KR101657051B1 - Marker composition for diagnosis of chronic obstructive pulmonary disease - Google Patents

Abstract

The present invention relates to a method for measuring the expression level of mRNA of mRNA of SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Protein, SnoN) A composition for the diagnosis of chronic obstructive pulmonary disease (COPD), a kit for diagnosing chronic obstructive pulmonary disease including the composition, and a method for providing information for diagnosis of chronic obstructive pulmonary disease using the composition . SKIL, which is a marker gene of the present invention, is remarkably increased in patients with chronic obstructive pulmonary disease as compared with normal individuals, so SKIL can be used to accurately and quickly diagnose the onset of COPD.

Description

[0001] The present invention relates to a marker composition for diagnosis of chronic obstructive pulmonary disease

The present invention relates to a method for measuring the expression level of mRNA of mRNA of SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Protein, SnoN) A composition for the diagnosis of chronic obstructive pulmonary disease (COPD), a kit for diagnosing chronic obstructive pulmonary disease comprising the composition, and a method for providing information for diagnosis of chronic obstructive pulmonary disease using the marker .

Chronic Obstructive Pulmonary Disease (COPD) is a chronic obstructive pulmonary disease (COPD) that is caused by abnormal inflammation of the lungs caused by inhalation of harmful particles or gases, to be. Emphysema, and chronic bronchitis.

It is estimated that chronic obstructive pulmonary disease is the fifth leading cause of death in the world and the fourth leading cause of death in 2020. In Korea, the prevalence rate is 7.9%, and the national health insurance expenditure due to chronic obstructive pulmonary disease for one year reaches 60 billion won, which is a health and economically important disease. Chronic obstructive pulmonary disease (COPD) is an ongoing disease that requires early diagnosis and prevention as other chronic diseases. There are studies that have clear diagnostic criteria such as hypertension and diabetes mellitus, which are typical chronic diseases, and lowered complications and mortality when they are administered, and there is active effort for early diagnosis. However, there is much debate about the early diagnosis of chronic obstructive pulmonary disease, and no specific guidelines are available in Korea.

Thus, chronic obstructive pulmonary disease is a preventable and treatable disease, and proper diagnosis and treatment are very important. However, biomarkers for diagnosing chronic obstructive pulmonary disease are rarely studied.

In addition, proteins encoded by SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN) It is known as a component of the SMAD pathway that regulates differentiation, but the relationship between SKIL gene and chronic obstructive pulmonary disease has not been elucidated.

Accordingly, the present inventors have continued to develop a marker for diagnosing chronic obstructive pulmonary disease (COPD). As a result, the present inventors have found that SKIL, a gene whose expression is remarkably increased in patients with chronic obstructive pulmonary disease Thereby completing the present invention.

It is an object of the present invention to provide a method of treating chronic obstructive pulmonary disease (COPD) including SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, pulmonary disease (COPD)) diagnostic biomarker composition.

It is an object of the present invention to provide a composition for diagnosing chronic obstructive pulmonary disease, which comprises an agent for measuring an expression level of mRNA of SKIL gene or a protein thereof.

Another object of the present invention is to provide a kit for the diagnosis of chronic obstructive pulmonary disease comprising the above composition.

It is still another object of the present invention to provide a method for providing information for diagnosis of chronic obstructive pulmonary disease using the marker.

In order to solve the above-mentioned problems, the present invention provides a method for preventing chronic obstructive lung disease including SKIL (Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN) A biomarker composition for the diagnosis of chronic obstructive pulmonary disease (COPD) is provided.

The present invention also provides a composition for the diagnosis of chronic obstructive pulmonary disease, which comprises an agent for measuring an expression level of mRNA of SKIL gene or a protein thereof.

The present invention also provides a kit for the diagnosis of chronic obstructive pulmonary disease comprising the above composition.

In addition, the present invention provides a method for providing information for diagnosis of chronic obstructive pulmonary disease using the marker.

SKIL-like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Protein, SnoN) Since the expression is remarkably increased, it can be usefully used for accurately and quickly diagnosing the onset of COPD.

FIG. 1 is a schematic view of a lung tissue of a normal person, chronic obstructive bronchitis and a lung obstructive bronchitis, which is a kind of chronic obstructive pulmonary disease (COPD), and chronic obstructive pulmonary disease (COPD) FIG. 2 is a graph showing the expression level of SKIL protein in the lung tissue of a bronchial asthma patient, which is a kind of non-lung disease.
2 is a graph showing the expression level of SKIL protein in plasma of a normal human and chronic obstructive pulmonary disease patients.

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

The present invention relates to a method of treating a chronic obstructive pulmonary disease (SKN) including SKIL (Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, , COPD) diagnostic biomarker composition.

In the present invention, "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to ascertain whether or not the onset of chronic obstructive pulmonary disease is feasible.

The types of chronic obstructive pulmonary disease of the present invention include chronic obstructive bronchitis, chronic bronchiolitis, and emphysema.

