Screening method of mRNA participating in 396, 404 site serine phosphorylation tau protein aggregation and application thereof

Abstract

The invention discloses mRNA participating in 396, 404 site serine phosphorylation tau protein aggregation and a screening method and application thereof, and belongs to the technical field of biological medicine. According to the invention, RIP sequencing and RNA sequencing are carried out on a 5 * FAD transgenic mouse, and several mRNAs combined with p-tau396 and 404 proteins are determined through combined analysis of the RIP sequencing and the RNA sequencing; the method comprises the following steps: carrying out RT-qPCR and mRNA fluorescence in-situ hybridization identification on a plurality of mRNAs, and verifying the influence function of the mRNAs on expression and / or aggregation of p-tau396 and 404 proteins on HT22 neuronal cells by knocking down mRNA expression, thereby finally obtaining the target mRNA-NM001313929.1. The mRNA can inhibit aggregation of p-tau396 and 404 proteins so as to improve synaptic injury and cognitive impairment of 5 * FAD transgenic mice, and has a very wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an mRNA involved in the aggregation of tau protein phosphorylated at sites 396 and 404, and a screening method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease whose main pathological features are amyloid β protein (Aβ) deposition and neurofibrillary tangles (NFTs). Compared with Aβ deposition, NFTs are considered to be more related to cognitive impairment and positively correlated with the development of AD. The main component of NFTs is aggregates formed by hyperphosphorylation of tau protein (p-tau). Currently, more than 85 phosphorylation sites have been found on tau protein. Among them, p-tau (p-tau) at serine 396 and 404 sites has a high phosphorylation rate. 396,404 ) is one of the types of p-tau protein. After binding to other types of p-tau protein, a series of events are initiated, 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 shown 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 occurrence of cognitive impairment. However, the key factors that cause p-tau396, 404 protein to aggregate into NFTs are still unclear.

[0003] Studies have shown that after tau protein is phosphorylated, its microtubule binding domain (MTBD) and disordered regions (IDRs) are usually rich in charged amino acids, which can bind to RNA molecules through electrostatic interactions, thereby causing tau protein to undergo liquid-liquid phase separation (LLPS) and aggregate into a tighter fibrous structure, and eventually form neurofibrillary tangles. In this regard, some studies have further 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 stress response and metabolic process in the cell 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 As one of the main types of phosphorylated tau proteins in NFTs, the involvement of RNA may be a key factor in the formation of NFTs. However, how to determine the involvement of p-tau 396,404 There is no relevant report on the key RNA for protein aggregation in the existing technology. Summary of the invention

[0004] In order to solve the above technical problems, the present invention conducts RIP sequencing and RNA sequencing on 5×FAD transgenic mice, and determines the p-tau related genes through the combined analysis of the two. 396,404 These mRNAs were then identified by RT-qPCR and mRNA fluorescence in situ hybridization, and their effects on p-tau were verified by knocking down mRNA expression in HT22 neuronal cells. 396,404 The mRNA involved in the phosphorylation of tau protein at serine 396,404 was NM_001313929.1, and the corresponding gene name was Crhr1, which was located on chromosome 11; this mRNA can inhibit p-tau 396,404 Protein aggregation can improve synaptic damage and cognitive impairment in 5×FAD transgenic mice, and has broad application prospects in the detection and treatment of Alzheimer's disease.

[0005] To achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide an mRNA expression level regulating product for use in preparing a product for regulating the aggregation of tau protein phosphorylated at serine 396,404 sites, wherein the mRNA sequence is shown in SEQ ID NO.1.

[0006] The mRNA screening method specifically comprises the following steps: Step 1: Phosphorylation of tau protein at serine 396,404 (p-tau 396,404 ) Combined with RNA extraction The hippocampal tissues of 8-month-old 5×FAD transgenic mice and wild-type mice were collected and ground in a lysis buffer containing RNA and DNA enzyme inhibitors. After centrifugation at 16,100 g for 10 min, the ground supernatants of the hippocampal tissues of the two mice were divided into three parts: 0.5 mL, 0.5 mL, and 0.1 mL. 50 μg of p-tau 396,404 The antibody and IgG antibody were incubated with Protein A / G immunoprecipitation magnetic beads for 2 hours. Subsequently, each antibody / magnetic bead complex was added to the grinding supernatant of the hippocampal tissue of the two groups of mice and incubated overnight at 4°C under a vertical shaker to obtain the immunoprecipitated complex. Thereafter, the immunoprecipitated complex was washed three times with lysis buffer, and the RNA-p-tau in the immunoprecipitated complex was separated with elution buffer. 396,404 Finally, TRIzol reagent was used to extract RNA-p-tau 396,404The protein complex and RNA in the supernatant of the grinded hippocampal tissues of the two groups of mice were stored at -80°C for later use.

