Novel biomarker for prognosis of oral lichen planus and uses thereof

Abstract

The present invention relates to a biomarker composition for prognosis or pathological status determination of oral lichen planus, comprising at least one selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD. According to the present invention, it is possible to predict or determine the pathological condition or prognosis of oral lichen planus on the basis of a non-invasive and easily accessible sample such as saliva, and the invention can be utilized for establishing treatment strategies or developing therapeutics.

Description

Novel biomarkers and their applications for predicting the prognosis of oral lichen planus

[0001] The present invention relates to a novel biomarker for predicting the prognosis of oral lichen planus and a method for providing information on predicting the prognosis of oral lichen planus using the same.

[0002] Oral lichen planus (OLP) is a persistent (chronic) inflammatory condition affecting the mucous membranes inside the mouth. It may appear as white, mesh-like patches; red, swollen tissue; or open sores. These lesions may cause burning, pain, or other discomfort.

[0003] While symptoms are generally manageable, those with oral lichen planus are at risk for developing oral cancer in the affected area, requiring regular monitoring. Furthermore, due to the nature of oral lichen planus as an inflammatory disease, its cause and specific treatment remain unknown, and only symptomatic treatment is currently available.

[0004] Furthermore, oral lichen planus is clinically difficult to distinguish from other conditions with similar symptoms, such as white lesions in the oral cavity, which can lead to misdiagnosis and mistreatment. In particular, some forms of oral lichen planus are considered precancerous, requiring special attention in diagnosis and treatment.

[0005] Accordingly, the inventors of the present invention have discovered a biomarker related to the pathological condition of oral lichen planus and have completed and provided the present invention to use it for clinical prognosis prediction or confirmation of the pathological condition.

[0006] 본 발명이 이루고자 하는 기술적 과제는 BPIFA1 (BPI Fold Containing Family A Member 1, UniProt accession No. Q9NP55), ISG15 (Ubiquitin-like protein ISG15, UniProt accession No. P05161), APOBEC3B (DNA dC->dU-editing enzyme APOBEC-3B, UniProt accession No. Q9UH17), STAT1 (Signal Transducer And Activator Of Transcription 1-alpha / beta, UniProt accession No. P42224), WARS1 (Tryptophan--tRNA ligase, cytoplasmic, UniProt accession No. P23381), VPS11 (Vacuolar protein sorting-associated protein 11 homolog, UniProt accession No. Q9H270), MX1 (Interferon-induced GTP-binding protein Mx1, UniProt accession No. P20591), SSR1 (Translocon-associated protein subunit alpha, UniProt accession No. P43307), TMED2 (Transmembrane emp24 domain-containing protein 2, UniProt accession No. Q15363), UBA7 (Ubiquitin-like modifier-activating enzyme 7, UniProt accession No. P41226), NCSTN (Nicastrin, UniProt accession No. Q92542), STX3 (Syntaxin-3, UniProt accession No. Q13277), ARF3 (ADP-Ribosylation Factor 3, UniProt accession No.P61204), PRH1 / 2 (Salivary acidic proline-rich phosphoprotein 1 / 2, UniProt accession Nos. P02810 / P04278 for PRH1 and PRH2 respectively), TFF3 (Trefoil Factor 3, UniProt accession No. Q07654), CPE (Carboxypeptidase E, UniProt accession No. P16870), SAA1 (Serum Amyloid A1 protein, UniProt accession No. P0DJI8), NUCB2 (Nucleobindin-2, UniProt accession No. P80303), NUCB1 (Nucleobindin-1, UniProt accession No. Q02818), FOLH1 (Glutamate carboxypeptidase 2, UniProt accession No. Q04609), FAM3D (Protein FAM3D, The present invention provides a biomarker composition for predicting the prognosis of oral lichen planus, comprising at least one selected from the group consisting of: CPD (Carboxypeptidase D, UniProt accession No. Q96BQ1) and CPD (Carboxypeptidase D, UniProt accession No. O75976).

[0007] Another technical problem to be achieved by the present invention is to provide a composition for predicting the prognosis of oral lichen planus, comprising an agent capable of measuring the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same.

