KR102282342B1 - Biomarker composition for diagnosing or predicting prognosis of behcet's diseases - Google Patents

Abstract

The present invention provides a biomarker composition for diagnosing Behcet's disease or predicting prognosis. The biomarker composition includes one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein encoded by the gene as an active ingredient. The present invention provides a composition and kit for diagnosing or prognosis of Behcet's disease, a method for providing information for diagnosing or predicting the prognosis of Behcet's disease, and a drug responsive diagnostic marker composition and drug screening method for preventing or treating Behcet's disease. The present invention also provides a composition for treating Behçet's disease. By using this, it is possible to effectively and easily predict the diagnosis or risk of Behçet's disease, and accordingly, it is possible to obtain a better therapeutic effect by enabling appropriate treatment.

Description

Biomarker composition for diagnosis or prognosis of Behcet's disease {BIOMARKER COMPOSITION FOR DIAGNOSING OR PREDICTING PROGNOSIS OF BEHCET'S DISEASES}

The present invention relates to a biomarker composition for diagnosing or predicting Behcet's disease.

Behcet's disease originated in 1937 when a Turkish dermatologist, Hulusi Behcet, reported two patients with recurrent oral and genital ulcers and recurrent eye inflammation. Behcet's disease is a disease that occurs frequently in the Mediterranean coast, the Middle East, and Asian regions such as China, Japan, and Korea. It is one of the possible chronic inflammatory diseases.

Although the exact cause or pathophysiology of Behcet's disease has not been elucidated, it is thought that the immune response is activated as a result of the addition of environmental factors to patients with genetic predisposition for a long time, resulting in various symptoms. A special gene called HLA-B51 was found in 50-60% of patients, and this gene is presumed to be related to Behcet's disease. It is also possible that the immune response to some viruses and bacteria is related to the inflammatory response of Behçet's disease.

The clinical symptoms of Behcet's disease are diverse, and the severity of the disease ranges from mild symptoms such as skin mucosal symptoms or arthritis to severe uveitis or serious sequelae that invade major organs such as the brain, lung, heart, and kidneys. As such, Behcet's disease shows various clinical features and prognosis according to the invasion of various organs, and thus suffers from considerable difficulties in diagnosis and treatment. Therefore, it is very important to accurately diagnose Behcet's disease at an early stage in order to minimize complications and disability caused by Behcet's disease.

However, since there are no symptoms or test results that are conclusively helpful in diagnosing Behcet's disease, currently, Behcet's disease is diagnosed using the classification criteria consisting of those with high diagnostic value among various clinical symptoms of Behcet's disease, and the treatment is also Behcet's disease. Without specific treatment for rheumatoid arthritis, drugs used for rheumatoid disease or inflammatory bowel disease are only empirically used. There is a problem that no test can reliably confirm or rule out Behcet's disease, and it cannot predict the response to treatment.

Korean Patent Registration No. 10-1568632 (Registered on Nov. 5, 2015)

In order to solve the above problems, the present invention provides a biomarker composition for diagnosing or prognosis of Behcet's disease including a differentially expressed gene.

The present invention provides a composition and kit for diagnosing or predicting Behcet's disease.

The present invention provides an information providing method for diagnosing Behcet's disease or predicting a prognosis.

The present invention provides a drug screening method for preventing or treating Behcet's disease and a composition for diagnosing drug reactivity.

In addition, the present invention provides a pharmaceutical composition for preventing or treating Behcet's disease, and a health functional food composition for preventing or improving Behcet's disease, targeting the gene.

The biomarker composition for diagnosing Behcet's disease or predicting prognosis according to the present invention comprises at least one gene selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein encoded by the gene as an active ingredient can be included as

The composition for diagnosis or prognosis of Behçet's disease according to the present invention is one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or the expression or activity level of a protein encoded by the gene It may include a formulation for measuring as an active ingredient.

The kit for diagnosis or prognosis of Behçet's disease according to the present invention may include the composition.

One or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 of a patient sample suspected of Behcet's disease or the above measuring the expression or activity level of a protein encoded by the gene; and comparing the expression or activity level of the gene or protein with the expression or activity level of the corresponding gene or protein in a normal control sample.

A drug screening method for preventing or treating Behcet's disease according to the present invention comprises the steps of treating a test substance in a biological sample isolated from the human body; Measuring the expression or activity level of one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 in the sample treated with the test substance or a protein encoded by the gene; and comparing the expression or activity level of the gene or the protein with a control untreated with the test substance to select a test substance with reduced or increased expression or activity level of the gene or the protein.

