KR102480069B1 - Composition for diagnosing diabetic retinopathy using biomarkers, method for providing information for diagnosing diabetic retinopathy, and screening method for substances for treatment of diabetic retinopathy - Google Patents

Abstract

The present invention relates to: a composition for diagnosing diabetic retinopathy using biomarkers; a method for providing information for diagnosing diabetic retinopathy; and a screening method for substances for the treatment of diabetic retinopathy, wherein the biomarkers are described in detail in the specification. In addition, the composition of the present invention includes an agent for measuring the level of the biomarkers related to diabetic retinopathy (DR).

Description

Composition for diabetic retinopathy diagnosis using biomarkers, method for providing information for diagnosis of diabetic retinopathy, and method for screening substances for treatment of diabetic retinopathy FOR SUBSTANCES FOR TREATMENT OF DIABETIC RETINOPATHY}

The present invention relates to a composition for diagnosing diabetic retinopathy using a biomarker, a method for providing information for diagnosing diabetic retinopathy, and a method for screening a substance for treating diabetic retinopathy.

Diabetic retinopathy (DR) is a metabolic disease involving inflammation and alteration of mitochondria, which is the leading cause of blindness in adults worldwide. Diabetic retinopathy is a complication of diabetes, and is mainly diagnosed as pathological changes in microvessels, showing mechanisms such as abnormal blood vessel leakage in the retina, development of new and fragile blood vessels, hemorrhage, body fluid leakage, inflammation, and retinal cell death.

Early stages of diabetic retinopathy include synaptic protein changes, neurodegeneration, retinal pigment epithelial (RPE) cell death and thinning of the inner nuclear layer, mitochondrial dysfunction, increased oxidative stress, increased advanced glycation end products, and apoptosis of photoreceptor cells. includes the phenomenon of In addition, the pathogenesis of diabetic retinopathy involves activation of the polyol pathway, excessive production of reactive oxygen species, VEGF activation, neovascularization and blood-retinal barrier (BRB) damage, mitochondrial dysfunction, and insulin-metabolites including PI3K. Including interactions between proteomes. However, it is difficult to detect these changes, and the molecular mechanisms involved have not been fully elucidated.

The present inventors identified the mechanism at the molecular level of diabetic retinopathy through proteomic analysis, such as extracting and analyzing proteomes and metabolites, and creating metabolomic maps for the diabetic retinopathy model. The present invention was completed by discovering biomarkers that can be used for diagnosis of disease or screening for therapeutic agents.

Hyo Jung Gye, et al., New Modalities for the Diagnosis and Treatment of Diabetic Retinopathy, The Korean Journal of Medicine: Vol. 89, no. 3, 2015

Accordingly, the problem to be solved by the present invention is to provide a composition and method capable of effectively diagnosing diabetic retinopathy, and providing a method capable of screening a substance for treating diabetic retinopathy.

The tasks of the present invention are not limited to the technical tasks mentioned above, and other technical tasks not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a composition for diabetic retinopathy (DR) diagnosis according to an embodiment.

A composition for diagnosing diabetic retinopathy includes an agent for measuring the level of biomarkers associated with diabetic retinopathy, and the biomarkers include one or more of the biomarkers shown in Table 1 below.

division Kinds change First biomarker Protein of SEQ ID NO: 1 (CAC2PBS) increase Protein of SEQ ID NO: 2 (VIMIII) increase Protein of SEQ ID NO: 3 (FAM81B) increase Protein of SEQ ID NO: 4 (ADAM18) increase Adenylate cyclase 4 (ADCY4) increase Myogenic regulatory factor 2 (MRF2) increase Vinculin (VCL) increase Toll-like Receptor 7 (TLR7) increase SH3 domain-binding protein 1 (SH3BP1) increase Thioredoxin domain containing protein 17 (TXNDC17) increase Microtubule-actin cross linking factor 1 (MACF1) increase Dynamin-1 like protein (DNM1L) increase Regulator of G-protein signaling 3 (RGS3) increase Activated protein C (APC) increase Ras-related C3 botulinum toxin substrate 1 (RAC1/2) increase RhoA (Ras homolog family member A) increase Calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) increase Complement protein 3 (Complement 3, C3) increase Disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) increase Vascular cell adhesion molecule 1 (VCAM1) increase Estrogen receptor 1 (ESR1) (Estrogen receptor alpha) increase SOS1 (Son of Sevenless homolog 1) increase Second biomarker Prohibitin (PHB) decrease T-cell receptor beta (TCRB) decrease Insulin-like growth factor 2 receptor A (IGF2R) decrease Junction plakoglobin (JUP) decrease E3 ubiquitin-protein ligase (RNF135) decrease Prohibitin 2 (PHB2) decrease