[0007] Step 2: RNA Immunoprecipitation Sequencing (RIP-Seq) For RIP sequencing, all RNA samples were first quality assessed by Agilent 2200 TapeStation and Qubit and fragmented to approximately 200 bp. Subsequently, RNA fragments were sequenced on the Illumina platform with double-end 150 bp. For RNA sequencing analysis, HTSeq was used to calculate the read count value of each transcript and estimate the TPM (Transcripts Per Kilobase of exon model per Million mapped reads) value of the transcript. 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 adjusted P values ​​less than 0.05 were considered significant.

[0008] Step 3: Analysis of differential binding peaks and differentially expressed genes Analysis of differential binding peaks: p-tau in hippocampal tissues of two groups of mice 396,404 Data quality control of protein-binding RNA; sequence gene alignment; binding peak detection; motif prediction and annotation; binding peak annotation and related statistical analysis; differential binding peak analysis; KEGG and GO analysis of differential binding peak annotated genes; Analysis of differentially expressed genes: The total RNA in the hippocampal tissues of the two groups of mice was analyzed for significantly differentially expressed genes by difference fold and significance level; KEGG and GO analysis was performed on the differentially expressed genes, with P<0.05 of enrichment as the significance threshold, and the differentially expressed genes with the function of regulating inflammatory response were selected for further analysis; Screening and p-tau 396,404 Protein-bound RNA: Combine the results of differentially expressed gene analysis and differential binding peak analysis to discover common inflammation-related genes as markers for p-tau 396,404 Protein-binding RNA candidate target genes, and select RNAs corresponding to the candidate target genes that are related to p-tau 396,404 mRNAs whose protein binding regions are exons or 3'UTRs are candidate RNAs; Step 4: p-tau 396,404 Verification of protein-bound RNA Step 4.1 For the above with p-tau 396,404For candidate RNAs bound to proteins, primers were designed using Primer5, RIP and RNA sequencing samples were reverse transcribed into cDNA, and RNA expression was verified using real-time quantitative PCR; Step 4.2 For the above with p-tau 396,404 We designed FISH fluorescent probes labeled with Alexa Fluor 647 to identify candidate RNAs that bind to proteins. We incubated frozen sections of the brain of 5×FAD transgenic mice with FISH probes of different RNAs overnight, and then used antibodies to detect p-tau. 396,404 Protein was stained, and finally laser confocal microscopy was used to observe different RNAs and p-tau in hippocampal tissue 396,404 Protein co-localization.

[0009] Step 5: Exploring the effect of RNA on p-tau in HT22 neuronal cells 396,404 Effects of protein aggregation and cell viability For the above and p-tau 396,404 Protein-binding candidate RNAs were selected by real-time quantitative PCR to associate with p-tau 396,404 The candidate RNAs that significantly bind to p-tau were further explored in the HT22 hippocampal neuronal cell line. 396,404 The pLKO.1-shRNA vector was prepared and co-transfected with the lentiviral plasmid into HEK293T cells. The viral supernatant was collected and filtered to infect HT22 cells and the RNA stable knockdown cell line was obtained after puromycin selection. Subsequently, the expression level of the candidate RNA in the RNA stable knockdown cells was detected by real-time quantitative PCR. Finally, p-tau was detected by Western blot and MTT reagent in the stable knockdown cells induced by okadaic acid (OA). 396,404 Protein levels and cell viability to validate candidate RNAs for p-tau 396,404 Protein aggregation and cell viability. Further selection of p-tau in candidate RNA stable knockdown cells 396,404 The cell lines most affected by protein aggregation were then tested for expression levels of other phosphorylated forms of tau protein and p-tau 396,404 The protein expression levels were compared.