The pharmaceutical composition for preventing or treating Behcet's disease according to the present invention comprises at least one gene selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein encoded by the gene as an active ingredient can be included as

The pharmaceutical composition for preventing or treating Behcet's disease according to the present invention comprises one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein expression or activity modulator encoded by the gene. It may contain as an active ingredient.

The health functional food composition for preventing or improving Behcet's disease according to the present invention is one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or protein expression or activity encoded by the gene. It may contain a modulator as an active ingredient.

The biomarker composition for diagnosis or prognosis of Behçet's disease according to the present invention can be used to very effectively and easily diagnose Behcet's disease and predict its risk using differentially expressed genes. Accordingly, by diagnosing Behçet's disease early, it is possible to reduce the time taken to confirm Behcet's disease and to provide appropriate treatment for the onset, thereby minimizing complications due to worsening of the patient's symptoms. In addition, a better treatment effect can be achieved by avoiding expensive and unnecessary treatment through rapid diagnosis and providing the patient with customized treatment and accurate information on the prognosis related to the disease.

In addition, it is possible to effectively prevent, improve and treat Behçet's disease by using a pharmaceutical composition or a health functional food composition targeting the gene as a therapeutic target.

1 is a heat map of a differentially expressed gene (DEG) list according to Example 1 of the present invention.
2 is a Venn diagram for a differentially expressed gene (DEG) according to Example 1 of the present invention.
3 shows an important canonical pathway of Ingenuity Pathway Analysis (IPA) according to Example 1 of the present invention.
4 shows one of the important canonical pathways of the ininity pathway analysis (IPA) according to Example 1 of the present invention.
5 is a result of validation (validation) of genes differentially expressed in RNA-sequencing according to Example 1 of the present invention.

Hereinafter, the present invention will be described in detail.

In a previous study, the present inventors confirmed that the frequency of senescent CD8+ T cells was increased in the peripheral blood of Behcet's disease patients, and based on this, non-senescent and senescent CD8+ T cells isolated from the peripheral blood of Behcet's disease patients and normal controls. The present invention was completed by discovering the difference in gene expression in

As used herein, "diagnosis" refers to determining whether or not the onset of Behçet's disease or the possibility (risk) of the onset, and determining the susceptibility of the subject to at least one or more symptoms of Behçet's disease or Behcet's disease, determining whether a subject currently has Behcet's disease, or therametrics (eg, monitoring the subject's condition to provide information about treatment efficacy). Also includes primary diagnosis of clinical condition or diagnosis of recurrent disease.

As used herein, the term “prognosis” refers to the prospect of future symptoms or progress determined by diagnosing a disease, and includes a positive prognosis such as a disease-free state and a negative prognosis such as exacerbation, metastasis, and recurrence of the disease. That is, after diagnosis of Behcet's disease or after treatment according to diagnosis, predicting and judging progress within a certain period of time, treatment progress, etc., includes prediction and judgment of Behcet's disease recurrence, metastasis, drug reactivity, tolerance, and the like. Prediction of prognosis is a very important clinical task as it provides clues for future treatment directions, including whether Behcet's disease patients are treated.

As used herein, the term "biomarker" is an indicator that can detect changes in the body, and can diagnose the normal or pathological state of a living organism, that is, diagnosis of Behçet's disease, the degree of response to drugs, etc. by distinguishing it from the normal control group. Organic substances such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in Behçet's disease cells compared to normal cells biomolecules and the like.

As used herein, the term “normal control” refers to a normal individual not afflicted with Behcet's disease.

The present invention provides a biomarker composition for diagnosing Behcet's disease or predicting prognosis.

The biomarker composition according to the present invention may include one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein encoded by the gene as an active ingredient.

In the present invention, RGS11 (Regulator of G protein signaling 11) is a G protein signal transduction regulator, and the protein encoded by this gene belongs to the RGS (G protein signaling regulator) family. Members of the RGS family act as GTP hydrolase (hereinafter, GTPase)-activating proteins in the alpha subunit of the heterotrimeric signal-transducing G protein, thereby increasing the GTPase activity of the G protein alpha subunit, thereby transducing signals. to induce an inactive GDP-bound form. In addition, RGS11 is a protein belonging to the R7 RGS family and is overexpressed in metastatic tumor cells, and induces an increase in Rac-1-dependent tumor cell migration through c-RAf/Erk/FAK activity.

