCGGGGACGAGAACGAGCATGCCCATTTCCAGAAAGCCAAAGAGAGGCTGGAAGCCAAGCACCGAGAGAGAATGTCCCAGGTCATGAGAGAATGGGAAGAGGCAGAGCGTCAAGCCAAGAACTTGCCCAAAGCTGACAAGAAGGCCGTTATCCAGCATTTCCAGGAGAAAGTGGAATCTCTGGAACAGGAAGCAGCCAATGAGAGACAGCAGCTTGTAGAGACACACATGGCCAGAGTTGAAGCCATGCTCAATGACCGCCGCCGCCTGGCCCTCGAGAATTACATCACTGCACTGCAGGCGGTGCCCCCAAGGCCTCATCATGTGTTCAACATGCTGAAGAAGTACGTCCGTGCGGAGCAGAAAGACAGACAGCACACCCTAAAGCATTTTGAACATGTGCGCATGGTGGACCCCAAGAAAGCTGCTCAGATCCGGTCCCAGGTTATGACACACCTCCGTGTGATCTACGAGCGCATGAACCAGTCTCTGTCCCTGCTCTACAATGTCCCTGCGGTGGCTGAGGAGATTCAAGATGAAGTCG SEQ ID NO.4: MSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPHHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVVPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEE Example 2; Human circ-sAPPα (hsa_circ_0115728), murine mmu- circ-sAPPα (mmu_circ_0000705) and its translated protein "mmu-sAPPα-199aa" detection method circ-sAPPαPrimer sequences for (hsa_circ_0115728) (hsa_circ_0115728-F1: 5'-CAGTGGCCGAGGAGATTCAG-3', hsa_circ_0115728-R1: 5'-ACTTGTCAACGGCATCAGGG-3'). mmu- circ-sAPPα Primer sequences for (mmu_circ_0000705) (mmu_circ_0000705-F1: 5'-TCAAGATGAAGTCGTTCCCAC-3', mmu_circ_0000705-R1: 5'-ACATTCTCTCTCGGTGCTTG-3'). Rabbit anti-human or mouse sAPPα-199aa polyclonal antibody, antibody name anti-APP-199aa, antigen sequence IQDEVVPTTAASTPC. Antibody validation was performed using mouse brain tissue and human whole blood. Figure 1 ).

[0036] Example 3; Clinical AD patients circ-sAPPα Its sAPPα-199aa content decreased Inclusion criteria: The diagnosis of AD patients adopted the NIA-AA diagnostic criteria jointly developed by the National Institute on Aging and the Alzheimer's Disease Association in 2011. All patients were screened by physical examination, auxiliary examinations and the Mini-Mental State Examination (MMSE).

[0037] Exclusion criteria: ① Vascular dementia, mixed dementia, Lewy body dementia, and other types of dementia were excluded from the AD group, as were Parkinson's disease, depression, anxiety, and other mental illnesses; ② Patients with brain diseases were excluded, such as intracranial inflammatory diseases, traumatic brain injury, and hydrocephalus; ③ Patients who had previously taken or were currently taking drugs that affect peripheral inflammatory factors were excluded. Fifty Alzheimer's patients were collected as study subjects. Fifty healthy individuals of the same age group undergoing physical examinations during the same period were selected as the control group. 3 ml of fasting venous blood was drawn from both groups in the morning. Total RNA was extracted using TRIzol LS Reagent from Gisele Biotechnology. The blood of AD patients... circ-sAPPα The expression of sAPPα-199aa (using the ELISA kit prepared with the above-mentioned sAPPα-199aa antibody) was lower than that of the general healthy population. Figure 2-3 ).

[0038] Example 4; The effect of OV-circ-sAPPα in delaying the progression of Alzheimer's disease Construction and identification of overexpression vectors The full-length 597bp sequence of the gene mmu_circ_0000705 and the full-length 598bp sequence of mut-mmu_circ_0000705 were amplified by PCR and ligated into the modified pLC5-ciR (EcoRI and BamHI sites were not preserved) using In-Fusion cloning.