[0010] Step 6: Exploring the effect of RNA on p-tau in 5×FAD transgenic mice 396,404 Protein aggregation, synaptic plasticity, and cognitive function For the above screening, the participation of p-tau 396,404Protein aggregation mRNA-Crhr1, further constructed sh-Crhr1 lentiviral silencing vector with EGFP fluorescence. The constructed sh-Crhr1 lentiviral vector was injected into the hippocampus of 5×FAD transgenic mice by stereotaxic injection and mRNA-Crhr1 and p-tau were detected 396,404 Protein expression and changes in learning, memory and synaptic plasticity in mice.

[0011] Furthermore, the mRNA expression level regulating product is a product for downregulating the mRNA expression level.

[0012] Furthermore, the product of down-regulating the mRNA expression level includes sh-Crhr1, whose sequence is shown in SEQ ID NO.2.

[0013] Alternatively, the product for downregulating the mRNA expression level comprises a recombinant expression vector, and the recombinant expression vector is a recombinant expression vector for knocking down the mRNA.

[0014] Alternatively, the product for downregulating the mRNA expression level comprises a host cell containing a recombinant expression vector for knocking down the mRNA.

[0015] Furthermore, the above-mentioned application of the product for downregulating 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.

[0016] Furthermore, the mRNA expression level regulating product is a product for upregulating the mRNA expression level.

[0017] Furthermore, the above-mentioned application of the product for increasing 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.

[0018] The second object of the present invention is to provide an application of an mRNA expression level regulating product in the preparation of a product for preventing and treating Alzheimer's disease, wherein the mRNA expression level regulating product includes an mRNA expression level downregulating product in any of the above applications.

[0019] Furthermore, 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.

[0020] The product for preventing and treating Alzheimer's disease of the present invention can be administered by central nervous system-specific administration methods such as intraventricular injection, intrathecal injection or intranasal administration, and the dosage form is any dosage form of common gene targeted therapy products.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention conducts RIP sequencing and RNA sequencing on 5×FAD transgenic mice, and determines the relationship between p-tau through the combined analysis of the two. 396,404 These mRNAs were then identified by RT-qPCR and mRNA fluorescence in situ hybridization, and their effects on p-tau were verified by knocking down mRNA expression in HT22 neuronal cells. 396,404 The mRNA involved in the phosphorylation of tau protein at serine 396,404 was NM_001313929.1, and the corresponding gene name was Crhr1, which was located on chromosome 11; this mRNA can inhibit p-tau 396,404 Protein aggregation can improve synaptic damage and cognitive impairment in 5×FAD transgenic mice, and has broad application prospects in the detection and treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the p-tau bound to the Protein A / G magnetic beads in Example 1 of the present invention 396,404 Dot plot and Western blot analysis results of proteins; Figure 2 It is a logo image corresponding to a specific motif and an annotation analysis image of a difference peak in Example 1 of the present invention; Figure 3 The number of genes significantly up-regulated or down-regulated in RIP and RNA sequencing in Example 1 of the present invention and the enrichment pathway result diagram with the most significant differences between KEGG and GO; Figure 4 In Example 1 of the present invention, the differential peak of RIP sequencing and the inflammation-related gene data of RNA sequencing are jointly analyzed to screen out genes related to p-tau 396,404 The result graph of mRNA with obvious protein binding; Figure 5 is p-tau in Example 1 of the present invention 396,404 Figure 1 shows the identification results of protein-bound mRNA; Figure 6 The knockdown of mRNA (Cacna1a, Crhr1) in Example 1 of the present invention can inhibit OA-induced p-tau in HT22 neuronal cells. 396,404 The results of protein aggregation and enhanced OA-induced HT22 neuronal cell activity; Figure 7The knockdown of mRNA-Crhr1 in Example 1 of the present invention specifically inhibits p-tau in HT22 neuronal cells 396 ,404 Result diagram of protein aggregation; Figure 8 The sh-Crhr1 lentiviral vector with EGFP fluorescence injected into the hippocampus in Example 1 of the present invention can be normally expressed in the hippocampus tissue and can significantly reduce the mRNA of Crhr1 and p-tau 396,404 Result graph of protein expression level; Fig. 9 This is a result diagram showing that hippocampal injection of sh-Crhr1 lentiviral vector with EGFP fluorescence can improve cognitive function of 5×FAD transgenic mice in Example 1 of the present invention; Fig.10 This is a result diagram showing that hippocampal injection of sh-Crhr1 lentiviral vector with EGFP fluorescence can improve synaptic plasticity in 5×FAD transgenic mice in Example 1 of the present invention. DETAILED DESCRIPTION

[0023] The following examples will further illustrate the specific steps and features of the invention, which are for illustrative purposes only and are not intended to limit the present invention. The methods used in the present invention are conventional methods in the art unless otherwise specified. The reagents, materials, and instruments involved in the present invention are commercially available unless otherwise specified.