In the present invention, SHANK1 (SH3 and multiple ankyrin repeat domains 1) encodes a protein of the SHANK family including the SH3 domain and ankyrin repeat. Proteins of this family serve as scaffold proteins necessary for the development and function of connecting synapses. The protein expression of SHANK1 is highest in the cerebellum, moderately expressed in the spleen and bone marrow, and low in the cerebral lymph nodes. It is also expressed in the human mononuclear cell line THP-1 and the human mononuclear leukemia cell line U937, and its expression is increased in ovarian cancer. This family of proteins is involved in signal transduction by forming complexes with various cell membrane receptors and cytoplasmic proteins using the PDZ domain.

In the present invention, ARHGEF10 (Rho guanine nucleotide exchange factor 10) encodes a Rho guanine nucleotide exchange factor (hereinafter, Rho GEF). Rho GEFs can modulate the activity of small Rho GTPases by stimulating the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP).

ARHGEF10 binds to RAB6A and RAB8A in exocytic vesicles and regulates membrane trafficking to participate in breast cancer cell invasion and platelet function, single nucleotide polymorphism (SNP) and ischemic atherosclerosis. It is also associated with an ischemic atherothrombotic stroke. In addition, variants of this gene affect changes in the activity of delta-6 desaturase, and Charcot-Marie-Tooth disease (Charcot-Marie-Tooth), a hereditary neurological disorder in which lower extremity muscle atrophy and sensory disturbance occur. disease) is also associated with

In the present invention, UBE2F-SCLY is a ubiquitin-conjugating enzyme E2F (ubiquitin-conjugating enzyme E2F; UBE2F) neighboring on chromosome 2 and a selenocysteine lyase; -through transcription). The over-translational transcription is a phenomenon in which transcription is not normally terminated and transcription continues to an adjacent transcription unit. The RNA polymerase that initiated transcription from the promoter, which is one transcription unit, is a transcription termination site present at the end of the transcription unit. It occurs when the termination signal is not recognized for deletion or for some reason. The UBE2F-SCLY fusion protein is expected to regulate cholesterol metabolism through selenium metabolism.

In the present invention, LOC102724428 is a serine / threonine-protein kinase similar to SIK1 (serine / dthreonine-protein kinase SIK1), an important paralog of this gene is SIK1 (salt inducible kinase 1). This gene encodes a serine/threonine protein kinase comprising a ubiquitin-associated (UBA) domain. In addition, ATP binding, magnesium ion binding, and protein phosphorylation functions are predicted, and mRNA expression is observed in most tissues, including lymph nodes and spleen, but there are still few studies on their functions.

In the present invention, COL5A1 is a collagen type V alpha 1 chain gene, and encodes an alpha chain, which is one of fibrin collagen.

In the present invention, TRPV3 (transient receptor potential cation channel subfamily V member 3) is a subfamily of temperature-sensitive channels and belongs to a family of non-selective cation channels.

Table 1 below is for the genes selected in the present invention, more details will be described later through the following examples.

gene ID gene symbol Transcript ID 8786 RGS11 NM_001286485,NM_001286486,NM_003834,NM_183337 50944 SHANK1 NM_0116148 9639 ARHGEF10 NM_001308152,NM_001308153,NM_014629 100533179 UBE2F-SCLY NR_037904 102724428 LOC102724428 NM_001320643 1289 COL5A1 NM_000093,NM_001278074 162514 TRPV3 NM_001258205,NM_145068

In the present invention, the expression or activity of the gene or the protein encoded by the gene may be increased or decreased in a patient with Behçet's disease. That is, a difference occurs with the expression or activity level of the gene or the protein of a normal person who does not have Behcet's disease, and thus it can be used as a biomarker composition for diagnosis or prognosis of Behcet's disease.

In the present invention, the RGS11, SHANK1 or COL5A1 gene, or the protein encoded by the gene, may have a difference in expression or activity in senescent CD8+ T (CD8+ CD27- CD28-) cells compared to a normal control. That is, the gene or the protein may be increased in expression or activity in senescent CD8+ T cells.

