CN114225035B - Application of circular RNA Cwc27 down-regulator in the preparation of drugs for preventing and/or treating Alzheimer's disease - Google Patents

Abstract

The invention discloses application of a cyclic RNA Cwc27 down regulator in preparing a medicament for preventing and/or treating Alzheimer disease. The cyclic RNACwc27 down-regulator is a cyclic RNACwc27 inhibitor and/or a cyclic RNACwc27 modifier. The annular RNAcwc27 down-regulator can enter the nucleus by inhibiting Pur-alpha, thereby reducing the transcription level of the A beta precursor protein APP, reducing the expression level of the APP protein, improving the learning and memory ability and having clinical application prospect for treating AD.

Description

Circular RNA Cwc27 down-regulator in the preparation of prevention and/or treatment of Alzheimer's disease application in medicine

technical field

The invention belongs to the fields of biotechnology and medicine, and specifically relates to the application of circular RNA Cwc27 (circular RNA Cwc27, circCwc27 for short) and its analogues as nucleotide drugs for the treatment and/or prevention of Alzheimer's disease.

Background technique

Alzheimer's disease (AD) is the most common neurodegenerative disease, and it has become a major disease that seriously affects human health in today's society due to its high prevalence, high disability rate and high mortality rate. . Existing studies have found that the main pathological changes of AD are senile plaques (SP) formed by the aggregation of β-amyloid protein (Aβ), neurofibrillary tangles (NFT) formed by abnormal aggregation of Tau protein, and a large number of neurons in the brain. lost. For AD, there is still a lack of effective early diagnosis and treatment measures clinically. In developed countries and my country, AD has become the fourth cause of death after heart disease, cancer and stroke. At present, the pathogenesis of AD is still unclear, but it is generally believed that the activation of the amyloid precursor protein degradation pathway, leading to the abnormal increase and aggregation of Aβ, is the key factor leading to the occurrence of AD. Therefore, elucidating the pathogenesis of AD and finding new therapeutic strategies and targets have become the key points that need to be solved urgently.

Circular RNA (circRNA) is a type of endogenous non-coding RNA molecule that does not have a 5' end cap structure and a 3' end poly A tail structure, and forms a circular structure with covalent bonds. It can be derived from exons or introns of the coding gene. In the 1970s, scientists discovered the existence of circRNA for the first time in the research on RNA viruses, and then with the help of RNA sequencing technology and bioinformatics, a large number of circRNA molecules were discovered one after another. Initial studies believed that circRNA was a by-product of RNA shearing, but in recent years scientists have gradually discovered that circRNA has important research value due to its conservation, stability, and expression specificity. Recent studies have shown that circRNAs distributed in the nucleus can regulate the transcription of their parental genes, circRNAs in the cytoplasm can act as miRNA sponges, insulators of RNA-binding proteins, etc., and even a small part of circRNAs can also translate proteins. In recent years, international studies on the role of circRNAs in the pathogenesis of AD have been increasing. At present, research at home and abroad is mainly focused on the establishment of circRNA expression profiles to find abnormally expressed circRNAs in AD, and the functions of these abnormally expressed circRNAs in AD There are few reports on role research. In the central nervous system, the role of circCwc27 remains unclear. At the same time, no research report on circRNA (including circCwc27) and AD treatment has been found so far through domestic and foreign literature searches of the prior art.

Contents of the invention

In view of the above problems, the object of the present invention is to provide a circular RNACwc27 target or a drug based on the circular RNACwc27 target in the treatment and/or prevention of Alzheimer's disease.

In the first aspect, the present invention provides the use of a circular RNACwc27 down-regulator in the preparation of a drug for preventing and/or treating Alzheimer's disease, wherein the circular RNACwc27 down-regulator is a circular RNACwc27 inhibitor and/or a circular RNACwc27 Modifiers.