[0024] The present invention is further described in detail below with reference to the embodiments and drawings: Example 1

[0025] This embodiment provides a screening method for obtaining RNA involved in the aggregation of tau protein phosphorylated at serine 396,404, comprising the following steps: 1. Collection of tissue samples 5×FAD transgenic and wild-type mice were purchased from AlphaGo, and all mice were kept in the SPF animal room of Wuhan National Optoelectronics Research Center, Huazhong University of Science and Technology. This experiment selected 8-month-old mice, which are in the period of accelerated development of Alzheimer's disease pathology. 396,404 Protein expression was significantly increased. Three 5×FAD transgenic mice and three wild-type mice were selected, and the mice were killed by cervical dislocation and the mouse brains were removed. The hippocampal tissues of each group of mice were collected and placed in cryopreservation tubes, quickly frozen in liquid nitrogen, and stored in a -80℃ refrigerator for subsequent RIP sequencing and RNA sequencing.

[0026] 2. Main reagents and instruments Fluorescence quantification kit and reverse transcription kit were purchased from Nanjing Novezan Co., Ltd.; RNA binding protein immunoprecipitation kit was purchased from Boxin Biotechnology Co., Ltd.; TRIzol reagent (Life Technologies, Carlsbad, CA, USA), Nanodrop2000 (Termo Fisher Scientific, Waltham, MA, USA), and Agilent 2200 bioanalyzer (Agilent Technologies, CA, USA).

[0027] 3. RNA Immunoprecipitation Sequencing 3.1 Extraction of p-tau 396,404 Protein-bound RNA and total RNA The hippocampal tissue was removed and ground in a lysis buffer containing RNA and DNA enzyme inhibitors. After centrifugation at 16,100 g for 10 min, the ground supernatant of the hippocampal tissue of the two mice was divided into three parts: 0.5 mL (IP), 0.5 mL (IgG), and 0.1 mL (input). 50 μg of p-tau was added at 4°C. 396,404 The antibody and IgG antibody were incubated with Protein A / G immunoprecipitation magnetic beads for 2 hours. Subsequently, the antibody / magnetic bead complex was added to the grinding supernatant of the hippocampal tissue of the corresponding IP and IgG groups of mice, and incubated overnight at 4°C on a vertical shaker to obtain the immunoprecipitation complex. Thereafter, the immunoprecipitated complex was washed three times with lysis buffer, and the RNA-p-tau in the immunoprecipitation complex was separated with elution buffer. 396,404 Protein complex.

[0028] Take out a small portion of RNA-p-tau 396,404 The protein complexes were boiled in 5× SDS loading buffer for 10 min and analyzed by western blot. Figure 1 As shown, p-tau in the IP group was higher than that in the IgG group. 396,404 It can bind more significantly to Protein A / G magnetic beads, indicating that RNA-p-tau on the magnetic beads 396,404 The protein complex can be used for subsequent RNA extraction. Afterwards, most of the remaining RNA-p-tau 396,404 The protein complex and the remaining hippocampal tissue grinding supernatant were used to extract p-tau using TRIzol reagent 396,404 Protein-bound RNA and total RNA were stored at -80°C until use.

[0029] 3.2 Library construction and high-throughput sequencing First, the RNA samples for RIP sequencing were quality assessed by Agilent 2200 TapeStation (Agilent Technologies, USA) and Qubit (Thermo Fisher Scientific, USA). Briefly, RNA was fragmented to approximately 200 bp. Subsequently, the RNA fragments were subjected to first-strand and second-strand cDNA synthesis, and adapter ligation and low-cycle enrichment were performed according to the instructions of NEBNext® Ultra RNA Library Prep Kit for Illumina (NEB, USA). The final library product was quality assessed by Agilent 2200 TapeStation and Qubit® (Life Technologies, USA), and paired-end 150 bp sequencing was performed on the Illumina platform (Illumina, USA).