[0008] Another technical problem to be achieved by the present invention is to provide a method for providing information for predicting the prognosis of oral lichen planus, comprising a step of measuring the expression level of at least one protein selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] 상기 과제를 해결하기 위하여, 본 발명자들은 BPIFA1 (BPI Fold Containing Family A Member 1, UniProt accession No. Q9NP55), ISG15 (Ubiquitin-like protein ISG15, UniProt accession No. P05161), APOBEC3B (DNA dC->dU-editing enzyme APOBEC-3B, UniProt accession No. Q9UH17), STAT1 (Signal Transducer And Activator Of Transcription 1-alpha / beta, UniProt accession No. P42224), WARS1 (Tryptophan--tRNA ligase, cytoplasmic, UniProt accession No. P23381), VPS11 (Vacuolar protein sorting-associated protein 11 homolog, UniProt accession No. Q9H270), MX1 (Interferon-induced GTP-binding protein Mx1, UniProt accession No. P20591), SSR1 (Translocon-associated protein subunit alpha, UniProt accession No. P43307), TMED2 (Transmembrane emp24 domain-containing protein 2, UniProt accession No. Q15363), UBA7 (Ubiquitin-like modifier-activating enzyme 7, UniProt accession No. P41226), NCSTN (Nicastrin, UniProt accession No. Q92542), STX3 (Syntaxin-3, UniProt accession No.Q13277), ARF3 (ADP-Ribosylation Factor 3, UniProt accession No. P61204), PRH1 / 2 (Salivary acidic proline-rich phosphoprotein 1 / 2, UniProt accession Nos. P02810 / P04278 for PRH1 and PRH2 respectively), TFF3 (Trefoil Factor 3, UniProt accession No. Q07654), CPE (Carboxypeptidase E, UniProt accession No. P16870), SAA1 (Serum Amyloid A1 protein, UniProt accession No. P0DJI8), NUCB2 (Nucleobindin-2, UniProt accession No. P80303), NUCB1 (Nucleobindin-1, UniProt accession No. P80303). Q02818), FOLH1 (Glutamate carboxypeptidase 2, The present invention provides a biomarker composition for predicting the prognosis of oral lichen planus, comprising at least one selected from the group consisting of FAM3D (Protein FAM3D, UniProt accession No. Q04609), FAM3D (Protein FAM3D, UniProt accession No. Q96BQ1), and CPD (Carboxypeptidase D, UniProt accession No. O75976).

[0011] According to another embodiment of the present invention, a composition for predicting the prognosis of oral lichen planus is provided, comprising an agent capable of measuring the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD, or a gene encoding the same.

[0012] According to one aspect, the agent capable of measuring the expression level of the protein may be an antibody or an antigen-binding fragment thereof that specifically binds to the protein; or an aptamer that specifically binds to the protein.

[0013] According to one aspect, the agent capable of measuring the expression level of the gene may be a primer or probe that specifically binds to a nucleic acid molecule of the gene.

[0014] According to another embodiment of the present invention, a kit for confirming the prognosis of oral lichen planus is provided, comprising any one of the prognostic predictive compositions.

[0015] According to another embodiment of the present invention, a method for providing information for predicting the prognosis of oral lichen planus is provided, comprising the step of measuring the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same, from a biological sample isolated from a subject.

[0016] According to one aspect, the method may further include a step of measuring the expression level of one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same, from a biological sample isolated from a control group; and a step of comparing the expression levels of the proteins or the gene encoding the same in the subject and the control group.

[0017] According to one aspect, when the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3 and ARF3 or a gene encoding the same of the subject is higher than that of the control group, it may be predicted that the prognosis of the subject is poor.

[0018] According to one aspect, when the expression level of any one or more proteins selected from the group consisting of PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same of the subject is higher than that of the control group, it can be predicted that the subject has a good prognosis.

[0019] According to one side, the biological sample may be saliva.

[0020] According to the present invention, it is possible to rapidly and accurately predict the prognosis of oral lichen planus and confirm the pathological condition using saliva, a sample that can be collected non-invasively and is highly accessible.

[0021] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0022] Figure 1 is a schematic diagram showing a sample collection method and analysis method in the present invention.