In addition, the RGS11, ARHGEF10, UBE2F-SCLY, LOC102724428 or TRPV3 gene, or the protein encoded by the gene, may exhibit differences in expression or activity in non-senescent CD8+ T (CD8+ CD27+ CD28+) cells compared to normal controls, in particular The expression or activity of the RGS11 or TRPV3 gene, or a protein encoded by the gene, is increased, and the ARHGEF10, UBE2F-SCLY or LOC102724428 gene, or a protein encoded by the gene, expression or activity may decrease. The RGS11 may have a difference in expression or activity in both non-senescent cells and senescent cells, and may increase expression or activity.

As for a marker for diagnosing a disease, the selection and application of a significant marker acts as a very important factor in determining the reliability of the diagnostic result. It means a marker with high reliability to show consistent results.

The biomarker according to the present invention shows the same results even in repeated experiments with the above genes, and since the difference in expression or activity level shows a significant difference compared to the control group, it can be regarded as a highly reliable marker. Therefore, a diagnosis result based on the result obtained by measuring the expression or activity level of a significant marker of the present invention can be reasonably trusted.

The present invention provides one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or an agent for measuring the expression or activity level of a protein encoded by the gene as an active ingredient. A composition for diagnosing Behcet's disease or predicting a prognosis is provided.

The agent may include a primer or probe that specifically binds to the gene, an antibody, peptide, peptide mimetics, aptamer, or compound that specifically binds to the protein.

In the present invention, "primer" is a nucleic acid sequence having a short free 3'hydroxl group, a short nucleic acid sequence capable of forming a base pair with a complementary template and functioning as a starting point for template strand copying. it means. Primers are capable of initiating DNA synthesis in the presence of different 4 nucleotide triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature.

The primers may be sense and antisense nucleic acids having a sequence of 7 to 50 nucleotides as primers specific for the gene, and incorporate additional features as long as they do not change the basic properties of the primers serving as the starting point of DNA synthesis. can do. PCR conditions and lengths of sense and antisense primers may be appropriately selected according to techniques known in the art.

In the present invention, “probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible, which can specifically bind to mRNA, and is labeled so that the presence or absence of a specific mRNA; You can check the expression level. The probe may be formed in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. Appropriate probes and hybridization conditions may be appropriately selected according to techniques known in the art.

As used herein, “antibody” is a term known in the art, and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the protein encoded by the gene, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and any immunoglobulin antibody may be included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. Moreover, the said antibody also includes special antibodies, such as a humanized antibody.

In the present invention, "peptide" has the advantage of high binding force to the target material, and does not undergo denaturation even during heat/chemical treatment. In addition, since the molecular size is small, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.

In the present invention, “aptamer” refers to a special kind of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure by itself and having a characteristic capable of binding to a target molecule with high affinity and specificity. It refers to a kind of polynucleotide composed of As described above, the aptamer can specifically bind to an antigenic substance in the same way as an antibody, but has higher stability than a protein, has a simple structure, and is composed of a polynucleotide that is easy to synthesize. can

The present invention provides a kit for diagnosing or prognosis of Behcet's disease comprising the composition.

The kit for diagnosis or prognosis according to the present invention may include an antibody selectively recognizing a marker for the diagnosis or prognosis of Behçet's disease, as well as one or more other component compositions, solutions, or devices suitable for the analysis method. can The kit may be a primer kit, a DNA chip kit, or a protein chip kit, but is not limited thereto.

In the kit for diagnosis or prognosis prediction according to the present invention, the kit for measuring the protein expression level includes a substrate, an appropriate buffer, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody. may include The substrate may include a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, and a glass slide glass, and the chromogenic enzyme is peroxidase, alkaline phosphorus It may include phatase (alkaline phosphatase) and the like, and the fluorescent material may be FITC, RITC, or the like. In addition, the chromogenic substrate solution contains ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (O-phenylenediamine), or TMB (tetramethyl benzidine). can be used

The present invention provides an information providing method for diagnosing Behcet's disease or predicting a prognosis.

The information providing method according to the present invention provides expression of one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein encoded by the gene in a patient sample suspected of Behcet's disease or measuring the level of activity; and comparing the expression or activity level of the gene or protein with the expression or activity level of the corresponding gene or protein in a normal control sample.

For example, the RGS11, SHANK1, COL5A1 or TRPV3 gene in the suspected patient sample; or the expression or activity of the protein encoded by the gene is increased compared to the normal control sample, or the expression or activity of the ARHGEF10, UBE2F-SCLY or LOC102724428 gene, or the protein encoded by the gene, is increased in the normal control sample If it decreases further, information can be provided, and the suspected patient can be diagnosed as having Behcet's disease.