[0039] C705-UnF:CATTAATATTTCTTCTTCGAATTCTAATACTTTCAGTTCCCACGACAGCAGCCAGCAC705-UnR:AGTATGGAGTTGTTAGCTAGGATCCAGTTGTTCTTACCGACTTCATCTTGAATCTCCT (1) mmu_circ_0000705 overexpression circular lentivirus plasmid system >mmu_circ_0000705|NM_001198824|App SEQ ID NO.5: TTCCCACGACAGCAGCCAGCACCCCCGACGCCGTCGACAAGTACCTGGAGACACCCGGGGACGAGAACGAGCATGCCCATTTCCAGAAAGCCAAAGAGAGGCTGGAAGCCAAGCACCGAGAGAGAATGTCCCAGGTCATGAGAGAATGG GAAGAGGCAGAGCGTCAAGCCAAGAACTTGCCCAAAGCTGACAAGAAGGCCGTTATCCAGCATTTCCAGGAGAAAGTGGAATCTCTGGAACAGGAAGCAGCCAATGAGAGACAGCAGCTTGTAGAGACACACATGGCCAGAGTTGAAGC CATGCTCAATGACCGCCGCCGCCTGGCCCTCGAGAATTACATCACTGCACTGCAGGCGGTGCCCCCAAGGCCTCATCATGTGTTCAACATGCTGAAGAAGTACGTCCGTGCGGAGCAGAAAGACAGACAGCACACCCTAAAGCATTTTG AACATGTGCGCATGGTGGACCCCAAGAAAGCTGCTCAGATCCGGTCCCAGGTTATGACACACCTCCGTGTGATCTACGAGCGCATGAACCAGTCTCTGTCCCTGCTCTACAATGTCCCTGCGGTGGCTGAGGAGATTCAAGATGAAGTCG (2) Point mutation mut-mmu_circ_0000705 circular lentiviral plasmid system SEQ ID NO.6: TTCCCACGACAGCAGCCAGCACCCCCGACGCCGTCGACAAGTACCTGGAGACACCCGGGGACGAGAACGAGCATGCCCATTTCCAGAAAGCCAAAGAGAGGCTGGAAGCCAAGCACCGAGAGAGAATGATCCCAGGTCATGAGAG AATGGGAAGAGGCAGAGCGTCAAGCCAAGAACTTGCCCAAAGCTGACAAGAAGGCCGTTATCCAGCATTTCCAGGAGAAAGTGGAATCTCTGGAACAGGAAGCAGCCAATGAGAGACAGCAGCTTGTAGAGACACACATGGCCAGAGTTGA AGCCATGCTCAATGACCGCCGCCGCCTGGCCCTCGAGAATTACATCACTGCACTGCAGGCGGTTGCCCCAAGGCCTCATCATGTGTTCAACATGCTGAAGAAGTACGTCCGTGCGGAGCAGAAAGACAGACAGCACACCCTAAAGCATTTT GAACATGTGCGCATGGTGGACCCCAAGAAAGCTGCTCAGATCCGGTCCCAGGTTATGACACACCTCCGTGTGATCTACGAGCGCATGAACCAGTCTCTGTCCCTGCTCTACAATGTCCCTGCGGTGGCTGAGGAGATTCAAGATGAAGTCG The sequencing peaks were normal, with no extraneous peaks or overlapping bands, and the sequence comparison showed good agreement, indicating that mmu_circ_0000705 and mut-mmu_circ_0000705 were successfully inserted into pLC5-ciR, and the overexpression vector was successfully constructed.

