Application of RNF14 in diagnosis and treatment of cerebral infarction

Abstract

The invention discloses an application of RNF14 in diagnosis and treatment of cerebral infarction, provides an application of a reagent for detecting the expression level of a biomarker in preparation of a product for diagnosing cerebral infarction, and provides an application of the biomarker or an accelerant thereof in preparation of a pharmaceutical composition for treating and / or preventing cerebral infarction. The invention also provides a method for screening candidate drugs for treating cerebral infarction.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of RNF14 in diagnosing and treating cerebral infarction. Background Art

[0002] Cerebral infarction, also known as ischemic stroke, is one of the most common cerebrovascular diseases. Data surveys show that the incidence, disability and mortality of cerebral infarction are very high, which has a serious impact on the quality of life of patients. Cerebral infarction is a cerebrovascular disease caused by cerebral blood supply disorder, with lacunar obstruction being the most common. The main characteristics of cerebral infarction are acute onset, often accompanied by a history of hypertension or emotional stimulation before the onset, dizziness, unsteady walking, limb weakness, severe impaired consciousness, sensory and motor dysfunction, neurodegenerative diseases, and even morbidity. When a medium-sized infarction occurs, it often manifests as sudden, fixed-position severe throbbing pain, often accompanied by frequent vomiting and nausea, and some are also accompanied by aphasia. It is reported that due to the high incidence and mortality of cerebral infarction, it has become the main cause of disability and death worldwide. According to statistics from the World Health Organization in 2015, on average one person dies from cerebral infarction every 225 seconds worldwide. Even in the United States, where medical care is relatively advanced, deaths from cerebral infarction account for about 5.6% of the total number of deaths. Cerebral infarction not only seriously affects the quality of life of patients, but also brings tremendous burden and pressure to society and families.

[0003] Ring finger protein 14 (RNF14) is composed of 474 amino acids. Because it contains the androgen receptor binding domain, it is also called androgen receptor-associated 54 (ARA54). The protein structure of RNF14 is highly conserved. Previous studies have shown that RNF14 can affect the transcriptional function of mitochondria and is widely expressed in various tissues throughout the body. The TiGER database (Tissue-specific Gene Expression and Regulation, TiGER) also shows that it is widely expressed in human liver and other tissues. Studies have shown that it may be related to the occurrence and development of various human digestive tract tumors, such as esophageal cancer and colorectal cancer, but the relationship between RNF14 and cerebral infarction has not yet been reported. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides the use of the biomarker RNF14 in the diagnosis and treatment of cerebral infarction.

[0005] To achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides use of a reagent for detecting the expression level of a biomarker in the preparation of a product for diagnosing cerebral infarction, wherein the biomarker is RNF14.

[0006] The term "biomarker" refers to a gene or gene product that is a target for regulating one or more target phenotypes (e.g., a target phenotype in a myeloid cell). In this case, the term "biomarker" is synonymous with "target". However, in some embodiments, the term also encompasses measurable entities that have been determined to be targets that indicate target outputs such as one or more diagnostic, prognostic, and / or therapeutic outputs (e.g., for regulating inflammatory phenotypes, cancer states, etc.). In other embodiments, the term also encompasses compositions that regulate genes or gene products, including anti-gene product antibodies and antigen-binding fragments thereof. Therefore, biomarkers may include, but are not limited to, nucleic acids (e.g., genomic nucleic acids and / or transcribed nucleic acids), proteins, and antibodies (and antigen-binding fragments thereof). In a specific embodiment of the present invention, the biomarker is preferably RNF14.

[0007] In the present invention, the "level" of a biomarker refers to the absolute (quantitative) or relative (qualitative) value or amount of the biomarker, and is used interchangeably with the terms "amount" and "concentration".

[0008] In some embodiments, the expression level of the biomarkers of the present invention can be determined by a variety of techniques. In particular, the expression at the nucleic acid level can be determined by measuring the amount of RNA, mRNA or any other RNA species representing the biomarker in question using methods well known in the art. Non-limiting examples of suitable methods include digital PCR and real-time (RT) quantitative or semi-quantitative PCR. Primers suitable for these methods can be easily designed by technicians. In other embodiments, other suitable techniques for determining the expression level of the biomarkers of the present invention at the nucleic acid level include but are not limited to fluorescence activated cell sorting (FACS) and in situ hybridization.