The circular RNA Cwc27 down-regulator can reduce the transcription level of Aβ precursor protein APP by inhibiting Pur-α into the nucleus, reduce the expression level of APP protein, improve learning and memory ability, and has a clinical application prospect for treating AD.

The circular RNA Cwc27 down-regulator is: an RNA fragment including the base sequence shown in SEQ ID NO.1; an RNA fragment including a base sequence complementary to the base sequence shown in SEQ ID NO.2; based on Circular RNACwc27 modified by substitution, deletion or addition of one or more nucleotides in SEQ ID NO.3; or a polypeptide, protein or compound capable of inhibiting the expression of circular RNACwc27. Wherein, SEQ ID NO.1 is the nucleotide sequence of the down-regulator of circular RNACwc27, SEQ ID NO.2 is the nucleotide sequence at the junction of circular RNACwc27, and SEQ ID NO.3 is the nucleoside of circular RNACwc27 itself acid sequence.

The sequences of circCwc27 in mouse, rat and human brain are highly conserved, and basically contain the base sequence shown in SEQ ID NO.3. The research results of circCwc27 in mice have more clinical significance, so circCwc27 Can be used as a reliable target for the treatment and/or prevention of Alzheimer's disease.

Preferably, the modified circular RNACwc27 is obtained by performing at least one of the following modifications on the circular RNACwc27: ribose modification of any nucleotide, base modification, phosphate backbone modification, augmentation of any nucleotide Subtract, replace.

Preferably, the drug includes a circular RNA Cwc27 down-regulator, and a pharmaceutically acceptable carrier or adjuvant.

In a second aspect, the present invention provides the use of circular RNA Cwc27 as a target in the development, screening or preparation of drugs for the prevention and/or treatment of Alzheimer's disease.

In a third aspect, the present invention provides the use of circular RNA Cwc27 as a target in preparing a drug screening model for preventing and/or treating Alzheimer's disease.

In a fourth aspect, the present invention provides a method for screening drugs for the prevention and/or treatment of Alzheimer's disease, the method comprising:

Treating a system expressing circular RNA Cwc27 with a candidate substance; and

Detecting the expression of circular RNA Cwc27 in the system;

If the expression of the circular RNA Cwc27 is reduced, it indicates that the candidate substance is a potential substance for preventing and/or treating Alzheimer's disease.

Description of drawings

Figure 1 shows the level of circCwc27 in APP/PS1 mouse cortex, hippocampus and plasma of AD patients, where A is the expression level of each circRNA in APP/PS1 and control wild-type mouse cortex, B is APP/PS1 and control wild-type The expression levels of each circRNA in the mouse hippocampus; C is the level of circCwc27 in the plasma of AD patients and healthy controls; it can be seen from Figure 1 that the level of circCwc27 in the plasma of AD patients and the brain tissue of APP/PS1 mice was significantly increased.

Figure 2 shows the effect of circCwc27 interference virus on the expression of APP protein in the brain of APP/PS1 mice; where A is the electrophoresis of APP protein in the hippocampus of APP/PS1 mice injected with circCwc27 interference virus or control virus, and B is the APP protein in A Statistical graph of protein expression; from Figure 2, it can be seen that the circCwc27 interference virus can significantly reduce the expression of APP protein in the brain of APP/PS1 mice.

Figure 3 shows the learning and memory ability of AD model mice APP/PS1 supplemented with circCwc27 interference virus LV-sh-circCwc27 for two months after brain stereotaxic injection, where A is the latency time for the mice in the treatment group and the control group to find the target quadrant, B is the area under the curve of the time for the mice in the treatment group and the control group to find the target quadrant, C is the swimming speed of the mice in the treatment group and the control group, D is the residence time of the mice in the treatment group and the control group in the target quadrant, E is the treatment The trajectory diagram of the mice in the group and the control group; as can be seen from Figure 3, compared with the control group, the learning and memory abilities of the treatment group were significantly improved.