In Table 1, the CAC2PBS protein, the VIMIII protein, the FAM81B protein, and the ADAM18 protein are each independently 80% or more, 85% or more, 90% or more, 95% or more, 96 % or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity. Alternatively, the CAC2PBS protein, VIMIII protein, FAM81B protein, and ADAM18 protein may be proteins having amino acid sequences of SEQ ID NOs: 1, 2, 3, and 4, respectively. In the present specification, a protein having an amino acid sequence of a specific sequence number includes a functional equivalent thereof. The "functional equivalent" is a result of addition, substitution or deletion of amino acids, which is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more of the amino acid sequence of the specific SEQ ID NO. % or more, or more than 99% sequence homology, which means that it has substantially the same physiological activity as the protein having the amino acid sequence of the specific sequence number. In a protein having an amino acid sequence of a specific sequence number, one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, or five or more amino acids may be substituted.

The "substantially homogeneous physiological activity" means a preventive or therapeutic activity of diabetic retinopathy due to the structural and functional homology of the protein. In addition, the protein may or may not carry physiochemical properties as a result of amino acid additions, substitutions or deletions. In an exemplary embodiment, the amino acid substitution may improve the thermostability of the protein, alter the substrate specificity, or change the pH optimum. However, it is not limited thereto.

A specific functional group may be bound to the N or C-terminus of a protein having an amino acid sequence of a specific SEQ ID NO. The protein may have improved stability by the modification. The stability is a concept including storage stability as well as in vivo stability. The protecting group can protect the protein from attack by proteolytic enzymes in vivo.

Measuring the level of the biomarker may be to measure the amount or concentration of the biomarker, or to directly express the biomarker or measure the expression level of mRNA or protein of a gene involved in the increase of the biomarker. may be A gene involved in the increase of a biomarker may mean a case in which a substance such as a protein generated through expression of the corresponding gene increases the amount of the corresponding biomarker.

As an agent for measuring the level of a biomarker as shown in Table 1, when measuring the mRNA expression level of a gene related to the biomarker, it includes at least one of a primer pair, a probe, and an antisense nucleotide that specifically binds to the gene. can do. Since nucleic acid information of at least one or more genes is known in GeneBank or the like, a person skilled in the art can design primer pairs, probes, or antisense nucleotides thereof based on the sequences.

The probe refers to a material that can confirm the presence of a target material by specifically binding to a target material to be detected in a sample, and includes PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA Or it may be DNA, but is not limited thereto.

As a method for measuring mRNA expression levels, reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting, DNA chip, etc., but are not limited thereto.

Alternatively, it may include one or more of antibodies, interacting proteins, ligands, nanoparticles, and aptamers that specifically bind to the biomarker.

The antibody may include both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

In order to solve the above other problems, the present invention provides a diagnostic kit for diabetic retinopathy according to an embodiment. A diagnostic kit for diabetic retinopathy includes the composition for diagnosing diabetic retinopathy as described above. The kit for diabetic retinopathy diagnosis may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. there is.

In addition, the kit can be used for mass spectrometry. In this case, the specific amino acid residue of the protein is myristoylation, isoprenylation, prenylation, glypiation, lipoylation, acylation, alkylation, methylation, demethylation, amidation, It may have modifications such as ubiquitination, phosphorylation, deamidation, glycosylation, oxidation, or acetylation.

A kit for diabetic retinopathy can be prepared by a method known to those skilled in the art. A kit for diabetic retinopathy diagnosis may include, for example, a freeze-dried antibody, a buffer, a stabilizer, an inactive protein, and the like. The antibody may be labeled with radionuclides, fluorescors, enzymes, and the like.

In order to solve the above another problem, the present invention provides an information providing method for diagnosing diabetic retinopathy according to an embodiment. An information providing method for diabetic retinopathy diagnosis includes (a) measuring the level of a biomarker related to diabetic retinopathy in a biological sample of a subject; (b) comparing the measured level to a control sample; and (c) diagnosing that there is a possibility of diabetic retinopathy when the measured level increases or decreases compared to the control sample. The biomarkers associated with diabetic retinopathy are one or more of the biomarkers in Table 1 above.

Specifically, among the biomarkers in Table 1, the first biomarker is characterized by an increased (or more) mRNA (or protein) expression level or abundance in cells or patients with diabetic retinopathy compared to normal controls. Accordingly, if the level of the first biomarker increases (or is higher) compared to the control sample, the subject can be diagnosed as having a possible prevalence of diabetic retinopathy. That is, it is possible to diagnose (or determine, determine) that the subject has a high possibility of having or developing diabetic retinopathy.