[0009] In some embodiments, a variety of techniques can be used to determine the expression level of the biomarkers of the present invention at the protein level. Non-limiting examples of suitable methods include mass spectrometry-based quantitative proteomics techniques, such as isobaric tags (iTRAQ) and label-free analysis of relative and absolute quantitative reagents, and any other techniques of selected reaction monitoring (SRM) mass spectrometry and targeted proteomics. Moreover, the level or amount of protein markers can be determined by, for example, immunoassays, Western blotting, spectrophotometry, enzyme assays, ultraviolet assays, kinetic assays, electrochemical assays, colorimetric assays, turbidimetric assays, atomic absorption assays, flow cytometry, mass spectrometry flow cytometry, or any combination thereof.

[0010] Furthermore, the reagents include reagents used to detect biomarkers in samples through protein immunoassay technology, sequencing technology, nucleic acid hybridization technology, and nucleic acid amplification technology.

[0011] Protein immunoassays of the invention include sandwich immunoassays, such as sandwich ELISA, in which two antibodies that recognize different epitopes on a biomarker are used to detect the biomarker: radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), Western blotting, immunoprecipitation, and any particle-based immunoassay (such as using gold particles, silver particles or latex particles, magnetic particles or quantum dots). Immunoassays can be implemented, for example, in the form of microtiter plates or strips.

[0012] Illustrative non-limiting examples of nucleic acid sequencing methods of the present invention include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that, because RNA is less stable in cells and more susceptible to nuclease attack in experiments, RNA is usually reverse transcribed into DNA before sequencing.

[0013] Another exemplary non-limiting example of the nucleic acid sequencing method of the present invention includes next-generation sequencing (deep sequencing high-throughput sequencing). High-throughput sequencing technology is a sequencing-by-synthesis technology based on single-molecule clusters, based on a proprietary reversible termination chemical reaction principle. During sequencing, random fragments of genomic DNA are attached to an optically transparent glass surface. After extension and bridge amplification, these DNA fragments form hundreds of millions of clusters on the glass surface, each of which is a single-molecule cluster with thousands of copies of the same template. Then, four special deoxyribonucleotides with fluorescent groups are used to sequence the template DNA to be tested through a reversible sequencing-by-synthesis technology.

[0014] The nucleic acid hybridization method in the present invention includes but is not limited to in situ hybridization (ISH), microarray and Southern or Northern blot. In situ hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a part or slice of a tissue (in situ) or if the tissue is small enough, the whole tissue (whole tissue embedding ISH) using a labeled complementary DNA or RNA chain as a probe. 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., cRNA) in tissue sections or whole tissue embedding. Sample cells and tissues are usually treated to fix the target transcript in situ and increase the entry of the probe. The probe hybridizes with the target sequence at high temperature, and then the excess probe is washed away. Use radioautography, fluorescence microscopy or immunohistochemistry, respectively, to locate and quantify probes labeled with radioactive, fluorescent or antigen-labeled bases in tissues. ISH can also use two or more probes labeled with radioactive or other non-radioactive markers to detect two or more transcripts simultaneously.

[0015] Southern and Northern blotting are used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from a 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 with a labeled probe complementary to the sequence of interest. The hybridized probe bound to the filter is detected. A variation of this procedure is the reverse Northern blotting, in which the substrate nucleic acid fixed to the membrane is a collection of isolated DNA fragments, and the probe is RNA extracted from the tissue and labeled.

[0016] The nucleic acid amplification technique in the present invention is selected from 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).

[0017] In some embodiments, the reagents also include reagents used to detect biomarkers in a sample by chromatography, spectroscopy, mass spectrometry, or liquid chromatography-mass spectrometry.

[0018] Furthermore, the sample includes blood and tissue.

[0019] In the present invention, the sample may refer to a biological sample, usually a clinical sample, including but not limited to peripheral blood, serum, plasma, urine and saliva, and solid tissue samples. The sample of the present invention also includes samples that have been operated or processed in any appropriate manner after purchase, including but not limited to centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of a certain component of the sample, such as a cell population.

[0020] In a specific embodiment of the invention, the sample is selected from a tissue.

[0021] Furthermore, the reagents include a reagent for detecting the mRNA expression level of the biomarker RNF14 and a reagent for detecting the protein expression level of the biomarker RNF14.

[0022] Furthermore, the reagent for detecting the mRNA expression level of the biomarker RNF14 includes a probe that specifically recognizes RNF14 and a primer that specifically amplifies RNF14.

[0023] The term "primer" is a chain of a short nucleic acid sequence that recognizes a target gene sequence, which includes a pair of forward and reverse primers. Specifically, the "primer" includes a pair of primers that provide specific and sensitive analysis results. Primers are believed to provide a high degree of specificity when used to amplify a target gene sequence, but they do not cause amplification of non-target sequences that are inconsistent with or complementary to the target gene sequence.

[0024] In the present invention, the term "probe" refers to a nucleic acid sequence comprising at least 5 nucleotides, for example, 5 to 100 nucleotides, which can hybridize with the expression product of the target gene or the amplified product of the expression product to form a complex under specified conditions.

