A screening method for mRNA involved in the aggregation of serine-phosphorylated tau protein at site 396,404 and its application

Abstract

The present invention discloses an mRNA involved in the aggregation of phosphorylated tau protein at serine sites 396 and 404, as well as a screening method and application thereof, belonging to the technical field of biomedicine. The present invention conducts RIP sequencing and RNA sequencing on 5×FAD transgenic mice, and determines several mRNAs bound to p-tau<supgt;396,404< / supgt; protein through the combined analysis of the two; then, RT-qPCR and mRNA fluorescence in situ hybridization are performed on these several mRNAs to identify them, and the function of their influence on the expression and / or aggregation of p-tau<supgt;396,404< / supgt; protein is verified by knocking down the mRNA expression in HT22 neuronal cells. Finally, the target mRNA - NM_001313929.1 is obtained; this mRNA can inhibit the aggregation of p-tau<supgt;396,404< / supgt; protein, thereby improving synaptic damage and cognitive impairment in 5×FAD transgenic mice, and has very broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to an mRNA participating in the aggregation of serine-phosphorylated tau protein at positions 396 and 404, a screening method thereof, and applications thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease, and its main pathological features are amyloid-β protein (Aβ) deposition and neurofibrillary tangles (NFTs). Compared with Aβ deposition, NFTs are considered to have a higher correlation with cognitive impairment and are positively correlated with the development of AD disease. The main component of NFTs is aggregates formed by hyperphosphorylation (p-tau) of tau protein. Currently, more than 85 phosphorylation sites have been found on tau protein. Among them, p-tau at serine 396 and 404 sites (p-tau 396,404 ), as one type of p-tau protein, initiates a series of events after binding to other types of p-tau proteins, leading to the formation of NFTs. In addition, the expression level of p-tau396, 404 protein increases with the increase of NFTs and has been proven to be closely related to the development of neurodegenerative diseases. Therefore, the aggregation of p-tau396, 404 protein is a key step in the formation of NFTs and the emergence of cognitive impairment. However, the key factors causing the aggregation of p-tau396, 404 protein into NFTs are still unclear at present.

[0003] Studies have shown that after tau protein is phosphorylated, its microtubule-binding domain (MTBD) and intrinsically disordered regions (IDRs) are usually rich in charged amino acids, which can bind to RNA molecules through electrostatic interactions, thereby causing the tau protein to undergo liquid-liquid phase separation (LLPS) and aggregate into a more compact fibrillar structure, and ultimately form neurofibrillary tangles. Further studies in this regard have found that different types of RNA (such as mRNA, long non-coding RNA, small RNA, etc.) can participate in the aggregation of p-tau protein and regulate the function of p-tau protein through different protein-binding modes. Among them, lncRNA affects the intracellular stress response and metabolic processes by participating in the aggregation of p-tau protein. In addition, abnormal splicing of RNA can also change the structure or function of p-tau protein, thereby affecting its interaction with RNA. Therefore, p-tau 396,404 protein, as one of the main phosphorylated tau protein types in NFTs, the participation of RNA may be a key factor leading to the formation of NFTs. However, how to determine the key RNA participating in the aggregation of p-tau 396,404 protein has not been reported in the prior art. Summary of the Invention

[0004] To solve the above technical problems, the present invention conducts RIP sequencing and RNA sequencing on 5×FAD transgenic mice, and determines several mRNAs that bind to p-tau protein through the combined analysis of the two; then, these several mRNAs are identified by RT-qPCR and mRNA fluorescence in situ hybridization, and their effects on the expression and / or aggregation of p-tau protein are verified by knocking down mRNA expression on HT22 neuronal cells. Finally, the mRNA involved in the aggregation of phosphorylated tau protein at serine 396 and 404 sites is NM_001313929.1, and the corresponding gene name is Crhr1, which is located on chromosome 11; this mRNA can inhibit the aggregation of p-tau protein, thereby improving synaptic damage and cognitive impairment in 5×FAD transgenic mice, and has very broad application prospects in the fields of detection and treatment of Alzheimer's disease. 396,404 After that, the expression and / or aggregation of p-tau protein are verified by knocking down mRNA expression on HT22 neuronal cells. Finally, the mRNA involved in the aggregation of phosphorylated tau protein at serine 396 and 404 sites is NM_001313929.1, and the corresponding gene name is Crhr1, which is located on chromosome 11; this mRNA can inhibit the aggregation of p-tau 396,404 protein and thus improve synaptic damage and cognitive impairment in 5×FAD transgenic mice, and has very broad application prospects in the fields of detection and treatment of Alzheimer's disease. 396,404 protein aggregation, thereby improving synaptic damage and cognitive impairment in 5×FAD transgenic mice, and has very broad application prospects in the fields of detection and treatment of Alzheimer's disease.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] One object of the present invention is to provide an application of a product for regulating mRNA expression level in the preparation of a product for regulating the aggregation of phosphorylated tau protein at serine 396 and 404 sites, and the mRNA sequence is as shown in SEQ ID NO.1.