[0023] Figure 2 shows a graph of the high pH fraction of a TMT labeled sample (x-axis: time (min), y-axis: absorbance (normalized intensity)).

[0024] The present inventors collected saliva samples from a group of patients with oral lichen planus, performed the same analysis on the saliva samples as on samples from a control group that received treatment, and identified proteins that were significantly higher or lower expressed in the samples from the patient group with oral lichen planus compared to the samples from the control group, and provided the invention of a biomarker and a composition for predicting the prognosis of oral lichen planus or confirming the pathological condition.

[0025] 본 발명자들은 BPIFA1 (BPI Fold Containing Family A Member 1, UniProt accession No. Q9NP55), ISG15 (Ubiquitin-like protein ISG15, UniProt accession No. P05161), APOBEC3B (DNA dC->dU-editing enzyme APOBEC-3B, UniProt accession No. Q9UH17), STAT1 (Signal Transducer And Activator Of Transcription 1-alpha / beta, UniProt accession No. P42224), WARS1 (Tryptophan--tRNA ligase, cytoplasmic, UniProt accession No. P23381), VPS11 (Vacuolar protein sorting-associated protein 11 homolog, UniProt accession No. Q9H270), MX1 (Interferon-induced GTP-binding protein Mx1, UniProt accession No. P20591), SSR1 (Translocon-associated protein subunit alpha, UniProt accession No. P43307), TMED2 (Transmembrane emp24 domain-containing protein 2, UniProt accession No. Q15363), UBA7 (Ubiquitin-like modifier-activating enzyme 7, UniProt accession No. P41226), NCSTN (Nicastrin, UniProt accession No. Q92542), STX3 (Syntaxin-3, UniProt accession No. Q13277), ARF3 (ADP-Ribosylation Factor 3, UniProt accession No.P61204), PRH1 / 2 (Salivary acidic proline-rich phosphoprotein 1 / 2, UniProt accession Nos. P02810 / P04278 for PRH1 and PRH2 respectively), TFF3 (Trefoil Factor 3, UniProt accession No. Q07654), CPE (Carboxypeptidase E, UniProt accession No. P16870), SAA1 (Serum Amyloid A1 protein, UniProt accession No. P0DJI8), NUCB2 (Nucleobindin-2, UniProt accession No. P80303), NUCB1 (Nucleobindin-1, UniProt accession No. Q02818), FOLH1 (Glutamate carboxypeptidase 2, UniProt accession No. Q04609), FAM3D (Protein FAM3D, UniProt The present invention provides a biomarker composition for predicting the prognosis of oral lichen planus, comprising at least one selected from the group consisting of: CPD (Carboxypeptidase D, UniProt accession No. Q96BQ1) and CPD (Carboxypeptidase D, UniProt accession No. O75976).

[0026] Information on proteins or genes encoding them according to the above UniProt Accession Number can be found at https: / www.UniProt.org.

[0027] In this specification, 'prognosis' means determining whether an individual has not yet been diagnosed or has recurrence, metastasis, drug response, resistance, treatment progress, pathological condition, etc. before / after treatment. In the present invention, the biomarkers were derived by comparing a patient group that did not receive treatment and a treatment group that did receive treatment, and the invention was completed by confirming that the biomarkers that were predominantly expressed in the patient group were indicators closer to a pathological condition. In other words, one of the most preferable examples in which the present invention can be utilized is to confirm the expression level of the biomarkers in a patient group that has already been diagnosed with or is being treated for oral lichen planus and to establish a treatment strategy based on whether the expression level is 1.5 times or more higher or lower than that of the control group, or whether the expression level of the biomarkers decreases or increases over time, or to use it as a histological marker that can quantitatively confirm whether appropriate treatment is being carried out, or to use it to distinguish it from other oral diseases with similar symptoms.

[0028] Here, “biomarker” generally includes all organic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, and sugars that can be detected in biological samples and that can detect biological changes.

[0029] For the purposes of the present invention, the above prognosis refers to the prognosis of oral lichen planus.

[0030] In the present invention, the term '(bio)marker for predicting prognosis, (bio)marker for determining prognosis or prognostic marker' means a marker including an agent capable of distinguishing the level of organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in the saliva of a patient group compared to a treated group, or a composition included therein, which can determine whether the pathological condition of oral lichen planus is improved, from normal cells or can measure the level thereof.

