WO2023172087A1 - Biomarker composition for diagnosing sjögren syndrome, comprising, as active ingredient, klk5, klk7, cstb, ube2v2, tmsb10 or pi3 - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing Sjögren syndrome, comprising, as an active ingredient, KLK5, KLK7, CSTB, UBE2V2, TMSB10 or PI3. Particularly, in order to diagnose Sjögren syndrome, proteins are detected in saliva through salivary liquid biopsy, and gene expression in tissue-derived organoids is identified through salivary gland tissue biopsy so that KLK5, KLK7, CSTB, UBE2V2, TMSB10 or PI3, each being a biomarker increased in a Sjögren syndrome patient, is identified as a Sjögren syndrome-specific biomarker. Therefore, KLK5, KLK7, CSTB, UBE2V2, TMSB10 or PI3 of the present invention can be effectively used for the diagnosis of Sjögren syndrome.

Description

Biomarker composition for diagnosing Sjögren syndrome comprising KLK5, KLK7, CSTB, UBE2V2, TMSB10 or PI3 as active ingredients

The present invention relates to Kallikrein-5 (KLK5), Kallikrein-7 (KLK7), Cystatin-B (CSTB), and Ubiquitin-conjugating enzyme E2 variant 2 (Ubiquitin-conjugating enzyme E2 variant 2). It relates to a biomarker composition for diagnosing Sjögren's syndrome containing enzyme E2 variant 2 (UBE2V2), Thymosin beta-10 (TMSB10), or Elafin (PI3) as an active ingredient.

Sjogren's syndrome is a type of autoimmune disease that occurs throughout the body, and the tissues where symptoms mainly appear are exocrine glands such as salivary and lacrimal glands. In addition to symptoms of dryness in the mouth, eyes, and skin, salivary gland swelling, discomfort, and joint pain were reported in more than 80% of patients. The exact cause of this syndrome is not known, but it is currently suggested that genetic factors and various environmental factors play a complex role. It is characteristically more common in women than men (male:female = 1:9), and is frequently observed in menopausal women in their 50s or older.

Diagnosis of Sjögren's syndrome is basically based on immunological findings in patients with dry mouth, and detection of autoimmune antibodies (anti-SSA) and immune cells through biopsy of the minor salivary gland. It is based on the infiltration (Focus score). However, the symptoms of Sjogren's syndrome are common symptoms that can be experienced depending on the physical condition even without the disease, and symptoms are also shared with other diseases, which makes accurate diagnosis of Sjögren's syndrome difficult.

Kallikrein is a type of serine protease and is known to be involved in immune responses, skin keratinization, and enamel formation. In diseases, there are many studies on the relationship with cancer, such as promoting cell growth through activation of hormones or protease-activated receptors (PAR), decomposition of extra-cellular matrix (ECM), and epithelial damage. -It is known to promote cancer by participating in mesenchymal transition (EMT, epithelial-mesenchymal transition). In particular, KLK7 is known as a prognostic marker in breast cancer, and in other diseases, its role in atopic dermatitis has been suggested in animal models and patient samples. However, nothing is known about the role of KLK in salivary glands and related diseases.

The object of the present invention is Kallikrein-5 (KLK5), Kallikrein-7 (KLK7), Cystatin-B (CSTB), Ubiquitin-conjugating enzyme E2 variant 2 (Ubiquitin - Contains one or more proteins selected from the group consisting of conjugating enzyme E2 variant 2; UBE2V2), Thymosin beta-10 (TMSB10), and Elafin (PI3), or a gene encoding the same as an active ingredient. The aim is to provide a biomarker composition for diagnosing Sjögren's syndrome.

In addition, another object of the present invention is Sjögren's syndrome, which includes as an active ingredient an agent capable of measuring the expression level of one or more proteins selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10 and PI3 or the genes encoding them. The object is to provide a diagnostic composition and a Sjögren's syndrome diagnostic kit containing the same.

In addition, another object of the present invention is to provide information for diagnosing Sjögren's syndrome, including measuring the expression level of one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 from a sample isolated from a patient. The goal is to provide a method to provide it.

In addition, another object of the present invention is to provide a screening method for Sjögren's syndrome treatment by measuring the expression level of one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3.