[0025] Furthermore, the reagents for detecting the protein expression level of the biomarker RNF14 include antibodies, antibody fragments, high-affinity polymers, peptides, peptide mimetics, and nucleic acid aptamers that specifically bind to the full-length protein encoded by RNF14 or its fragments.

[0026] In the present invention, the term "antibody" is well known in the art and refers to an immunoglobulin specific for an antigenic site.

[0027] In the present invention, the term "peptide" comprises a sequence of 3 or more amino acids and generally less than 50 amino acids, wherein the amino acids are naturally occurring or non-naturally occurring amino acids. Non-naturally occurring amino acids refer to amino acids that do not occur naturally in vivo, but which can be introduced into the peptide structures described herein.

[0028] Furthermore, the products include test kits, chips, test strips, and high-throughput sequencing platforms.

[0029] Further, the kit includes a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit and an electrochemiluminescence detection kit; preferably, the kit also includes a buffer and a preservative; the kit may also include necessary components for detecting detectable agents; the kit may also contain a control sample or a series of control samples, which can be measured and compared with the test samples contained therein; further, other components of the kit include but are not limited to: tools for collecting biological samples, tools for labeling detection agents (binders).

[0030] Further, the components of the test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit usually include at least a vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will usually also include a second, third or other additional container, in which additional components are placed separately. However, the components of different combinations can be contained in a vial. The test kit of the present invention will usually also include a container for accommodating reactants, sealed for commercial sale. This container can include a plastic container of injection molding or blow molding, in which the required vial can be retained.

[0031] Furthermore, the chip includes a gene chip and a protein chip; the gene chip includes a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, and the oligonucleotide probe includes an oligonucleotide probe for the RNF14 gene for detecting the transcription level of the RNF14 gene; the protein chip includes a solid phase carrier and a specific antibody for the RNF14 protein fixed on the solid phase carrier; the gene chip can be used to detect the expression level of multiple genes including the RNF14 gene (for example, multiple genes related to cerebral infarction). The protein chip can be used to detect the expression level of multiple proteins including the RNF14 protein (for example, multiple proteins related to cerebral infarction). By detecting multiple markers related to cerebral infarction at the same time, the accuracy of cerebral infarction diagnosis can be greatly improved.

[0032] Furthermore, the test paper includes a test paper carrier and an oligonucleotide fixed on the test paper carrier, and the oligonucleotide can detect the transcription level of the RNF14 gene.

[0033] Furthermore, the product includes reagents for processing the sample.

[0034] Furthermore, the product also includes instructions, which record that when the expression of the biomarker RNF14 is significantly downregulated, the subject suffers from cerebral infarction or is at risk of suffering from cerebral infarction.

[0035] The second aspect of the present invention provides the use of a biomarker or a promoter thereof in the preparation of a pharmaceutical composition for preventing or treating cerebral infarction, wherein the biomarker is RNF14.

[0036] Furthermore, the promoter promotes the expression of RNF14.

[0037] In the present invention, the promoter refers to any substance that can increase the activity of RNF14 protein, improve the stability of RNF14 gene or protein, upregulate the expression of RNF14 protein, increase the effective action time of RNF14 protein, or promote the transcription and translation of RNF14 gene, and these substances can be used in the present invention. For example, the promoter includes a vector that promotes overexpression of RNF14.

[0038] Furthermore, the pharmaceutical composition includes an agent for promoting the expression of RNF14, other drugs compatible with the agent for promoting the expression of RNF14, and pharmaceutically acceptable carriers and / or excipients.

[0039] In the present invention, pharmaceutically acceptable refers to those compounds, materials, compositions and / or dosage forms that are suitable, within the scope of sound medical judgment, for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems commensurate with a reasonable benefit / risk ratio.

[0040] Pharmaceutically acceptable carriers include any and all solvents, diluents, buffers (e.g., neutral buffered saline, or optional Tris-HCl, acetate or phosphate buffers), solubilizers (e.g., polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavoring agents, aromatiser, thickeners, substances that obtain storage effects, coating agents, antifungal agents, preservatives (e.g., ThimerosalTM, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium pyrosulfite), tonicity control agents, absorption delay agents, adjuvants, bulking agents (e.g., lactose, mannitol), etc. The use of such media and agents for preparing pharmaceutical compositions is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in pharmaceutical compositions may be considered.

[0041] Pharmaceutical compositions can be prepared in a manner known to those skilled in the art. The exact nature of the carrier or other raw materials depends on the route of administration. Such suitable administration forms can be solid, semisolid, or liquid (this depends on the mode of administration and the method and carrier for its preparation), which will be clear to the technician.