In the present invention, the term "marker" refers to a substance capable of diagnosing chronic obstructive pulmonary disease, including a polypeptide associated with chronic obstructive pulmonary disease, a nucleic acid (e.g., mRNA), a lipid, a glycolipid, a glycoprotein, a sugar (monosaccharide, And the like), and the like. For the purposes of the present invention, the marker is SKIL, a gene whose expression is markedly increased in patients with chronic obstructive pulmonary disease.

The present invention also provides a composition for diagnosing chronic obstructive pulmonary disease (COPD) comprising an agent for measuring an expression level of mRNA of SKIL (SKI like oncogene, Gene ID: 6498) gene or a protein thereof .

In the present invention, " measurement of mRNA expression level " is a process of confirming the presence and expression level of mRNA of SKIL, a chronic obstructive pulmonary disease-related gene, in a biological sample in order to diagnose chronic obstructive pulmonary disease. For example, RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA). RNase protection assay, Northern blotting, DNA chip, and the like. The agent for measuring the mRNA level of a gene in the present invention is preferably an antisense oligonucleotide, a primer pair or a probe.

In the present invention, "antisense" means that the antisense oligomer is hybridized with the target sequence in the RNA by Watson-Crick base pairing to form the sequence of the nucleotide base, typically within the target sequence, And an oligomer having a backbone between subunits. Oligomers may have an exact sequence complement or approximate complementarity to the target sequence. This antisense oligomer can alter the processing of mRNA that blocks or inhibits translation of mRNA and produces splice variants of mRNA.

"Primer" in the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

In the present invention, "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to mRNA, and is labeled to detect the presence or absence of a specific mRNA Can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

Since the nucleic acid sequence of SKIL which is a gene related to chronic obstructive pulmonary disease of the present invention is registered in the gene bank, a person skilled in the art will design an antisense oligonucleotide, a primer pair or a probe that specifically amplifies a specific region of these genes based on the sequence .

The antisense oligonucleotides, primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, " measurement of protein expression level " is a process of confirming the presence and expression level of protein expressed from SKIL, a gene related to chronic obstructive pulmonary disease, in a biological sample in order to diagnose COPD. . Examples of the assay methods include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis But are not limited to, tissue immuno staining, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), protein chips, and the like. The agent for measuring the protein expression level in the present invention is preferably an antibody.

In the present invention, " antibody " means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since the marker of the present invention is remarkably increased in patients with chronic obstructive pulmonary disease as compared with normal subjects, the marker can be used to accurately and quickly diagnose the incidence of COPD.

The present invention also provides a kit for the diagnosis of chronic obstructive pulmonary disease comprising the above composition.

The kit of the present invention can diagnose chronic obstructive pulmonary disease by confirming the expression level of the SKIL gene mRNA or its protein. The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method as well as primers, probes, and antibodies for diagnosing COPD, preferably RT-PCR A kit, a microarray chip kit, a DNA chip kit, and a protein chip kit.

As a specific example, in the present invention, the kit for measuring the mRNA expression level of the marker genes may be a kit containing essential elements necessary for performing RT-PCR. RT-PCR kits contain enzymes such as test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC Water (DEPC-water), sterile water, and the like.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing the microarray. The microarray kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof, Can be easily produced by a production method commonly used in the art. In order to fabricate a microarray, a micropipetting method using a piezo electric method or a micropipetting method using a pin-shaped method to immobilize the detected marker on a substrate of a DNA chip using the probe as a probe DNA molecule A method using a spotter or the like is preferably used, but the present invention is not limited thereto. The substrate of the microarray chip is preferably coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but is not limited thereto. In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.

In addition, the kit of the present invention may be a kit containing essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate on which a cDNA or an oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

In addition,

(a) measuring the level of expression of an SKIL gene mRNA or protein thereof from a biological sample; And

(b) comparing the expression level of mRNA of SKIL gene or its protein with the level of mRNA of SKIL gene or protein thereof in a normal control sample, and do.

In the present invention, the term "biological sample" includes whole blood, serum, plasma, saliva, urine, sputum, lymph fluid, cells and the like isolated from an individual, preferably lung tissue.

Methods for measuring mRNA expression levels in the present invention include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA RNase protection assay, Northern blotting, DNA chip, and the like.

Methods for measuring the expression level of a protein in the present invention include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip. However, there are limitations on the use of immunoassays, It does not.

Since the marker gene SKIL of the present invention is remarkably increased in patients with chronic obstructive pulmonary disease as compared with that of normal individuals, the presence or absence of COPD can be accurately and quickly diagnosed through comparison with a normal control sample.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

Example 1. Measurement of SKIL gene expression level in lung tissue of patients with chronic obstructive pulmonary disease (COPD) using RNA sequencing

1-1. Preparation of mRNA samples

MRNA was obtained from lung tissues of 100 patients with chronic obstructive pulmonary disease (COPD) and 100 normal controls (control) of Asan Hospital (Seoul, Korea) according to conventionally known methods.