Conversely, the second biomarker among the biomarkers in Table 1 has a characteristic in that the mRNA (or protein) expression level or abundance in cells or patients with diabetic retinopathy is reduced (or less) compared to normal controls. Accordingly, if the level of the second biomarker decreases (or is less) compared to the control sample, the subject can be diagnosed as having a possible prevalence of diabetic retinopathy. That is, it is possible to diagnose (or determine, determine) that the subject has a high possibility of having or developing diabetic retinopathy.

In addition, the more types of the first biomarker are measured as increased (or the more types of the second biomarker are measured as decreased), the higher the possibility of prevalence may be diagnosed.

The biological sample may be derived from a vascular region or a non-vascular region of the subject.

A blood vessel region is a tissue including blood vessels of a target, and a biological sample derived from a blood vessel region may be, for example, a sample including blood, plasma, and/or vascular tissue (vascular endothelium, vascular mesothelium, vascular sheath, etc.) . More specifically, a biological sample derived from a blood vessel region may be derived from a blood vessel region in a tissue related to a disease to be diagnosed. For example, in the case of diabetic retinopathy, the retina, macula, retinal nerve, and retina It may be blood, plasma, and/or vascular tissue collected from pigment epithelium or the like.

The non-vascular region is a tissue that does not contain blood vessels of a target, and the biological sample derived from the non-vascular region may be a sample containing tissue of extravascular cells. More specifically, a biological sample derived from a non-vascular region may be an extravascular tissue of a tissue related to a disease to be diagnosed. For example, in the case of diabetic retinopathy, the retina, macula, retinal nerve, and retinal pigment It may be a tissue of an extravascular region taken from an epithelium or the like.

As a more specific example, in the case of retinal blood vessels, whole retinal cells may be collected and biomarkers related to diabetic retinopathy may be analyzed. In the case of diabetes, brown adipose tissue or adipose tissue of white adipose tissue can be separated and analyzed. However, embodiments of the present invention are not limited thereto.

In some embodiments, in the method for providing information for diagnosing diabetic retinopathy of the present invention, step (a) is a step of measuring the level of at least one of the first biomarkers and at least one of the second biomarkers in a biological sample of the subject. can As such, by measuring and comparing the first and second biomarkers together, the accuracy of diagnosis of diabetic retinopathy can be further increased. In this case, it can be diagnosed as having a higher prevalence probability when the first biomarker increases and the second biomarker decreases than when only the first biomarker is evidenced or only the second biomarker decreases.

Measurement and comparison of the level of the biomarker can be performed using the above-described diagnostic composition or kit.

To analyze the level of the biomarker, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), MALDI-TOF (Matrix-assisted laser desorption / ionization), GC-MS (Gas chromatography-mass spectrometry) analysis, LC-MS (Liquid chromatography-mass spectrometry) analysis, cholesterol oxidase analysis, lipid panel analysis, etc. may be used alone or additionally, but the embodiments of the present invention are not limited thereto.

In addition, the analysis of steps (a) to (c) can be analyzed using statistical methods or algorithms to improve the accuracy of diagnosis, and linear or nonlinear regression analysis methods, prior or nonlinear classification analysis methods, logistic Regression analysis method (logistic regression), Analysis of Variance (ANOVA), neural network analysis method, genetic analysis method, support vector machine analysis method, hierarchical analysis or clustering analysis method, hierarchical algorithm using decision tree or kernel principal component ( Kernel principal component) analysis method, Markov Blanket analysis method, recursive feature elimination or entropy-based regression feature elimination analysis method, and forward floating search or backward floating search analysis One or more analytical methods selected from the group consisting of methods may be used.

In order to solve the above problems, the present invention according to another embodiment (d) administering a therapeutic agent capable of decreasing (or increasing) the level of the biomarker measured in step (a) It provides a method for treating diabetic retinopathy further comprising. That is, when a specific biomarker is increased through steps (a) to (c) and it is determined that there is a possibility of prevalence of the disease, by administering a therapeutic agent that can directly or indirectly inhibit the corresponding biomarker, etc. The target can be cured. On the other hand, if the biomarker whose level is measured is the first biomarker, a therapeutic agent capable of decreasing it is administered, and if it is the second biomarker, a therapeutic agent capable of increasing it is administered.

The therapeutic agent may be administered in the form of a pharmaceutical composition, and the therapeutic agent may be mixed with a pharmaceutically acceptable carrier, excipient, diluent, etc. by a method known in the pharmaceutical field to prepare a formulation such as an injection. It can be prepared and administered intravenously.

The method of treating diabetic retinopathy of the present invention may further include (e) measuring the level of the biomarker again after administration of the pharmaceutical agent in step (d).