[0007] The screening method of the mRNA specifically includes the following steps:

[0008] Step 1: Extraction of RNA bound to phosphorylated tau protein at serine 396 and 404 sites (p-tau 396,404 )

[0009] The hippocampal tissues of the brains of 8-month-old 5×FAD transgenic mice and wild-type mice are collected separately, and ground in a lysis buffer containing RNA and DNA enzyme inhibitors. Then, centrifuged at a rate of 16,100 g for 10 min, and the ground supernatant of the hippocampal tissues of the two types of mice is divided into three parts of 0.5 mL, 0.5 mL and 0.1 mL respectively. At 4°C, 50 μg of p-tau 396,404 antibody and IgG antibody are co-incubated with Protein A / G immunoprecipitation magnetic beads for 2 hours. Subsequently, each antibody / magnetic bead complex is added to the ground supernatant of the hippocampal tissues of the two groups of mice, and incubated overnight at 4°C under a vertical shaker to obtain an immunoprecipitation complex. Thereafter, the immunoprecipitated complex is washed three times with the lysis buffer, and the RNA-p-tau in the immunoprecipitation complex is separated with the elution buffer 396,404Protein complex. Finally, TRIzol reagent was used to extract RNA-p-tau 396,404 from the protein complex and the RNA in the ground supernatant of the hippocampal tissues of the two groups of mice, and stored at -80 °C for later use.

[0010] Step 2: RNA immunoprecipitation sequencing (RIP-seq)

[0011] For RIP-seq, all RNA samples were first evaluated for quality by Agilent 2200 TapeStation and Qubit and fragmented to approximately 200 bp. Subsequently, the RNA fragments were sequenced in paired-end 150 bp on the Illumina platform. For RNA-seq analysis, HTSeq was used to calculate the read count values of each transcript, and the TPM (Transcripts Per Kilobase of exon model per Million mapped reads) values of the transcripts were estimated. For GO and KEGG pathway enrichment analysis, the results were limited to GO biological processes and KEGG pathway entries, and GO and KEGG pathway entries with an adjusted P-value less than 0.05 were considered significant.

[0012] Step 3: Analysis of differentially bound peaks and differentially expressed genes

[0013] Analysis of differentially bound peaks: Data quality control was performed on the p-tau 396,404 protein-bound RNA in the hippocampal tissues of the two groups of mice; sequence gene alignment; binding peak detection; motif prediction and annotation; binding peak annotation and related statistical analysis; differentially bound peak analysis; KEGG and GO analysis of genes annotated by differentially bound peaks;

[0014] Analysis of differentially expressed genes: Significantly differentially expressed gene analysis was performed on the total RNA in the hippocampal tissues of the two groups of mice by fold change and significance level; KEGG and GO analysis were performed on the differentially expressed genes, with a P < 0.05 for the enrichment level as the significance threshold, and the differentially expressed genes with the function of regulating the inflammatory response were selected for further analysis;

[0015] Screening for RNA bound to p-tau 396,404 Combining the results of differentially expressed gene analysis and differentially bound peak analysis, the common genes related to inflammation were explored as candidate target genes for RNA bound to p-tau 396,404 protein, and mRNAs with exon or 3’UTR in the RNA corresponding to the candidate target genes as the protein-binding regions of p-tau 396,404 were selected as candidate RNAs;

