KR20140090570A - Marker composition for diagnosis of chronic obstructive pulmonary disease - Google Patents

Abstract

The present invention relates to: a composition, for diagnosing a chronic obstructive pulmonary disease (COPD), which includes the mRNA of one or more genes or proteins thereof which are selected from a group which is composed of a homeodomain-interacting protein kinase 3 (HIPK3), a guanine nucleotide binding protein G protein q polypeptide (GNAQ), a max dimerization protein 1 (MXD1), an ets variant 3 (ETV3), and a neuroblastoma breakpoint family, member 10 (NBPF10); a COPD diagnosis kit which includes the composition; and an information providing method for diagnosing the COPD using the composition. The present invention can be usefully used when accurately and quickly diagnosing whether a COPD occurs or not since the expression of a marker gene (HIPK3, GNAQ, MXD1, ETV3, or NBPF10) of the present invention remarkably increases in a COPD patient more than a normal person.

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 screening a compound of the present invention for use in the treatment or prevention of diseases such as HIPK3 (homeodomain interacting protein kinase 3), GNAQ (Guanine nucleotide binding protein, G protein q polypeptide), MXD1 (Max dimerization protein 1), ETV3 (ets variant 3) and NBPF10 A composition for the diagnosis of chronic obstructive pulmonary disease (COPD) comprising an agent for measuring an expression level of mRNA of the at least one gene selected from the group consisting of or a protein thereof, And a method for providing information for diagnosis of chronic obstructive pulmonary disease using the composition.

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 national health insurance expenditure for COPD 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 early diagnosis of COPD, and there is no specific guidance 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.

Therefore, the present inventors have continued to develop a marker for diagnosing chronic obstructive pulmonary disease. As a result, HIPK3, GNAQ, MXD1, ETV3 and NBPF10, which are remarkably increased in expression of COPD in patients with chronic obstructive pulmonary disease Thereby completing the present invention.

It is an object of the present invention to provide a method for screening a compound of the present invention for the treatment and / or prophylaxis of HIPK3 (homeodomain interacting protein kinase 3), GNAQ (Guanine nucleotide binding protein, G protein q polypeptide), MXD1 (Max dimerization protein 1), ETV3 (ets variant 3) and NBPF10 ) For the diagnosis of chronic obstructive pulmonary disease (COPD) comprising the mRNA of one or more genes selected from the group consisting of:

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

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

In order to solve the above-described problems, the present invention provides a method for diagnosing chronic obstructive pulmonary disease (COPD), comprising the step of measuring the mRNA of one or more genes selected from the group consisting of HIPK3, GNAQ, MXD1, ETV3 and NBPF10, obstructive pulmonary disease < RTI ID = 0.0 > (COPD) < / RTI >

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 above composition.

Since the expression of the marker gene of the present invention (HIPK3, GNAQ, MXD1, ETV3 or NBPF10) is significantly increased in patients with chronic obstructive pulmonary disease compared with normal individuals, it is useful for accurate and rapid diagnosis of the incidence of COPD .

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

The present invention also relates to a method for screening for Hodgkin's disease (HIPK3), GDNQ (Gene ID: 10114), GNAQ (Guanine nucleotide binding protein, Gene ID 2776), MXD1 (Max dimerization protein 1, Gene ID: and an agent for measuring an expression level of mRNA of the at least one gene selected from the group consisting of NBPF10 (ets variant 3, Gene ID: 2117) and NBPF10 (Neuroblastoma breakpoint family, member 10, Gene ID: 100132406) A composition for the diagnosis of chronic obstructive pulmonary disease (COPD) is provided.

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 a gene whose expression is markedly increased in chronic obstructive pulmonary disease patients with HIPK3, GNAQ, MXD1, ETV3 or NBPF10.

In the present invention, " measurement of mRNA expression level " is a process for determining the presence and expression level of mRNA of a chronic obstructive pulmonary disease-related gene (HIPK3, GNAQ, MXD1, ETV3 or NBPF10) in a biological sample in order to diagnose chronic obstructive pulmonary disease , and measuring the amount of mRNA. 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 serving 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.

The term "probe" in the present invention 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 so that 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 nucleotide sequence of the COPD gene (HIPK3, GNAQ, MXD1, ETV3 or NBPF10) of the present invention is registered in the gene bank, a person skilled in the art will be able to identify the nucleotide sequence of the antisense oligonucleotide Nucleotides, primer pairs or probes can be designed.

