CN111363808B

CN111363808B - Biomarker related to Alzheimer disease and application thereof

Info

Publication number: CN111363808B
Application number: CN202010383293.1A
Authority: CN
Inventors: 杨庆河
Original assignee: Second People's Hospital Of Liaocheng
Current assignee: Second People's Hospital Of Liaocheng
Priority date: 2019-06-21
Filing date: 2020-05-08
Publication date: 2022-09-20
Anticipated expiration: 2040-05-08
Also published as: CN111363808A; CN110144397A

Abstract

The invention discloses a biomarker related to Alzheimer and application thereof, and relates to application of biomarkers TES and PPP1R18 in Alzheimer, in particular to application of TES or PPP1R18 in diagnosis and treatment of Alzheimer.

Description

Biomarker related to Alzheimer disease and application thereof

Technical Field

The invention relates to the field of biomedicine, and relates to a biomarker related to Alzheimer and application thereof, wherein the molecular marker is TES or PPP1R 18.

Background

Alzheimer's Disease (AD) is the most common cause of dementia, accounting for 75% of all dementia cases. AD is insidious in onset and clinically manifested as slow and progressive memory loss, cognitive impairment, and personality and behavior changes. Typical pathological features of AD include mainly the formation of Neuritic Plaques (NP) and neurofibrillary tangles (NFT) and the loss of neurons and synapses. Research on AD has progressed rapidly in recent years, including studies on the etiology and pathology of AD, the pathogenesis of AD, cerebrospinal fluid and imaging markers, and a range of therapeutic strategies developed therefrom. However, the etiology of AD is not completely clear, the pathogenesis of AD is complex, and it is widely believed that the onset of AD occurs under the influence of environmental factors, genetic factors, and the interaction between the two. Thanks to the continuous promotion of gene detection technology, the genetic research on the onset of AD has become the focus of attention worldwide, and new sites of some new risk genes are discovered through a large number of large-scale genome-wide association analysis researches.

The onset form of alzheimer's disease is insidious and mainly divided into pre-dementia and dementia stages. The pre-dementia stage can be further divided into pre-mild cognitive impairment and mild cognitive impairment stages (Scheltens P, Blennow K, Breteler MM, et al, Alzheimer's disease [ J ]. Lancet,2016,388(10043): 505-17). Patients in the pre-dementia stage may not have any cognitive dysfunction or may show only mild memory impairment, and other language, executive and visual space abilities may also be slightly impaired. If the patient is not found in time and does not have active and effective intervention, the patient can enter a dementia stage along with the further aggravation of the disease. Impairment of memory, impairment of executive performance and decline of social contact are the main clinical features of the dementia stage. Memory impairment is mainly manifested as a disorder of near memory in the early stage and impaired distant memory in the later stage. The decline of the executive ability and the social contact ability of the patient is mainly manifested by the obvious reduction of the abilities of computing ability, language, visual space and the like, and the occurrence of character changes such as irritability, autism and the like. In the late stage of dementia, in addition to the above-mentioned symptoms, patients may also develop certain psychoneurosis symptoms such as anger abnormality, apathy, and vertebral body system and autonomic nerve dysfunction such as tetanic limbs, sphincter muscle disorder, etc., gradually losing their ability to daily life. In addition, there are also diagnoses of AD that patients have been overlooked because the first symptoms are speech, vision, and executive dysfunction, rather than typical memory dysfunction (Barnes J, Dickerson BC, Frost C, et al, Alzheimer's disease first systems area dependency: evolution from the NACC dataset [ J ]. Alzheimer's & deterioration: the journel of the Alzheimer's Association 2015,11(11): 1349-57). With the development of medical technology, the clinical diagnosis of AD is no longer limited to inquiring patient and family medical history, neuropsychological tests and the like, and the detection of AD markers provides a new basis for the diagnosis of AD.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, the object of the present invention is to provide molecular markers associated with alzheimer for the diagnosis and treatment of alzheimer.

