CN109234387B

CN109234387B - Application of C14orf129 as molecular target in Alzheimer's disease diagnosis and treatment

Info

Publication number: CN109234387B
Application number: CN201811429104.9A
Authority: CN
Inventors: 肖枫; 黄露宁; 常鹏
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2021-08-13
Anticipated expiration: 2038-11-27
Also published as: CN109234387A

Abstract

The invention discloses application of C14orf129 as a molecular target in Alzheimer's disease diagnosis and treatment. The QPCR experiment proves that the expression of C14orf129 in the blood of the Alzheimer's disease patient is reduced, and the fact that C14orf129 can be used as a detection index to be applied to early diagnosis of the Alzheimer's disease is suggested. Cell experiments prove that the survival rate of the Alzheimer's disease mode cells can be influenced by changing the expression level of C14orf129, and the fact that C14orf129 can be used as a drug target for treating Alzheimer's disease is suggested.

Description

Application of C14orf129 as molecular target in Alzheimer's disease diagnosis and treatment

Technical Field

The invention relates to the technical field of biology, and relates to application of C14orf129 as a molecular target in Alzheimer's disease diagnosis and treatment.

Background

Alzheimer's Disease (AD) is a degenerative disease of the nervous system, mainly manifested by progressive memory impairment, cognitive decline, behavioral changes and language disorders, the most common type of senile dementia (Matsumoto Y, Niimi N, Kohyama K. development of a new DNA vaccine for Alzheimer's disease targeting a wide range of ap specific and amyloid peptides [ J ]. PLoS One,2013,8(9): e 75203.). The disease is unconscious in onset and slow in development, the cognitive function of a patient is continuously reduced, the disease course is generally 5-10 years, and researches show that the physiological change of the neuron disease exists more than 10 years before clinical symptoms appear in the patient. It is currently believed that the disease is caused by abnormal deposition of beta-amyloid (beta-amyloid, Abeta) and hyperphosphorylation of tau protein, which ultimately leads to pathological changes such as Senile Plaque (SP), formation of Neurofibrillary tangles (NFT), and vacuolar degeneration and neuronal loss of hippocampal pyramidal cell granules (Bloom GS. amyloid-p and tau: the trigger and bellet in Alzheimer disease pathogenesis [ J ]. MA Neurol,2014,71(4): 505-508.).

With the increasing aging progress of the population in China, the incidence rate of AD is on the trend of rising year by year. The disease can cause the old people to lose the ability of work, social intercourse and life self-care, which not only seriously harms the mental health of the old people, but also brings heavy economic burden to the family members and the society of the patients. The incidence of 10% in elderly over the age of 65 years and 50% over the age of 85 years (Gonsalves D, Jovanovic K, Da Costa Dias B, et al. Global Alzheimer Research Summit: basic and clinical Research [ J ]. Prion,2012,6(l): 7-10.). It is estimated that 2400 million AD patients are currently worldwide and increase by 460 million each year, with the number expected to break through 8100 million in 2040. At present, no effective treatment measures for AD can reverse the pathological process, and only medicaments can be used for slowing down the disease process and carrying out symptomatic treatment aiming at corresponding symptoms, so that the improvement of the early diagnosis level of AD and effective intervention and treatment in the early stage of the disease are urgently needed.

With the development of biotechnology, genes play an important role in the occurrence and development process of alzheimer, and become a hotspot for researching the pathogenesis of alzheimer and realizing personalized clinical diagnosis and treatment. At present, few biomarker species for diagnosing AD are available, the sensitivity and specificity are not ideal, and only a few biomarker species are used as auxiliary diagnostic methods for clinical application. Therefore, the search for a new biomarker applied to the clinical detection of Alzheimer's disease has important significance in improving the sensitivity and specificity of diagnosis.

Disclosure of Invention

In order to make up for the deficiencies of the prior art, the invention aims to provide a molecular target for clinical diagnosis and treatment of Alzheimer's disease.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a reagent for detecting the C14orf129 level in preparing a product for diagnosing Alzheimer disease.

Further, the product comprises: a chip, a preparation, a kit or a nucleic acid membrane strip. Wherein, the chip comprises a gene chip and a protein chip; the kit comprises a gene detection kit and a protein immunodetection kit.