In order to solve the above problem, the present invention provides Kallikrein-5 (KLK5), Kallikrein-7 (KLK7), Cystatin-B (CSTB), and ubiquitin-conjugating enzymes. One or more proteins or genes encoding the same selected from the group consisting of Ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), Thymosin beta-10 (TMSB10), and Elafin (PI3) Provided is a biomarker composition for diagnosing Sjogren's syndrome containing as an active ingredient.

In addition, the present invention provides a composition for diagnosing Sjogren's syndrome comprising as an active ingredient an agent capable of measuring the expression level of one or more proteins selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10 and PI3 or the genes encoding them. to provide.

Additionally, the present invention provides a kit for diagnosing Sjogren's syndrome comprising the composition.

In addition, the present invention provides (1) the mRNA expression level of one or more genes selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 or KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 from samples isolated from patients; Measuring the expression level of one or more proteins selected from the group consisting of; (2) comparing the mRNA expression level of the gene or the expression level of the protein with a control sample; and (3) determining that the sample has Sjögren's syndrome when the mRNA expression level of the gene or the protein expression level is higher than that of the control sample.

In addition, the present invention includes the steps of (1) contacting a test substance with minor salivary gland tissue or minor salivary gland organoids; (2) measuring the expression or activity level of one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 in minor salivary gland tissue or minor salivary gland organoids in contact with the test substance; and (3) a Sjogren's syndrome treatment screening method comprising the step of selecting a test substance with a reduced level of expression or activity of any one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 compared to the control sample. to provide.

The present invention relates to a biomarker composition for diagnosing Sjögren's syndrome containing KLK5, KLK7, CSTB, UBE2V2, TMSB10 or PI3 as an active ingredient. Specifically, for diagnosing Sjögren's syndrome, proteins are detected in saliva through salivary liquid biopsy. , Gene expression in tissue-derived organoids was confirmed through salivary gland tissue biopsy, and KLK5, KLK7, CSTB, UBE2V2, TMSB10, or PI3, which are biomarkers increased in Sjogren's patients, were identified as Sjögren's syndrome-specific biomarkers. Accordingly, it is expected that KLK5, KLK7, CSTB, UBE2V2, TMSB10 or PI3 of the present invention can be usefully used in the diagnosis of Sjögren's syndrome.

Figure 1 shows the results of KLK gene expression analysis of organoids derived from a Sjögren's syndrome patient.

Figure 2 shows the results of proteomic analysis (confirmation of differences by group) of saliva derived from Sjögren's syndrome patients.

Figure 3 shows the results of proteomic analysis (volcano plot of different proteins) of saliva derived from a Sjögren's syndrome patient.

The present invention relates to Kallikrein-5 (KLK5), Kallikrein-7 (KLK7), Cystatin-B (CSTB), and Ubiquitin-conjugating enzyme E2 variant 2 (Ubiquitin-conjugating enzyme E2 variant 2). Sjögren containing as an active ingredient one or more proteins selected from the group consisting of enzyme E2 variant 2 (UBE2V2), Thymosin beta-10 (TMSB10), and Elafin (PI3), or a gene encoding the same. Provided is a biomarker composition for diagnosing a syndrome.

“Kallikrein-7 (KLK7)” of the present invention is NCBI accession no. It may be NM_005046.4 (mRNA), NP_005037.1 (Protein), but is not limited thereto.

“Cystatin-B (CSTB)” of the present invention is NCBI accession no. It may be NM_000100 (mRNA), NP_000091 (Protein), but is not limited thereto.

“Ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2)” of the present invention is NCBI accession no. It may be NM_003350_(mRNA), NP_003341 (Protein), but is not limited thereto.

“Thymosin beta-10 (TMSB10)” of the present invention is NCBI accession no. It may be NM_021103 (mRNA), NP_066926_(Protein), but is not limited thereto.

“Elafin (PI3)” of the present invention is NCBI accession no. It may be NM_002638 (mRNA), NP_002629_(Protein), but is not limited thereto.

In this specification, the term “diagnosis” refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, and determining whether an object currently has a specific disease or condition. Includes determining the subject's prognosis, or therametrics (e.g., monitoring the subject's condition to provide information about treatment efficacy).

In addition, the present invention provides a composition for diagnosing Sjogren's syndrome comprising as an active ingredient an agent capable of measuring the expression level of one or more proteins selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10 and PI3 or the genes encoding them. to provide.