[0030] 3.3 Data Preprocessing Adapter sequences and low-quality bases were trimmed using the Trimmomatic tool (version: 0.36) and rRNA removal was performed by RNAcentral to obtain valid read sequences. Genome alignment (version from UCSC genome browser) was performed using Hisat2 (version: 2.2.1) to obtain uniquely mapped read sequences.

[0031] 3.4 Expression calculation The valid read sequences of the input samples were used for RNA-seq analysis, and HTSeq (version: 0.6.0) was used to calculate the read count value of each transcript and estimate the TPM value of the transcript reads.

[0032] 3.5 Peak calling and motif identification Peak calling was performed using Piranha (version: 1.2.1). Subsequently, peaks were annotated using HOMER (version: 4.8). Consensus motifs were detected for nucleotide sequences in the peak regions using STREME (version: 5.5.1) and MEME (version: 5.5.1). Motif enrichment analysis was performed using CentriMo (version: 5.5.1).

[0033] 3.6 Functional enrichment analysis GO and KEGG pathway enrichment analysis was performed using KOBAS3.0 or ClusterProfiler. The enrichment results were limited to GO biological processes and KEGG pathway entries, and GO biological processes and KEGG pathway entries with adjusted P values ​​less than 0.05 were considered significant.

[0034] 4 p-tau 396,404 Screening and identification of protein-binding RNA 4.1 Quality control of RIP and RNA sequencing data After the data is downloaded, the quality control of the raw sequencing data (Raw Data) is first required. Valid data is obtained after removing the adapter sequence and low-quality sequence from the raw sequencing. After that, data quality control is performed based on the error rate (Error), data volume, sequence length and base quality (Q20 / Q30) in the valid data. After that, the RNAcentral database is used to filter known rRNA sequences, and the sequencing data is compared with the reference genome using the HISAT2 software to make a comprehensive evaluation of the coverage area and coverage depth of the sequencing data. For each sample, the coverage of reads in each 100,000bp window on the whole genome and the coverage depth of the unique aligned reads in the whole genome are counted.

[0035] 4.2 RIP sequencing data analysis The Piranha software package was used to search for binding peaks in the entire transcriptome. Motifs were identified using the motif discovery algorithms STREME and MEME. The logos corresponding to specific motifs are shown in the figure below. Figure 2 As shown in A. Based on the alignment results and the obtained peak regions, the htseq software was used to calculate the expression values ​​of the peak regions of the comparison group samples, and the edger software was used to calculate the difference peaks between the comparison groups. The results of the annotation analysis of the difference peaks are shown in Figure 2 As shown in B, Exon, 3'UTR and 5'UTR are key regions for regulating mRNA transcription and translation. Figure 3 As shown in A, the number of significantly upregulated peaks is 17302, and the number of significantly downregulated peaks is 6505, indicating that the p-tau 396,404 The protein-bound peaks were mainly expressed as upregulated. In order to identify the functions of these differentially methylated genes, KEGG pathway analysis was performed on the differentially methylated genes. Figure 3 As shown in B, the pathways with the most significant differences in KEGG analysis are mainly concentrated in Axon guidance, MAPK signaling pathway and Phosphatidylinositol signaling system pathways, among which the MAPK signaling pathway has the largest number of enriched genes, and this pathway is related to inflammatory response and tau protein phosphorylation.

[0036] 4.3 RNA sequencing data analysis RNA-seq data analysis showed that a total of 2318 genes were significantly differentially expressed, of which 1398 were significantly upregulated and 920 were significantly downregulated ( Figure 3 C). GO enrichment analysis found that the most significantly different pathways were mainly concentrated in Regulation of inflammatory response, Translational elongation, and Myeloid cell activation involved in immune response pathways, which are also pathways related to inflammation and immunity ( Figure 3 D).