The invention also provides a product for diagnosing Alzheimer's disease, which comprises a reagent for detecting the level of C14orf 129. Such products include, but are not limited to, chips, formulations, kits, or nucleic acid membrane strips.

Further, the reagent comprises:

a probe that specifically recognizes the C14orf129 gene; or

A primer for specifically amplifying the C14orf129 gene; or

An antibody or ligand that specifically binds to a protein encoded by C14orf 129.

As a preferred embodiment, the primer sequence for specifically amplifying the C14orf129 gene is shown as SEQ ID NO. 1-2.

The invention provides application of C14orf129 in screening potential drugs for treating Alzheimer disease.

The invention provides a method for screening potential drugs for treating Alzheimer disease, which comprises the following steps:

treating a culture system expressing or containing the C14orf129 gene or a protein encoded thereby with a substance to be screened; and

detecting the expression or activity of the C14orf129 gene or protein encoded thereby in the system;

wherein, if the substance to be screened can increase the expression or activity of C14orf129, the substance to be screened is a potential drug for treating Alzheimer disease.

Such culture systems include, but are not limited to, cell systems, subcellular systems, solution systems, tissue systems, organ systems, or animal systems (e.g., animal models, preferably non-human mammalian animal models, such as mice, rabbits, sheep, monkeys, etc.), and the like.

The invention provides application of a promoter of a C14orf129 gene and/or an expression product thereof in preparing a medicament for treating Alzheimer disease.

Further, the promoter includes a substance that improves the stability of the C14orf129 gene or an expression product thereof, up-regulates the expression level of the C14orf129 gene or an expression product thereof, and increases the effective acting time of the C14orf129 gene or an expression product thereof.

Further, the promoter of C14orf129 is an expression vector comprising C14orf 129.

The invention provides a pharmaceutical composition for treating alzheimer's disease, comprising an enhancer against C14orf 129.

The invention has the advantages and beneficial effects that:

the invention discovers that the expression of the C14orf129 gene is related to the Alzheimer disease for the first time, and the expression of the C14orf129 in blood of a subject can be detected to judge whether the subject suffers from the Alzheimer disease or not or judge whether the subject is at risk of suffering from the Alzheimer disease or not, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject.

The invention firstly discovers that the expression level of C14orf129 can be changed, the proliferation of nerve cells can be changed, and the C14orf129 can be used as a potential molecular target to be applied to the treatment of Alzheimer disease.

Drawings

FIG. 1 is a graph showing the detection of the expression of the C14orf129 gene in the blood of patients with Alzheimer's disease by QPCR;

FIG. 2 is a graph showing the detection of C14orf129 gene overexpression using QPCR;

FIG. 3 is a graph showing the effect of C14orf129 gene expression on the growth of Alzheimer's nerve cells using MTT.

Detailed Description

According to the invention, through extensive and intensive research, the gene expression levels in the blood of an Alzheimer patient and normal human are detected by a high-throughput sequencing method, genes with obvious differences are found, and the relationship between the genes and the occurrence of Alzheimer is discussed, so that a better way and a better method are found for early detection and targeted treatment of Alzheimer. Through screening, the invention discovers the remarkable down-regulation of C14orf129 in Alzheimer's disease for the first time. Experiments prove that the expression level of C14orf129 is promoted, the proliferation of nerve cells can be effectively promoted, and a new way is provided for the personalized treatment of Alzheimer's disease.

C14orf129 gene

The C14orf129 gene is located on region 2 of chromosome 14 long arm 3, and the gene ID currently in GeneBank, the International public nucleic acid database, is 51527. In the present invention, C14orf129 includes wild type, mutant type or fragment thereof. As a non-limiting example, a representative C14orf129 gene has the sequence shown in NM _ 001271904.1.

The present invention may utilize any method known in the art for determining gene expression. 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 expression level of the biomarker can be detected at the transcriptional level.

The genes of the invention can be detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technology, and protein immunodetection technology.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

The present invention can amplify nucleic acids (e.g., ncRNA) 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).

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using RNA replicase (commonly known as Q.beta.replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Protein immunological techniques include, but are not limited to, 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.

Any direct (e.g., using a sensor chip) or indirect method may be used in the detection of the biomarkers of the invention.