[0042] The pharmaceutical compositions of the present invention can be administered parenterally (e.g., by intravenous, intracerebral, intracerebroventricular, intramuscular, subcutaneous (intraperitoneal) injection, or intravenous infusion) in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability.

[0043] The pharmaceutical compositions according to the invention can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example in the form of suppositories, or transdermally or topically, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or by inhalation, for example in the form of nasal sprays or aerosol mixtures, or in the form of microcapsules, implants or rods.

[0044] For the production of pills, tablets, sugar-coated tablets and hard gelatin capsules, it is possible to use, for example, lactose, starches such as corn starch or starch derivatives, stearic acid or its salts, etc. Carriers for soft gelatin capsules and suppositories such as fats, waxes, semisolid and liquid polyols, natural oils or hardened oils, etc. Suitable carriers for preparing solutions such as injection solutions or emulsions or syrups such as water, physiological sodium chloride solutions, alcohols such as ethanol, glycerol, polyols, sucrose, invert sugar, glucose, mannitol, vegetable oils, etc. It is also possible to freeze-dry the active ingredient and use the resulting lyophilizate, for example to prepare a preparation for injection or infusion. Suitable carriers for microcapsules, implants or rods such as copolymers of glycolic acid and lactic acid.

[0045] For oral administration forms, the pharmaceutical composition of the present invention can be mixed with suitable additives such as excipients, stabilizers or inert diluents, and suitable administration forms are produced by conventional methods. Examples of suitable inert carriers include gum arabic, magnesium oxide, magnesium carbonate, potassium phosphate, lactose, glucose or starch, particularly corn starch. In this case, dry granules and wet granules can be prepared. Suitable oily excipients or solvents are vegetable oils or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous solutions or alcoholic solutions are water, ethanol, sugar solutions or mixtures thereof. Polyethylene glycol and polypropylene glycol are also used as additional adjuvants for other administration forms. As immediate release tablets, these compositions can contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

[0046] Orally administered pharmaceutical compositions comprising at least one biomarker component as disclosed herein are suitably prepared by uniformly and intimately mixing together appropriate amounts of the components in powdered form (optionally also including a finely dispersed solid carrier) and encapsulating the blend in, for example, a hard gelatin capsule. The solid carrier may include one or more substances used as a binder, lubricant, disintegrant, colorant, etc. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinyl pyrrolidine, low melting point waxes, and ion exchange resins.

[0047] For subcutaneous or intravenous administration, the biomarker components of the present invention are prepared into solutions, suspensions or emulsions with, for example, solubilizers, emulsifiers or other adjuvants. The active ingredients can also be lyophilized and the lyophilisates obtained can be used, for example, for the preparation of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solutions or alcohols such as ethanol, propanol, glycerol, and also sugar solutions such as glucose or mannitol solutions, or mixtures of the various solvents mentioned. Injectable solutions or suspensions can be formulated according to known techniques using suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution or suitable dispersants or wetting agents and suspending agents (e.g., sterile, bland fixed oils, including synthetic monoglycerides or diglycerides, and fatty acids, including oleic acid).

[0048] When administered rectally in the form of suppositories, these preparations can be prepared by mixing the biomarker components described herein with suitable non-irritating excipients such as cocoa butter, synthetic glycerides or polyethylene glycols, which are solid at ordinary temperatures but liquefy and / or dissolve in the rectal cavity to release the drug.

[0049] The third aspect of the present invention provides a method for screening candidate drugs for treating cerebral infarction, the method comprising the following steps: Treating a system expressing or containing RNF14 with a test substance; Detecting the expression level of RNF14 in the system; Test substances that can promote the expression of RNF14 are selected as candidate drugs.

[0050] Furthermore, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system.

[0051] In the present invention, diagnosis, diagnostic, diagnosing and variations of these terms refer to the discovery, judgment or cognition of the health status or condition of an individual based on one or more signs, symptoms, data or other information related to the individual. The health status of an individual can be diagnosed as healthy / normal (i.e., the absence of a disease or illness), or can be diagnosed as unhealthy / abnormal (i.e., the presence of a disease or illness or an assessment of characteristics). Diagnosis, diagnostic, diagnosing, etc. include early detection of a disease in relation to a specific disease or illness; the characteristics or classification of a disease; the discovery of the progression, cure or recurrence of a disease; and the discovery of the response to a disease after treatment or treatment of an individual.

[0052] In the present invention, including or comprising is inclusive or open-ended and does not exclude additional, unrecited members, elements or method steps. The term also includes consisting of and consisting essentially of.