[0037] 4.4 RIP and RNA sequencing association analysis The differential peaks of RIP sequencing and the inflammation-related gene data in the GO and KEGG enriched pathways of RNA sequencing were jointly analyzed, such as Figure 4 As shown in A, there are four types of common genes: the first type is the common genes of WT mouse-specific genes in RIP sequencing and 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 inflammation-related genes enriched in RNA sequencing, with a total of 3; the third type is the common genes of 5×FAD mouse-specific genes in RIP sequencing and 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 2826, but because these genes are not related to inflammation, only the Cacna1a gene with the most significant differential expression was selected for subsequent screening. The above 12 common genes were sorted according to protein binding regions and differential expression trends. The results showed that only the binding regions of 5 mRNAs and proteins, namely Cacna1a, Flt1, Kif9, CD4 and Crhr1, were key regions for regulating mRNA transcription and translation, and they were all significantly upregulated in 5×FAD samples. Subsequently, the expression results of these five mRNAs in WT and 5×FAD samples were imported into IGV software to view the distribution of reads in the genome for further analysis of differences between samples. Figure 4 As shown in B, the number of binding peaks of these five mRNAs in the 5×FAD sample was significantly higher than that in the WT sample, which also indicated that these five mRNAs were involved in p-tau 396,404 The above results indicate that these five mRNAs may be related to p-tau 396,404 There is obvious binding of proteins.

[0038] 5 p-tau 396,404 Identification of protein-bound mRNA 5.1 p-tau396,404 Protein binding to mRNA expression For the above screened p-tau 396,404 Protein-binding mRNA (Cacna1a, Flt1, Kif9, CD4, Crhr1) was further verified by real-time quantitative PCR and 1% agarose gel electrophoresis, indicating that the above-screened mRNAs can bind to p-tau 396,404 Protein binding ( Figure 5 AB).

[0039] 5.2 mRNA fluorescence in situ hybridization (FISH) and p-tau 396,404 Protein immunofluorescence For the above screened p-tau 396,404 Protein-binding mRNA (Cacna1a, Flt1, Kif9, CD4, Crhr1), several mRNAs screened in the brain of 5×FAD transgenic mice were distributed and labeled with Alexa Fluor 647-labeled FISH probes, and then p-tau was detected by immunofluorescence. 396,404 Protein was stained, and finally laser confocal microscopy was used to observe the expression of several screened mRNAs and p-tau in hippocampal tissue. 396,404 The co-localization of proteins showed that the above-screened mRNAs can colocalize with p-tau 396,404 Protein binding ( Figure 5 C).

[0040] 6 Knockdown of mRNA expression can specifically inhibit p-tau 396,404 Protein aggregation 6.1 Construction of mRNA (Cacna1a, Crhr1) stable knockdown cell lines According to the above real-time quantitative PCR results, we selected 396,404 The candidate mRNAs (Cacna1a and Crhr1) that were significantly bound by the protein were further explored in the HT22 hippocampal neuronal cell line to investigate the effects of the two candidate mRNAs on p-tau. 396,404 The effect of protein aggregation function. Two pLKO.1-shRNA vectors were prepared, and the pLKO.1-shRNA vectors and lentiviral plasmids were co-transfected into HEK293T cells. The viral supernatant was collected and filtered to infect HT22 cells. Subsequently, HT22 cells with stable knockdown of two mRNAs (Cacna1a and Crhr1) were selected by puromycin, and the expression levels of the two mRNAs in the cells were detected by real-time quantitative PCR. The results showed that the expression levels of the mRNAs (Cacna1a and Crhr1) corresponding to the two stable knockdown HT22 cells were significantly reduced ( Figure 6A) HT22 cells with stable knockdown of two mRNAs (Cacna1a and Crhr1) were successfully constructed.

[0041] 6.2 Effect of mRNA knockdown (Cacna1a, Crhr1) on p-tau 396,404 Effects of protein aggregation and cell viability To investigate the effect of mRNA (Cacna1a, Crhr1) knockdown on p-tau 396,404 The effect of protein aggregation was investigated by Western blot and MTT reagent in two stable knockdown cell lines induced by OA. 396,404 The results showed that knockdown of mRNA-Crhr1 more significantly inhibited the expression of p-tau in cells 396,404 Protein aggregation ( Figure 6 B) and can also significantly improve OA-induced decrease in cell viability ( Figure 6 C), indicating that mRNA-Crhr1 is involved in p-tau 396,404 Key mRNA for protein aggregation.

[0042] Afterwards, for HT22 cells with stable knockdown of mRNA-Crhr1, p-tau was further detected by Western blot. 181 , p-tau 217 , p-tau 231 The results showed that knockdown of mRNA-Crhr1 could only significantly reduce p-tau 396,404 Protein aggregation, while other phosphorylated forms of tau protein did not change significantly ( Figure 7 ), which indicates that mRNA-Crhr1 has an important effect on p-tau 396,404 The effects of protein aggregation are specific.