Preferably, the expression level may be measured from minor salivary gland tissue or saliva, but is not limited thereto.

In detail, the agent capable of measuring the expression level is a primer or probe that specifically binds to any one or more genes selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3, KLK5, KLK7, It may be an antibody, peptide, aptamer, or compound that specifically binds to one or more proteins selected from the group consisting of CSTB, UBE2V2, TMSB10, and PI3, but is not limited thereto.

Additionally, the present invention provides a kit for diagnosing Sjogren's syndrome comprising the composition.

As used herein, the term "primer" refers to a nucleic acid sequence with a short free 3' hydroxyl group that can form base pairs with a complementary template and acts as a starting point for copying the template strand. refers to a nucleic acid sequence. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, of as short as a few bases or as long as several hundreds of bases, capable of binding specifically to mRNA, and is labeled to determine the presence or absence of a specific mRNA and its expression. You can check the amount. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art.

As used herein, the term “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 biomarker of the present invention, and the antibody can be produced according to conventional methods in the art. The types of antibodies include polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant to be intact, having two full-length light chains and two full-length heavy chains. Additionally, the above antibodies also include special antibodies such as humanized antibodies.

In addition, the kit of the present invention includes an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with a substrate, a chromogenic substrate solution that will undergo color development with the label, a washing solution, and It may contain an enzyme reaction stopping solution, etc., and may be manufactured into a number of separate packaging or compartments containing the reagent components used.

As used herein, the term “peptide” has the advantage of high binding capacity to the target substance and does not undergo denaturation even during heat/chemical treatment. Additionally, because the molecule size is small, it can be used as a fusion protein by attaching it to another protein. Specifically, it can be used by attaching it to a polymer protein chain, so it can be used as a diagnostic kit and drug delivery material.

As used herein, the term "aptamer" refers to a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and the ability to bind to a target molecule with high affinity and specificity. ) refers to a type of polynucleotide composed of As mentioned above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easier to synthesize, so they can be used as a replacement for antibodies. You can.

In addition, the present invention provides (1) the mRNA expression level of one or more genes selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 or KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 from samples isolated from patients; Measuring the expression level of one or more proteins selected from the group consisting of; (2) comparing the mRNA expression level of the gene or the expression level of the protein with a control sample; and (3) determining that the sample has Sjögren's syndrome when the mRNA expression level of the gene or the protein expression level is higher than that of the control sample.

In detail, methods for measuring the mRNA expression level include RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA). ), Northern blotting, and DNA chips are used, but are not limited to these.

In detail, methods for measuring the protein expression level include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip are used, but are not limited to these.

As used herein, the term "sample isolated from a patient" includes samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differ from the control group in the expression levels of the biomarkers. It is not limited to this. Preferably, the sample may be minor salivary gland tissue or saliva.

In addition, the present invention includes the steps of (1) contacting a test substance with minor salivary gland tissue or minor salivary gland organoids; (2) measuring the expression or activity level of one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 in minor salivary gland tissue or minor salivary gland organoids in contact with the test substance; and (3) a Sjogren's syndrome treatment screening method comprising the step of selecting a test substance with a reduced level of expression or activity of any one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 compared to the control sample. to provide.

The term "test substance" used while referring to the screening method of the present invention refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. do. The samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA), and natural product extracts.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Reagents

The following reagents were used in the present invention.

Advanced DMEM/F12 (ADF12) (#12634010, Gibco), HEPES (#15630-080, Gibco), GlutaMAX (#35050-061, Gibco), Y-27632 dihydrochloride (#1254, Tocris), 1×HBSS solution ( #14025092, Gibco), Collagenase, type II (#4176, Worthington), Matrigel (#356231, Corning), TrypLE express (#12605-010, Life technologies), CellBanker 1 (Zenoaq), BSA (Bovine Serum Albumin; # BSA-68700-1kg, LSP), Primocin (#ant-pm, Invivogen), B-27 Supplement (50X), serum free (#17504-044, Gibco), NAC (N-acetyl-cysteine; #A9165, Sigma ), Nicotinamide (#N0636, Sigma), A83-01 (#2939, Tocris), Prostaglandin E2 (#2296, Tocris), Recombinant RSPO3-Fc fusion protein conditioned medium (hRSPO3-CM) (R001, U-Protein Express BV ), Noggin-Fc Fusion Protein conditioned medium (hNOG-CM) (#N002, U-Protein Express BV), Nrg1 (Neuregulin-1; #100-03, Peprotech), FGF2 (#100-18B, Peprotech), FGF10 (#100-26, Peprotech), TRIzol™ Reagent (Invitrogen™ #15596026), PrimeScript™ RT reagent Kit (Takara #RR037A), SensiFAST™ SYBR® Lo-ROX Kit (Bioline #BIO-94020), Pierce™ BCA Protein Assay Kit (Thermo, #23227)