Figure 4 shows the amyloid pathology of AD model mice APP/PS1 via brain stereotaxic injection supplemented with circCwc27 interfering virus LV-sh-circCwc27 for two months, where A is after APP/PS1 mice were injected with circCwc27 interfering virus or control virus Fluorescent images of amyloid plaques in the hippocampus and cortex, B is the statistical map of amyloid plaque deposition in image A; it can be seen from Figure 4 that compared with the control group, the AD model was smaller after localized injection of circCwc27 interference virus Amyloid pathology in mice was significantly alleviated.

detailed description

The present invention will be further described below in conjunction with the drawings and the following embodiments. It should be understood that the drawings and the following embodiments are only used to illustrate the present invention rather than limit the present invention.

The inventors have found through research that the non-coding RNA circular RNA Cwc27 (circCwc27 for short, its base sequence is shown in SEQ ID NO.3. Its expression is significantly increased in the brain tissue of Alzheimer's disease patients and model mice, And participate in the generation of amyloid pathology in the brain. Giving circCwc27 interference virus to the brain of AD model mice can significantly alleviate the occurrence of amyloid pathology in the brain and improve the learning and memory ability of mice, indicating that circCwc27 has clinical application in the treatment of AD Prospect, complete the present invention thus.

Disclosed herein is the application of the circCwc27 down-regulator in the preparation of drugs for the prevention and/or treatment of Alzheimer's disease.

The term "prevention of a disease" refers, for example, to the non-development of clinical symptoms of a disease in mammals that may have been exposed or pre-treated to the disease but have not yet experienced or shown symptoms of the disease.

The term "treating a disease" can refer to inhibiting a disease, such as arresting or reducing the development of a disease or its clinical symptoms, or ameliorating a disease, such as regressing a disease or its clinical symptoms.

circCwc27 down-regulators include substances capable of reducing circCwc27 levels. The circular RNACwc27 down-regulator is a circular RNACwc27 inhibitor and/or a circular RNACwc27 modifier. For example, the interference RNA of circCwc27 (including the RNA fragment of the base sequence shown in SEQID NO.1.

An example of the circCwc27 inhibitor includes an RNA fragment having a nucleotide sequence complementary to the circCwc27 linker in its nucleotide sequence, that is, including a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.2.

CircCwc27 modifications refer to the design of nucleic acid sequences and modifications based on the nucleotide sequence of circCwc27, such as circular RNACwc27 modifications based on SEQ ID NO.3 with substitution, deletion or addition of one or more nucleotides. The modification includes ribose modification, base modification and phosphate backbone modification of any nucleotide or a combination of several or addition, deletion or replacement of any nucleotide. As long as the modified nucleotide sequence can satisfy the similar functional activity of reducing circCwc27.

In addition, it should be understood that circCwc27 down-regulators also include substances that can reduce the activity and stability of circCwc27, and substances that can reduce the effective action time of circCwc27. For example, circCwc27 down-regulators also include other polypeptides, proteins or compounds that can inhibit the expression of circular RNA Cwc27.

The effect of the circCwc27 down-regulator on inhibiting the pathology of amyloid in the AD brain is disclosed here, and the application of the circCwc27 down-regulator can effectively inhibit the progression of pathological phenotypes in AD model mice and improve learning and memory abilities. The study found that circCwc27 was significantly elevated in peripheral plasma of AD patients and brain tissue of APP/PS1 mice. For AD model mice, inhibiting circCwc27 in the brain can significantly reduce the expression levels of Aβ precursor protein APP and key cleavage enzyme BACE1, thereby inhibiting the production of Aβ and the generation of amyloid pathology. The application of the present invention can be used to prepare a nucleotide analog drug for treating Alzheimer's disease.

The circCwc27 downregulator can reduce APP and BACE1 transcription levels, inhibit the amyloid precursor protein pathway, and serve as a new type of nucleotide drug.