[0043] Effect of sh-Crhr1 lentiviral vector injection into the hippocampus on p-tau in 5×FAD transgenic mice 396,404 Effects of protein aggregation For the mRNA-Crhr1 involved in the aggregation of phosphorylated tau protein at sites 396 and 404, a 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 hippocampus of 5×FAD transgenic mice by stereotaxic injection, and the tissues of the hippocampus were subjected to fluorescence quantitative PCR and brain section fluorescence confocal imaging. The results showed that the sh-Crhr1 lentiviral vector with EGFP fluorescence could be expressed normally and could significantly reduce the mRNA expression level of Crhr1 in the hippocampus ( Figure 8 A). In addition, Western blot analysis of mouse hippocampal tissues showed that injection of sh-Crhr1 lentiviral vector could significantly reduce p-tau in the hippocampus of 5×FAD transgenic mice compared with injection of control viral vector. 396,404 Protein aggregation ( Figure 8 B), which further indicates that mRNA-Crhr1 is involved in p-tau in 5×FAD transgenic mice 396,404 Key mRNA for protein aggregation.

[0044] 8 Injection of sh-Crhr1 lentiviral vector into the hippocampus can improve synaptic plasticity and cognitive function in 5×FAD transgenic mice Due to p-tau 396,404 Protein aggregation can cause cognitive impairment by forming a large number of neurofibrillary tangles. In order to further verify the efficacy of sh-Crhr1 lentiviral vector in inhibiting p-tau in the hippocampus 396,404 Effects of protein aggregation on cognitive function of 5×FAD transgenic mice. One month after injection of lentiviral vectors, the learning and memory function of mice was observed by Morris water maze behavioral experiment. The results showed that compared with injection of control viral vectors, injection of sh-Crhr1 lentiviral vectors can shorten the escape latency of 5×FAD transgenic mice and increase the number of platform crossings and the time spent in the target quadrant ( Fig. 9 ), which showed an improvement in learning and memory function. In addition, immunofluorescence and Western blot analysis of mouse hippocampal tissues further showed that compared with injection of control viral vectors, injection of sh-Crhr1 lentiviral vectors can increase the density of dendritic spines 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 ( Fig.10 ). This suggests that inhibition is involved in p-tau396,404 Protein-aggregated mRNA-Crhr1 can improve synaptic plasticity and cognitive function in 5×FAD transgenic mice.

[0045] It can be seen that in this example, an RNA, NM_001313929.1, which can participate in the aggregation of phosphorylated tau protein at serine 396,404 sites, and thus improve synaptic damage and cognitive impairment in 5×FAD transgenic mice with Alzheimer's disease, and can be further applied to the fields of Alzheimer's disease detection and treatment, is finally obtained. It is derived from 5×FAD mice, the corresponding gene name is Crhr1, and the gene is located on chromosome 11. The RNA sequence information is as follows: >NM_001313929.1 Mus musculus corticotropin releasing hormone receptor1 (Crhr1), transcript variant 3, mRNA The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 the preparation of a product for regulating the aggregation of tau protein phosphorylated at serine 396,404, characterized in that: The mRNA sequence is shown as SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The mRNA expression level regulating product is a product for downregulating the mRNA expression level.

3. The use according to claim 2, characterized in that: The mRNA expression level down-regulating product includes sh-Crhr1, and its sequence is shown in SEQ ID NO.

2.

4. The use according to claim 2, characterized in that: The mRNA expression level down-regulation product comprises a recombinant expression vector, and the recombinant expression vector is a recombinant expression vector for knocking down the mRNA.

5. The use according to claim 4, characterized in that: The product for downregulating the mRNA expression level comprises a host cell containing a recombinant expression vector for knocking down the mRNA.

6. The use according to any one of claims 2 to 5, characterized in that: The application is the application in preparing a product for inhibiting the aggregation of phosphorylated tau protein at serine 396 and 404 sites.

7. The use according to claim 1, characterized in that: The mRNA expression level regulating product is a product for upregulating the mRNA expression level.

8. The use according to claim 7, characterized in that: The application is the application in preparing a product for promoting the aggregation of phosphorylated tau protein at serine 396 and 404 sites.

9. 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 expression level regulating product includes the mRNA expression level downregulating product in any one of the applications of claims 2 to 5.

10. The use according to claim 9, 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.

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