[0016] Step 4: p-tau 396,404Verification of Protein-Bound RNA

[0017] Step 4.1 For the candidate RNAs that bind to p-tau 396,404 protein, use Primer5 for primer design, reverse transcribe the RIP and RNA sequencing samples into cDNA, and verify the expression of the RNA using real-time quantitative PCR;

[0018] Step 4.2 For the candidate RNAs that bind to p-tau 396,404 protein, design FISH fluorescent probes labeled with Alexa Fluor 647. Incubate the frozen sections of the brains of 5×FAD transgenic mice overnight with the FISH probes for different RNAs, and then stain the p-tau 396,404 protein with an antibody. Finally, observe the co-localization of different RNAs and p-tau 396,404 protein in the hippocampal tissue using a laser confocal microscope.

[0019] Step 5: Explore the effects of RNA on p-tau 396,404 protein aggregation and cell viability in HT22 neuronal cells

[0020] For the candidate RNAs that bind to p-tau 396,404 protein, select the candidate RNAs that show significant binding to p-tau 396,404 protein through real-time quantitative PCR, and further explore the effects of the candidate RNAs on the p-tau 396,404 protein aggregation function in the HT22 hippocampal neuronal cell line. Prepare the pLKO.1-shRNA vector, co-transfect the pLKO.1-shRNA vector and the lentiviral plasmid into HEK293T cells, collect and filter the resulting viral supernatant, and use it to infect HT22 cells and obtain a stable RNA knockdown cell line through puromycin screening. Subsequently, detect the expression level of the candidate RNA in the stable RNA knockdown cells by real-time quantitative PCR. Finally, detect the p-tau 396,404 protein level and cell viability in the stable knockdown cells induced by okadaic acid (OA) using Western blot and MTT reagent to verify the effects of the candidate RNA on p-tau 396,404 protein aggregation and cell viability. Further select the cell line with the most significant effect on p-tau 396,404 protein aggregation in the candidate RNA stable knockdown cells, and then detect the expression levels of other phosphorylated forms of tau protein and compare them with the p-tau 396,404 protein expression level.

[0021] Step 6: Explore the effects of RNA on p-tau 396,404Effects on protein aggregation, synaptic plasticity, and cognitive function

[0022] Regarding the mRNA-Crhr1 involved in p-tau 396,404 protein aggregation screened above, a sh-Crhr1 lentiviral silencing vector with EGFP fluorescence was further constructed. The constructed sh-Crhr1 lentiviral vector was injected into the hippocampal brain region of 5×FAD transgenic mice by stereotaxic injection, and the expression of mRNA-Crhr1 and p-tau 396,404 proteins, as well as the changes in learning and memory and synaptic plasticity of the mice, were detected.

[0023] Furthermore, the mRNA expression level regulation product is a product for downregulating the mRNA expression level.

[0024] Even further, the product for downregulating the mRNA expression level includes sh-Crhr1, and its sequence is shown in SEQ ID NO.2.

[0025] Alternatively, the product for downregulating the mRNA expression level includes a recombinant expression vector, and the recombinant expression vector is a recombinant expression vector for knocking down the mRNA.

[0026] Or, the product for downregulating the mRNA expression level includes a host cell, and the host cell contains a recombinant expression vector for knocking down the mRNA.

[0027] Still further, the above application of the product for downregulating the mRNA expression level is specifically an application in the preparation of a product for inhibiting the aggregation of phosphorylated tau protein at serine 396 and 404 sites.

[0028] Furthermore, the mRNA expression level regulation product is a product for upregulating the mRNA expression level.

[0029] Even further, the above application of the product for upregulating the mRNA expression level is specifically an application in the preparation of a product for promoting the aggregation of phosphorylated tau protein at serine 396 and 404 sites.

[0030] The second object of the present invention is to provide an application of an mRNA expression level regulation product in the preparation of a product for preventing and treating Alzheimer's disease. The mRNA expression level regulation product includes the product for downregulating the mRNA expression level in any of the above applications.

[0031] Furthermore, the product for downregulating the mRNA expression level inhibits p-tau 396,404 protein aggregation by downregulating the mRNA expression level, thereby improving synaptic damage and / or cognitive impairment in Alzheimer's disease patients.