In order to achieve the above objects, the present invention has been extensively and intensively studied to detect differentially expressed genes in alzheimer patients and normal persons by a high throughput sequencing method, and investigate the relationship between the differentially expressed genes and the occurrence of alzheimer, thereby providing a marker for early detection of alzheimer.

The invention discovers that TES and PPP1R18 are remarkably upregulated in Alzheimer patients for the first time, and based on the discovery, the invention provides application of biomarkers in preparing products for diagnosing Alzheimer, wherein the biomarkers are selected from one or two of the following: TES, PPP1R 18.

Further, the product comprises a reagent for detecting TES or PPP1R 18.

Further, the reagent comprises: a probe that specifically recognizes the TES or PPP1R18 gene; or a primer for specifically amplifying the TES or PPP1R18 gene; or a binding agent that specifically binds TES or a protein encoded by PPP1R 18.

Further, the primer sequence pair of the specific amplification TES or PPP1R18 gene is respectively shown in SEQ ID NO. 1-2 and SEQ ID NO. 3-4.

The invention provides a product for diagnosing Alzheimer's disease, which comprises a reagent for detecting the level of TES or PPP1R 18.

Further, the product comprises a nucleic acid membrane strip, a chip or a kit.

Further, the chip includes a gene chip including a primer or an oligonucleotide probe for TES or PPP1R18, a protein chip including a binding agent that specifically binds to TES or PPP1R18 protein.

Further, the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a primer, an oligonucleotide probe or a chip which is specifically directed at TES or PPP1R 18; the protein detection kit comprises a binding agent that specifically binds to TES or PPP1R18 protein.

Furthermore, the primer sequence specific to TES or PPP1R18 is shown in SEQ ID NO. 1-4.

The invention provides application of TES or PPP1R18 in preparing a pharmaceutical composition for treating Alzheimer disease.

Further, the pharmaceutical composition comprises an inhibitor of TES or PPP1R 18. The inhibitor comprises nucleic acid inhibitor, protein inhibitor, proteolytic enzyme and protein binding molecule. Wherein the nucleic acid inhibitor is selected from: an interfering molecule which has TES or PPP1R18 or a transcript thereof as a target sequence and is capable of inhibiting TES or PPP1R18 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. The protein binding molecule is selected from: a substance that specifically binds to TES or PPP1R18 protein, such as an antibody or ligand that inhibits the activity of TES or PPP1R18 protein.

Further, the inhibitor is an agent that reduces the expression of TES or PPP1R 18.

Further, the inhibitor is siRNA.

Further, the sequences of siRNA for reducing TES expression are shown in SEQ ID NO. 7-8, and the sequences of siRNA for reducing PPP1R18 expression are shown in SEQ ID NO. 9-10.

The invention provides a pharmaceutical composition for treating Alzheimer's disease, which comprises an inhibitor of TES or PPP1R 18. The inhibitor comprises nucleic acid inhibitor, protein inhibitor, proteolytic enzyme and protein binding molecule. Wherein the nucleic acid inhibitor is selected from: an interfering molecule which has TES or PPP1R18 or a transcript thereof as a target sequence and is capable of inhibiting TES or PPP1R18 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. The protein binding molecule is selected from: a substance that specifically binds to TES or PPP1R18 protein, such as an antibody or ligand that inhibits the activity of TES or PPP1R18 protein.

Further, the inhibitor is siRNA.

Furthermore, the sequence of siRNA for reducing TES expression is shown in SEQ ID NO. 7-8, and the sequence of siRNA for reducing PPP1R18 expression is shown in SEQ ID NO. 9-10.

In the present invention, TES (Gene ID: 26136) includes the human TES gene and its encoded protein. As a non-limiting example, the gene sequence of TES is shown in any transcript NM-015641.4, NM-152829.3, and the corresponding amino acid sequence is shown in NP-056456.1, NP-690042.1. In the present invention, the gene sequence of a representative TES is shown as NM-015641.4, and the amino acid sequence is shown as NP-056456.1.