2. Digestion and storage of tissue derived from salivary glands of patients with Sjögren's syndrome

(1) Sjögren's patient's salivary gland tissue is placed in buffer containing 1% BSA in 1× HBSS and stored at 4°C until the experiment on the same day or the next day.

(2) Add 1 mL of digestion solution containing 5 mg/mL of Collagenase type II and 10 μM of Y-27632 to Advanced DMEM/F12 per 50 mg of salivary gland tissue.

(3) Digest the tissue with a blade and transfer it to a new conical tube.

(4) Set the shaking incubator to 37℃ and 200rpm and react for 1 hour.

(5) Add 4 mL of Advance DMEM/F12 and centrifuge at 500g for 5 minutes.

(6) Dissolve the pellet in 2mL of TrypLE express containing 10μM of Y-27632 and react at 37°C for 10 minutes.

(7) After filtering the decomposed tissue through a 100μm strainer, add at least 2mL of Advanced DMEM/F12 to stop the reaction, then centrifuge at 500g for 5 minutes to obtain cells.

(8) After measuring the number of cells, dissolve a cell pellet of at least ^2.5 Store in the freezer for 2-3 days and then transfer to a nitrogen tank for long-term storage.

3. Derived from salivary gland cells of patients with Sjögren’s syndrome of organoids Production and culture maintenance

(1) After thawing the cells stored in the nitrogen tank, dilute them in 5 mL of Advanced DMEM/F12 and centrifuge at 500g for 5 minutes.

2. Mix 40 μL of Matrigel per well with the pellet of centrifuged cells in a 24-well plate that has been placed in a 37°C incubator for at least 1 hour, and then dispense into the wells in the form of a dome.

(3) Turn the plate over and react in a 37°C incubator for more than 20 minutes to harden the dome. Afterwards, add 0.5 ml of culture medium to each well.

(4) Culture conditions for organoids derived from salivary glands of patients with Sjögren's syndrome are shown in Table 1.

(5) Replace with new culture medium every 2-3 days.

(6) Subculture is performed in new wells at two-week intervals.

(7) Remove the culture medium from the plate where the organoids are growing and wash once with 500 μl of Advanced DMEM/F12.

(8) Dissolve in 2 mL of TrypLE express containing 10 μM of Y-27632 and react at 37°C for 10 minutes.

(9) After diluting in 2 ml of Advanced DMEM/F12, centrifuge at 500g for 5 minutes.

( ¹⁰ ) Calculate 5.0

(11) Repeat steps 2-5 to subculture the organoids.

Advanced DMEM/F12
Glutamax	1×
HEPES	1×
Primocin	1×
B27vit A+	1×
NAC	1mM
Nicotinamide	5mM
A83-01	5μM
PGE2	3μM
hNOG-CM	2%
hFGF2	5ng/mL
hFGF10	10ng/mL
hNRG1	5ng/mL
hRSPO3-CM	One%
Y-27632*	10μM

* Y-27632 is removed after treatment for 7 days from the start of organoid culture.

4. Salivary glands of patients with Sjögren’s syndrome organoid RNA expression analysis

(1) After removing the culture medium, leaving only the dome containing the organoids, the solution was dissolved in 500 μl of Advanced DMEM/F12, and then centrifuged at 500g for 5 minutes.

(2) After discarding the supernatant, dissolve the pellet containing matrigel in 1 ml of TRIZOL.

(3) After isolating RNA according to the protocol provided by the company, dissolve the RNA in RNase-free D.W.

(4) After quantifying the amount of RNA with Nanodrop, calculate 500 ng of RNA per tube, and then synthesize cDNA using PrimeScript™ RT reagent Kit. The synthesis method follows the protocol provided by the company.