In addition, the present invention provides a drug screening method for preventing or treating Behcet's disease. This is a method of comparing the increase or decrease in the expression or activity of the gene or the protein in the presence and absence of a therapeutic candidate, and can be usefully used for screening a therapeutic agent for Behcet's disease.

The drug screening method according to the present invention comprises the steps of treating a test substance in a biological sample isolated from the human body; Measuring the expression or activity level of one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 in the sample treated with the test substance or a protein encoded by the gene; and comparing the expression or activity level of the gene or the protein with a control that is not treated with the test substance to select a test substance in which the expression or activity level of the gene or the protein is changed.

That is, when the expression or activity of the RGS11, SHANK1, COL5A1 or TRPV3 gene, or the protein encoded by the gene, is decreased compared to the control by the treatment with the test substance, or the ARHGEF10, UBE2F-SCLY or LOC102724428 gene , or when the expression or activity of the protein encoded by the gene is increased compared to the control, the test substance may be determined as a therapeutic substance for Behçet's disease.

In the drug screening method according to the present invention, the biological sample isolated from the human body may include a tissue, cell, serum, etc. sample that is different from the normal control in the expression or activity level of the gene or the protein, The present invention is not limited thereto.

In the drug screening method according to the present invention, the test substance is a substance used in screening to test whether it affects the expression or activity of the gene or the protein, and may be a candidate drug for the treatment of Behcet's disease.

In the present invention, the expression level of the gene is RT-PCR, competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR), RNase protection assay (RPA: RNase protection assay), Northern The measurement may be performed by any one or more methods selected from the group consisting of Northern blotting and a DNA microarray chip, but is not limited thereto.

In the present invention, the protein expression or activity level is determined by Western blot, Enzymelinked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Radioimmunodiffusion, Oukteroni immune diffusion method, rocket ( rocket) immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, flow cytometry (FACS), and protein chip can be measured by any one or more methods selected from the group consisting of, The present invention is not limited thereto.

The present invention relates to one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein encoded by the gene as an active ingredient for the prevention or treatment of Behcet's disease for diagnosing drug reactivity A composition is provided.

Using the diagnostic composition, it is possible to determine the reactivity of a drug for preventing or treating Behçet's disease through the expression or activity of the gene or the protein.

In addition, one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein encoded by the gene may be a therapeutic target.

Accordingly, the present invention provides one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3, or a protein expression or activity regulator encoded by the gene. Prevention of Behcet's disease containing an active ingredient Or it provides a pharmaceutical composition for treatment.

The expression or activity modulator of the gene or protein may activate or inhibit the expression or activity of the gene. For example, the expression or activity of the RGS11, SHANK1, COL5A1, or TRPV3 gene, or a protein encoded by the gene, can be inhibited, and the ARHGEF10, UBE2F-SCLY or LOC102724428 gene, or the protein encoded by the gene, is expression or activity may be enhanced.

The modulator is an antisense nucleotide, small interfering RNA (siRNA) or a short one or more genes that complementarily binds to the mRNA of one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3. It may be short hairpin RNA (shRNA), and may be a compound, peptide, peptide mimetics, aptamer, antibody, or natural product that specifically binds to the protein, but is not limited thereto.

The pharmaceutical composition according to the present invention may be prepared according to a conventional method in the pharmaceutical field. The pharmaceutical composition may be combined with a suitable pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. may be prepared further comprising there is. The appropriate carrier and the like do not impair the properties of the modulator, and may be selected differently depending on the dosage form and dosage form.

The pharmaceutical composition according to the present invention can be applied in any dosage form, and more specifically, it can be used by formulating oral dosage forms, external preparations, suppositories, and parenteral dosage forms of sterile injection solutions according to conventional methods.

The solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may be included. In addition, the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Among the oral dosage forms, liquid formulations include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. there is.

The parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount.

As used herein, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects.

The effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered to any animal capable of developing Behcet's disease, and the animal may include, for example, not only humans and primates, but also livestock such as cattle, pigs, horses, and dogs. .

The pharmaceutical composition according to the present invention may be administered by an appropriate administration route according to the form of the formulation, and may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The administration method does not need to be particularly limited, and for example, oral, rectal or intravenous, muscle, skin application, subcutaneous, respiratory inhalation, intrauterine dural or intracerebroventricular injection, etc. are administered in a conventional manner. can be

The pharmaceutical composition according to the present invention may be used alone for preventing or treating Behcet's disease, or may be used in combination with surgery or other drug treatment.