[0040] Vector sequencing primers: pC5-seqF: TGTGAATTTGACCCTTAAGA Four groups of cells—Alu705, mut-Alu705, NC (pLC5-ciR), and control—were transfected into 293T cells. Cells were collected 48 h later for qPCR detection. Total RNA was extracted from cell samples, reverse transcribed, and then analyzed by qPCR using specific primers for the internal control GAPDH and mmu_circ_0000705. Sequencing primers were also designed, and the bands were recovered after PCR and sequenced to verify the circularized adapter sites.

[0041] The RNA electrophoresis image showed clear 28S and 18S bands, indicating good RNA integrity, which is suitable for subsequent experiments. Figure 4 ).

[0042] Table 1. RNA Concentration and Purity Measurement ; Table 2. Relative expression differences of mmu_circ_0000705 ; qPCR primers GAPDH GAPDH-F: AGAAGGCTGGGGCTCATTTG GAPDH-R: GCAGGAGGCATTGCTGATGAT Amplified fragment size 140bp mmu_circ_0000705 mmu_circ_0000705-F1: TTCAAGATGAAGTCGTTCCC mmu_circ_0000705-R1: CTTTGGCTTTCTGGAAATGG The amplified fragment size of mmu_circ_0000705 is 111bp. qPCR product sequencing results Note: The circularized site sequence of mmu_circ_0000705 in circBase is AGATGAAGTCGTTCCCACGACA ( Figure 5 ) qPCR product sequencing primers mmu_circ_0000705-F2:GATCTACGAGCGCATGAACC mmu_circ_0000705-R1: CTTTGGCTTTCTGGAAATGG The amplified fragment size is 176bp. Note: The circularized site sequence of mut-mmu_circ_0000705 in circBase is AGATGAAGTCGTTCCCACGACA ( Figure 5 ) The amplified fragment size is 176bp. Compared to the control group, the overexpression fold increase of Alu705 was 135,504, while that of the mut-Alu705 group was 91,533. Figure 6 The PCR product sequencing peaks were normal, with no extraneous peaks or overlapping bands, and the sequence matched the reference sequence at the circularization site. These results indicate that mmu_circ_0000705 can successfully overexpress the target circRNA in eukaryotic cells.

[0043] An overexpression system of circ-sAPPα adeno-associated virus (OV-circ-sAPPα AAV) was injected via tail vein into 2-month-old 3×Tg-AD mice (B6;129-Tg (APPSwe, tauP301L) 1Lfa). After 8 months, the mice were treated as follows: (1) their physiological potentials were measured to evaluate their memory and learning abilities; (2) their differential gene expression in brain tissue was detected using transcriptome sequencing technology; (3) their levels of AD-related marker proteins Tau, p-Tau, APOE, Aβ42, sAPPα, sAPPβ, BACE, and APP in brain tissue were detected using Western blot (WB) and immunohistochemistry (IHC) techniques. The expression of microglia (IBA1), astrocytes (GFAP), and inflammatory factors such as NF-κB, TNF-α, IL-6, and TGF-β1 / 3, as well as c-Fos, NPAS4, Hsp40, Hsp60, Hsp70, Hap90, LC3A / B, and LAMP1 / 2 was also detected.

[0044] The detection revealed that the expression levels of AD-related pathological markers Tau, p-Tau, APOE, and Aβ42 in the OV-circ-sAPPα group were significantly lower than those in the 3×Tg-AD group. P <0.05, n=3), while the expression level of APP protein did not differ significantly between the two groups ( Figure 7-8 () P >0.05, n=3). Ov- circ-sAPPα The expression level of APOE in the CA2 region of the hippocampus of the group mice was lower than that in the 3×Tg-AD group mice. Figure 9 () P <0.05, n=2). Two groups of Aβ 42 The expression of [something] differs significantly in the thalamic region. Figure 9 () P<0.05, n=5) These results indicate overexpression circ-sAPPα It has the ability to degrade the accumulation of AD pathological proteins or reduce the expression of AD pathological proteins, thereby improving the course of AD. No difference in APP expression may indicate... circ-sAPPα It cannot regulate the expression of the APP gene, nor does it affect gene expression elements such as the promoter of the APP gene.

