KR102293148B1 - Biomarker for early diagnosing diabetes and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for early diagnosis of diabetes and its use, and more particularly, to an NGF-β biomarker for early diagnosis of diabetes, a composition for early diagnosis of diabetes using the biomarker, a diagnostic kit, and necessary for early diagnosis of diabetes How to provide information.
Since it was confirmed that the expression of the NGF-β biomarker for early diagnosis of diabetes of the present invention is specifically decreased compared to that of normal subjects in the pre-diabetes stage, it can be usefully used for early diagnosis of diabetes.

Description

Biomarker for early diagnosis of diabetes and use thereof

The present invention relates to a biomarker for early diagnosis of diabetes and its use, and more particularly, an NGF-β biomarker for early diagnosis of diabetes, a composition for early diagnosis of diabetes using the biomarker, a diagnostic kit, and necessary for early diagnosis of diabetes How to provide information.

Diabetes mellitus (糖尿病; diabetes mellitus) is a metabolic disease characterized by hyperglycemia caused by defects in insulin secretion or insulin action. It is a chronic degenerative disease accompanied by

In particular, type 2 diabetes occurs when the blood sugar control fails because insulin secretion is smooth but insulin does not function properly due to acquired factors caused by lack of exercise, obesity, or stress.

Currently, the prevalence of diabetes is increasing year by year due to changes in diet according to rapid economic development, reaching about 5 to 10% in Korea. As of 2005, the number of diabetic patients exceeded 4 million, 83 out of 100, and by 2025. It is estimated that the number will increase to about 6.8 million in the next year (Korea Diabetes Information Center, 2005) Diabetes is the third most serious disease worldwide and the fourth leading cause of death in Korea. will be shortened to some extent.

It is estimated that the main cause of such a rapid increase in prevalence is changes in environmental factors, such as improved nutritional status and lack of exercise. In Korea, non-insulin-dependent type 2 diabetes patients account for 90-95% of all diabetes patients. This is increasing at a frightening rate. In the existing diabetes diagnosis, only the blood glucose level or the glycated hemoglobin level is diagnosed, but since this is a clinical sign after the progress of diabetes is sufficiently advanced, it is difficult to use as a tool for early diagnosis. can't see that

Accordingly, the present inventors made efforts to discover blood protein markers with high sensitivity and specificity for early diagnosis of diabetes. As a result, NGF-β (Nerve Growth Factor-beta) biomarkers were selected, and in the case of NGF-β, specifically diabetes mellitus. In the previous step, it was confirmed that the expression decreased compared to normal subjects, and the present invention was completed.

An object of the present invention is to provide a biomarker for early diagnosis of diabetes.

Another object of the present invention is to provide a composition or diagnostic kit for early diagnosis of diabetes using the biomarker.

Another object of the present invention is to provide a method for providing information necessary for early diagnosis of diabetes using the biomarker.

In order to achieve the above purpose,

The present invention provides a biomarker for early diagnosis of diabetes comprising NGF-β (Nerve Growth Factor-beta).

In addition, the present invention provides a composition for early diagnosis of diabetes comprising an agent for measuring the mRNA or protein level of NGF-β.

In another preferred embodiment of the present invention, the agent for measuring the mRNA level of NGF-β may be a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene of the marker.

In another preferred embodiment of the present invention, the agent for measuring the protein level of NGF-β is an antibody that specifically binds to the protein or peptide fragment, an interacting protein, a ligand, nanoparticles, or a pressure It may contain an aptamer.

In addition, the present invention provides a kit for early diagnosis of diabetes comprising the composition for early diagnosis of diabetes.

In a preferred embodiment of the present invention, the kit is RT-PCR (Reverse transcription polymerase chain reaction) kit, DNA chip kit, ELISA (Enzymelinked immunosorbent assay) kit, protein chip kit, rapid (rapid) kit or MRM (Multiple) kit reaction monitoring) kit.

In addition, the present invention comprises the steps of (a) measuring the mRNA or protein level of NGF-β from a biological sample of a patient; and

(b) comparing the mRNA or protein expression level with a normal subject sample; provides an information providing method for early diagnosis of diabetes, including.

In a preferred embodiment of the present invention, the mRNA expression level measurement is performed using a reverse transcriptase polymerase reaction, a competitive reverse transcriptase polymerase reaction, a real-time reverse transcriptase polymerase reaction, an RNase protection assay, Northern blotting or a DNA chip. can do.

In another preferred embodiment of the present invention, the measurement of the protein expression level is performed using an antibody that specifically binds to a protein or peptide fragment, an interacting protein, a ligand, nanoparticles or an aptamer. can do.