[0031] According to another embodiment of the present invention, a composition for predicting the prognosis of oral lichen planus is provided, comprising an agent capable of measuring the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD, or a gene encoding the same.

[0032] The term "measuring expression level" in this specification refers to measuring the presence, expression, or expression level of a specific protein (peptide) or a gene encoding the protein, and specifically, may be measuring the expression level of one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD, or mRNA or genes encoding the same.

[0033] According to one aspect, the agent capable of measuring the expression level of the protein may be an antibody or an antigen-binding fragment thereof that specifically binds to the protein; or an aptamer that specifically binds to the protein.

[0034] The term "antibody" as used herein is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. For example, the antibody can specifically bind to one or more proteins or fragments thereof selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD. The fragment refers to a protein fragment having one or more epitopes that can be recognized by an antibody against the protein, and may be, for example, an immunogenic fragment. The form of the antibody includes a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, and includes all immunoglobulin antibodies. Additionally, the above antibodies include special antibodies such as humanized antibodies.

[0035] Using these antibodies, it is possible to determine whether the protein is expressed in a biological sample by methods known in the art, such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting on polyacrylic gel, or immunoblotting.

[0036] The term "antibody fragment" as used herein refers to a polypeptide that does not have the structure of an intact antibody, peptide, or protein, but has a specific antigen-binding site or binding domain directed against an antigenic site. The fragment includes a functional fragment of an antibody molecule other than a complete antibody having two light chains and two heavy chains. A functional fragment of an antibody molecule means a fragment that retains at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, or Fv. The binding fragment may comprise at least 7 amino acids, for example, 9 amino acids, or 12 amino acids.

[0037] Analysis methods for detecting the above protein include, but are not limited to, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoassay, immunohistochemistry assay, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chip, etc.

[0038]

[0039] According to one aspect, the agent capable of measuring the expression level of the gene may be a primer or probe that specifically binds to the nucleic acid molecule of the gene. As an analysis method, for example, one or more methods selected from the group consisting of reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Realtime RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chips may be used.

[0040] The term "primer" as used herein refers to a nucleic acid sequence having a free 3' hydroxyl group, which can form base pairs with a template complementary to a specific base sequence and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. For example, PCR amplification is performed using sense and antisense primers having sequences of 7 to 50 nucleotides as specific primers for a gene or mRNA encoding one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD, and by measuring the amount of the desired product produced, it is possible to determine whether an individual has a pathological condition related to oral lichen planus or to predict the progression of the pathological condition. PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art. The above primers may have 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 50, or 20 to 30 nt.

[0041] The term "probe" in this specification refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a target nucleic acid, for example, mRNA, and may be labeled so as to enable detection of the presence, content, and expression level of a specific mRNA. The probe may be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. For example, by performing hybridization using a probe having a nucleic acid sequence complementary to a gene or mRNA encoding one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD, the expression level of mRNA can be measured through the degree of hybridization, thereby determining whether an individual is in a pathological state related to oral lichen planus or predicting the progression of the pathological state. The selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art. The probe may have 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 50, or 20 to 30 nt.

[0042] The primer or probe can be chemically synthesized using phosphoramidite solid support synthesis or other well-known methods. In addition, such nucleic acid sequences can be modified using various methods known in the art. Examples of such modifications can include methylation, capping, substitution with one or more homologs of a natural nucleotide, or modification between nucleotides, such as modification with an uncharged linker (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or a charged linker (e.g., phosphorothioate, phosphorodithioate, etc.). In addition, the primer or probe can be modified using a label that can directly or indirectly provide a detectable signal. Examples of such labels can include radioisotopes, fluorescent molecules, or biotin.

[0043] According to another embodiment of the present invention, a kit for determining the prognosis of oral lichen planus, comprising any one of the prognostic compositions described above, is provided. Determining the prognosis may include determining whether a subject is in a pathological condition or predicting the course of treatment.

[0044] For example, in a biological sample of an individual for which it is necessary to confirm the prognosis of oral lichen planus or to confirm improvement in the pathological condition, a composition including at least one protein detection agent selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD can be used to confirm the expression level of the corresponding protein in the sample, thereby providing information for predicting or determining the prognosis of oral lichen planus. The kit may be an immunoassay kit.