Chip, preparation, nucleic acid membrane strip and kit

The present invention provides products, including but not limited to chip chips, formulations, nucleic acid membrane strips or kits, for detecting the expression level of the C14orf129 gene. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown in C14orf 129.

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 term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, and may be polynucleotides obtained by replacing nucleotides in a part or all of them with artificial Nucleic acids such as PNA (Polyamide Nucleic Acid), LNA (registered trademark, locked Nucleic Acid, bridge Nucleic Acid, crosslinked Nucleic Acid), ENA (registered trademark, 2 '-O, 4' -C-Ethylene-Bridged Nucleic acids), GNA (Glycerol Nucleic Acid), and TNA (Threose Nucleic Acid).

In the present invention, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies. Antibodies specific for the C14orf129 protein include intact antibody molecules, any fragment or modification of an antibody (e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc., so long as the fragment retains the ability to bind to the C14orf129 protein.

In the present invention, a nucleic acid membrane strip comprises a substrate and oligonucleotide probes 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 plate, a silica gel wafer, a micro magnetic bead, or the like.

The present invention provides a kit that can be used to detect the expression of C14orf 129. The kit comprises a specific primer pair for amplifying C14orf 129; a standard DNA template; and (3) PCR reaction liquid.

In a preferred embodiment, the specific primer pair comprises an upstream primer and a downstream primer, and the sequences are shown as SEQ ID NO. 1-2.

As a more preferable embodiment, the kit is a fluorescent quantitative PCR detection kit, and the primer is suitable for detection of SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes and composite probes.

In a more preferred embodiment, the PCR reaction solution in the kit is a fluorescent quantitative PCR reaction solution, and further comprises a fluorescent dye.

In a more preferred embodiment, the fluorescent quantitative PCR reaction solution comprises dNTP and Mg²⁺Taq enzyme and buffer solution, wherein the fluorescent dye is SYBR Green II, and the Taq enzyme is hot startAn enzyme.

Accelerator and pharmaceutical composition

Based on the inventors' findings, the present invention provides a promoter for C14orf129, which comprises a substance that improves stability of the C14orf129 gene or an expression product thereof, up-regulates an expression level of the C14orf129 gene or an expression product thereof, and increases an effective acting time of the C14orf129 gene or an expression product thereof.

Typically, these enhancers will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of about 5 to about 8, preferably a pH of about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

In a preferred embodiment of the present invention, the promoter of C14orf129 is an expression vector of C14orf 129. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.

In the present invention, there are various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like. The expression vector can be introduced into the host cell by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.

In the present invention, a "host cell" cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, or 293 cell.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The invention also provides a pharmaceutical composition, which contains an effective amount of the C14orf129 promoter and a pharmaceutically acceptable carrier. The composition can be used for treating Alzheimer disease. Any of the aforementioned C14orf129 accelerators may be used in the preparation of the composition. The pharmaceutically acceptable carrier and/or adjuvant includes (but is not limited to) diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, and disintegrant.

Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.

Statistical analysis

In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data of the results are expressed as mean ± standard deviation, and the statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is considered to have statistical significance by using t test when P is less than 0.05.

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. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

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

1. Sample collection

Samples of 45 normal blood and Alzheimer's disease patients were collected, all known to have informed consent, and were obtained with the consent of the ethical Committee. Samples of 5 normal human blood and blood from alzheimer patients were taken for high throughput sequencing and all samples were used for validation.

2. Preparation of RNA samples

The RNA sample was extracted using the blood RNA extraction kit from Invitrogen, and the detailed operation is described in the specification.

3. Mass analysis of RNA samples

The concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The concentration is more than or equal to 200 ng/mul, and the OD260/280 is between 1.8 and 2.2.

4. Removal of rRNA

Ribosomal RNA was removed from total RNA using Ribo-Zero kit.

5. Construction of cDNA library

The construction of cDNA library was carried out using Illumina Truseq RNA sample Prep Kit, the specific procedures were as described in the specification.

6. Sequencing on machine

And (3) sequencing the cDNA library by using an Illumina X-Ten sequencing platform, wherein the specific operation is carried out according to the instruction.