[0032] The product for preventing and treating Alzheimer's disease in the present invention can be administered by central nervous system-specific administration methods such as intracerebroventricular injection, intrathecal injection, or intranasal administration, and the dosage form is any dosage form in common gene-targeted therapy products.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention conducts RIP sequencing and RNA sequencing on 5×FAD transgenic mice, and determines several mRNAs that bind to p-tau 396,404 protein through the combined analysis of the two; then, RT-qPCR and mRNA fluorescence in situ hybridization are used to identify these several mRNAs, and the function of their effects on p-tau 396,404 protein expression and / or aggregation is verified by knocking down mRNA expression on HT22 neuronal cells. Finally, the mRNA involved in the aggregation of phosphorylated tau protein at serine 396 and 404 sites is NM_001313929.1, and the corresponding gene name is Crhr1, which is located on chromosome 11; this mRNA can inhibit the aggregation of p-tau 396,404 protein, thereby improving synaptic damage and cognitive impairment in 5×FAD transgenic mice, and has very broad application prospects in the fields of detection and treatment of Alzheimer's disease. Description of the Drawings

[0035] Figure 1 is the Dot plot and Western blot analysis results of p-tau 396,404 protein bound to Protein A / G magnetic beads in Example 1 of the present invention;

[0036] Figure 2 is the logo diagram corresponding to the specific motif and the annotation analysis diagram of differential peaks in Example 1 of the present invention;

[0037] Figure 3 is the result diagram of the number of genes significantly up-regulated or down-regulated in RIP and RNA sequencing in Example 1 of the present invention, as well as the most significantly enriched pathways of KEGG and GO differences;

[0038] Figure 4 is the result diagram of jointly analyzing the differential peaks of RIP sequencing and the gene data related to inflammation of RNA sequencing in Example 1 of the present invention and screening out mRNAs that have obvious binding to p-tau 396,404 protein;

[0039] Figure 5 is the identification result diagram of p-tau 396,404 protein-binding mRNA in Example 1 of the present invention;

[0040] Figure 6 In Example 1 of the present invention, knocking down mRNA (Cacna1a, Crhr1) can inhibit OA-induced p-tau 396,404 protein aggregation and increase the activity of HT22 neuronal cells induced by OA; the result diagram

[0041] Figure 7 In Example 1 of the present invention, knockdown of mRNA-Crhr1 specifically inhibits p-tau 396 ,404 protein aggregation in HT22 neuronal cells; the result diagram

[0042] Figure 8 In Example 1 of the present invention, injecting a lentiviral vector of sh-Crhr1 with EGFP fluorescence into the hippocampal brain region can be normally expressed in the hippocampal tissue and can significantly reduce the mRNA of Crhr1 and p-tau 396,404 protein expression level; the result diagram

[0043] Figure 9 In Example 1 of the present invention, injecting a lentiviral vector of sh-Crhr1 with EGFP fluorescence into the hippocampus can improve the cognitive function of 5×FAD transgenic mice; the result diagram

[0044] Figure 10 In Example 1 of the present invention, injecting a lentiviral vector of sh-Crhr1 with EGFP fluorescence into the hippocampus can improve the synaptic plasticity of 5×FAD transgenic mice; the result diagram Detailed implementation method

[0045] The following examples will further illustrate the specific steps and features of the invention. These examples are for illustrative purposes only and do not limit the present invention. The methods used in the present invention are all conventional methods in the art unless otherwise specified. The reagents, materials, and instruments involved in the present invention are all commercially available unless otherwise specified

[0046] The following further describes the present invention in detail in combination with examples and drawings Example 1

[0047] This example provides a screening method for obtaining RNA involved in the aggregation of phosphorylated tau protein at serine 396 and 404 sites, including the following steps

[0048] 1. Sampling of tissue samples

[0049] Both 5×FAD transgenic and wild-type mice were purchased from Mousecome Biotechnology Co., Ltd. All mice were housed in the SPF animal facility of the Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology. In this experiment, 8-month-old mice were selected. At this age, the 5×FAD mice are in the period of accelerated pathological development of Alzheimer's disease, and the expression of p-tau 396,404 protein is significantly increased. Three 5×FAD transgenic mice and three wild-type mice were selected. After decapitating the mice, their brains were removed, and the hippocampal tissues of each group of mice were collected and placed in cryotubes, quickly frozen in liquid nitrogen, and transferred to an -80°C refrigerator for storage, for subsequent RIP sequencing and RNA sequencing.