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 " refers to the presence and expression level of a protein expressed from a chronic obstructive pulmonary disease-related gene (HIPK3, GNAQ, MXD1, ETV3 or NBPF10) in a biological sample to diagnose chronic obstructive pulmonary disease And measuring the amount of protein. 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 mRNA or its protein of one or more genes selected from the group consisting of HIPK3, GNAQ, MXD1, ETV3 and NBPF10. 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. Using the marker of the present invention, 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 to which a cDNA or 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 expression level of mRNA of the at least one gene selected from the group consisting of HIPK3, GNAQ, MXD1, ETV3 and NBPF10 or a protein thereof, from the biological sample; And

(b) comparing the expression level of the mRNA or a protein thereof with the mRNA of the normal control sample or the expression level of the protein of the mRNA or the protein thereof, to provide information for diagnosing chronic obstructive pulmonary disease.

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 are examples of the immunoassay method. It is not limited.

Since the expression of the marker gene (HIPK3, GNAQ, MXD1, ETV3 or NBPF10) of the present invention is significantly increased in patients with chronic obstructive pulmonary disease as compared with that of normal individuals, comparison with normal control samples shows the presence or absence of chronic obstructive pulmonary disease Can be accurately and quickly diagnosed.

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  One. mRNA  Preparation of 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.

Example  2. RNA  Sequencing

RNA sequencing (QIAGEN, RNA-Seq Experiment) was performed from the sample obtained in Example 1 according to a conventionally known method to identify a gene that specifically increases in the lung tissue of patients with COPD.

The results are shown in Table 1 below.

gene In patients with normal COPD
Increase rate of gene expression HIPK3 (Homeodomain interacting protein kinase 3) 180% GNAQ (Guanine nucleotide binding protein, G protein q polypeptide) 160% MXD1 (Max dimerization protein 1) 160% ETV3 (ets variant 3) 160% NBPF10 (Neuroblastoma breakpoint family, member 10) 157%

As shown in Table 1, about 180% of the HIPK3 (Homeodomain interacting protein kinase 3) gene and the GNAQ (Guanine nucleotide binding protein, G protein q polypeptide) were detected in the lung tissues of patients with chronic obstructive pulmonary disease ) 160%, MXD1 (Max dimerization protein 1) gene about 160%, ETV3 (ets variant 3) gene about 160% and NBPF10 (Neuroblastoma breakpoint family, member 10) gene about 157% Respectively.

The above results indicate that the expression level of mRNA of the gene or the protein thereof can be measured to diagnose chronic obstructive pulmonary disease.

Claims (10)

A group consisting of HIPK3 (homeodomain interacting protein kinase 3), GNAQ (Guanine nucleotide binding protein, G protein q polypeptide), MXD1 (Max dimerization protein 1), ETV3 (ets variant 3) and NBPF10 (Neuroblastoma breakpoint family, member 10) A composition for the diagnosis of chronic obstructive pulmonary disease (COPD), comprising an agent that measures the level of expression of mRNA or a protein of the selected one or more genes.
The composition for the diagnosis of chronic obstructive pulmonary disease according to claim 1, wherein the chronic obstructive pulmonary disease is chronic obstructive bronchitis, chronic bronchiolitis, or Emphysema.
2. The method according to claim 1, wherein the agent for measuring the expression level of the mRNA of the gene is an antisense oligonucleotide that specifically binds to at least one gene selected from the group consisting of HIPK3, GNAQ, MXD1, ETV3 and NBPF10, Wherein the composition is for the diagnosis of chronic obstructive pulmonary disease.
The method according to claim 1, wherein the agent for measuring the expression level of the protein comprises an antibody specific to at least one protein selected from the group consisting of HIPK3, GNAQ, MXD1, ETV3 and NBPF10. Diagnostic composition.
A kit for the diagnosis of chronic obstructive pulmonary disease comprising the composition of any one of claims 1 to 4.
6. The kit for diagnosing chronic obstructive pulmonary disease according to claim 5, wherein the kit is an RT-PCR kit, a microarray chip kit, a DNA kit or a protein chip kit.
(a) measuring the expression level of mRNA of the at least one gene selected from the group consisting of HIPK3, GNAQ, MXD1, ETV3 and NBPF10 or a protein thereof, from the biological sample; And
(b) comparing the expression level of the mRNA or a protein thereof with the mRNA of the normal control sample or the expression level of the protein of the mRNA or the protein thereof.
The method according to claim 7, wherein the biological sample is at least one sample selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph, / RTI >
The method according to claim 7, wherein the mRNA expression level is measured by RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase Wherein the method is one or more selected from the group consisting of RPA (RNase protection assay), Northern blotting and DNA chip.
8. The method according to claim 7, wherein the method for measuring the expression level of the protein comprises the steps of Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip (FACS) were performed by immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Wherein the method is one or more methods selected from the group consisting of: (1) a method for providing information for diagnosis of chronic obstructive pulmonary disease;