[0045] According to another embodiment of the present invention, a kit for predicting the prognosis of oral lichen planus includes an agent for detecting a gene encoding at least one protein selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD, thereby providing information utilized for confirming the prognosis of oral lichen planus. For example, a composition including an agent for extracting a nucleic acid encoding at least one protein selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD from a biological sample of an individual for which confirmation of the prognosis of oral lichen planus or improvement of the pathological condition is required, and detecting the nucleic acid, can be used to determine the expression level of the nucleic acid encoding the corresponding protein in the sample (when utilizing a sample in which mRNA is reverse transcribed, cDNA and an agent for detecting cDNA are applied), thereby providing information for predicting the prognosis of oral lichen planus. The kit may be a DNA chip.

[0046] According to another embodiment of the present invention, a method for providing information for predicting the prognosis of oral lichen planus is provided, comprising the step of measuring the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same, from a biological sample isolated from a subject.

[0047] The term "subject" in this specification means any living organism that is or is likely to be affected by oral lichen planus, and may include, but is not limited to, mammals such as dogs, cats, mice, rats, monkeys, cows, pigs, miniature pigs, livestock, and humans.

[0048] The term "sample" in this specification means a material derived from the subject, and may specifically include tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, etc., and preferably may include oral mucosal tissue such as saliva, oral swab, nasopharyngeal swab, etc., but is not limited thereto.

[0049]

[0050] According to one aspect, the method may further include a step of measuring the expression level of one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same, from a biological sample isolated from a control group; and a step of comparing the expression levels of the proteins or the gene encoding the same in the subject and the control group.

[0051] The term "control group" in this specification may preferably be a group that does not exhibit a pathological condition related to oral lichen planus, and most preferably may be a group of oral lichen planus patients whose pathological condition has improved through appropriate treatment commonly performed in the art.

[0052] According to one aspect, when the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3 and ARF3 or a gene encoding the same of the subject is higher than that of the control group, preferably higher by 1.5 times or more, it may be predicted or determined that the prognosis of the subject is poor.

[0053] According to one aspect, when the expression level of any one or more proteins selected from the group consisting of PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same of the subject is higher than that of the control group, preferably lower by 0.66 times or less, it may be predicted or determined that the prognosis of the subject is good.

[0054]

[0055] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0056] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0057] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0058] Example 1. Collection of samples

[0059] Saliva samples were collected from 11 patients with oral lichen planus and 10 patients in the oral lichen planus treatment group at Seoul National University Bundang Hospital (IRB No. B-2103-675-301). The treatment group was selected after clinically confirming symptom improvement after gargling with 5-10 cc of 0.05% dexamethasone solution twice daily for 5 minutes for 4 weeks. After spitting to remove mucus and foreign substances from the oral cavity, the gargle was gargled with 10 mL of distilled water for 1 minute. The spit-out gargle was used as a saliva sample. cOmplete, Mini EDTA-free protease inhibitor cocktail (Roche) was added as a protease inhibitor to prevent protein degradation or modification in the saliva samples. After adding the inhibitor to each sample to a final concentration of 1.5X, the samples were stored at -80°C until use in the experiment.

[0060]

[0061] Example 2. Quantification of collected saliva samples

[0062] The collected saliva samples were quantified using the bicinchoninic acid (BCA) assay. The protein concentration was measured after diluting the samples with water at a ratio of 1:2. Figure 1 illustrates a schematic diagram of the experimental process for protein biomarker discovery using saliva samples.