[0050] 2. Main reagents and instruments

[0051] The fluorescence quantitative kit and reverse transcription kit were purchased from Nanjing Novizan Co., Ltd.; the RNA-binding protein immunoprecipitation kit was purchased from Boxin Biotechnology Co., Ltd.; TRIzol reagent (Life Technologies, Carlsbad, CA, USA), Nanodrop2000 (Termo Fisher Scientifc, Waltham, MA, USA), Agilent 2200 Bioanalyzer (Agilent Technologies, CA, USA).

[0052] 3. RNA immunoprecipitation sequencing

[0053] 3.1 Extraction of p-tau 396,404 protein-bound RNA and total RNA

[0054] The hippocampal tissues were taken out and ground in lysis buffer containing RNA and DNA enzyme inhibitors. Then, centrifuged at a rate of 16,100 g for 10 min, and the ground supernatants of the hippocampal tissues of the two types of mice were each divided into three parts: 0.5 mL (IP), 0.5 mL (IgG), and 0.1 mL (input). At 4°C, 50 μg of p-tau 396,404 antibody and IgG antibody were respectively co-incubated with Protein A / G immunoprecipitation magnetic beads for 2 hours. Subsequently, the antibody / magnetic bead complexes were respectively added to the ground supernatants of the hippocampal tissues of the IP and IgG group mice, and incubated overnight at 4°C on a vertical shaker to obtain immunoprecipitation complexes. Thereafter, the immunoprecipitated complexes were washed three times with lysis buffer, and the RNA-p-tau 396,404 protein complex in the immunoprecipitation complex was separated with elution buffer.

[0055] A small part of the RNA-p-tau 396,404The protein complex was boiled in 5×SDS loading buffer for 10 minutes and subjected to western blot analysis. As Figure 1 shown, compared with the IgG group, p-tau in the IP group 396,404 could bind more significantly to Protein A / G magnetic beads, indicating that the RNA-p-tau 396,404 protein complex on the magnetic beads could be used for subsequent RNA extraction. After that, most of the remaining RNA-p-tau 396,404 protein complex and the previously remaining hippocampal tissue grinding supernatant were extracted with TRIzol reagent for p-tau 396,404 protein-bound RNA and total RNA, and stored at -80 °C for later use.

[0056] 3.2 Library construction and high-throughput sequencing

[0057] First, the RNA samples for RIP sequencing were subjected to quality assessment using an Agilent 2200 TapeStation (Agilent Technologies, USA) and Qubit (Thermo Fisher Scientific, USA). Briefly, the RNA was fragmented to approximately 200 bp. Subsequently, first-strand and second-strand cDNA synthesis of the RNA fragments was performed, and adapter ligation and low-cycle number enrichment were carried out according to the instructions of the NEBNext® Ultra RNA Library Prep Kit for Illumina (NEB, USA). The final library product was subjected to quality assessment using an Agilent 2200 TapeStation and Qubit® (Life Technologies, USA), and paired-end 150 bp sequencing was performed on an Illumina platform (Illumina, USA).

[0058] 3.3 Data preprocessing

[0059] The Trimmomatic tool (version: 0.36) was used to trim adapter sequences and low-quality bases, and rRNA removal was performed through RNAcentral to obtain effective read sequences. Genome alignment (version from the UCSC Genome Browser) was performed using hisat2 (version: 2.2.1) to obtain uniquely mapped read sequences.

[0060] 3.4 Expression quantification

[0061] The effective read sequences of the input samples were used for RNA-seq analysis. HTSeq (version: 0.6.0) was used to calculate the read count values of each transcript and estimate the TPM values of transcript reads.

[0062] 3.5 Peak calling and motif recognition

[0063] Peak calling was performed using Piranha (version: 1.2.1). Subsequently, the peaks were annotated using HOMER (version: 4.8). The nucleotide sequences of the peak regions were used to detect consensus motifs using the STREME (version: 5.5.1) and MEME (version: 5.5.1) tools. Motif enrichment analysis was performed using CentriMo (version: 5.5.1).