7. High throughput transcriptome sequencing data analysis

And (3) performing bioinformatics analysis on a sequencing result, performing RNA-seq reading positioning by using TopHat v1.3.1, standardizing the number of RNA-seq fragments by using Cufflinks v1.0.3 to calculate the relative abundance of the transcript, detecting differential expression by using cuffdiff, and considering that the gene is significantly differentially expressed when the p value is less than 0.05.

8. Results

The RNA-seq result shows that the expression level of the C14orf129 gene in the blood of the Alzheimer disease patient is obviously lower than that of the normal human blood, and the C14orf129 is possibly used as a biomarker of the Alzheimer disease.

Example 2 QPCR sequencing verification of differential expression of the C14orf129 Gene

1. And selecting the C14orf129 gene according to the detection result of high-throughput sequencing for carrying out QPCR verification on the large sample.

2. The RNA extraction procedure was as in example 1.

3. Reverse transcription:

2. mu.g of total RNA was subjected to reverse transcription, and 2. mu.l of oligo (dT) was added thereto and mixed well. Water bath is carried out for 5 minutes at 70 ℃, and ice bath is carried out for 2-3min immediately; mu.l of 5 XTRT, 5. mu.l of dNTP (2.5mM), 40U/. mu.l of RNase, 200U/. mu.l of M-MLV, and the ribozyme-free water were added thereto, and after 60 minutes of water bath at 42 ℃, 5 minutes of water bath at 95 ℃ were carried out to inactivate M-MLV.

4. QPCR amplification

(1) Primer design

QPCR amplification primers were designed based on the coding sequences of C14orf129 gene and GAPDH gene in Genbank and synthesized by Shanghai Biotechnology engineering services, Inc. The specific primer sequences are as follows:

c14orf129 gene:

the forward primer is 5'-AGTTGTAAATGATGTTCTCT-3' (SEQ ID NO. 1);

the reverse primer was 5'-GTTCTAGGCAATATCTGTT-3' (SEQ ID NO. 2).

GAPDH gene:

the forward primer is 5'-AACTCTGGTAAAGTGGATATTG-3' (SEQ ID NO. 3);

the reverse primer was 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 4).

(2) Preparing a PCR reaction system:

each of the forward primer and the reverse primer is 1 μ l, 12.5 μ l of SYBR Green polymerase chain reaction system and 2 μ l of template are added with deionized water to make up to 25 μ l.

(3) And (3) PCR reaction conditions:

95 ℃ 10min, (95 ℃ 15s, 60 ℃ 60s) 45 cycles. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.

5. Statistical method

The experiment was repeated 3 times, the data were expressed as mean ± sd, and the statistical analysis was performed using SPSS13.0 statistical software, and the difference between the two was considered statistically significant when P <0.05 using the t-test.

6. Results

The results are shown in fig. 1, compared with the normal human blood, the expression of the C14orf129 gene in the blood of the Alzheimer disease patients is significantly reduced, and the difference has statistical significance (P <0.05), which is consistent with the RNA-sep results.

Example 3 overexpression of the C14orf129 Gene

1. Cell culture

Dopamine neuron cell SH-SY5Y, in DMEM culture solution (pH7.2-7.4) containing 10% fetal calf serum and 1% penicillin/streptomycin, at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. Changing the culture solution once every 2 days, carrying out passage when the cells grow to 90% contact, washing with PBS, adding 0.25% -EDTA trypsin for digestion to separate the cells from the bottle wall, stopping pancreatin digestion reaction with DMEM culture solution containing fetal calf serum, centrifuging for 2min at 1000g, discarding supernatant, re-suspending with newly configured culture solution, carrying out passage at a ratio of 1: 3-1: 4, changing the culture solution when the cells enter a logarithmic phase after 24 hours, and carrying out different interventions according to experimental requirements.

2. Transfection

1) Treatment of cells prior to transfection

One day before transfection, 6-well culture plates are seeded with 3-5 multiplied by 10⁵And (3) culturing each cell/hole in an antibiotic-free culture medium for one day, wherein the cell density is 30-50% during transfection, and the cell/hole is replaced by a serum-free culture medium before transfection.