In order to solve the above another problem, the present invention provides a screening method for a substance for treating diabetic retinopathy according to an embodiment. A method for screening a substance for treating diabetic retinopathy includes (a) processing a candidate substance in an experimental model; (b) measuring the level of one or more of the aforementioned biomarkers in the treated experimental model; and (c) determining whether the biomarker is decreased or increased by comparing the measured level with a control model.

Since the biomarker of the present invention is a substance that increases (first biomarker) or decreases (second biomarker) compared to the normal control, the biomarker decreases (first biomarker) or increases (second biomarker) by treating a specific candidate substance. marker), the candidate substance can be judged as a substance with the potential to treat diabetic retinopathy and used to conduct next-stage experiments such as preclinical and clinical trials.

The step of treating the candidate material may be performed in one or more of in vitro, in vivo, ex vitro, ex vivo and in silico, and the experimental model may be a cell, animal model, human, or isolated or derived from an animal or human. It may be a cell or tissue, and may be a vascular region or a non-vascular region.

The experimental model may be a cell model or an animal model in which diabetic retinopathy is induced, or a cell, animal, human, or a cell or tissue isolated from or derived from an animal or human having diabetic retinopathy.

Other embodiment specifics are included in the detailed description.

According to embodiments of the present invention, diabetic retinopathy can be effectively diagnosed and a substance for treating diabetic retinopathy can be screened by using specific biomarkers related to diabetic retinopathy.

Effects according to the embodiments of the present invention are not limited by the contents exemplified above, and more diverse effects are included in the present specification.

1 is a graph confirming that mitochondrial prohibitin (PHB) decreases in hyperglycemia through an experimental example of the present invention.

Advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only the embodiments make the disclosure of the present invention complete, and those skilled in the art in the art to which the present invention belongs It is provided to fully inform the person of the scope of the invention, and the invention is only defined by the scope of the claims.

Terminology used herein is for describing the embodiments and is not intended to limit the present invention. In this specification, 'and/or' includes each and every combination of one or more of the recited items. In addition, singular forms also include plural forms unless otherwise specified in the text. The terms 'comprises' and/or 'comprising' used in the specification do not exclude the presence or addition of one or more other elements other than the mentioned elements. Numerical ranges expressed using '-' or 'to' indicate numerical ranges including the values listed before and after them as lower and upper limits, respectively, unless otherwise specified. 'About' or 'approximately' means a value or range of values within 20% of the value or range of values set forth thereafter.

Also, terms such as first, second, A, B, (a), and (b) may be used to describe components of an embodiment of the present invention. These terms are only used to distinguish the component from other components, and the nature, order, or order of the corresponding component is not limited by the term.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used in a meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless explicitly specifically defined.

And, in describing the embodiments of the present invention, if it is determined that a detailed description of a related known configuration or function hinders understanding of the embodiments of the present invention, the detailed description will be omitted.

As used herein, 'prevention' means suppressing or delaying the occurrence of a symptom or disease in a subject who does not yet have the symptom or disease, but is susceptible to such a symptom or disease.

As used herein, 'treatment' refers to any action that improves or beneficially changes a symptom in an individual, and includes, for example, (a) inhibition of the development (exacerbation) of a symptom or disease, (b) alleviation or improvement of a symptom or disease. , or (c) elimination of a symptom or disease.

In the present specification, 'individual' or 'subject' means a cell, tissue, animal, or human to diagnose, prevent, or treat a specific disease or disorder using the present invention.

In the present specification, 'diagnosis' refers to an act of determining whether or not a specific disease or disease has occurred or is at risk of developing using the composition or kit of the present invention, as well as providing information related to the disease or disease, such as identifying the characteristics of a pathological state. It means everything you can do.

In the present specification, 'probability of prevalence' is meant to include both the possibility that a specific disease or disorder has occurred and the possibility that it will occur.

In the present specification, 'biological sample' refers to blood, plasma, serum, cells, tissues, body fluids, saliva, urine, aqueous humor, aqueous humor, and the like of a subject.

In this specification, 'derived' may mean not only a thing isolated from a specific object or a specific site, but also the object or site itself.

As used herein, 'biomarker' refers to peptides, polypeptides, nucleic acids, organic biomolecules such as lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.); and the like.

In the present specification, 'control group' refers to normal cells or patients that do not have or are not induced by a specific disease or disease.

In the present specification, 'level' may refer to the mRNA or protein expression level of a specific factor including a biomarker, or may also refer to a content, concentration, etc. that can be confirmed by measuring or analyzing the abundance of a specific factor.