[0053] In the present invention, unless the context clearly indicates otherwise, the term "or" is used herein to mean the term "and / or" and can be used interchangeably with the terms.

[0054] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention proposes for the first time a new use of RNF14 in the diagnosis and treatment of cerebral infarction. The present invention provides a theoretical basis and a new treatment idea for the diagnosis and treatment of cerebral infarction, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the result of proteomics testing of the cerebral infarction group; Figure 2 The results of WB analysis of RNF14 expression in mice after MCAO surgery, A is the WB result of RNF14 in mice after sham MCAO surgery, and B is the quantitative analysis result of WB; Figure 3 The results of WB analysis of RNF14 expression in astrocytes after OGD / R, where A is the WB result of RNF14 in astrocytes after OGD / R, and B is the quantitative analysis result of WB; Figure 4 The figure is a graph of RNF14 expression in the astrocyte control group and the OGD / R group detected by immunofluorescence; A is the immunofluorescence detection graph of the glial cell control group, and B is the immunofluorescence detection graph of the glial cell OGD / R group; Figure 5 The figure is a graph of RNF14 low expression constructed by astrocyte transfection verified by WB analysis, wherein A is the WB result of RNF14 after astrocyte transfection, and B is the quantitative analysis result of WB; Figure 6 Figure 1 is the result of ROS production in the glial cell control group and the RNF14 low expression group; Figure A is the result of ROS detection by fluorescence microscopy, and Figure B is the result of quantitative analysis of ROS; Figure 7 Figure 1 is the result of ROS production in the glial cell control group and the RNF14 overexpression group; Figure A is the result of ROS detection by fluorescence microscopy, and Figure B is the result of quantitative analysis of ROS; Figure 8 The results of CCK8 detection of cell survival rates in the glial cell control group, RNF14 low expression group and overexpression group are shown in Figure 1, where A is the cell survival rate result graph of the glial cell control group and the RNF14 low expression group, and B is the cell survival rate result graph of the glial cell control group and the RNF14 overexpression group; Fig. 9 This is the TTC staining result of the control group and RNF14 overexpression group of brain tissue after MCAO in mice; Fig.10 This is the result of measuring the cerebral infarction volume in the control group and RNF14 overexpression group after MCAO surgery in mice; Fig.11 This is a graph of neurological function score results. DETAILED DESCRIPTION

[0056] The present invention demonstrates the application of RNF14 in the diagnosis and treatment of cerebral infarction by constructing a mouse MCAO model and an oxygen-glucose deprivation / reoxygenation-reglycosylation model.

[0057] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention.

[0058] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0059] Example 1 Proteomics detection of gene changes in cerebral infarction tissue 1. Experimental methods 1.1 Protein extraction and quantitative quality inspection 1.1.1 Protein extraction The total protein was extracted using the mammalian tissue total protein extraction kit (AP0601-50) from Beijing Bangfei Biotechnology Co., Ltd.

[0060] 1.1.2 Protein quantification (1) Preparation of standard curve.

[0061] (2) Prepare the protein solution according to the calculation, with a total volume of 20 μL, and add it to the detection ELISA plate.

[0062] (3) Add 200 μL of quantitative working solution to each well and mix by gently pipetting.

[0063] (4) Leave the sample at room temperature for 10 min and observe with the naked eye whether there is a purple gradient. If the gradient is not obvious, terminate the next step of testing.

[0064] (5) Detect using an ELISA instrument, deduce the total amount of the detected protein based on the standard curve, and calculate the corresponding concentration.

[0065] 1.1.3 SDS-PAGE electrophoresis (1) Take 30 μg of protein sample at a ratio of 5:1 (v / v) and add 5× loading buffer. Boil in water for 5 min and centrifuge at 14,000 g for 10 min. Take the supernatant and perform 10% SDS-PAGE electrophoresis.

[0066] (2) Electrophoresis conditions: constant current 14 mA, electrophoresis time 90 min.

[0067] (3) Coomassie Brilliant Blue staining showed that the total amount of protein met the requirements of subsequent experiments.

[0068] 1.2 Protein Trypsin enzymatic hydrolysis (1) After protein quantification, take 60 μg of protein solution and place it in a centrifuge tube. Add 5 μL of 1M DTT solution, mix well, and keep warm at 37℃ for 1 hour; add 20 μL of 1M IAA solution, mix well, and react at room temperature in the dark for 1 hour.

[0069] (2) Pipette all samples into the ultrafiltration tube, centrifuge and discard the collected liquid.

[0070] (3) Add 100 μL UA (8M urea, 100 mM Tris-HCl, pH 8.0) to the ultrafiltration tube, centrifuge and discard the collected liquid. Repeat twice.