In addition, the present invention is one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3 or a protein expression or activity regulator encoded by the gene. Prevention of Behçet's disease containing an active ingredient Or it provides a health functional food composition for improvement.

The expression or activity modulator of the gene or protein may activate or inhibit the expression or activity of the gene. For example, the expression or activity of the RGS11, SHANK1, COL5A1, or TRPV3 gene, or a protein encoded by the gene, can be inhibited, and the ARHGEF10, UBE2F-SCLY or LOC102724428 gene, or the protein encoded by the gene, is expression or activity may be enhanced.

The modulator is an antisense nucleotide, small interfering RNA (siRNA) or a short one or more genes that complementarily binds to the mRNA of one or more genes selected from the group consisting of RGS11, SHANK1, ARHGEF10, UBE2F-SCLY, LOC102724428, COL5A1 and TRPV3. It may be short hairpin RNA (shRNA), and may be a compound, peptide, peptide mimetics, aptamer, antibody, or natural product that specifically binds to the protein, but is not limited thereto.

In the health functional food composition according to the present invention, the health functional food may be prepared as a powder, granules, tablets, capsules, syrups or beverages for the purpose of preventing or improving Behcet's disease. There is no limitation in the form that the health functional food can take, and it can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods.

In the health functional food composition according to the present invention, the health functional food may include all food in a conventional sense. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.

The health functional food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives (food additives) and other suitable auxiliary ingredients commonly used in the art. Unless otherwise specified, the suitability as a food additive can be judged according to the standards and standards for the relevant item in accordance with the general rules and general test methods of the Food Additives Code approved by the Food and Drug Administration. The items listed in the 'Food Additives Codex' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar dye preparation, etc. are mentioned.

The other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. may further contain. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used. , as the sweetener, natural sweeteners such as taumatine and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.

The health functional food composition has the advantage that there are no side effects that may occur during long-term administration of general drugs using food as a raw material, and has excellent portability, and can be taken as an adjuvant for preventing or improving Behcet's disease.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

[Example 1]

The following experiment was performed for transcript analysis of 18 patients with Behçet's disease (BD) and healthy controls (hereafter HCs), respectively.

1. Experimental method

1) Cell Preparation

Peripheral blood mononuclear cells (hereinafter, PBMCs) were isolated from heparinized blood samples using a Ficoll Hypaque density gradients centrifugation (Ficoll paqueTM plus; StemCell Technologies, Vancouver, BC, Cananda). prepared. Cells were washed with phosphate buffered saline (hereinafter, PBS, Sigma, St. Louis, MO, U.S.A.).

2) CD8+ T cell isolation

Positive selection was performed using microbeads for CD8 T cell isolation (human CD8+ T cell isolation kit, MACS, Miltenyi Biotec). CD8 microbeads were used according to the manufacturer's instructions.

3) Sorting

The isolated CD8+ T cells were labeled with monoclonal antibodies conjugated with fluorescent substances as follows: FITC-anti-CD8, allophycocyanin (APC)-H7-anti-CD27, APC-anti-CD28 (BD Pharmingen; BD Biosciences, San Jose, CA, USA).

Labeled cells were analyzed by flow cytometry (Fluorescence Activated Cell Sorting, FACS) to senescent CD8+ T cells (CD8+/CD27-CD28-cells) and non-senescent CD8+ T cells (CD8+/CD27+CD28+ cells), and purified ( purity) greater than 90%.

4. Cell Culture and Stimulation Sorting Cells

The isolated senescent or non-senescent CD8+ T cells were cultured in a medium containing 10% FBS (RPMI 1640 medium supplemented with 2mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin _{at 37° C., 5% CO 2 ;} -BRL, Grand Island, NY, USA). Cells were stimulated with anti-CD3 antibody (500 ng/μl, clone OKT3, eBiosciences, San Diego, CA, USA) for 72 hours.

5. RNA Extraction

Total RNA was extracted from cells stimulated with anti-CD3 antibody using an RNA isolation kit (NocleoSpin RNA XS kit (Macherey-nagel, Duren, Germany)). Extraction was performed according to the protocol provided by the manufacturer.