[0045] Figure 7 In the table: A: Western blot (WB) bands in brain tissue of mice in the 3×Tg-AD group and the OV-circ-sAPPα group; B: Quantitative statistical plot of WB bands; C: Relative quantitative quantification of circ-sAPPα in brain tissue of mice in the 3×Tg-AD group and the OV-circ-sAPPα group by qPCR. *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

[0046] Figure 8 In: A: Mice in the 3×Tg-AD group and OV- circ-sAPPα Western blot (WB) bands of AD marker proteins in the brain tissue of mice in group A; B, C, and D are quantitative statistical graphs of the WB bands of tau, p-tau, and APOE, respectively. *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

[0047] Figure 9 In the middle: A, B, C: 100x and 400x magnification brain slices and quantitative images of APOE, Aβ, and APP in OV-circ-sAPPα and 3-Tg-AD mice detected by immunohistochemical staining. Immunohistochemical brain slices were statistically quantified using ImageJ software. 3×Tg-AD mice represent triple-transgenic mice, OV- circ-sAPPα The three transgenic mice overexpressing circ-sAPPα are shown. *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

[0048] The study selected OV- circ-sAPPα Six brain tissue samples were collected from mice and six from 3×Tg-AD mice. These samples were then pooled pairwise to form six samples each for transcriptome sequencing analysis. The sequencing results revealed 429 differentially expressed genes between the two groups of mice, among which... Circ-sAPPα The effects affected 35 upregulated genes and 394 downregulated genes. Figure 10 Of these differentially expressed genes, 321 can be annotated using KEGG, and 11 of these annotated genes are related to learning and memory. Figure 10 45 genes are involved in intracellular phagocytosis and autophagy. Figure 10B, C). In the environmental information processing module of the KEGG pathway classification, 14 and 21 genes were enriched in the MAPK signaling pathway and the PI3k-Akt signaling pathway, respectively. Figure 10 C). Differential gene expression analysis using the KEGG pathway revealed a significant downregulation of the phagocytic-autophagy pathway, while osteoclast differentiation, transcriptional errors in cancer, and apoptosis pathways were significantly upregulated. Figure 10 D).

[0049] Figure 10 In: A: 3×Tg-AD group and OV- circ-sAPPα Volcano plot of differentially regulated genes in mouse brain tissue, red indicates upregulated genes, green indicates downregulated genes; B: 3×Tg-AD group and OV- circ-sAPPα C: Statistical diagram of differentially expressed genes in the brain tissue of mice; D: Number of differentially expressed genes enriched in the learning, memory, phagocytosis and autophagy pathways; E: KEGG differential gene classification diagram; F: KEGG differential gene enrichment analysis diagram.