In another preferred embodiment of the present invention, the protein expression level measurement is protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced) Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D electrophoresis analysis, liquid chromatography- Liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry/Mass Spectrometry (LCMS/MS), Western blot, enzyme linked immunosorbent assay (ELISA), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), selected reaction monitoring (SRM) or immuno multiple reaction monitoring (iMRM) It can be done using

In another preferred embodiment of the present invention, the information providing method for the early diagnosis of diabetes further comprises the step of determining that the gene expression level or protein expression level of NGF-β is reduced compared to that of a normal subject, determining that it is an early stage of diabetes. may include

In another preferred embodiment of the present invention, the analysis of step (b) may be performed by a statistical analysis method.

In another preferred embodiment of the present invention, the statistical analysis method is a linear or non-linear regression analysis method, a linear or non-linear classification analysis method, a logistic regression analysis method, an analysis of variance (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 analysis method, Markov Blanket analysis method , recursive feature elimination or entropy-based regression feature elimination analysis method, forward floating search or backward floating search analysis method, and combinations thereof. .

Since it was confirmed that the expression of the NGF-β biomarker for early diagnosis of diabetes of the present invention is specifically decreased compared to that of normal subjects in the pre-diabetes stage, it can be usefully used for early diagnosis of diabetes.

1 is data visualized as clusters and heat maps of results of performing antibody arrays on blood of normal people, pre-diabetes, and diabetic patients.
Figure 2 is data analyzing the level of NGF-β expression in the blood of normal people, pre-diabetes and diabetic patients.

Hereinafter, the present invention will be described in detail.

The present invention relates to a biomarker for early diagnosis of diabetes comprising NGF-β (Nerve Growth Factor-beta).

In the present invention, the diabetes is characterized in that type 2 diabetes.

As used herein, the term “diagnosis” refers to ascertaining the presence or characteristics of a pathological condition. For the purpose of the present invention, the diagnosis is early diagnosis of diabetes and confirming whether diabetes is invented.

As used herein, the term "diagnostic biomarker" refers to a polypeptide or nucleic acid (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, and organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.), preferably NGF-β (Nerve Growth Factor-beta).

In a specific embodiment of the present invention, the results of performing the antibody array on the blood of normal people, pre-diabetes and diabetic patients were visualized as clusters and heat maps (FIG. 1). As a result, Nerve Growth Factor-beta (NGF-β) biomarkers were selected, and in the case of NGF-β, it was confirmed that the expression of NGF-β was significantly reduced compared to normal in the pre-diabetic stage specifically ( FIG. 2 ).

Specifically, when the expression of NGF-β is reduced by 3 to 4 times compared to normal people, it is possible to diagnose early stage diabetes (pre-diabetes stage), so the NGF-β biomarker of the present invention can be used for early diagnosis of diabetes.

In another aspect, the present invention relates to a composition for early diagnosis of diabetes comprising an agent for measuring the mRNA or protein level of NGF-β.

As used herein, the term “mRNA expression level measurement” refers to a process of determining the presence and expression level of mRNA of a biomarker for early diagnosis of diabetes in a biological sample, and measures the amount of mRNA. Analysis methods for this are 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; RNaseprotection assay) ), Northern blotting, DNA chip, and the like, but is not limited thereto.

The agent for measuring the mRNA level of NGF-β is characterized in that it is a primer pair, probe, or antisense nucleotide that specifically binds to the NGF-β gene. Based on these primer pairs, probes or antisense nucleotides can be designed.

As used herein, the term “primer” refers to a fragment recognizing a target gene sequence, including a pair of forward and reverse primers, but preferably a primer pair that provides analysis results with specificity and sensitivity.

As used herein, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and means a substance capable of specifically confirming the presence of a target substance in the sample through the binding. do. The type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably It is PNA.

As used herein, the term "antisense" means that an antisense oligomer hybridizes with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of a mRNA and RNA:oligomeric heteroduplex within the target sequence, a nucleotide base It refers to an oligomer having a sequence and a backbone between subunits. An oligomer may have exact sequence complementarity or approximate complementarity to a target sequence.

In the present invention, the agent for measuring the protein level of NGF-β is an antibody that specifically binds to the protein or peptide fragment, an interacting protein, a ligand, nanoparticles, or an aptamer can be done with

As used herein, the term "measurement of protein expression level" is a process of confirming the presence and expression level of a protein expressed from a biomarker for early diagnosis of diabetes or diagnosis of diabetic complications in a biological sample. The amount of the protein can be confirmed using an antibody, an interacting protein, a ligand, nanoparticles, or an aptamer that specifically binds to a protein or peptide fragment of the gene.

The protein expression level measurement or comparative analysis method includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time) analysis. of Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (liquid chromatography) -Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbent assay), multiple reaction monitoring (MRM), parallel reaction monitoring (parallel reaction monitoring: PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), selected reaction monitoring (SRM) or immuno multiple reaction monitoring (iMRM) It is not limited thereto.