PPP1R18 (Gene ID: 170954) includes the human PPP1R18 gene and the protein encoded thereby. As a non-limiting example, the PPP1R18 gene sequence is shown in NM _001134870.2, NM _133471.4 transcript, its corresponding amino acid sequence is shown in NP _001128342.1, NP _ 597728.1. In the present invention, a representative PPP1R18 gene sequence is shown in NM _001134870.2, and an amino acid sequence is shown in NP _ 001128342.1.

The utility of the present invention is not limited to quantifying gene expression of any particular variant of the target gene of the present invention. Those skilled in the art will appreciate that when performing bioinformatic analysis, the sequenced sequence will generally be aligned with a known gene, and the expression of that gene will be considered as long as the sequence can be aligned with the gene of interest (e.g., ID: 170954 or ID: 26136), and therefore, when referring to a differentially expressed gene, different transcripts, mutants or fragments thereof of that gene are also encompassed by the present invention.

It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The present invention may utilize any method known in the art to determine the expression level of a gene.

TES or PPP1R18 of the present invention is detected using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and protein immunization technology.

The present invention can amplify the nucleic acid prior to or simultaneously with detection. Illustrative, non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.

Protein immunization techniques include sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

The term "differential expression" as used herein means the difference in the amount or level of mRNA determined, in one sample, in the expression level of RNA of one or more biomarkers of the invention and/or one or more splice variants of said biomarker mRNA compared to the expression level of the same one or more biomarkers of the invention in a second sample. "differentially expressed" may also include the determination of a protein encoded by a biomarker of the invention in a sample or sample population as compared to the amount or level of protein expression in a second sample or sample population. Differential expression can be determined as described herein and as understood by one of skill in the art. The term "differential expression" or "change in expression level" means that an increase or decrease in the expression level of a given biomarker in a sample can be determined by determining the amount of RNA and/or the amount of protein as compared to the determinable expression level of the given biomarker in a second sample. The term "differential expression" or "change in expression level" may also mean an increase or decrease in the measurable expression level of a given biomarker in a sample population as compared to the measurable expression level of the biomarker in a second sample population. As used herein, "differential expression" can be determined as the ratio of the expression level of a given biomarker relative to the average expression level of the given biomarker in a control, wherein the ratio is not equal to 1.0. Differential expression can also be measured using p-values. When using a p-value, biomarkers are identified as differentially expressed between the first and second populations when the p-value is less than 0.1. More preferably, the p-value is less than 0.05. Even more preferably, the p-value is less than 0.01. Still more preferably, the p-value is less than 0.005. Most preferably, the p value is less than 0.001. When differential expression is determined based on the ratio, the RNA or protein is differentially expressed if the ratio of the expression levels in the first and second samples is greater than or less than 1.0. For example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, such as 0.8, 0.6, 0.4, 0.2, 0.1, 0.05. In another embodiment of the invention, the nucleic acid transcript is differentially expressed if the ratio of the average expression level of the first population to the average expression level of the second population is greater than or less than 1.0. For example, a ratio greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, such as 0.8, 0.6, 0.4, 0.2, 0.1, 0.05. In another embodiment of the invention, a nucleic acid transcript is differentially expressed if the ratio of the expression level in the first sample to the average expression level in the second population is greater than or less than 1.0, for example including ratios greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or ratios less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1, 0.05.

By "differential expression increase" or "upregulation" is meant that gene expression (as measured by RNA expression or protein expression) exhibits an increase of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold or more, of the gene relative to a control.

By "differential expression reduction" or "down-regulation" is meant a gene whose expression (as measured by RNA expression or protein expression) exhibits a reduction in gene expression relative to a control of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or less than 1.0-fold, 0.8-fold, 0.6-fold, 0.4-fold, 0.2-fold, 0.1-fold or less. For example, an up-regulated gene includes a gene that has an increased level of expression of mRNA or protein in blood isolated from an individual characterized as having Alzheimer's disease, as compared to the expression of mRNA or protein isolated from a normal individual. For example, a down-regulated gene includes a gene that has a reduced level of mRNA or protein expression in blood isolated from an individual characterized as having alzheimer's disease as compared to blood isolated from a normal individual.