(5) Real-time PCR assay is performed using SensiFAST™ SYBR® Lo-ROX Kit. The experiment was performed according to the protocol provided by the company, and the primer sequences used are shown in Table 2.

Gene	Forward Primer	Reverse Primer
KLK5	GGA GAA ACG ACG CAT CCA CTA C	GAA CCT CCA GTC GCA GCC TTC
KLK6	GGT CCT TAT CCA TCC ACT GTG G	GAA CTC TCC CTT TGC CGA AGG T
KLK7	CAG GCT GTC ATC CAT GGT GAA G	ATC CAC GCA CAT GAG GTC AGA G
KLK8	TAC TCT GTG GCG GTG TCC TTG T	TGG GAT GGA CTG AAC CAC AGG T
KLK11	CGG CAA CAT CAC AGA CAC CAT G	CCA GGA GAT AAT GCC TTG AAG AG
KLK13	CCA ACT TCG CTC AGA TGA GGA G	CAG GAG ACG ATG CCA TAC AGT G
KLK14	GAG TGT CAG GCT GGG GAA CTA T	AAC TCC TGC ACA GAC CAT GCC A
KLK15	TAA CTG TGG CGC TTC CCT CAT C	AGA CGT GGT CCG TAG TTG CTC T
CSTB	CGT GTC ATT CAA GAG CCA GGT G	CTA CCA GAC CAA CAA AGC CAA GC
UBE2V2	AAG GAG TAG GCG ACG GTA CAG T	GCC TGA AAG TAG AAT GTG GAC CT
TMSB10	AAA TCG CCA GCT TCG ATA AG	AGT GGG AGC ACC AGG ATC T
PI3	CGC TGC TTG AAA GAT ACT GAC TG	TCC TTG CTG CAC CTG TGC CGT

5. Sjögren’s syndrome patient saliva sampling

(1) Avoid eating or smoking 2 hours before collecting saliva.

(2) Have the patient sit in a comfortable position and place a vitamin tablet under the tongue.

(3) After 1 minute, insert a catheter connected to a 1cc syringe into the entrance to the parotid conduit.

(4) Carefully inject the physiological saline solution filled in the 1cc syringe.

(5) Massage the parotid area and apply negative pressure to the syringe to wash the injected saline solution and aspirate the saliva in the parotid duct.

(6) Continue massaging for 30 seconds to 1 minute to collect at least 0.1cc of saliva.

(7) Collect saliva from the other side in the same way.

(8) Close the mouth of the syringe from which saliva was collected and store it in a liquid nitrogen container.

6. Proteome analysis in saliva

(1) The amount of protein in saliva was analyzed using BCA assay, and the total protein amount of each sample was adjusted to the minimum amount.

(2) Process trypsin and perform in-solution digestion.

(3) The proteome was analyzed using single shot LC-MS (Liquid chromatography - Mass spectrometry) using 1 μg of sample.

(4) Proteins were identified from peptide sequences using the MaxQuant platform, and those with FDR (False Discovery Rate) greater than 1% were excluded from the analysis. Through this, a total of 727 proteins were identified.

<Example>

Patients who visited the hospital with dry mouth were divided into two groups: those diagnosed with Sjogren's syndrome (SS) and those not diagnosed with Sjögren's syndrome (non-SS). Minor salivary glands and saliva derived from them were obtained from each patient. Afterwards, the minor salivary glands were used for organoid culture, and the saliva was used for proteomic analysis. When organoids were cultured from minor salivary glands, it was confirmed that there was no significant difference in organoid shape or growth between the SS and non-SS groups. Therefore, to identify markers with differences in gene expression, cultured organoids were obtained and RNA analysis was performed. Among genes, the level of gene expression was confirmed by focusing on the KLK family, which has been suggested as a biomarker related to cancer and immune response. The KLK family is known to have a total of 15 genes in the human genome. Among KLK1 to KLK15, quantitative analysis of RNA expression was performed on the remaining genes, excluding KLK9, KLK10, and KLK12, which do not have known physiological substrates (Figure 1). As a result, the expression of KLK1, KLK2, KLK3, and KLK4 was not observed in the minor salivary gland-derived organoids, and among the remaining genes, KLK5 and KLK7 were found to have significantly increased expression in the SS group.