In this specification, the general rules for naming amino acid sequences may be based on either three-letter or one-letter amino acid codes unless there are specifically indicated exceptions. For example, the center of the amino acid structure is indicated by a 3-letter code (eg, Ala, Lys), and the D-conformation (eg, D-Ala, D-Lys) is specifically indicated by writing "D-" in front of the 3-letter code. If not indicated, L-conformation can be assumed. Amino acid residues constituting a protein or peptide may be natural or non-natural amino acid residues.

Hereinafter, embodiments of the present invention will be described in detail through preparation examples and experimental examples, but it is obvious that the effects of the present invention are not limited by the following experimental examples.

Experimental Example: Biomarker Analysis Using Diabetic Retinopathy (DR) Model

Experiment method

1) Production of cell model and analysis of proteome and metabolome

Retinal pigment epithelial cells (ARPE-19) were obtained from ATCC (Manassas, VA). Retinal progenitor cells were donated by Professor Harold J. Sheeldo of the University of North Texas.

Cells were grown in 100 mm dishes (Nalge Nunc International, Naperville, IL) in a 5% CO ₂ incubator at 37 °C in Dulbecco's modified Eagle's medium (DMEM) with fetal bovine serum (10%) and penicillin/streptomycin (1%). and trypsinized (5-7 min at 37 °C). Cells were subjected to oxidative stress under H ₂ O ₂ (200 μM) and intense light (7,000-10,000 lx, 1-24 hours) or constant light (700 lx, 48 hours) for 2-4 days, and proteomic and metabolome changes were observed. Observed.

Cells were disrupted by periodic sonication (3 x 5) using PBS and IP lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol and protease inhibitors) and (0 °C). , pH 7.4, 5 min) and centrifuged (10 min, 13,000 x g). Separated proteins were separated by SDS-PAGE using Laemmli sample buffer (5X, 5% β-mercaptoethanol) and visualized (Coomassie blue (Pierce, IL) or silver staining).

2) Animal model manufacturing and proteomic analysis

Male Wistar rats (250-350 g/rat, 8 weeks old, Charles River Laboratories) were randomly assigned to either control or diabetic groups. All procedures followed Directive 2010/63/EU, NIH guidelines and ARVO guidelines. Mice were injected with streptozotocin (STZ in 10 mM sodium citrate, 65 mg/kg, pH 4.5, Sigma, St. Louis, MO, USA) and diabetic was induced, with blood glucose levels exceeding 250 mg/dl resulting in hyperglycemia. was regarded as It was confirmed through known literature that STZ administration is a verified model manufacturing method for the induction of diabetic retinopathy (Chen-Yuan Gong et al, Streptozotocin induced diabetic retinopathy in rat and the expression of vascular endothelial growth factor and its receptor, Int J Ophthalomol, Vol. 6, No. 5, Oct. 18, 2013 et al.). Two days after STZ injection, blood glucose was checked with a blood glucose meter (Elite, Bayer, Portugal), blood was collected for glucose analysis, and the animals were prepared as control and diabetic rats matched in weight and age. Thereafter, protein and metabolome analysis was performed as described below.

3) Extraction and analysis of retinal proteins from donated eyes of patients with diabetic retinopathy

Eyes from diabetic retinopathy patients were donated from Utah Eye Hospital and Georgia Eye Hospital. Retinal proteins were extracted from diabetic patients and age-matched control eyes.

Retina was dissected using phosphate-buffered saline (2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , 137 mM NaCl, pH 7.4, 4 °C) and incubated in RIPA buffer (0.1% SDS, 1 mM DTT, 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 0.5% DOC, pH 7.4), protease inhibitor cocktail tablets and phosphatase inhibitors (10 mM NaF and 1 mM Na ₃ VO ₄ ) were used. were sonicated and centrifuged to collect proteins in the supernatant (10 min, 16,000 xg, 4 °C) and analyzed using 1D and 2D SDS-PAGE, western blot, liquid chromatography-mass spectrometry, database search, and BLAST analysis. Retinal proteins were analyzed.