[0050] The study measured the 3×Tg-AD group and OV- circ-sAPPα The response of hippocampal slices from mouse brain tissue to electrical stimulation. Electrical stimulation was performed using voltage stimulation, ranging from 2-8V. Linear fitting was performed on the slopes of all stimulation intensities to obtain the following results: Figure 11 The linear graph of the stimulus-response relationship for A shows that the slope of the linear relationship for the 3×Tg-AD group is 0.09319±0.007519, and the slope of the linear relationship for OV- is 0.09319±0.007519. circ-sAPPα The linear slope of the two groups was 0.1064 ± 0.008774, and there was no significant difference between the slopes of the two groups. Figure 11 B) P >0.05, n=11). The above results indicate that 3×Tg-AD and OV- circ-sAPPα There were no significant differences in the excitability of neurons in the CA3-CA1 region of the hippocampus in mice. Synaptic plasticity is considered a biological mechanism for memory formation, and in order to study... circ- sAPPα The study investigated the effects on mouse memory by measuring the facilitation response to double-pulse stimulation. The facilitation response reflects short-term synaptic plasticity. The results are as follows: Figure 11 C, using double pulse intervals of 15ms, 50ms, 100ms, and 400ms, the 3×Tg-AD group and OV- circ-sAPPα There were no significant differences between the groups. P >0.05 (n=11), indicating no significant difference in short-term synaptic plasticity in the hippocampus between the two groups of mice. Long-term potentiation (LTP) is an important manifestation of synaptic transmission plasticity; this study compared the differences in LTP between the two groups to reflect... circ-sAPPα Impact on memory. Recorded data were obtained according to the relationship between time and response slope. Figure 11 Figure D shows a segment with the first 40 recordings of Theta wave stimulation as the baseline and a segment with the last 120 recordings of Theta wave stimulation. In the analysis of LTP results in these two groups of mice, the slope values ​​at 0-10 min and 50-60 min after Theta wave stimulation were compared. Statistical results showed that the slope value of the 3×Tg-AD group at 0-10 min was (1.679±0.02769, n=20), while that of the OV- circ-sAPPα The slope value of the two groups at 0-10 min was (1.883±0.0369, n=20), and there was a statistically significant difference between the two groups. P <0.05, n=20) and the LTP value of the OV-circ-sAPPα group was higher than that of the 3×Tg-AD group ( Figure 11 D). The slope of the 3×Tg-AD group at 50-60 min was (1.348±0.002478, n=20), while the slope of the OV- circ-sAPPα The slope value of the two groups at 0-10 min was (1.523±0.002237, n=20), and there was a statistically significant difference between the two groups. P <0.05, n=20) and OV-circ-sAPPα The LTP value of the group was higher than that of the 3×Tg-AD group ( Figure 11 D). The above data illustrates... circ-sAPPα It does not alter the neuronal excitability and short-term synaptic plasticity of CA3-CA1 in the hippocampus of 3×Tg-AD mice, but it does alter the long-term synaptic plasticity of 3×Tg-AD mice and improve the memory ability of AD model mice.

[0051] Figure 11 In the diagram: A: Slope of all brain slices under different voltage stimulation; B: IO slope distribution of each brain slice; C: Statistical results of facilitated response at different time intervals for double-pulse stimulation; D: Change in LTP over time in the 3×Tg-AD group and the OV-circ-sAPPα group. *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

[0052] Immediately early genes (OVs) are a class of genes that respond rapidly to extracellular stimuli, and their products include transcription factors and cytokines. This study found that OVs... -circ-sAPPα Nine genes for early detection were upregulated in the mice group. Arc, Junb, Nr4a1, Fos, Nr4a2, Fosl2, Nr4a3, Fosb, Npas4 Two early genes were downregulated ( Pik3cg, Thbs1 () Figure 12 A). c-FosThe gene is an immediate early gene and has been used as a marker of neuronal activity related to memory. NPAS4 is involved in synaptic remodeling, which is fundamental to memory development. Immunoblot assays show OV- circ-sAPPα c-Fos and NR4A1 genes in the brain tissue of the group of mice were significantly upregulated compared with those in the 3×Tg-AD group. P >0.05, n=7), while NPAS4 showed no significant difference between the two groups ( Figure 12 B) P >0.05, n=3).

[0053] Figure 12 In: A: 3×Tg-AD group and OV- circ-sAPPα Differentially expressed genes related to learning and memory in mice; B: 3×Tg-AD group and OV- circ-sAPPα Immunoblot bands and corresponding quantitative maps of the early-stage genes NPAS4, NR4A1, and c-Fos. P <0.05,** P <0.01, *** P <0.005, **** P <0.0001.