[0071] (4) Add 100 μL of 50 mM NH4HCO3, centrifuge and discard the collected liquid, and repeat three times.

[0072] (5) Update the collection tube, add trypsin to the ultrafiltration tube, add Trypsin in a ratio of 50:1 between protein and enzyme, and perform enzymatic hydrolysis at 37°C for 12-16 hours.

[0073] 1.3 Mass spectrometry analysis (1) Capillary high performance liquid chromatography: Each sample is separated using a nano-flow HPLC liquid phase system. The chromatographic column is balanced with 95% liquid A. The sample is loaded onto the mass spectrometer pre-column by an automatic sampler and then separated on the analytical column.

[0074] (2) Mass spectrometry identification: Each sample was separated by capillary high performance liquid chromatography and then analyzed by mass spectrometry using an Orbitrap Fusion mass spectrometer (Thermo Scientific).

[0075] 2. Experimental results The proteomics test results are as follows Figure 1 As shown, RNF14 was significantly downregulated when cerebral infarction occurred.

[0076] Example 2 In vivo and in vitro experiments verify the changes of RNF14 gene during cerebral infarction 1. Experimental methods (1) Construction of mouse MCAO model: Male C57BL / 6 mice weighing about 25 g (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were weighed and injected intraperitoneally with 0.1 mL of 1.25% avertin. After successful anesthesia, the mice were fixed on the operating table. After routine skin preparation and disinfection, hair removal was performed. A midline incision was made in the neck to separate the surrounding tissues. The common carotid artery, external carotid artery, and internal carotid artery were exposed and separated under a microscope. The pterygopalatine artery was separated and ligated to keep the internal carotid artery open. A 15 mm long silicone-coated suture of appropriate size was inserted from the external carotid artery into the internal carotid artery for about 10 mm and stopped at the beginning of the middle cerebral artery. After 1 hour, the suture was pulled out to complete reperfusion. The mice were placed on a heating pad. When the mice woke up, symptoms of hemiplegia were observed. After waking up, they were kept in an environment with suitable temperature and given sufficient feed and water. The corresponding samples were collected 24 hours after surgery.

[0077] (2) Primary astrocyte culture: Cortices were isolated from P1-3 C57BL / 6J mice and digested with trypsin (Gibco) at 37°C for 15 min; the dissociated cells were rinsed and resuspended in DMEM (Gibco) containing 10% fetal bovine serum and 1% antibiotic-antimycotic (Gibco). Subsequently, live cells (2×10 6 Astrocytes were seeded in T75 culture flasks. The cultures were maintained in a cell culture incubator at 37°C and 5% CO2, and the medium was changed every 3 days during the entire study. When the confluence reached 80%-90%, astrocytes were seeded in 60 mm culture dishes for subsequent studies.

[0078] (3) Construction of cellular oxygen-glucose deprivation / reoxygenation-reglycogenation (OGD / R) model: The original culture medium of the astrocyte OGD / R group was discarded, and the cells were rinsed once with an appropriate amount of PBS. An equal volume of sugar-free DMEM culture medium was added to the cells. The cells were cultured in a cell culture incubator pre-set with 95% nitrogen and 5% carbon dioxide at 37°C for 6 hours, and then replaced with normal culture medium. The cells were placed in a normal cell culture incubator for a further 24 hours to complete the reoxygenation-reglycogenation process.

[0079] (4) Immunoblotting: Brain tissue and astrocytes were homogenized in RIPA lysis buffer (Beyotime) containing a mixture of protease and phosphatase inhibitors (Beyotime) and centrifuged at 14,000 × g for 30 min at 4°C; the supernatant was transferred to a fresh tube and the concentration was determined by a BCA kit (Beyotime). Equal amounts of protein were separated by 4-15% SDS-PAGE and electroblotted onto a PVDF membrane; after blocking, the membrane was incubated with the primary antibody overnight at 4°C. After incubation with an appropriate secondary antibody conjugated to horseradish peroxidase (anti-rabbit antibody or anti-mouse antibody, 1:1000-1:2000) at room temperature for 1 hour, the protein bands were observed using a fluorescent secondary antibody, developed using an Odyssey developer, and the density of the bands was measured using ImageJ; changes in target protein expression were normalized using control protein expression.

[0080] (5) Immunofluorescence detection: Cells were seeded in groups and treated with oxygen-glucose deprivation / reoxygenation / re-glucose treatment in advance; then, they were fixed with 4% paraformaldehyde for 10 minutes and soaked in 0.5% Triton X-100 in PBS for 15 minutes; in this study, goat serum was used to avoid nonspecific binding at room temperature; after treatment for 30 minutes, the cells were incubated with the primary antibody (RNF14 mouse monoclonal antibody (IgG1), immunoway, dilution of 1:200) at 4°C overnight, and then incubated with the secondary antibody (goat anti-mouse IgG1-HRP, Bio-Tech, dilution of 1:200) at 20-37°C for 1 hour; nuclear staining was performed with DAPI, and images were captured with a Nikon inverted epifluorescence microscope.