For AD animal model APP/PS1 mice, intracerebral injection of circCwc27 interfering lentivirus was used as a therapeutic drug. The results proved that inhibiting circCwc27 can significantly reduce the production of Aβ and the formation of amyloid pathology, and promote the alleviation of learning and memory ability.

circCwc27 can be used as a target for developing, screening or preparing drugs for preventing and/or treating Alzheimer's disease. CircCwc27 can be used as the target to screen candidate substances. The screened substances that can reduce the expression of circCwc27, inhibit the activity and stability of circCwc27, and/or reduce the effective action time of circCwc27 can be used as alternative drugs for preventing and/or treating Alzheimer's disease.

A drug screening model targeting circCwc27 can be established to screen drugs targeting circCwc27.

In one embodiment, the system expressing circCwc27 is treated with a candidate substance, and the expression of circCwc27 in the system is detected. If the expression of circCwc27 decreases, it indicates that the candidate substance is a potential substance for preventing and/or treating Alzheimer's disease.

Disclosed herein is a pharmaceutical composition, which includes an effective amount of a circCwc27 down-regulator, and a pharmaceutically acceptable carrier or adjuvant.

"Effective amount" means that a circCwc27 down-regulator (i) treats a particular disease, condition or disorder, (ii) attenuates, improves or eliminates one or more symptoms of a particular disease, disorder or disorder, or (iii) prevents or delays the present invention The amount of onset of one or more symptoms of the particular disease, condition or disorder.

)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。"Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gel substances, which are suitable for human use and must have sufficient purity and low toxicity. "Compatibility" here means that each component in the composition can mix with the active ingredient without significantly reducing the efficacy of the active ingredient. Pharmaceutically acceptable carriers or excipients include but are not limited to cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as Stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyalcohol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as

), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

The medicines or pharmaceutical compositions in the present disclosure can be made into injection preparations, oral preparations, nasal drops, spray preparations, ointment preparations or patches, etc. according to common methods for preparation of medicines.

The circCwc27 down-regulator can also be combined with other drugs for the prevention and/or treatment of Alzheimer's disease.

Examples are given below to describe the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention, and should not be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above contents of the present invention all belong to the present invention scope of protection. The specific process parameters and the like in the following examples are only examples of suitable ranges, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values exemplified below.

Example 1: In the peripheral plasma of AD patients and the brain tissue of model mice, the level of circCwc27 was significantly increased.

Select 6-month-old APP/PS1 transgenic mice and 3 wild control mice of the same age and background. After anesthesia, the brain was perfused and taken out. The brain tissue was divided into two halves, one half was used for RNA extraction for circRNA sequencing analysis, and the other half was polymerized. Paraffin sections were made after formaldehyde fixation for in situ hybridization experiments. Relevant operations conform to ethical operating standards and procedures.

circRNA sequencing analysis: 1-2 μg RNA was selected for library construction through NanoDrop ND-100 concentration measurement, the quality of the constructed library was identified by Agilent2100, and library quantification was performed by qPCR. The mixed sample libraries were sequenced using Illumina 4000 sequencer , including NaOH alkali denaturation single-strand generation, Illumina flowcell in situ amplification, 150 paired-end cycle sequencing and other steps. Solexa pipeline version 1.8 software was used for image processing and base recognition, FastQC software was used to evaluate the quality of reads after splice removal, STAR software was used to compare to the reference genome, CIRCexplorer2 was used to detect Backsplice junction reads, and R software edgeR was used to detect expression differences calculate.

RT-PCR: In the experiment, total RNA was extracted using the instructions of the RNAeasy ^TM Plus kit from Biyuntian Company, and real-time fluorescent quantitative PCR was used. The RNA was reverse-transcribed into cDNA according to the instructions of the PrimeScript ^TM RT Master Mix kit provided by TAKARA, and the RNA was reverse-transcribed into cDNA according to the instructions of TB Green ^TM Premix Ex The method provided in the Taq ^TM kit manual was carried out on the RT-PCR ABI7300 instrument for real-time fluorescent quantitative PCR reaction, with GAPDH as the internal reference. After the reaction is completed, the CT value in each reaction tube is calculated by the supporting computer software. Relative quantification method 2 ^ΔΔCt was used to calculate the relative expression levels of circRNAs.