6. Request for NGS analysis (Macrogen, Korea)

from 4 different groups (each group divided into Behcet's disease (BD) patients vs. normal controls, senescent CD8+ T cells (CD8+/CD27-CD28- cells) vs. non-senescent CD8+ T cells (CD8+/CD27+CD28+ cells)) Differentially expressed genes (DEG) were analyzed through transcriptome analysis. The differential expression gene analysis is a method of analyzing whether the average expression level of the same gene is significantly different under different conditions.

· Library Kit: TruSeq Stranded mRNA LT Sample Prep Kit

· Type of Sequencer: HiSeq 2500

· Sequencing Control Software: HCS 2.2.70

7. Ingenuity Pathway Analysis (hereinafter, IPA, Qiagen, Germany)

Ingenuity Pathway Analysis (IPA) is a web-based software tool that visually expresses biological functions, pathways, and molecular networks. A functional relationship can be derived and molecular factors that can be the cause can be found.

IPA was performed on differentially expressed genes (DEGs) of 2 or more fold change (fc).

8. Validation through Real-time PCR

cDNA was prepared using oligo-dT primers (Invitrogen, Carlsbad, CA, USA) with Superscript™ III kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Real-time PCR was performed with primers and internal probes for RGS11 (Hs01594651_m1), SHANK1 (Hs00211718_m1), AHRGEF10 (Hs00405154_m1), LOC102724428 (Hs00545020_m1), and UBE2F-SCLY (Hs00996436_m1) (Thermo Scientific), COL5A1, CD302 gene, and COL5A1 gene. was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster, CA, USA) according to the manufacturer's instructions with SYBR Green Supermix for

Primer sequences are shown in Table 2 below.

TRPV3 5’-GTTAGCTACCCGCATTAAGCCTGA-3’ 5’-AGCAATTCTGGAATTCCCAGCTC-3’ COL5A1 5’-CCTGCATTGTCTGTGGTGTGA-3’ 5’-AGCGCATGGCATGACTGAA-3’ CD302 5’-GCCCAGATGATATCCTACTAGGC-3’ 5’-GCAGAAAAGCACAGGTGTCAACT-3’ NBL1 5’-TCCACAGAGTCCCTGGTTCACT-3’ 5’-GCTACAGTGCAGGATCTTCTCC-3’

2. Experimental results

1 is a heat map of a differentially expressed gene (DEG) list according to Example 1 of the present invention. Referring to FIG. 1 , the heat map shows various information that can be expressed in color as a visual graphic in the form of a heat distribution on a certain image. The region in which the differentially expressed genes occur a lot is yellow, and the region in which the differential expression gene occurs is blue. appears as

2 is a Venn diagram for a differentially expressed gene (DEG) according to Example 1 of the present invention. 2, A is the number of differentially expressed genes in senescent cells of Behcet's disease patients and senescent cells of normal persons, and B is the number of differentially expressed genes in non-senescent cells of Behcet's disease patients and non-senescent cells of normal persons. . There are 912 genes corresponding only to A, 461 genes corresponding only to B, and 191 genes are commonly differentially expressed between A and B.

3 shows an important canonical pathway of IPA according to Example 1 of the present invention. Referring to FIG. 3 , A is a comparative analysis of senescent cells of a Behcet patient and a normal person, and B is a comparative analysis of non-senescent cells of a Behcet patient and a normal person.

11 important canonical pathways showed increased activity in senescent CD8+ T cells of Behcet's disease patients compared to senescent CD8+ T cells in normal controls, and this pathway in non-senescent CD8+ T cells in Behcet's disease patients compared to non-senescent CD8+ T cells in normal controls. activity was decreased.

Gene ID RNA-seq fold change 4681 131.366677 8786 107.751843 50944 72.962133 102465252 37.189333 1289 36.918116 4199 32.903108 57282 30.737880 6571 29.400024 5783 27.901175 100846999 -88.716100

Table 3 is a list of the top 10 genes whose fold change is up- or down-regulated in senescent CD8+ T cells compared to normal in Behcet patients. The fold gradient is used to measure a change in gene expression level when analyzing gene expression data in a microarray or RNA-sequencing (RNA-Seq) experiment.

Referring to Table 3, it can be seen that the fold change (fc) of RGS11, which is Gene ID 8786, is 107.751843, and that of SHANK1, which is Gene ID 50944, is highly expressed as 72.962133.

Table 4 below is a list of the top 10 genes with up- or down-regulated fold gradients in Behcet patients versus normal subjects in non-senescent CD8+ T cells.