[0054] The study examined 3×Tg-AD mice and OV- circ-sAPPα Mouse transcriptome, transcriptome results suggest circ-sAPPα The genes related to phagocytosis / autophagy in 3×Tg-AD mice were altered. Figure 13 A). circ-sAPPα Upregulate 3×Tg-AD mice Hspa1a and Hspa1b Both genes are members of the heat shock protein 70 (HSP70) family, and KEGG pathway analysis showed that they are involved in phagocytosis. Phagocytosis is crucial for maintaining protein homeostasis and is involved in the degradation of Aβ amyloid in AD. To verify... circ-sAPPα To determine whether these proteins directly affect phagocytosis in brain cells, the study used Western blotting and immunohistochemistry to detect phagocytosis-related marker proteins, such as LC3A / B, LAMP2, Hsp90, Hsp70, and Hsp40. The results are as follows... Figure 13 B, heat shock protein family Hsp90 ( P <0.05, n=5), Hsp70 ( P <0.05, n=5), Hsp40 ( P <0.05, n=3) in OV- circ-sAPPα The expression level in the brains of mice in the OV group was significantly higher than that in the brain tissue of mice with 3×Tg-AD. circ-sAPPα The expression levels of LC3A / B and LAMP2 in the group were significantly higher than those in the 3×Tg-AD group. Figure 14() P <0.05, n=3). TREM2 is mainly expressed on microglia in the brain and is a recognition receptor. This study found that OV- circ-sAPPα TREM2 expression levels in the group were significantly higher than those in the 3×Tg-AD group ( Figure 14 D) P <0.05, n=3). The above data illustrates... circ-sAPPα It can activate phagocytosis in 3×Tg-AD mice.

[0055] Figure 13 A: Heatmap of differentially expressed genes related to phagocytosis and autophagy in brain tissue of mice in the 3×Tg-AD group and the OV-circ-sAPPα group; B: Immunoblotting bands and quantitative maps of Hsp90, Hsp70, and Hsp40 in brain tissue of mice in the 3×Tg-AD group and the OV-circ-sAPPα group.

[0056] Figure 14 In the middle: A, B, C, D: 3×Tg-AD group and OV- circ-sAPPα Immunoblot bands and quantitative images of LAMP2, Hsp40, LC3A / B, and TREM2 in the brain tissue of mice in the group. P <0.05,** P <0.01, *** P <0.005, **** P <0.0001.

[0057] Therefore, this application provides circ-sAPPα This research also outlines novel applications of the sAPPα-199aa molecule in predicting AD risk, assessing AD pathogenesis, and evaluating AD treatment efficacy in subjects, and provides corresponding diagnostic kits. These advancements suggest… around sAPPα The detection and intervention methods of sAPPα-199aa are expected to become a new growth point in the diagnosis and treatment of AD.

[0058] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting Circ-sAPPα The application of substances at the sAPPα-199aa level in the preparation of products for predicting and assessing the severity or grade of Alzheimer's disease and for diagnosing Alzheimer's disease, wherein the sequence of the circ-sAPPα nucleic acid fragment is shown in SEQ ID NO.1 and the sequence of the sAPPα-199aa is shown in SEQ ID NO.

2.

2. Use according to claim 1, characterized in that, The detection Circ-sAPPα Substances that measure sAPPα-199aa expression levels are used to detect at the gene and / or protein levels. Circ-sAPPα And substances containing sAPPα-199aa.

3. The application according to claim 1, characterized in that, The means for detecting Circ-sAPPα and sAPPa-199aa expression levels are means for one or more detection techniques or methods selected from the group consisting of Northern blotting, PCR, biochip methods.

4. Use according to claim 1, characterized in that, The Circ-sAPPα Decreased sAPPα-199aa levels indicate that the subject is susceptible to or has developed AD, or that the subject has a poor prognosis, or that the subject already has AD.

5. A substance for detecting Circ-sAPPα and use of the substance for predicting the level of aggregated amyloid protein, wherein the sequence of the circ-sAPPa nucleic acid fragment is shown as SEQ ID NO. 1, and the sequence of the sAPPa-199aa is shown as SEQ ID NO. 2.

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