Proteins separated on SDS-PAGE gels (8-16%) were transferred to PVDF membranes (Millipore, Billerica, MA, USA), and non-specific proteins were blocked using low-fat milk (5%) in Tris-buffered saline (TBS- T, 20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween-20, pH 7.6, 1 hour, 25 °C). The membrane was reacted with primary antibody (rabbit polyclonal, Genemed Synthesis, San Antonio, TX) at 4 °C for 16 hours at room temperature, and after membrane washing (TBS-T, 0.5% low-fat milk, 1 hour), anti-mouse or anti-chlorine alkali Incubation with phosphatase-conjugated IgG secondary antibody (1:10,000, GE Healthcare, Buckinghamshire, UK or 1:10,000 dilution, anti-rabbit, Agrisera, Vannas, Sweden) TBS-T (1% low-fat milk, 1 hour, 25 °C) The membrane was washed (TBS-T, 0.5% low-fat milk, 1 hour). Retinal proteins were imaged using chemiluminescence (ECF, GE) (Typhoon FLA 9000, GE Healthcare, Image Quant 5.0 software Molecular Dynamics, Inc., Sunnyvale. CA, USA). As a control group, β-actin (1:5,000, Sigma), β-III tubulin (1:5,000, Covance) and HRP-conjugated secondary antibody (1:7,000, anti-mouse, Santa Cruz Biotechnology, Santa Cruz, CA) were used. Quantitative analysis was performed using

Intracellular protein analysis was performed using immunohistochemistry and immunocytochemistry techniques. Cells were reacted with primary antibody (2 hours, 25 °C), washing (PBS), and secondary antibody (1 hour, 25 °C). Cell nuclei were analyzed using DAPI staining (1:5,000), and mitochondria were analyzed using MitoTracker Red (250 nM). Coverslips were mounted on glass slides using a Dako fluorescence mount (Dako, Denmark) and cells were analyzed using a laser. Quantitative analysis was performed using a scanning confocal microscope LSM 710 META (Zeiss, Germany) and ImageJ.

4) Proteomic and metabolome analysis methods

Pieces (1 mm ² cubes) cut from protein bands/spots (1D/2D) were used in Coomassie destaining buffer (200 μL of 50% MeCN in 25 mM NH ₄ HCO ₃ , pH 8.0, 20 min, 25 °C) or 30 °C. Silver decolorization buffer containing mM potassium ferricyanide (50%) and 100 mM sodium thiosulfate (50%) solution was used. Gels were dehydrated using acetonitrile (200 μL).

Diabetic retinopathy-related proteins were reduced (100 mM NH ₄ HCO ₃ , 10 mM DTT, 56 °C, 30 minutes), and alkylated (100 mM NH ₄ HCO ₃ , 55 mM iodoacetamide, 20 minutes, 25 °C, dark). and trypsin reaction (13 ng/μL sequencing grade trypsin, Promega, 10 mM NH ₄ HCO ₃ , 10% MeCN, 37° C., 12 hours). Peptides were extracted with buffer (50 μL of 50% MeCN in NH ₄ HCO ₃ , 5% formic acid, 20 min, 37 °C), dried and dissolved in mass spectrometry sample buffer (5-10 μL in NH ₄ HCO ₃ 75% MeCN, 1% trifluoroacetic acid). Organic matrix (alpha-cyano-4-hydroxycinnamic acid, 5 mg/mL, MW 189.04, Sigma-Aldrich, St. Louis, MO) buffer (50% MeCN, 50% NH4HCO3, 1% trifluoroacetic acid) and after centrifugation (13,000 xg, 5 min), matrix peptides (0.5 μL) were prepared using a MALDI plate (Ground steel, Bruker Daltonics, Germany) and the peptide mass was 800-3,000 Da (Flex MALDI-TOF mass spectrometer, Bruker Daltonics, Germany) 70-75% laser intensity and 100-300 shots were used. All spectra were analyzed using tryptic peptide (842.5099, 2211.105 Da) Flex analysis software.

Mascot software (Matrix Science) and proteins were searched using NCBI/SwissProt. A database with the following criteria was used: 50 ppm mass tolerance, zero missing truncation, carbamidomethyl cysteine, methionine oxidation, and 4 minimum peptide matches. Protein identification was verified based on protein sequence coverage, using the number of matching peptides, the MOWSE score, and the tandem mass amino acid sequence, and the MOWSE score was expressed as a probability value for calculating -10 logP.

More than 1,000 proteins were analyzed in the retina of normal eyes and diabetic retinopathy donor eyes.

SwissProt search (http://www.expasy.ch/spot/), HPRD (http://hprd.org) and Gene Ontology (GO) database (http://www.geneontology.org), TargetP (cbs. Gene fusion using software such as dtu.dk/service/TargetP/) and Psort (http://psort.ims.utokyo.ac.jp/), STRING 11 (http://string-db.org/) (red), co-occurrence (dark blue), co-expression (black), binding experiments (purple), databases (blue), text mining (lime) and homology (turquoise), etc. .uth.edu/retnet/; RPGeNet 2.0; RD5000 DB; RetinoBase) was used.

Diabetic retinopathy metabolites were analyzed with OmicsNet.ca software using KEGG (Kyoto Encyclopedia of Genes and Genomes) and PubChem accessions (https://pubchem.ncbi.nlm.nih.gov/rest/pug). In order to discover the hidden interactions of diabetic retinopathy, the degree of association between proteome and metabolome was analyzed.