Western blot experiment: 72 hours after PC12 cells (purchased from Jiangsu KGI Biotechnology Co., Ltd.) were transfected with si-circCwc27 (purchased from Jiman Biotechnology (Shanghai) Co., Ltd.) and the control, the cells were lysed with RIPA lysate, After 20 min, the lysate was transferred to a 1.5 mL centrifuge tube and labeled, and the protein was quantified with a BCA kit. After protein denaturation, separated by SDS-PAGE electrophoresis, transfer to PVDF membrane, 5% skimmed milk powder (0.1% TBST diluted) blocked at room temperature for 1 hour, washed 3 times with TBST, each time for 5 minutes, and incubated APP and internal reference protein GAPDH antibody at 4°C respectively ( Purchased from Abcam, APP antibody product number ab32136, GAPDH antibody product number ab8245) (1:1000) overnight, washed 3 times with TBST, 5 min each time, incubated secondary antibody HRP-labeled antibody (purchased from Biyuntian) (1:5 000) for 2 hours, Wash 3 times with TBST, add ECL luminescent solution, expose, develop, and use Image J software for grayscale analysis.

In Example 1, circRNA sequencing analysis and RT-PCR experiments showed that, as shown in Figure 1, in the peripheral plasma of AD patients and in the brain tissue (hippocampus and cortex) of APP/PS1 model mice, the level of circCwc27 was significantly increased ( ^* p <0.05, ^** p<0.01 vs. WT group; n=6, ^*** p<0.001 vs. control group; n(control group)=18, n(AD)=20, data are expressed as mean ± SEM).

Example 2: Interfering with the expression of circCwc27 reduces APP protein levels

AD model mouse APP/PS1 was injected with circCwc27 interfering virus LV-sh-circCwc27 (purchased from Shanghai Jiying Biotechnology Co., Ltd.) via brain stereotaxic injection. The circCwc27 interfering RNA is coated with lentivirus for long-term and stable reduction of circCwc27 expression in the mouse brain. The circCwc27 interfering RNA sequence is shown in SEQ ID NO.1, and the virus concentration is 1×10 ⁹ virus/microliter, injected The volume is 5 μL. The control group was the group of APP/PS1 mice stereotaxically injected with the control virus LV-sh- ^circCon (purchased from Shanghai Jiying Biotechnology Co., Ltd.). 5 μL. Then, Western blot experiments found that the circCwc27 interference virus could significantly reduce the expression of APP protein in the brains of APP/PS1 mice, which indicated that circCwc27 played an important role in regulating Aβ production.

Figure 2 shows that the circCwc27 interference virus can significantly reduce the expression of APP protein in the brain of APP/PS1 mice (***p<0.001APP/PS1-LV-sh-circ compared with the control group; n=4, the data are averaged Values ± SEM represent).

Example 3: circCwc27 inhibitor alleviates pathological and behavioral characterization of amyloid in the brain of AD model mice