Referring to Table 4 below, it can be seen that the fold change (fc) of RGS11, which is Gene ID 8786, is 20.348263, and the expression difference is small compared to Table 3, and ARHGEF10, which is Gene ID 9639, is -14.521649, Gene ID 100533179 It can be seen that UBE2F-SCLY is down-regulated to -26.323132, and LOC102724428, which is Gene ID 102724428, is down-regulated to -1634.675298.

Gene ID RNA-seq fold change 100996741 35.168681 162514 23.557014 8786 20.348263 100033416 16.539254 9639 -14.521649 100526767 -15.502246 100533179 -26.323132 414062 -31.670879 9936 -47.069576 102724428 -1634.675298

Figure 4 shows one of the important canonical pathways of IPA according to Example 1 of the present invention, cAMP-mediated signaling of Behcet patients versus normal people in senescent cells.

5 is a result of validation (validation) of genes differentially expressed in RNA-sequencing according to Example 1 of the present invention.

Referring to Figure 5, to verify the RNA sequencing results, 8 genes (RGS11, SHANK1, AHRGEF10, LOC102724428, CD302, COL5A1, TRPV3, and UBE2F-SCLY) selected from the upper DEG except for miRNA and uncharacterized genes. As a result of performing real-time PCR, it was possible to confirm the gene showing consistency with the transcript analysis result.

Specifically, in FIG. 5A , COL5A1 and ARHGEF10 showed the same pattern as in the transcript analysis of senescent CD8+ T cells in BD patients compared to HC, and COL5A1 showed statistical significance. 5B , TRPV3, ARHGEF10, UBE2F-SCLY, CD302 and SHANK1 showed the same pattern as in the transcript analysis of non-senescent CD8+ T cells in BD patients compared to HC, and TRPV3 and ARHGEF10 showed statistical significance.

So far, specific embodiments of the present invention have been described. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

A biomarker composition for diagnosing Behcet's disease, comprising the COL5A1 gene or a protein encoded by the gene as an active ingredient. The method of claim 1,
The gene, or the protein,
A biomarker composition for diagnosing Behcet's disease, characterized in that expression or activity is increased in senescent CD8+ T cells as compared to a normal control. The method of claim 1,
The composition is
One or more genes selected from TRPV3 or ARHGEF10, or one or more proteins encoded by the gene, characterized in that it further comprises a biomarker composition for diagnosing Behcet's disease. 4. The method of claim 3,
The TRPV3 gene, or a protein encoded by the gene, has increased expression or activity in non-senescent CD8+ T cells compared to a normal control, and the ARHGEF10 gene, or a protein encoded by the gene, is non-aging compared to a normal control. A biomarker composition for diagnosing Behcet's disease, characterized in that the expression or activity is reduced in senescent CD8+ T cells. A composition for diagnosing Behcet's disease, comprising, as an active ingredient, an agent for measuring the expression or activity level of the COL5A1 gene or a protein encoded by the gene. 6. The method of claim 5,
The composition is
One or more genes selected from TRPV3 or ARHGEF10, or an agent for measuring the expression or activity level of one or more proteins encoded by the gene, Behçet's disease diagnostic composition. 6. The method of claim 5,
The formulation is
A primer or probe that specifically binds to the gene, an antibody, peptide, peptide mimetics, aptamer or compound that specifically binds to the protein, the composition for diagnosing Behcet's disease. A kit for diagnosing Behcet's disease, comprising the composition according to any one of claims 5 to 7. measuring the expression or activity level of the COL5A1 gene or a protein encoded by the gene in senescent CD8+ T cells of a biological sample isolated from a patient suspected of having Behcet's disease;
comparing the expression or activity level of the gene or protein with the expression or activity level of the gene or protein in a normal control sample; and
When the expression or activity level of the gene or the protein is higher than that of a normal control sample, determining as Behcet's disease; Containing, a method for providing information for diagnosing Behcet's disease. 10. The method of claim 9,
The method of providing the information,
Further comprising measuring the expression or activity level of one or more genes selected from TRPV3 or ARHGEF10, or one or more proteins encoded by said genes, in non-senescent CD8+ T cells of a biological sample isolated from said patient,
when the expression or activity level of the TRPV3 gene, or a protein encoded by the gene, is increased compared to a normal control sample; or when the expression or activity of the ARHGEF10 gene, or a protein encoded by the gene, is decreased compared to that of a normal control sample; A method for providing information for diagnosing Behcet's disease, characterized in that it is determined as Behcet's disease delete delete

Images