[0064] 3.6 Functional enrichment analysis

[0065] GO and KEGG pathway enrichment analyses were performed using KOBAS 3.0 or ClusterProfiler. The enrichment results were limited to GO biological processes and KEGG pathway entries, and GO biological processes and KEGG pathway entries with an adjusted P-value less than 0.05 were considered significant.

[0066] 4 p-tau 396,404 Screening and identification of proteins binding to RNA

[0067] 4.1 Quality control of RIP and RNA sequencing data

[0068] After the data was downloaded, quality control of the raw sequencing data (Raw Data) was first required. After removing adapter sequences and low-quality sequences from the raw sequencing, valid data was obtained. Thereafter, data quality control was performed based on the error rate (Error), data volume, sequence length, and base quality (Q20 / Q30) in the valid data. Subsequently, the RNAcentral database was used to filter known rRNA sequences, and the HISAT2 software was used to align the sequencing data with the reference genome to comprehensively evaluate the coverage area and coverage depth of the sequencing data. For each sample, the reads coverage in windows of 100,000 bp in size across the whole genome and the coverage depth of uniquely mapped reads across the whole genome were separately counted.

[0069] 4.2 Analysis of RIP sequencing data

[0070] The Piranha software package was used to find binding peaks across the whole transcriptome. Motifs were identified using the motif discovery algorithms STREME and MEME, and the logo graph corresponding to a specific motif is shown in Figure 2 Figure A. According to the alignment results and the obtained peak regions, the htseq software was used to calculate the expression values of the peak regions of the sample in the comparison group, and the edger software was used to calculate the differential peaks between the comparison groups. The results of the annotation analysis of the differential peaks are shown in Figure 2As shown in Figure B, where Exon, 3’UTR, and 5’UTR are key regions that regulate mRNA transcription and translation. As Figure 3 shown in Figure A, the number of significantly upregulated peaks was 17,302, and the number of significantly downregulated peaks was 6,505, indicating that the peaks bound to the p-tau 396,404 protein were mainly upregulated in expression. To identify the functions of these differentially expressed genes, KEGG pathway analysis was performed on the differentially methylated genes. As Figure 3 shown in Figure B, the pathways with the most significant differences in the KEGG analysis mainly concentrated in the Axon guidance, MAPK signaling pathway, and Phosphatidylinositol signaling system pathways. Among them, the MAPK signaling pathway had the largest number of enriched genes, and this pathway was related to inflammatory responses and tau protein phosphorylation.

[0071] 4.3 RNA sequencing data analysis

[0072] RNA-seq data analysis showed that a total of 2,318 genes had significant expression differences, among which 1,398 were significantly upregulated and 920 were significantly downregulated ( Figure 3 Figure C). GO enrichment analysis found that the pathways with the most significant differences mainly concentrated in the Regulation of inflammatory response, Translational elongation, and Myeloid cell activation involved in immune response pathways, which were also pathways related to inflammation and immunity ( Figure 3 Figure D).

[0073] 4.4 RIP and RNA sequencing correlation analysis

[0074] A combined analysis was performed on the differential peaks of RIP sequencing and the gene data related to inflammation in the GO and KEGG enrichment pathways of RNA sequencing. As Figure 4As shown in A, the common genes are divided into four types: the first type is the common genes of the genes unique to WT mice in RIP sequencing and the inflammation-related genes enriched in RNA sequencing, with a total of 3; the second type is the common genes of WT and 5×FAD samples in RIP sequencing and the inflammation-related genes enriched in RNA sequencing, with a total of 3; the third type is the common genes of the genes unique to 5×FAD mice in RIP sequencing and the inflammation-related genes enriched in RNA sequencing, with a total of 5; the fourth type is the common genes of WT and 5×FAD samples in RIP sequencing, with a total of 2,826. However, since these genes are not related to inflammation, only the most significantly differentially expressed Cacna1a gene is selected for subsequent screening. The above 12 common genes are sorted according to the protein-binding region and the trend of differential expression. It is found that only the binding regions of the 5 mRNAs of Cacna1a, Flt1, Kif9, CD4, and Crhr1 with proteins are the key regions for regulating mRNA transcription and translation, and they are all significantly up-regulated in the 5×FAD samples. Subsequently, the expression results of these 5 mRNAs in WT and 5×FAD samples are respectively imported into the IGV software to view the distribution of Reads in the genome for further differential analysis between samples. As Figure 4 shown in B, the number of binding peaks of these 5 mRNAs in the 5×FAD samples is significantly more than that in the WT samples, which also indicates that these 5 mRNAs are up-regulated in the 5×FAD samples with high expression of p-tau 396,404 protein. The above results show that these 5 mRNAs may have an obvious binding to p-tau 396,404 protein.