[0081] 2. Experimental results (1) WB analysis results after MCAO in mice: The results of WB analysis of mouse brain tissue are as follows Figure 2 As shown, RNF14 was downregulated after cerebral infarction in mice.

[0082] (2) WB analysis results after microglia OGD / R: After microglia OGD / R, the expression results of RNF14 are as follows Figure 3 As shown, RNF14 expression was significantly downregulated in the microglial OGD / R model.

[0083] (3) Immunofluorescence test results: Immunofluorescence test results are as follows Figure 4 Compared with the control group ( Figure 4 A), OGD / R group ( Figure 4 B) The green fluorescence becomes less and lighter, further indicating that RNF14 expression is downregulated after glial cells undergo OGD / R.

[0084] Example 3 In vivo and in vitro experiments verify the role of RNF14 gene in cerebral infarction 1. Experimental methods (1) Astrocyte transfection: One day before transfection, cells were plated in 500 μL of growth medium without antibiotics to achieve a confluency of 30% to 50% at the time of transfection. For each transfection sample, polyplus complexes were prepared as follows: 50 nmol of siRNA or DNA plasmid was dissolved in 200 μL of jetPRIME buffer and gently mixed; 4 μL of jetPRIME was added, gently mixed and incubated at room temperature for 10 minutes; the mixture was added to each well containing cells and serum-containing medium, and gently mixed by shaking the plate back and forth; the medium was replaced after 24 hours, and the cells were incubated at 37°C in a CO2 incubator for 72 hours.

[0085] (2) ROS measurement: ROS measurement was performed using the non-fluorescent cell-permeable compound 2'-7'-dichlorofluorescein diacetate (DCFH-DA) according to the manufacturer's instructions (Biyuntian). DCFH-DA is hydrolyzed by intracellular esterases to dichlorofluorescein (DCFH), which is trapped inside the cell; this non-fluorescent molecule is then oxidized to fluorescent dichlorofluorescein by the action of cellular oxidants; primary astrocytes treated under different conditions were washed twice with PBS 1× solution and loaded with 5 μM DCFH-DA diluted in serum-free medium at 37°C for 30 minutes; cells were washed three times with PBS, and fluorescent images were captured using a Nikon inverted epifluorescence microscope equipped with a 20× objective.

[0086] (3) CCK8 method for measuring cell survival rate: Collect cells in good growth state, inoculate them in 96-well plates (density 5000 / well), and treat them according to experimental groups, with 6 replicate wells in each group; wash with PBS twice and replace with new culture medium, then add 10 μL CCK8 solution to each well, incubate at 37℃ for 4 hours, set zero wells and control wells; adjust the microplate reader to 450 nm wavelength, and measure the absorbance value (OD value) of each well. Calculation: relative cell survival rate % = (OD value of experimental group) / (OD value of solvent group) × 100%, OD value of experimental group = OD value of experimental group - OD value of zero well, OD value of solvent group = OD value of solvent group - OD value of zero well.

[0087] (4) TUNEL staining: Wash the cells that have been cultured on the slides three times in PBS; fix them in 4% formaldehyde solution, and wash them three times in PBS after 25 minutes; incubate them in 0.2% Triton X-100 at room temperature for 5 minutes, and wash them three times in PBS. Then, add 50-100 μL of TUNEL reaction mixture and incubate them in a 37℃ wet box in the dark for 1 hour; wash them three times in PBS; add a drop of anti-fluorescence quencher containing DAPI and cover the slide with a coverslip; observe under a fluorescence microscope and take pictures with a camera; under ultraviolet excitation, the cell nucleus stained with DAPI appears blue, and TUNEL is labeled with FITC fluorescent dye, and positive apoptotic cells appear green.

[0088] (5) TTC staining and determination of cerebral infarction volume: Mice were stained with 2,3,5-triphenylterazolium chloride (TTC) and the percentage of cerebral infarction volume was determined one day after MCAO surgery. The brains of the experimental animals in each group were removed and placed in the groove of the brain mold. The front and back ends were removed. The coronal sections were cut from the front to the back with a distance of 2 mm from the frontal pole as the origin. The thickness of each section was 2 mm. The sections were immersed in 2% TTC staining solution and kept at 37°C in the dark for 10 minutes. The color change was observed and the sections were turned over and immersed for another 10 minutes. The brain slices were arranged neatly in the front-to-back order of the brain tissue. The normal brain tissue was red, and the cerebral infarction area was not stained (white). After taking pictures and collecting images, the volume of the white area and the total volume of the brain tissue were measured using Image J image analysis software. The percentage of infarct volume was calculated according to the formula [(B1+B2+…+Bn)x2 / (A1+A2…+An)x2]×100%, where B is the volume of the infarct area (white) and A is the total volume of brain tissue.