2) Construction of Gene overexpression vectors

Specific PCR amplification primers were synthesized based on the sequence of C14orf129 in GeneBank, and the primer sequences were as follows:

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

Reverse primer: 5'-CGGCTCGAGTGACTGTCCATCTCTTTTCAAAGC-3' (SEQ ID NO.6)

Two restriction sites KpnI and XhoI are added to the 5 'end primer and the 3' end primer respectively. cDNA extracted and reverse transcribed from Alzheimer patients is used as an amplification template, the cDNA sequence is inserted into eukaryotic cell expression vector pcDNA3.1 which is subjected to double enzyme digestion by restriction enzymes KpnI and XhoI, and the obtained recombinant vector pcDNA3.1-1 is connected for subsequent experiments.

3) Transfection

The nerve cells were divided into 3 groups, namely a control group (SH-SY5Y), a blank control group (transfected pcDNA3.1-NC) and an experimental group (transfected pcDNA3.1-1). Transfection of the vector was performed using liposome 3000, and the specific transfection method was performed as indicated in the specification.

3. QPCR detection of transcription level of C14orf129 Gene

3.1 extraction of Total RNA from cells

Total RNA of SH-SY5Y cells was extracted using TRIzol Reagent (Invitrogen Cat. No.15596-018) according to the method provided in the specification.

3.2 reverse transcription procedure as in example 2.

3.3QPCR amplification step as in example 2.

4. Statistical method

The experiment was repeated 3 times, the data were expressed as mean ± sd, and the statistical analysis was performed using SPSS18.0 statistical software, and the difference between the C14orf129 gene overexpression group and the control group was considered statistically significant when P <0.05 using t-test.

5. Results

The results are shown in fig. 2, in which the expression level of C14orf129 gene was significantly increased in the experimental group compared to the control group, while there was no significant difference between the transfection-unloaded group and the blank control group.

Example 4 Effect of C14orf129 Gene on nerve cells

MTT experiment is adopted to detect the influence of C14orf129 gene on the cell survival rate of SH-SY5Y Alzheimer disease cell model.

1. The cell culture transfection procedure was as in example 3.

2. Grouping cells:

SH-SY5Y group: in the blank control group, after SH-SY5Y cells are normally cultured for 48h, PBS with a control amount is added, and detection and analysis are carried out after incubation for 24 h;

Aβ_1-42group (2): after SH-SY5Y cells are normally cultured for 48h, 5 mu M A beta is added_1-42(GL Biochem), incubation for 24h and detection analysis;

pcDNA3.1-1 group: SH-SY5After the Y cell is transfected into pcDNA3.1-148 h, the concentration of 5 mu M A beta is added_1-42(GL Biochem), incubation for 24h and detection assay

3. MTT assay

Dissolving MTT with PBS to a final concentration of 5mg/ml, discarding the solution in the wells, adding 100. mu.l of culture medium, adding 10. mu.l of MTT with 5mg/ml in each well, continuing to culture at 37 ℃ for 4h, discarding the solution in the wells, adding 100. mu.l of DMSO in each well, and incubating for 10min in a shaker at room temperature. The absorbance value (OD value) of the microplate reader is measured at 490nm, and the percentage of the absorbance value of the microplate reader to that of the control group is the percentage of cell activity. Calculated according to the following formula:

percent cell activity%

4. Statistical method

The experiment was performed in 3 replicates using SPSS18.0 statistical software for statistical analysis, and the difference between the two was considered statistically significant when P <0.05 using the t-test.

5. Results

The results shown in fig. 3 show that: cell survival Rate comparison of pcDNA3.1-1 group to Abeta_1-42Group, with significant increase, the difference was statistically significant (P)<0.05). The above results indicate that the low expression of C14orf129 is not favorable for the growth of SH-SY5Y cells, and nerve cells can be protected by promoting the expression of C14orf129 gene.

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.

Sequence listing

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Application of <120> C14orf129 as molecular target in Alzheimer's disease treatment

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Claims

1. Application of a reagent for detecting the transcription level of C14orf129 in preparing a product for diagnosing Alzheimer's disease.

2. Use according to claim 1, characterized in that the product comprises: a chip, a kit or a nucleic acid membrane strip.

3. The use according to claim 1, wherein the agent comprises:

a probe that specifically recognizes the C14orf129 gene; or

A primer for specifically amplifying the C14orf129 gene.

4. The use according to claim 3, wherein the primer sequence for specifically amplifying the C14orf129 gene is shown in SEQ ID No. 1-2.