As used herein, the term "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a biomarker of the present invention. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

In another aspect, the present invention relates to a kit for early diagnosis of diabetes comprising the composition for early diagnosis of diabetes.

The kit may be prepared by a conventional manufacturing method known in the art. The kit may include, for example, a freeze-dried antibody, a buffer, a stabilizer, an inactive protein, and the like.

The kit may further include a detectable label. The term "detectable label" refers to an atom or molecule that allows for specific detection of a molecule comprising a label among molecules of the same type without a label. The detectable label may be attached to an antibody, an interacting protein, a ligand, a nanoparticle, or an aptamer that specifically binds to the protein or fragment thereof. The detectable label may include a radioactive species (radionuclide), a fluorophore (fluorophore), and an enzyme (enzyme).

The kit may be used according to various immunoassays or immunostaining methods known in the art. The immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining and immunoaffinity purification. Preferably, the kit 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 MRM (multiple reaction monitoring). have.

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

In another aspect, the present invention comprises the steps of (a) measuring the mRNA or protein level of NGF-β from a biological sample of a patient; and

(b) comparing the mRNA or protein expression level with a normal subject sample; relates to a method for providing information for early diagnosis of diabetes comprising a.

In the above method, "biological sample" means a tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid or urine sample, etc. in which the protein expression level or gene expression level is different due to the onset of diabetes, Preferably, it means blood, plasma, and serum.

In the present invention, the method for providing information for early diagnosis of diabetes may further include determining that the gene expression level or protein expression level of NGF-β is decreased compared to that of a normal subject, determining that the diabetes is in the early stage.

The mRNA expression level measurement in step (a) is measured or compared to the mRNA expression level measurement or comparison is reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc. may be used, but the present invention is not limited thereto, and the specific details are as described above.

In addition, the protein expression level measurement in step (a) can be measured and compared using an antibody, an interacting protein, a ligand, nanoparticles or an aptamer that specifically binds to a protein or peptide fragment. , the details are the same as described above.

The analysis in step (b) may be analyzed using a statistical method or algorithm to improve the accuracy of diagnosis, and a linear or non-linear regression analysis method, a preceding or non-linear classification analysis method, a logistic regression analysis method (logistic regression analysis method) 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 or kernel principal component analysis using decision tree method, Markov Blanket analysis method, recursive feature elimination or entropy-based regression feature elimination analysis method, forward floating search or backward floating search analysis method, and their methods An analysis method selected from the group consisting of combinations may be used. Preferably, in the present invention, the statistical method uses a logistic regression analysis method, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Patient selection and blood collection

In the present invention, in order to select biomarkers for early diagnosis of diabetes, normal persons, pre-diabetes and diabetic patients were selected and blood of the patients was collected. A total of 43 patients were selected, and the clinical characteristics of the patients are shown in Table 1 below.

Patient's clinical information Normal(16) IFG(14) DM(13)
Shin Hwan Age 54.00±2.73 66.43±4.64 59.31±9.20 height 158.41±5.32 154.82±6.11 156.76±5.70 weight 55.15±5.27 54.41±6.56 57.92±5.19 BMI 22.04±1.54 22.61±1.90 23.53±1.50 waist 74.58±5.57 78.38±4.69 82.50±5.70(10) FBS 86.44±5.01 105.86±5.65 126.92±20.79 HbA1c 5.33±0.17 6.04±0.17 7.36±0.94 M/C ratio -(0) 9.77±8.91 14.24±7.79 hypertension [1/0] 0/16 3/11 2/11 Hyperlipidemia[1/0] 0/16 2/12 5/8 Smoking[1/0] 0/16 0/14 0/13 Drinking[1/0] 3/13 0/14 1/12

In Table 1, the number in parentheses is the number of samples, "Normal" means normal, "IFG" means pre-diabetes, and "DM new patient" means a patient who has just been diagnosed with diabetes and has never taken a diabetes treatment.

for early diagnosis of diabetes biomarker Selection

2-1: protein isolation

Protein extraction buffer (Fullmoon biosystems, Canada) and lysis beads (Fullmoon biosystems, Canada) containing 1% protease inhibitor cocktail (Sigma, USA) and 1% phosphatase inhibitor cocktail (Sigma, USA) was used to extract the protein.

The extracted protein solution was purified using a gel matrix column included in an antibody array assay kit (Fullmoon biosystems, Canada). The column was vortexed for 5 seconds, and after hydration treatment at room temperature for 60 minutes, the column was centrifuged at 750 × g for 2 minutes. After centrifugation, the column was placed in a collection tube and 100 μl of a protein sample was added to the column, followed by centrifugation at 750 × g for 2 minutes to purify the protein.