[0063]

[0064] Example 3. Sample preparation and in-solution digestion for mass spectrometry

[0065] For mass spectrometry, 11 patient samples and 10 individual samples from the treatment group collected in Example 1 were prepared into 200 μg samples based on the results of the protein concentration measurement described above, concentrated to 100 μL, and then added to the samples to make 8 M urea and 2 M thiourea. After reducing disulfide bonds at 37°C using 0.5 μmol of TCEP (tris(2-carboxyethyl)phosphine), alkylation was performed using 1 μmol of IAA (2-iodoacetamide) in a dark environment at 25°C. After that, 25 mM ammonium bicarbonate was further added to 8 M urea to make 1 M urea. LysC (Endoproteinase Lys-C) was added at a ratio of 1:25 and digested at 30℃ for 2 hours, and then trypsin was added at a ratio of 1:50 and digested overnight at 25℃. Subsequently, the peptide was purified using a SepPak® tC18 cartridge (Sep-Pak tC18 1cc Vac cartridge, 100mg, Waters) and dried by vacuum drying. The dried peptide was dissolved in 100 mM tetraethylammonium bromide (TEAB) and the peptide was quantified at a ratio of 1:25.

[0066]

[0067] Example 4. Tandem Mass Tag (TMT) labeling of peptide samples

[0068] TMT labeling reagent (Thermo Fisher Scientific) was dissolved in 80 μL of acetonitrile, and 36 μL of TMT labeling reagent was added to each sample prepared in Example 3, 42 μg of peptide sample. The reaction mixture was incubated at 20°C for 1 hour to label the peptides, and then 5 μL of 5% (w / v) hydroxylamine was added and incubated at 20°C for 15 minutes to terminate the labeling reaction. The TMT-labeled samples were combined into one tube, purified using a SepPak® tC18 cartridge, and then vacuum-dried.

[0069] The dried sample was dissolved in 10 mM ammonium formate. 500 μg of the sample was injected onto an X-Bridge peptide BEH C18 column (4.6 mm id × 250 mm length; pore size 130 Å; particle size 3.5 μm, Waters Corporation, Milford, MA, USA) using Agilent 1290 Infinity liquid chromatography to perform high pH reversed-phase peptide fractionation. The column was equilibrated with 100% buffer A of 10 mM ammonium formate (pH 10). Buffer B was 90% acetonitrile of 10 mM ammonium formate (pH 10). The operating flow rate was 0.5 mL / min under the following gradient conditions.

[0070] 0 min: 100% buffer A and 0% buffer B,

[0071] 0 to 10 minutes: 0% to 5% buffer B,

[0072] 10 to 48.5 minutes: 5% to 40% buffer B,

[0073] 48.5 to 62.5 minutes: 40% to 70% buffer B,

[0074] 62.5 to 72.5 minutes: 70% buffer B,

[0075] 72.5 to 82.5 minutes: 70% to 5% buffer B,

[0076] 82.5 to 92.5 minutes: 5% Buffer B

[0077] The sample was divided into 96 peptide fractions ranging from 10 to 82.5 minutes. The fractionation graph is shown in Figure 2. The 96 fractions were combined into 24 fractions, and the fractions were vacuum-dried. The dried peptide samples were resuspended in 20.83 μL of 0.1% formic acid and dissolved.

[0078]

[0079] Example 5. Peptide mass spectrum analysis

[0080] 7 μL of each fractionated peptide sample was injected onto a reversed-phase PepMap RSLC C18 column (50 cm X 75 μm) on an Ultimate 3000 system. The column was equilibrated with 100% Buffer A (100% water containing 0.1% (v / v) formic acid). Buffer B was 100% acetonitrile containing 0.1% (v / v) formic acid. The operating flow rate was 300 nL / min under the following gradient conditions:

[0081] 0 min: 95% buffer A and 5% buffer B,

[0082] 0 to 4 minutes: 5% buffer B,

[0083] 4 to 13 minutes: 5% to 10% buffer B,

[0084] 13 to 150 minutes: 10% to 25% buffer B,

[0085] 150 to 155 minutes: 25% to 28% buffer B,

[0086] 155 to 160 minutes: 28 to 40% buffer B,

[0087] 160 to 165 minutes: 40% to 80% buffer B,

[0088] 165 to 167 minutes: 80% buffer B,

[0089] 167 to 170 minutes: 80% to 5% buffer B,

[0090] 170 to 180 minutes: 5% Buffer B

[0091] The nanoLC system was equipped with a Tribrid Orbitrap Eclipse. Survey full-scan MS spectra (300–16,000 m / z) were acquired at 1 microscan and a resolution of 120,000, and a preview mode was set for precursor selection and charge state determination. MS / MS spectra of the 20 most intense ions from the preview survey scan were acquired in the ion trap for a full scan with the following options: isolation window, 1.4 m / z; CID collision energy, 35%; and dynamic exclusion duration, 30 sec.