[0075] 5 p-tau 396,404 Identification of mRNA bound to p-tau

[0076] 5.1 Expression of mRNA bound to p-tau 396,404 For the above-screened p-tau

[0077] mRNA bound to protein (Cacna1a, Flt1, Kif9, CD4, Crhr1), real-time quantitative PCR and 1% agarose gel electrophoresis were used for further verification, indicating that the above-screened mRNAs can all bind to p-tau 396,404 protein ( 396,404 A-B). Figure 5 A-B).

[0078] 5.2 mRNA fluorescence in situ hybridization (FISH) and p-tau 396,404 protein immunofluorescence

[0079] For the above-screened p-tau 396,404The protein binds to mRNAs (Cacna1a, Flt1, Kif9, CD4, Crhr1). Several mRNAs screened in the brains of 5×FAD transgenic mice were labeled by the distribution of FISH probes labeled with Alexa Fluor 647. Then, p-tau 396,404 protein was stained by immunofluorescence. Finally, the co-localization of several screened mRNAs and p-tau 396,404 protein in the hippocampal tissue was observed with a laser confocal microscope. The results showed that the above-screened mRNAs could all bind to p-tau 396,404 protein ( Figure 5 C).

[0080] 6 Knockdown of the expression of mRNA can specifically inhibit the aggregation of p-tau 396,404 protein

[0081] 6.1 Construction of cell lines with stable knockdown of mRNAs (Cacna1a, Crhr1)

[0082] According to the above real-time quantitative PCR results, candidate mRNAs (Cacna1a, Crhr1) that showed significant binding to p-tau 396,404 protein were selected to further explore the effects of the two candidate mRNAs on the aggregation function of p-tau 396,404 protein in the HT22 hippocampal neuron cell line. By preparing two pLKO.1-shRNA vectors and co-transfecting the pLKO.1-shRNA vectors with the lentiviral plasmid into HEK293T cells, the resulting viral supernatant was collected and filtered for infecting HT22 cells. Subsequently, two HT22 cell lines with stable knockdown of mRNAs (Cacna1a, Crhr1) were screened out by puromycin, and the expression levels of the two mRNAs in the cells were detected by real-time quantitative PCR respectively. The results showed that the expression levels of the corresponding mRNAs (Cacna1a, Crhr1) in the two stably knocked-down HT22 cell lines were significantly decreased ( Figure 6 A), and the two HT22 cell lines with stable knockdown of mRNAs (Cacna1a, Crhr1) were successfully constructed.

[0083] 6.2 Effects of knockdown of mRNAs (Cacna1a, Crhr1) on the aggregation and cell viability of p-tau 396,404 protein

[0084] To explore the effects of knockdown of mRNAs (Cacna1a, Crhr1) on the aggregation of p-tau 396,404 protein, in the two stably knocked-down cell lines induced by OA, p-tau 396,404Protein levels. The results showed that knockdown of mRNA-Crhr1 more significantly inhibited the aggregation of cellular p-tau 396,404 protein ( Figure 6 B) and also significantly improved the OA-induced decrease in cell viability ( Figure 6 C), indicating that mRNA-Crhr1 is a key mRNA involved in p-tau 396,404 protein aggregation.

[0085] Subsequently, for HT22 cells with stable knockdown of mRNA-Crhr1, the expression levels of p-tau 181 、p-tau 217 、p-tau 231 and total tau proteins were further detected by Western blot. The results showed that knockdown of mRNA-Crhr1 only significantly reduced the aggregation of p-tau 396,404 protein, while there were no significant changes in other phosphorylated forms of tau protein ( Figure 7 ), indicating that the effect of mRNA-Crhr1 on p-tau 396,404 protein aggregation is specific.