Nucleic acid membrane strip, chip and kit

In the present invention, the nucleic acid membrane strip comprises a substrate and an oligonucleotide probe specifically recognizing TES or PPP1R18 immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass slide, a silica gel wafer, a micro magnetic bead, or the like.

The chip of the invention comprises a gene chip and a protein chip; the gene chip comprises a solid phase carrier; and oligonucleotide probes immobilized on the solid carrier in order, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown in TES or PPP1R 18. The protein chip comprises a solid phase carrier and a specific binding agent of TES or PPP1R18 encoded protein fixed on the solid phase carrier.

Specific binders are for example receptors for protein TES or PPP1R18, lectins binding protein TES or PPP1R18, antibodies against protein TES or PPP1R18, peptide antibodies (peptidebody) against protein TES or PPP1R18, bispecific dual binders or bispecific antibody formats.

Examples of specific binding agents are peptides, peptidomimetics, aptamers, spiegelmers, dappin, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies and monovalent antibody fragments. In a particular embodiment of the invention, the specific binding agent is a TES or PPP1R18 specific antibody.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

The invention provides a kit, which can be used for detecting the expression level of TES or PPP1R18 gene or protein, and comprises primers, oligonucleotide probes, ligands and/or chips for detecting and/or quantifying TES or PPP1R 18. Optionally together with instructions for the kit.

Kits include one or more sterile containers, which may be in the form of a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.

The product for diagnosing the Alzheimer disease can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to the Alzheimer disease) including TES or PPP1R18 genes, and can greatly improve the accuracy of the diagnosis of the Alzheimer disease by simultaneously detecting a plurality of markers of the Alzheimer disease.

The invention has the advantages and beneficial effects that:

the invention discovers that the expression level of the TES or PPP1R18 gene is up-regulated in an Alzheimer patient for the first time, and the detection of the expression level of the TES or PPP1R18 gene can assist in diagnosing whether the subject suffers from Alzheimer disease or the risk of suffering from Alzheimer disease, thereby guiding doctors to provide prevention or treatment schemes.

Drawings

FIG. 1 is a graph showing the detection of TES expression levels in Alzheimer's patients by QPCR, wherein Panel A is a graph showing TES expression levels and Panel B is a graph showing PPP1R18 expression levels.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 screening of Gene markers associated with Alzheimer's disease

1. Sample collection

4 samples of healthy human blood and blood of Alzheimer's patients are collected, mixed with anticoagulant, and subjected to high-throughput sequencing.

2. Preparation and Mass analysis of RNA samples

RNA was extracted using a blood extraction kit from Takara, the procedure was as follows:

1) transferring 0.25ml of Blood sample into a centrifuge tube, adding 0.75ml of RNAlso Blood, and repeatedly sucking up and down by using a pipette gun until the cells are completely lysed;

2) adding chloroform (1/5 volume amount of sample solution + RNAlso Blood volume amount), covering the centrifugal tube cover tightly, mixing until the solution is milky white, and standing at room temperature for 5 min;

3) centrifuging at 4 deg.C for 15min at 12,000 Xg, sucking supernatant, and transferring to new centrifuge tube;

4) adding isopropanol with equal volume into the supernatant, turning the centrifuge tube upside down, mixing, and standing at room temperature for 10 min;

5) centrifuging at 4 deg.C for 10min at 12,000 Xg;

6) discarding the supernatant, adding 75% ethanol with the same amount, washing the precipitate by Vortex oscillation, centrifuging at 7,500 Xg and 4 ℃ for 5min, and discarding the supernatant;

7) drying the precipitate at room temperature, adding a proper amount of RNase-free water to dissolve the precipitate after drying the precipitate;

8) and detecting the concentration of the RNA, and identifying the yield and purity of the RNA.