[0092]

[0093] Example 6. Data Processing for Protein Identification and Label-Based Quantitative Analysis

[0094] Each LC-MS / MS file was analyzed using the SEQUEST algorithm in Proteome Discoverer 2.4. MS and MS / MS data were compared against the SwissProt human database (containing 20,423 proteins, updated June 2023). The search was restricted to allow two missed cleavages of tryptic peptides. Carboamidomethylation of cysteine ​​(+57.021 Da) and TMT labeling of lysine and the peptide N-terminus (+304.207 Da) were set as fixed modifiers, while methionine oxidation was set as a floating modifier (+15.995 Da). The mass tolerance was set to 0.6 Da for MS / MS data and 10 ppm for MS data.

[0095] The relative amounts of proteins present in two groups of samples (10 samples from the treatment group and 11 samples from the patient group) were analyzed using MS using Proteome Discoverer 2.4. 3 The signal-to-noise ratio (SNR) of the reporter ions labeled with peptides from each sample was calculated from the scan. To identify statistically significant proteins, Student's t test analysis was performed on the SNR-based abundance values ​​for two groups (oral lichen planus patient group and normal control group) using Perseus software. A P value less than 0.05 was considered statistically significant.

[0096]

[0097] Example 7. Identification of proteins differentially expressed in oral lichen planus

[0098] From the results of the identification of proteins present in the two groups of samples from the patient and treatment groups of oral lichen planus, a total of 4,609 proteins were identified with at least one unique peptide. Among the identified proteins, 3,145 proteins had signal-to-noise values ​​that allowed label-based quantitative analysis. To identify statistically significant proteins, statistical analysis was performed on the abundance values ​​based on the signal-to-noise values ​​of the 3,145 proteins for the two groups (patient and treatment groups), resulting in 50 proteins with a P-value less than 0.05.

[0099] Among the 51 statistically significant proteins, we aimed to select proteins with differential expression of 1.5-fold or more in the oral lichen planus patient group, and identified a total of 13 proteins. In addition, we identified 9 proteins whose expression was reduced by 0.66-fold or more in the oral lichen planus patient group compared to the treated group, and the results are presented in Tables 1 and 2, respectively.

[0100] UniProt Accession No.GeneDescription (단백질 명칭)단백질 발현 변화 ratio(환자군 / 치료군)Q9NP55BPIFA1BPI fold-containing family A member 17.50P05161ISG15Ubiquitin-like protein ISG152.59Q9UH17APOBEC3BDNA dC->dU-editing enzyme APOBEC-3B2.46P42224STAT1Signal transducer and activator of transcription 1-alpha / beta1.79P23381WARS1Tryptophan--tRNA ligase, cytoplasmic1.75Q9H270VPS11Vacuolar protein sorting-associated protein 11 homolog1.74P20591MX1Interferon-induced GTP-binding protein Mx11.72P43307SSR1Translocon-associated protein subunit alpha1.61Q15363TMED2Transmembrane emp24 domain-containing protein 21.58P41226UBA7Ubiquitin-like modifier-activating enzyme 71.56Q92542NCSTNNicastrin1.54Q13277STX3Syntaxin-31.53P61204ARF3ADP-ribosylation factor 31.50

[0101]

[0102] UniProt Accession No. Gene Description (Protein Name) Protein Expression Change Ratio (Patient Group / Treatment Group) P02810 PRH1 / 2 Salivary acidic proline-rich phosphoprotein 1 / 20.31 Q07654 TFF3 Trefoil factor 30.51 P16870 CPECarboxypeptidase E0.53 P0DJI8SAA1 Serum amyloid A-1 protein0.53 P80303 NUCB2 Nucleobindin-20.56 Q02818 NUCB1 Nucleobindin-10.58 Q04609 FOLH1 Glutamate carboxypeptidase 20.61 Q96BQ1 FAM3D Protein FAM3D0.63 O75976 CPD Carboxypeptidase D0.66

[0103] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0104] Therefore, other implementations, other manufacturing examples and equivalents to the patent claims also fall within the scope of the claims described below.