[0086] 7 Effect of injecting sh-Crhr1 lentiviral vector into the hippocampal brain region on p-tau 396,404 protein aggregation in 5×FAD transgenic mice

[0087] For the above-mentioned mRNA-Crhr1 involved in the aggregation of phosphorylated tau protein at sites 396 and 404, an sh-Crhr1 lentiviral silencing vector with EGFP fluorescence was further constructed (sh-Crhr1 sequence: 5’-CCGGCTACCACATTGCCGTCATCATCTCGAGATGATGACGGCAATGTGGTAGTTTTTG-3’, SEQ ID NO.2). The constructed sh-Crhr1 lentiviral vector was injected into the hippocampal brain region of 5×FAD transgenic mice by stereotaxic injection. Fluorescence quantitative PCR and fluorescence confocal imaging of brain sections were performed on the hippocampal brain tissue. The results showed that the sh-Crhr1 lentiviral vector with EGFP fluorescence could be normally expressed and significantly reduced the mRNA expression level of Crhr1 in the hippocampal brain region ( Figure 8 A). In addition, Western blot analysis of mouse hippocampal tissue showed that, compared with injection of the control viral vector, injection of the sh-Crhr1 lentiviral vector could significantly reduce the p-tau 396,404 protein aggregation in the hippocampal brain region of 5×FAD transgenic mice ( Figure 8 B), which further indicated that mRNA-Crhr1 is involved in p-tau 396,404Key mRNAs for protein aggregation.

[0088] Intrahippocampal injection of sh-Crhr1 lentiviral vector can improve synaptic plasticity and cognitive function in 5×FAD transgenic mice

[0089] Due to p-tau 396,404 protein aggregation can cause cognitive impairment by forming a large number of neurofibrillary tangles. To further verify the effect of sh-Crhr1 lentiviral vector on the cognitive function of 5×FAD transgenic mice after inhibiting p-tau 396,404 protein aggregation in the hippocampal region. One month after the injection of the lentiviral vector, the learning and memory functions of the mice were observed by the Morris water maze behavioral experiment. The results showed that, compared with the injection of the control virus vector, the injection of sh-Crhr1 lentiviral vector could shorten the escape latency of 5×FAD transgenic mice and increase the number of platform crossings and the residence time in the target quadrant ( Figure 9 ), thereby showing an improvement in learning and memory functions. In addition, immunofluorescence and Western blot analysis of the mouse hippocampal tissue further showed that, compared with the injection of the control virus vector, the injection of sh-Crhr1 lentiviral vector could increase the dendritic spine density and the expression levels of synaptic-related proteins PSD95, Synapsin-1, and synaptophysin in 5×FAD transgenic mice, thereby improving the synaptic plasticity of 5×FAD transgenic mice ( Figure 10 ). This indicates that inhibiting the mRNA-Crhr1 involved in p-tau 396,404 protein aggregation can improve synaptic plasticity and cognitive function in 5×FAD transgenic mice.

[0090] It can be seen that in this example, an RNA - NM_001313929.1 is finally obtained, which can participate in the phosphorylation of tau protein at serine 396 and 404 sites and then aggregate, thereby improving synaptic damage and cognitive impairment in 5×FAD transgenic mice with Alzheimer's disease, and can further be applied to fields such as the detection and treatment of Alzheimer's disease. It is derived from 5×FAD mice, the corresponding gene name is Crhr1, and this gene is located on chromosome 11. The RNA sequence information is as follows:

[0091] >NM_001313929.1 Mus musculus corticotropin releasing hormone receptor1 (Crhr1), transcript variant 3, mRNA

[0092]

[0093] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a product for regulating mRNA expression level in preparing a product for preventing and treating Alzheimer's disease, characterized in that: The mRNA sequence is shown in SEQ ID NO.1; the mRNA expression level regulation product is a product for downregulating the mRNA expression level; the mRNA expression level downregulation product includes sh-Crhr1, whose sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The mRNA expression level downregulation product inhibits p-tau by downregulating the expression level of the mRNA 396,404 Protein aggregation thereby improves synaptic damage and / or cognitive impairment in Alzheimer's disease patients.

Images