The experiment was approved by the Animal Experiment Ethics Committee of Shanghai Jiao Tong University School of Medicine and carried out in accordance with the animal experiment guidelines. Six-month-old APP/PS1 male mice were purchased from the Model Animal Center of Nanjing University. The mice were randomly divided into WT-LV-sh-circCon group (wild-type mice injected with control virus group), WT-LV-sh-circCwc27 group (wild-type mice injected with circCwc27 interfering virus group), APP/PS1-LV -sh-circCon (APP/PS1 mouse injection control virus group) group and APP/PS1-LV-sh-circCwc27 group (APP/PS1 mouse injection circCwc27 interference virus group). The mice were anesthetized by intraperitoneal injection of 3% pentobarbital sodium, and their heads were fixed on a stereotaxic injection apparatus in a prone position, and 5 μl of LV-sh-circCwc27 (purchased from Shanghai Jiying Biotechnology Co., Ltd.) solution was drawn with a micro-syringe. The concentration of the solution is 1×10 ⁹ virus/microliter, and the brain tissue is vertically inserted into the skull hole. Use a micro-injection pump to inject into the third ventricle of the mouse at a speed of 0.5 μl/min (posterior bregma: -0.3 mm, left/right sagittal suture: 1.0 mm, depth: 2.2 mm), and slowly withdraw the syringe after the injection is completed. After the stereotaxic injection, the mice were disinfected around the skull injection site, the skin was sutured, and then put back into the mouse cage for further feeding. Two months later, the learning and memory abilities of the mice were tested by Morris water maze. After behavioral studies, the brains were sacrificed and the brains were fixed and sliced to detect the pathology of amyloid protein in the brain (the amyloid protein Aβ antibody was purchased from Cell Signaling Technology, product number 2450T).

As shown in Figure 3, the results of behavioral experiments showed that circCwc27 inhibitors could significantly shorten the latency to reach the platform and increase the residence time in the target quadrant of APP/PS1 mice. It can be seen from the trajectory diagram of the mice in the control group that compared with the control group, the learning and memory abilities of the treatment group were significantly improved ( ^* p<0.05, ^** p<0.01, ^*** p<0.001WT-LV-sh -circ vs. control group; ^# p<0.05APP/PS1-LV-sh-cir vs. control group, n=7-10, data are expressed as mean ± SEM).

As shown in Figure 4, the results of brain histopathological examination showed that treatment with circCwc27 inhibitors could reduce the deposition of amyloid plaques in the brain and alleviate the severity of amyloid pathology in the brain ( ^*** p<0.001APP/PS1-LV -sh-circ compared with the control group, n = 4, data are expressed as mean ± SEM).

SEQ ID NO. 1

GGCUUCUCGUACUUCCCCU

SEQ ID NO. 2

GAAGCCGGCAAGCCGAAGAGCAUGAAGGGGAGAAGU

SEQ ID NO. 3

AGCATGAAGGGGAGAAGTAAGAGCAGCCATGACCTGCTCAAGGACGACCCGCATCTAAGCTCTGTCCCAGCAGTGGAAAGTGAAAAAGATGATGCAACAGGAGATTTAGAAGATGATGGTGAGGATGACAGTGCAGAGCGTGACGAATACATGGAAGATGATGAGAAGAACTTGATGAGAGAAAGAATTGCAAAACGGTTAAAGAAAGATGCAAGTGCCAGTGTGAAGTCAGCCGGAGACGGGGAGAAGAAGCCGGCAAGCCGAAG

Claims (4)

1. The application of the circular RNACwc27 down-regulator in the preparation of the medicine for preventing and/or treating Alzheimer's disease, characterized in that, the circular RNACwc27 down-regulator is a circular RNACwc27 inhibitor, and the nucleus of the circular RNACwc27 The nucleotide sequence is shown in SEQ ID NO.3. 2. application according to claim 1, is characterized in that, described circular RNACwc27 down-regulator is: An RNA fragment comprising the base sequence shown in SEQ ID NO.1; or A polypeptide, protein or compound capable of inhibiting the expression of circular RNA Cwc27. 3. The application according to claim 1 or 2, characterized in that the drug comprises a circular RNA Cwc27 down-regulator, and a pharmaceutically acceptable carrier or adjuvant. 4. A method of screening a drug for preventing and/or treating Alzheimer's disease, said method comprising: Treating a system expressing circular RNA Cwc27 with a candidate substance; and Detecting the expression of circular RNA Cwc27 in the system; If the expression of circular RNA Cwc27 is reduced, it indicates that the candidate substance is a potential substance for preventing and/or treating Alzheimer's disease; The nucleotide sequence of the circular RNA Cwc27 is shown in SEQ ID NO. 3.

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