3. construction and sequencing of cDNA libraries

The construction and sequencing of the cDNA library are completed by the Huada gene, and the steps are as follows:

1) total RNA DNase I digestion: digesting DNA fragments existing in a Total RNA sample by using DNase I, purifying and recovering reaction products by using magnetic beads, and finally dissolving the reaction products in DEPC water;

2) removing rRNA: taking a digested Total RNA sample, removing rRNA by using a Ribo-Zero kit of Epicentre, detecting Agilent 2100 after removing the rRNA, and verifying the rRNA removing effect;

3) RNA disruption: taking the sample in the previous step, adding an interrupt Buffer, and placing the sample in a PCR instrument for thermal interrupt until the sample is interrupted to 140-160 nt;

4) reverse transcription one-strand synthesis: adding a proper amount of primers into the broken sample, fully and uniformly mixing, reacting for a certain time at a proper temperature of a Thermomixer to open a secondary structure and combine with the primers, adding a one-chain synthesis reaction system Mix prepared in advance, and synthesizing one-chain cDNA on a PCR instrument according to a corresponding procedure;

5) synthesis of reverse transcription two-chain: preparing a double-chain synthesis reaction system, reacting on a Thermomixer at a proper temperature for a certain time to synthesize double-chain cDNA with dUTP, and purifying and recovering reaction products by using magnetic beads;

6) and (3) repairing the tail end: preparing a tail end repairing reaction system, reacting in a Thermomixer at a proper temperature for a certain time, repairing the viscous tail end of a cDNA double-chain obtained by reverse transcription under the action of enzyme, purifying and recovering a tail end repairing product by using magnetic beads, and finally dissolving a sample in EB Solution;

7) 3' end of cDNA plus "A": preparing an A reaction system, reacting in a Thermomixer at a proper temperature for a certain time, and adding A basic groups to the 3' end of a product cDNA with repaired end under the action of enzyme;

8) ligation of cDNA 5' adapter: preparing a joint connection reaction system, reacting in a Thermomixer at a proper temperature for a certain time, connecting a joint with the A base under the action of enzyme, and purifying and recovering a product by using magnetic beads;

9) UNG digested cDNA double strand: preparing a UNG digestion reaction system, digesting two strands in double-stranded DNA by UNG enzyme, and purifying and recovering a product by using magnetic beads;

10) PCR reaction and product recovery: preparing a PCR reaction system, selecting a proper PCR reaction program, amplifying the product obtained in the previous step, carrying out magnetic bead purification and recovery on the PCR product, dissolving the recovered product in EB solution, and labeling;

11) and (3) detecting the quality of the library: the library quality was checked using Agilent 2100 Bioanalyzer and ABI StepOneplus Real-Time PCR System;

12) and (3) machine sequencing: and (4) detecting a qualified library, adding NaOH to denature the library into a single chain, and diluting the single chain to a certain computer-loading concentration according to the expected computer-loading data quantity. Adding the library after denaturation and dilution into the FlowCell, hybridizing with a linker on the FlowCell, completing bridge PCR amplification on a cBot, and finally sequencing by using an Illumina Hiseq x-ten platform.

4. Bioinformatics analysis

RNA-seq read mapping was performed using TopHat v1.3.1, RNA-seq fragment numbers were normalized by Cufflinks v1.0.3 to calculate relative abundance of transcripts, differential expression was detected using cuffdiff, p values were pooled using invert normal method in meta assay, genes were considered significantly differentially expressed when FDR value < 0.01.

5. Results

Sequencing results show that compared with healthy people, the expression level of TES and PPP1R18 genes in Alzheimer patients is remarkably increased.

Example 2QPCR sequencing verification of differential expression of genes

1. Blood samples from 31 alzheimer patients and 24 healthy persons were collected for QPCR validation in the manner as described in example 1.