Claims

1. BPIFA1 (BPI Fold Containing Family A Member 1, UniProt accession No. Q9NP55), ISG15 (Ubiquitin-like protein ISG15, UniProt accession No. P05161), APOBEC3B (DNA dC->dU-editing enzyme APOBEC-3B, UniProt accession No. Q9UH17), STAT1 (Signal Transducer And Activator Of Transcription 1-alpha / beta, UniProt accession No. P42224), WARS1 (Tryptophan--tRNA ligase, cytoplasmic, UniProt accession No. P23381), VPS11 (Vacuolar protein sorting-associated protein 11 homolog, UniProt accession No. Q9H270), MX1 (Interferon-induced GTP-binding protein Mx1, UniProt accession No. P20591), SSR1 (Translocon-associated protein subunit alpha, UniProt accession No. P43307), TMED2 (Transmembrane emp24 domain-containing protein 2, UniProt accession No. Q15363), UBA7 (Ubiquitin-like modifier-activating enzyme 7, UniProt accession No. P41226), NCSTN (Nicastrin, UniProt accession No. Q92542), STX3 (Syntaxin-3, UniProt accession No. Q13277), ARF3 (ADP-Ribosylation Factor 3, UniProt accession No.P61204), PRH1 / 2 (Salivary acidic proline-richphosphoprotein 1 / 2, UniProt accession Nos. P02810 / P04278 for PRH1 and PRH2 respectively), TFF3 (Trefoil Factor 3, UniProt accession No. Q07654), CPE (Carboxypeptidase E, UniProt accession No. P16870), SAA1 (Serum Amyloid A1 protein, UniProt accession No.P0DJI8), NUCB2 (Nucleobindin-2, UniProt accession No. P80303), NUCB1 (Nucleobindin-1, UniProt accession No. Q02818), FOLH1 (Glutamate carboxypeptidase 2, UniProt accession No. Q04609), FAM3D (Protein FAM3D, UniProt A biomarker composition for predicting the prognosis of oral lichen planus, comprising at least one selected from the group consisting of: CPD (Carboxypeptidase D, UniProt accession No. Q96BQ1) and CPD (Carboxypeptidase D, UniProt accession No. O75976).

2. A composition for predicting the prognosis of oral lichen planus, comprising an agent capable of measuring the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD, or a gene encoding the same.

3. A composition for predicting the prognosis of oral lichen planus, wherein the agent capable of measuring the expression level of the protein in the second paragraph is an antibody or an antigen-binding fragment thereof that specifically binds to the protein; or an aptamer that specifically binds to the protein.

4. A composition for predicting the prognosis of oral lichen planus, wherein the agent capable of measuring the expression level of the gene in the second paragraph is a primer or probe that specifically binds to a nucleic acid molecule of the gene.

5. A method for providing information for predicting the prognosis of oral lichen planus, comprising the step of measuring the expression level of at least one protein selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD, or a gene encoding the same, from a biological sample isolated from a subject.

6. In the fifth paragraph, the method further comprises: a step of measuring the expression level of one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, ARF3, PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D, and CPD, or a gene encoding the same; and a step of comparing the expression levels of the proteins or the gene encoding the same in the subject and the control.

7. In the 6th paragraph, when the expression level of any one or more proteins selected from the group consisting of BPIFA1, ISG15, APOBEC3B, STAT1, WARS1, VPS11, MX1, SSR1, TMED2, UBA7, NCSTN, STX3, and ARF3 or a gene encoding the same of the subject is higher than that of the control group, an information providing method is provided, wherein the prognosis of the subject is predicted to be poor.

8. A method for providing information, wherein, in paragraph 6, if the expression level of any one or more proteins selected from the group consisting of PRH1 / 2, TFF3, CPE, SAA1, NUCB2, NUCB1, FOLH1, FAM3D and CPD or a gene encoding the same of the subject is higher than that of the control group, the prognosis of the subject is predicted to be good.

9. A method for providing information, wherein the biological sample is saliva in paragraph 5.

10. A kit for confirming the prognosis of oral lichen planus, comprising a composition for predicting the prognosis of any one of claims 2 to 4.

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