To perform proteomic analysis in saliva derived from minor salivary glands, a group with a focus score of 2 or more and positive for anti-SSA (n = 4, severe), a group with a focus score of 1 or less and positive or negative for anti-SSA (n = 4, mild) and comparison between groups was performed. As a result, it was confirmed that the proteome was clustered differently between the mild and severe groups on the PCA plot (Figure 2). When a volcano plot was drawn to identify proteins with statistically significant differences in comparison between groups, the protein that significantly increased with a p-value of 0.01 or less in the severe group was Kallikrein-7 (gene name: KLK7). was detected significantly (Figure 3). In addition, amylase 1-alpha was detected among several proteins that significantly decreased in the severe group compared to the mild group, so it can be said that the experiment shows a good correlation with the severity of Sjögren's disease. . When setting the P value below 0.05, KLK7 and Cystatin-B (gene name: CSTB), Ubiquitin-conjugating enzyme E2 variant 2 (gene name: UBE2V2), Thymosin beta-10 (gene name: TMSB10), Elafin ( Gene name: PI3) was observed to be significantly increased in patients with severe Sjögren syndrome. This was again verified in salivary gland-derived organoids to confirm whether it was increased in Sjögren's patients at the gene level. As a result, among the five genes with a p value of 0.05 or less, KLK7 was found to be significantly increased at the gene level.

By combining the above experimental results, it was confirmed that KLK5 and KLK7 increased in organoids derived from bovine salivary glands. Additionally, at the protein level, KLK7, Cystatin-B (gene name: CSTB), Ubiquitin-conjugating enzyme E2 variant 2 (gene name: UBE2V2), Thymosin beta-10 (gene name: TMSB10), and Elafin (gene name: PI3) It was confirmed that it was increasing. Through this, KLK5, KLK7, Cystatin-B (gene name: CSTB), Ubiquitin-conjugating enzyme E2 variant 2 (gene name: UBE2V2), and Thymosin beta-10 (gene name: CSTB) were identified in organoids derived from minor salivary glands and saliva derived from minor salivary glands. TMSB10) and Elafin (gene name: PI3) are proposed as Sjögren syndrome-specific markers.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

1. Kallikrein-5 (KLK5), Kallikrein-7 (KLK7), Cystatin-B (CSTB), Ubiquitin-conjugating enzyme E2 variant 2 2; UBE2V2), Thymosin beta-10 (TMSB10), and Elafin (PI3). A bio for diagnosing Sjögren's syndrome containing as an active ingredient one or more proteins selected from the group consisting of, or genes encoding the same. Marker composition.
2. A composition for diagnosing Sjögren's syndrome comprising, as an active ingredient, an agent capable of measuring the expression level of one or more proteins selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10 and PI3 or the genes encoding them.
3. The composition for diagnosing Sjogren's syndrome according to claim 2, wherein the expression level is measured from minor salivary gland tissue or saliva.
4. The method of claim 2, wherein the agent capable of measuring the expression level is a primer or probe that specifically binds to any one or more genes selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3, KLK5, A composition for diagnosing Sjogren's syndrome, which is an antibody, peptide, aptamer or compound that specifically binds to one or more proteins selected from the group consisting of KLK7, CSTB, UBE2V2, TMSB10 and PI3.
5. A kit for diagnosing Sjogren's syndrome comprising the composition of any one of claims 2 to 4.
6. (1) mRNA expression level of one or more genes selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10 and PI3 or selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10 and PI3 from samples isolated from patients Measuring the expression level of one or more proteins;

   (2) comparing the mRNA expression level of the gene or the expression level of the protein with a control sample; and

   (3) A method of providing information necessary for diagnosing Sjögren's syndrome, including the step of determining Sjögren's syndrome when the mRNA expression level of the gene or the protein expression level is higher than that of the control sample.
7. The method of claim 6, wherein the sample is minor salivary gland tissue or saliva.
8. (1) contacting the test substance with minor salivary gland tissue or minor salivary gland organoids;

   (2) measuring the expression or activity level of one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 in minor salivary gland tissue or minor salivary gland organoids in contact with the test substance; and

   (3) A screening method for a Sjögren's syndrome treatment comprising the step of selecting a test substance with a reduced level of expression or activity of one or more selected from the group consisting of KLK5, KLK7, CSTB, UBE2V2, TMSB10, and PI3 compared to the control sample.

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