Statistical values were expressed as the average of three independent experiments and two-group comparisons were performed using a 2-tailed t-test. Multiple comparisons were evaluated by ANOVA and P < 0.05 was considered statistically significant using Tukey or Dunnet's test where applicable.

Experiment result

1 is a graph confirming that mitochondrial prohibitin (PHB) is reduced in hyperglycemia through experimental examples of cell models, animal models, and donated eyes of patients with diabetic retinopathy.

It was confirmed that actin, tubulin βIII and vimentin were changed in diabetic retinopathy. Tubulin βIII was significantly decreased in the GCL, INL, and RPE layers, and these changes indicate that the mitochondria of diabetic retinopathy cells may be altered by actin, tubulin, and vimentin polymers.

As a result of comparative analysis of more than 1,000 retinal proteins in the proteomic body of diabetic retinopathy donor eyes with normal eyes, changes in PI3K-AKT, WNT protein, p53, VEGF, and HIF-1 protein were confirmed in the retinal cells of diabetic retinopathy. did As a result of these proteomic changes, an increase in complement proteins related to rhodopsin/retinoid reproduction, mitochondrial structure, and insulin resistance was confirmed.

The diabetic retinopathy-related biomarkers found in this experimental example and the changes in diabetic retinopathy of each biomarker are shown in Table 2 below.

No. Protein Gene MW pI change One Prohibitin PHB 29820 5.57 decrease 2 New protein A (CAC2PBS) CAC2 27756 5.37 increase 3 New protein B (VIMIII) VIMIII 47488 5.09 increase 4 New protein C (FAM81B) FAM81B 48781 6.78 increase 5 New protein D (ADAM18) ADAM18 135097 8.91 increase 6 Adenylate cyclase 4 ADCY4 118799 8.15 increase 7 Myogenic regulatory factor 2 MRF2 12630 9.75 increase 8 Vinculin VCL 116615 5.83 increase 9 T cell receptor beta TCRB 35227 8.78 decrease 10 Toll-like Receptor 7 TLR7 121299 6.38 increase 11 SH3 domain-binding protein 1 SH3BP1 74852 6.16 increase 12 Thioredoxin doamin containing protein 17 TXNDC17 14092 4.85 increase 13 Insulin like growth factor 2 receptor A IGF2R 174358 5.45 decrease 14 Junction plakoglobin JUP 81801 5.75 decrease 15 Microtubule-actin cross linking factor 1 MACF1 830743 5.29 increase 16 Dynamin-1like protein DNM1L 83908 6.64 increase 17 E3 ubiquitin-protein ligase RNF135 RNF135 46012 5.53 decrease 18 Regulator of G-protein signaling 3 CRA_d RGS3 68401 8.54 increase 19 Prohibitin 2 PHB2 33296 9.21 decrease 20 Activated protein C APC 56200 4.40 increase 21 Ras-related C3 botulinum toxin substrate 1 RAC1/2 21450 8.77 increase 22 RhoA RHOA 21768 5.83 increase 23 calcium/calmodulin-dependent protein kinase II alpha CAMK2A 54088 6.61 increase 24 Complement 3 C3 187148 5.75 increase 25 Disintegrin and metalloproteinase domain-containing protein 17 ADAM17 93021 5.6 increase 26 vascular cell adhesion molecule 1 VCAM1 81276 5.14 increase 27 Estrogen receptor alpha ESR1 66216 8.30 increase 28 Son of Sevenless homolog 1 SOS1 152464 6.38 increase

Four novel proteins were further analyzed using bioinformatics tools. Novel protein 1 was named CAC2PBS. Novel protein 2 was named disintegrin and metalloproteinase (ADAM18), novel protein 3 was a vimentin variant (VIMIII), and novel protein 4 was named FAM81B. 2 to 5 show the amino acid sequences of novel proteins A to D, respectively. In addition to these novel proteins, novel biomarkers for diagnosis and/or treatment of diabetic retinopathy include prohibitin (PHB) and vinculin. , VCL), tubulin beta III (tubulin beta, TUBB3), E3 ubiquitin-protein ligase (RNF135), microtubule-actin crosslinking factor 1 (MACF1), TCRB protein (TCRB), tolllike receptor 7 precursor (TLR7), Insulin-like growth factor 2 receptor (IGF2R) and myogenic regulatory factor 2 (MRF2) were discovered.