2. RNA extraction

RNA was extracted from blood using Takara RNA extraction kit, see example 1 for specific procedures.

3、QPCR

1) Design of primers

Primers were designed based on the gene sequences of TES, PPP1R18 and GADPH, and the primer sequences are shown below.

TES Gene:

SEQ ID NO.1 (forward primer): 5'-TTGAGCAATGAAGAGGAT-3'

SEQ ID NO.2 (reverse primer): 5'-GTAACTGTATTGATGGAGAC-3', respectively;

PPP1R18 gene:

SEQ ID NO.3 (forward primer): 5'-CAGGAACAGAGTTTGGTA-3'

SEQ ID No.4 (reverse primer): 5'-TCCGAGTAGTCTTGTCTT-3', respectively;

GAPDH gene:

SEQ ID No.5 (forward primer): 5'-AATCCCATCACCATCTTCCAG-3'

SEQ ID NO.6 (reverse primer): 5'-GAGCCCCAGCCTTCTCCAT-3'

2) Real-time quantitative PCR

TaKaRa One Step TB Green ^TM Prime Script ^TM The RT-PCR kit (Code No. RR066A) was used for PCR reaction, and the reaction system and reaction conditions are shown in Table 1.

TABLE 1 QPCR reaction System and reaction conditions

In the Thermal Cycler

PCR amplification is carried out on the Time System amplification instrument, and Real Time PCR is confirmed after the reaction is finishedThe amplification curve and the dissolution curve of (2), and the relative quantification by the Δ Δ CT method.

4. ROC analysis

ROC analysis was performed on the variables TES and PPP1R18 using SPSS to determine the diagnostic potency, sensitivity and specificity of the gene.

5. Results

The QPCR result is shown in figure 1, compared with a healthy person, TES and PPP1R18 are up-regulated in the Alzheimer patient, TES is up-regulated by about 4.5 times, PPP1R18 is up-regulated by about 5.3 times, and the difference has statistical significance (P <0.05), so that the fact that the TES or PPP1R18 gene can be used as a gene marker for early diagnosis of Alzheimer is suggested, and early discovery and early intervention are achieved. Meanwhile, by the relationship between TES and PPP1R18 and Alzheimer, siRNA, shRNA or other agents targeting TES and PPP1R18 can be designed to treat Alzheimer.

The ROC analysis results are shown in table 2, and AUC values of TES and PPP1R18 as detection variables are 0.919 and 0.902, respectively, which have higher area under the curve, indicating that TES and PPP1R18 as detection variables have higher efficacy for alzheimer diagnosis.

TABLE 2 ROC analysis results

Example 3 functional verification of Gene markers

1. Cell culture

PC12 cells were cultured in DMEM medium containing 10% fetal calf serum and 1% penicillin/streptomycin at 37 ℃ under 5% CO ₂ Culturing in an incubator with the relative humidity of 90 percent, carrying out passage at the ratio of 1:3, changing the culture solution after 24 hours when the cells enter a logarithmic growth phase, and carrying out different interventions according to experimental requirements.

2. Transfection

2.1 treatment of cells before transfection

One day before transfection, 5X 10 wells of 6-well culture plates were plated ⁵ One cell/well in antibiotic-free medium at a cell density of 60% at transfection for one daySerum-free medium was changed before transfection.

2.2 Gene overexpression vectors and design and Synthesis of interfering RNAs

Interfering RNA for TES and PPP1R18 was designed and synthesized by Shanghai Ji code pharmaceutical technology Limited: siRNA-TES and siRNA-PPP1R18, control was general siRNA-NC. The sequences of siRNA-TES and siRNA-PPP1R18 are as follows:

siRNA-TES：

SEQ ID NO.7：5’-UUCUUUCAUACUCAGUUUGCA-3’

SEQ ID NO.8：5’-CAAACUGAGUAUGAAAGAAGG-3’；

siRNA-PPP1R18：

SEQ ID NO.9：5’-AGUUUGAGCCUCUAUGUCCCU-3’

SEQ ID NO.10：5’-GGACAUAGAGGCUCAAACUCA-3’；

the experiment was divided into three groups: blank control, negative control: transfecting siRNA-NC; experimental groups: siRNA-TES and siRNA-PPP1R18 were transfected. Transfection was carried out using lipofectamine 2000 from Invitrogen, the detailed procedure was performed according to the instructions.