The concentration of the purified sample was measured using a BCA protein analysis kit (Pierce, USA) and a nanophotometer (NanoPhotometer™, Implen, UK), and the purity of the purified sample was confirmed by UV spectrum.

2-2: Antibody array assay

50 μg of protein sample was filled with 75 μl of labeling buffer and treated with 3 μl of 10 μg/μl biotin/DMF solution. The samples were mixed and reacted at room temperature for 90 minutes. After the reaction, 35 μl of stop solution was further mixed, followed by reaction at room temperature for 30 minutes.

An antibody microarray slide (Fullmoon biosystems, Canada) was placed on a Petri dish and treated with 30 ml of blocking solution, then incubated on a shaker at 60 rpm for 30 minutes at room temperature and washed with distilled water. , this was repeated 3 times. After blocking the slides were washed with Milli-Q grade water.

After mixing the labeled sample with 6 ml of a coupling solution, the mixed coupling solution was treated on a blocking slide, and then reacted on a shaker at 60 rpm for 5 minutes. After coupling, the slides were washed with 30 ml of washing solution on a 60 rpm shaker for 5 minutes, and this was repeated 6 times. Finally, the slides were washed with Milli-Q grade water.

30 μl of 0.5 mg/ml Cy3-streptavidin (Cy3-streptavidin, GE Healthcare, UK) was mixed with 30 ml of detection solution, then treated on the coupled array slides, and 20 min on a 60 rpm shaker. reacted while After the detection reaction, the slide was treated with 30 ml of a washing solution and washed for 5 minutes on a 60 rpm shaker, and this was repeated 6 times. Finally, the slides were washed with Milli-Q grade water.

2-3: Antibody Array Data Collection and Analysis

Slide scans were performed using a GenePix 4100A scanner (Axon Instrument, USA). Slides were completely dried prior to scanning and scanned within 24-48 hours. Slides were scanned with 10 μm resolution, optimal laser power and PMT. After the scan images were obtained, they were gridded and quantified with GenePix 7.0 software (Axon Instrument, USA).

Numerical data were analyzed using Genowiz 4.0TM (Ocimum Biosolutions, India), and after analysis, data on protein information were annotated using UniProt DB.

2-4: Clustering heat map visualization

Sample and gene clustering was performed using MeV 4.9.0. Hierarchical cluster analysis was performed using Euclidean distance correlation as a distance measure through mean linkage. Clusters and heatmaps were visualized using MeV 4.9.0. (Fig. 1).

2-5: biomarker selection

Based on the normalized data as described above, a fold change was obtained by comparing normal subjects, pre-diabetes and diabetic patients, and markers showing a specific pattern in pre-diabetes or diabetes were selected.

As a result, NGF-β (Nerve Growth Factor-beta) biomarkers were selected.

NGF -β degree of expression comparison analysis

In the present invention, based on the data normalized in Example 2, the expression level of NGF-β was compared and analyzed based on the fold change value by dividing into normal people, pre-diabetic patients and diabetic patients.

Comparison of expression level of NGF-β Normal IFG (pre-diabetes) DM (diabetes) fold change One -3.75 1.04

As a result, as shown in FIG. 2 , it was confirmed that, in the case of NGF-β, the expression of NGF-β was significantly reduced compared to normal in the pre-diabetic stage specifically. Specifically, it was confirmed that when the expression of NGF-β is reduced by 3 to 4 times compared to normal persons, it can be diagnosed as an early stage of diabetes (pre-diabetes stage).

Therefore, the NGF-β biomarker of the present invention can be utilized for early diagnosis of diabetes.

Claims (8)

delete delete A composition for diagnosing a fasting blood glucose disorder (IFG) comprising an agent for measuring the level of mRNA or protein of NGF-β.
The composition for diagnosing a fasting blood glucose disorder (IFG) according to claim 3, wherein the agent for measuring the mRNA level of NGF-β is a primer pair, a probe, or an antisense nucleotide that specifically binds to the NGF-β gene. .
The method according to claim 3, wherein the agent for measuring the protein level of NGF-β comprises an antibody, an interacting protein, a ligand, nanoparticles or an aptamer that specifically binds to the protein or peptide fragment. A composition for diagnosis of fasting blood glucose disorder (IFG), characterized in that
A kit for diagnosing fasting blood glucose disorder (IFG) comprising the composition for diagnosis of any one of claims 3 to 5.
(a) measuring the mRNA or protein level of NGF-β from the patient's biological sample;
(b) comparing the mRNA or protein expression level with a sample of a normal subject and a subject with type 2 diabetes; and
(c) determining that the level of mRNA or protein expression of NGF-β decreases as compared to normal subjects and type 2 diabetic subjects as impaired fasting blood glucose (IFG);
A method for providing information for diagnosing a fasting blood glucose disorder (IFG) comprising a. delete

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