That is, the novel biomarkers for the diagnosis and/or treatment of diabetic retinopathy identified in this experiment are ADAM18, FAM81B, PHB, TXNDC17, ADCY4, VCL, DNM1L, RNF135, CAC2PBS, SH3BP1, MRF2, TLR7, TCRB, MACF1, JUP and IGF2, etc. These biomarkers of diabetic retinopathy show that mitochondrial protein trafficking, crystalline aggregation, ubiquitin-based protein degradation, and complementary inflammatory responses may correspond to the pathogenesis mechanisms of diabetic retinopathy.

In addition, the four novel proteins found through this experimental example are confirmed to have the following functions, but are not limited thereto.

ADAM18 regulates phosphorylation signals including EGFR, PI3K, and APC, regulates RAC1/2 and RHO through TGFA interaction, and regulates calcium signaling kinase IIα/β (CAMK2A/B) and calcium channel (CACNG2) through calcium/calmodulin appears to be involved in one or more of the Indirectly, it appears to be involved in regulating estrogen receptor (ESR1) phosphorylation separately through PI3K catalytic subunits (PIK3CA, p110) and regulatory subunits (PIK3R1, p85), and regulating RAC1, CDC42 and RHOA molecules through EGFR. In addition, it seems that CAMK and MYO can be regulated through DLG1, and ubiquitin concentration can be controlled through ADAM18-UBE3A binding and ubiquitin ligase (E3A).

In addition, it seems that the ADAM18 protein complex can have bidirectional signals depending on ADAM18 phosphorylation, the first is the RHO kinase pathway through ADAM18-TGFA-EGFR-RHOA, and the second is ADAM18-ESR1-PI3K -PI3K-AKT signaling pathway leading to RAC1-CDC42-RAC2 pathway.

The CCAC2PBS protein has a calcium-binding C2 domain and a phospholipid binding switch, and appears to be involved in the formation of phospholipid proteomes due to Ca ²⁺ concentration.

VIMIII proteins appear to be able to build the cytoskeleton, including mitochondria along with intermediate filaments and actin microfilaments along with microtubules. This protein is involved in neurogenesis and cholesterol transport and can regulate retinal cell adhesion, migration and phosphorylation.

The FAM81B protein appears to be able to bind DNA.

Mechanisms of diabetic retinopathy confirmed through changes in the entire protein body of this experimental example as described above are changes in energy metabolism and tubulin phosphorylation, changes in the rhodopsin cycle and reduced retinoid concentration, mitochondrial death, angiogenic response, apoptosis and cholesterol changes It can be seen that it is related to at least one of the changes in lipid metabolism due to , the turn on of the retinal cell death switch, and the rise of inflammatory markers.

Although the above has been described with reference to the embodiments of the present invention, this is only an example and does not limit the present invention, and those skilled in the art to which the present invention belongs will be able to It will be appreciated that various modifications and applications not exemplified above are possible. For example, each component specifically shown in the embodiment of the present invention can be modified and implemented. And differences related to these modifications and applications should be construed as being included in the scope of the present invention as defined in the appended claims.

Claims (7)

Including an agent for measuring the level of a biomarker related to diabetic retinopathy (DR),
The biomarker includes SOS1 (Son of Sevenless homolog 1)
A composition for diagnosing diabetic retinopathy. The method of claim 1,
The biomarker is activated protein C (Activated protein C), Ras-related C3 botulinum toxin substrate 1 (Ras-related C3 botulinum toxin substrate 1), RhoA (Ras homolog family member A), calcium / calmodulin-dependent protein kinase II Further comprising at least one selected from the group consisting of alpha (calcium / calmodulin-dependent protein kinase II alpha) and complement protein 3 (Complement 3)
A composition for diagnosing diabetic retinopathy. The method of claim 1,
The biomarker further comprises vascular cell adhesion molecule 1
A composition for diagnosing diabetic retinopathy. (a) measuring the level of the biomarker in a biological sample isolated from the subject;
(b) comparing the measured level to a control sample; and
(c) diagnosing that there is a possibility of diabetic retinopathy if the measured level is increased compared to the control sample,
The biomarker is SOS1 (Son of Sevenless homolog 1)
Information provision method for diagnosis of diabetic retinopathy. The method of claim 4,
The biomarker is activated protein C (Activated protein C), Ras-related C3 botulinum toxin substrate 1 (Ras-related C3 botulinum toxin substrate 1), RhoA (Ras homolog family member A), calcium / calmodulin-dependent protein kinase II Further comprising at least one selected from the group consisting of alpha (calcium / calmodulin-dependent protein kinase II alpha) and complement protein 3 (Complement 3)
Information provision method for diagnosis of diabetic retinopathy. The method of claim 4,
The biomarker further comprises vascular cell adhesion molecule 1
Information provision method for diagnosis of diabetic retinopathy. delete

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