2.3 detection of transfection Effect

The expression levels of TES and PPP1R18 in the cells were measured using QPCR and real-time quantitative PCR was performed as in example 2.

3. CCK-8 tests for the Effect of TES and PPP1R18 on AD cells

Cells were collected in log phase and adjusted to 5X 10 cell concentration ⁴ Perml, 100. mu.L of the cell suspension was inoculated into a 96-well plate and placed at 37 ℃ in 5% CO ₂ Incubate overnight, and set five replicate wells per group. The experiments were divided into three groups, blank control group, model group, experimental group. Control group: the cells were not subjected to any treatment; negative control group: transfection of siRNA-NC but no drug treatment; model group: cells were subjected to A.beta.only _1-42 Processing; experimental groups: cells were transfected with siRNA-TES or siRNA-PPP1R18 and A.beta. _1-42 And (6) processing. After 48h of drug treatment, 10 mu LCCK-8 solution is added into each well, incubated at 37 ℃ for 4h, and the absorbance value (OD value) of each well is detected under the wavelength of 450nm of a microplate reader

4. Statistical analysis

All data were processed using graphpad software, data are expressed as mean ± standard deviation (mean ± SD), differences between groups were analyzed using paired sample t-test, and p <0.05 indicates statistical differences.

5. Results

The expression level of TES and PPP1R18 in the blank control group is taken as a reference and is set as 1, and the transfection results show that the siRNA in the experimental group has better silencing effect on TES or PPP1R18 as shown in Table 3.

TABLE 3 relative expression amounts of TES and PPP1R18

Note: p value is the negative control (experimental group) vs blank control group

The CCK-8 detection result is shown in Table 4, the OD value of the experimental group (siRNA-TES, siRNA-PPP1R18) is obviously increased relative to the model group, and the TES and PPP1R18 have a protective effect on nerve cells.

TABLE 4 OD values

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tccgagtagt cttgtctt 18

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatcccatca ccatcttcca g 21

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gagccccagc cttctccat 19

<210> 7

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

uucuuucaua cucaguuugc a 21

<210> 8

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caaacugagu augaaagaag g 21

<210> 9

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aguuugagcc ucuauguccc u 21

<210> 10

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggacauagag gcucaaacuc a 21

Claims

1. Application of a reagent for detecting the RNA expression level of a biomarker TES gene in preparation of a product for diagnosing Alzheimer's disease.

2. The use according to claim 1, wherein the agent comprises: oligonucleotide probes that specifically recognize the TES gene; or primers that specifically amplify the TES gene.

3. The use of claim 2, wherein the primer sequence for specifically amplifying the TES gene is shown in SEQ ID NO. 1-2.

4. The use of claim 1, wherein the product comprises a nucleic acid membrane strip, chip or kit.

5. The use of claim 4, wherein the chip comprises a gene chip, wherein the gene chip comprises oligonucleotide probes for the TES gene.

6. The use of claim 4, wherein the kit comprises a gene detection kit comprising primers, oligonucleotide probes or chips specific for the TES gene.

7. The use of claim 6, wherein the primer sequence specific for TES gene is shown in SEQ ID NO. 1-2.

Use of an inhibitor of TES, wherein the inhibitor is siRNA, for the preparation of a pharmaceutical composition for the treatment of alzheimer's disease.

9. The use of claim 8, wherein the inhibitor reduces the expression of TES.

10. The use of claim 8, wherein the siRNA has a sequence as shown in SEQ ID No. 7-8.

11. The use of any one of claims 8-10, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.