1-2. RNA sequencing

RNA sequencing (QIAGEN, RNA-Seq Experiment) was performed according to a conventionally known method from the sample obtained in 1-1. Then, the gene that expresses differentially compared to the lung tissue of the normal person was identified statistically significantly Respectively.

In the lung tissue of patients with chronic obstructive nitrification, the expression of SKIL gene was increased by about 163% compared to the lung tissue of the normal control group. This result was statistically significant (p = 8.95E-11). These results indicate that SKIL gene mRNA or its protein expression levels can be measured to diagnose chronic obstructive pulmonary disease.

Example 2 Confirmation of Expression of SKIL Protein in Pulmonary Tissue of Patients with COPD by Immunohistochemistry

In order to confirm whether the expression of SKIL protein increases specifically in the lung tissue of patients with COPD, it is necessary to evaluate the expression of SKIL protein in lung tissues of normal persons, chronic obstructive bronchitis (COPD) We performed immunohistochemistry on the lung tissue of patients with bronchial asthma, a type of pulmonary disease that is not a chronic obstructive pulmonary disease (COPD).

To perform the immunohistochemistry, a commercially available tissue array slide (Pantomics, LUD481) was purchased. Immunohistochemistry was performed on the paraffin tissue slides using an antibody against the SKIL gene (1:50, abcam) The law was carried out. The results are shown in Fig.

As shown in FIG. 1, the expression of SKIL gene was increased in the lung tissues of patients with chronic obstructive bronchitis and Emphysema, which is a kind of chronic obstructive pulmonary disease (COPD), compared with normal lung tissues, The expression of SKIL gene was not increased in pulmonary tissue of bronchial asthma, which is a kind of pulmonary disease other than pulmonary disease (COPD). The above results indicate that the protein expression level of the gene can be measured to diagnose chronic obstructive pulmonary disease.

Example 3 Measurement of SKIL Protein Level in Plasma of Patients with COPD Using ELISA

In order to confirm that the expression of SKIL protein increases specifically in the plasma of patients with COPD, an enzyme linked immunosorbent assay (ELISA) is performed on the plasma of normal human patients with COPD and patients with chronic obstructive pulmonary disease (COPD) Respectively.

To perform the ELISA method, an ELISA kit (Cloud-clone corp., SEM234Hu) for SKIL was purchased. Using the above ELISA kit, the amount of SKIL protein in plasma was measured for plasma obtained from 20 healthy persons and 20 COPD patients from Asan Medical Center (Seoul, Korea). Plasma was diluted to 1/8 concentration with the buffer provided in the ELISA kit. The results are shown in Fig.

As shown in FIG. 2, it was confirmed that the amount of SKIL protein in the plasma was significantly increased in the plasma of patients with COPD compared with the plasma of normal persons. The above results indicate that the protein expression level in the blood of the gene can be measured to diagnose chronic obstructive pulmonary disease.

Claims (11)

Chronic obstructive pulmonary disease (COPD), including SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Diagnostic biomarker composition. 2. The biomarker composition according to claim 1, wherein said chronic obstructive pulmonary disease comprises chronic obstructive bronchitis, chronic bronchiolitis or Emphysema. A composition for the diagnosis of chronic obstructive pulmonary disease, comprising an agent for measuring an expression level of mRNA of SKIL gene or a protein thereof. 4. The composition for the diagnosis of chronic obstructive pulmonary disease according to claim 3, wherein the agent for measuring the expression level of mRNA of the gene comprises an antisense oligonucleotide, a primer pair or a probe specifically binding to SKIL gene. The composition for diagnosing chronic obstructive pulmonary disease according to claim 3, wherein the agent for measuring the expression level of the protein comprises an antibody specific to SKIL protein. A kit for the diagnosis of chronic obstructive pulmonary disease comprising the composition of any one of claims 3 to 5. 7. The kit for diagnosing COPD according to claim 6, wherein the kit is an RT-PCR kit, a microarray chip kit, a DNA kit or a protein chip kit. (a) measuring the level of expression of an SKIL gene mRNA or protein thereof from a biological sample; And
(b) comparing the expression level of the mRNA of the SKIL gene or a protein thereof with the level of mRNA of the SKIL gene or a protein thereof in a normal control sample. The method according to claim 8, wherein the biological sample in step (a) is at least one sample selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph, Information Providing Method for Diagnosis of Obstructive Pulmonary Disease. The method of claim 8, wherein the mRNA expression level is measured by RT-PCR, competitive RT-PCR, real-time RT-PCR, Wherein the method is one or more methods selected from the group consisting of PCR, RNase protection assay (RPA), Northern blotting, and DNA chip, for the diagnosis of chronic obstructive pulmonary disease Way. The method of claim 8, wherein the level of expression of the protein in step (a) is measured by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip Assay are used for immunoassay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, wherein the method is one or more selected from the group consisting of a protein chip, and a method for diagnosing chronic obstructive pulmonary disease.

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