[0089] (6) Neurological function score: refer to the Zea-Longa method: 0 points, no defect; 1 point, mild defect, weakness of the contralateral forelimb; 2 points, moderate defect, difficulty walking, turning in circles to the contralateral side; 3 points, severe defect, falling to the contralateral side; 4 points, unable to walk, impaired consciousness.

[0090] 2. Experimental results (1) Cell transfection to construct RNF14 low expression experiment results: WB verification results are as follows Figure 5 As shown in A, the expression of RNF14 decreased in the Si (low expression) group, and the quantitative analysis results ( Figure 5 B) It also showed that the expression level of RNF14 in the Si group decreased, indicating that the cellular RNF14 low-expression model was successfully constructed.

[0091] (2) ROS detection results: The ROS detection results of the astrocyte control group and the RNF14 low expression group (Si-RNF14) are as follows Figure 6As shown in the figure, in the OGD / R glial cell model, the amount of ROS produced by cells increased after low expression of RNF14; while after overexpression of RNF14, the amount of ROS produced by OGD / R glial cells decreased significantly ( Figure 7 ).

[0092] (3) CCK8 detection results: In the OGD / R glial cell model, the cell survival rate was significantly reduced after low expression of RNF14 ( Figure 8 A), while overexpression of RNF14 significantly increased the cell survival rate ( Figure 8 B).

[0093] (4) TTC staining results: TTC staining results are as follows Fig. 9 As shown, the brain tissue of mice turned white after MCAO surgery, while after overexpression of RNF14, the color of the brain tissue of mice after MCAO surgery returned to normal and turned red.

[0094] (5) Measurement results of cerebral infarction tissue volume: The measurement results of cerebral infarction volume are as follows: Fig.10 As shown, in the mouse MCAO model, overexpression of RNF14 significantly reduced the cerebral infarct volume compared with the control group.

[0095] (6) Neurological function scoring results: Neurological function scoring was performed on the MCAO model control group of mice. The results are as follows: Fig.11 As shown, compared with the control group, the neurological function score was significantly decreased after overexpression of RNF14, indicating that the neurological function deficits of mice were reduced after overexpression of RNF14.

[0096] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting the expression level of a biomarker in the preparation of a product for diagnosing cerebral infarction, characterized in that: The biomarker is RNF14.

2. The use according to claim 1, characterized in that: The reagents include reagents used to detect biomarkers in samples through protein immunoassay technology, sequencing technology, nucleic acid hybridization technology, and nucleic acid amplification technology; Preferably, the sample includes blood or tissue.

3. The use according to claim 1, characterized in that: The reagents include a reagent for detecting the mRNA expression level of the biomarker RNF14 and a reagent for detecting the protein expression level of the biomarker RNF14.

4. The use according to claim 1, characterized in that: The products include test kits, chips, test strips, and high-throughput sequencing platforms; Preferably, the kit includes a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit, and an electrochemiluminescence detection kit; Preferably, the chip includes a gene chip and a protein chip; Preferably, the test paper comprises a test paper carrier and an oligonucleotide fixed on the test paper carrier, and the oligonucleotide can detect the transcription level of the RNF14 gene.

5. The use according to claim 1, characterized in that: The product includes reagents for processing a sample.

6. The use according to claim 1, characterized in that: The product also includes instructions, which record that when the expression of the biomarker RNF14 is significantly downregulated, the subject suffers from cerebral infarction or is at risk of suffering from cerebral infarction.

7. Use of a biomarker or a promoter thereof in the preparation of a pharmaceutical composition for treating and / or preventing cerebral infarction, characterized in that: The biomarker is RNF14.

8. The use according to claim 7, characterized in that: The promoter promotes the expression of RNF14; Preferably, the promoter comprises a vector that specifically promotes overexpression of RNF14.

9. The use according to claim 7, characterized in that: The pharmaceutical composition comprises an agent for promoting the expression of RNF14, other drugs compatible with the agent for promoting the expression of RNF14, and a pharmaceutically acceptable carrier and / or excipient.

10. A method for screening candidate drugs for treating cerebral infarction, characterized in that: The method comprises the following steps: Treating a system expressing or containing RNF14 with a test substance; Detecting the expression level of RNF14 in the system; Test substances that can promote the expression of RNF14 are selected as candidate drugs.

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