JP7803502B2 - Novel biomarkers for diagnosing oral lichen planus and their applications - Google Patents

Description

The present invention relates to a novel biomarker for diagnosing oral lichen planus, and a method for providing information for diagnosing oral lichen planus using the biomarker.

Oral lichen planus (OLP) is a persistent (chronic) inflammatory condition that affects the mucous membranes inside the mouth. Oral lichen planus can appear as white, reticular patches (red, swollen tissue or open sores). These lesions can cause burning, pain, or other inconveniences.

Although symptoms can generally be managed, people with oral lichen planus are at risk of developing oral cancer in the affected areas and therefore require regular monitoring. Furthermore, due to the characteristics of oral lichen planus as an inflammatory disease, the cause of its onset and a clear treatment method have not been identified, and only symptomatic treatment is currently available.

In addition, oral lichen planus is sometimes misdiagnosed and treated because it is clinically difficult to distinguish from other diseases that present with similar symptoms, such as white lesions in the oral cavity. In particular, some forms of oral lichen planus are also being considered precancerous lesions, requiring special care in their diagnosis and treatment.

The inventors hereby aim to discover biomarkers that can be used to diagnose oral lichen planus, and to complete and provide the present invention for clinical use.

Integrative analysis of mRNA and miRNA expression profiles in oral lichen planus: preliminary results, Junjun Chen et al., Oral Surg Oral Med Oral Pathol Oral Radiol;124(4):390-402.e17 Tumour Necrosis Factor Alpha (TNF-α) and Oral Squamous Cell Carcinoma, Gary Brierly et al., Cancers (Basel). 2023 Mar; 15(6): 1841. Salivary MMP-1, MMP-2, MMP-3 and MMP-13 Levels in Patients with Oral Lichen Planus and Squamous Cell Carcinoma, Tahereh Nosratzehi et al., Asian Pac J Cancer Prev. 2017; 18(7): 1947-1951.

The technical problem that this invention aims to achieve is to identify and characterize CLC (Galectin-10, UniProt Accession No. Q05315), TNFAIP8 (Tumor necrosis factor alpha-induced protein 8, UniProt Accession No. O95379), EVI5L (EVI5-like protein, UniProt Accession No. Q96CN4), ISG15 (Ubiquitin-like protein ISG15, UniProt Accession No. P05161), and PIGS (GPI transaminase component PIG-S, UniProt Accession No. Q96S52), SLC4A1 (Band 3 anion transport protein, UniProt Accession No. P02730), MYH1 (Myosin-1, UniProt Accession No. P12882), CLTA (Clathrin light chain A, UniProt Accession No. P09496), CCAR2 (Cell cycle and apoptosis regulator protein) 2, UniProt Accession No. Q8N163), GRK6 (G protein-coupled receptor kinase 6, UniProt Accession No. P43250), ATP5F1D (ATP synthesis subunit delta, mitochondrial, UniProt Accession No. P30049), ASRGL1 (Isoaspartyl peptide/L-asparaginase, UniProt Accession No. Q7L266), BST1 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, UniProt Accession No. Q10588), CYBA (Cytochrome b-245 light chain, UniProt Accession No. P13498), ASAP1 (Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1, UniProt Accession No. Q9ULH1), MAP2K4 (Dual specificity mitogen-activated protein kinase kinase 4, UniProt Accession No. P45985), RNASE2 (Non-secretory ribonuclease, UniProt Accession No. P10153), NIPSNAP2 (Protein NipSnap homolog 2, UniProt Accession No. O75323), MMP1 (Interstitial collagenase, UniProt Accession No. P03956), EWSR1 (RNA-binding protein EWS, UniProt Accession No. Q01844), CCL28 (CC motif chemokine 28, UniProt Accession No Q9NRJ3), LEG1 (Protein LEG1 homolog, UniProt Accession No Q6P5S2), ACBP (Acyl-CoA-binding protein, UniProt Accession No. P07108), APOM (Apolipoprotein M, UniProt Accession No. O95445), TFF3 (Trefoil factor 3, UniProt Accession No. Q07654), FOLR1 (Folate receptor alpha, UniProt Accession No. P15328), WFDC2 (WAP four-disulfide core domain protein 2, UniProt Accession No. Q14508), SERPINA1 (Alpha-1-antitrypsin, UniProt Accession No P01009), SLURP1 (Secreted Ly-6/uPAR-related protein 1, UniProt Accession No. P55000), DEFB4A (Defensin beta 4A, UniProt Accession No. O15263), SLPI (Antileukoproteinase, UniProt Accession No P03973), IGFBP7 (Insulin-like growth factor-binding protein 7, UniProt Accession No. Q16270), IGLV1-51 (Immunoglobulin lambda variable 1-51, UniProt Accession No. P01701), SERPINI1 (Neuroserpin, UniProt Accession No. Q99574), IGLV3-9 (Immunoglobulin lambda variable 3-9, UniProt Accession No. A0A075B6K5), SERPINA3 (Alpha-1-antichymotrypsin, UniProt Accession No P01011), SPATS2L (SPATS2-like protein, UniProt Accession No Q9NUQ6), APOC1 (Apolipoprotein C-I, UniProt Accession No P02654), IGKV2-29 (Immunoglobulin Provided is a biomarker composition for diagnosing oral lichen planus, comprising one or more selected from the group consisting of kappa variable 2-29, UniProt Accession No. A2NJV5) and KALRN (Kalirin, UniProt Accession No. O60229).

Other technical problems that the present invention aims to achieve are the following: CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, The present invention provides a diagnostic composition for oral lichen planus, which comprises a preparation capable of measuring the expression level of one or more proteins selected from the group consisting of SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or the genes encoding such proteins.

A further technical problem that the present invention aims to achieve is the detection of CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERP The present invention provides a method for providing information for diagnosing oral lichen planus, which includes measuring the expression level of one or more proteins selected from the group consisting of INA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or genes encoding such proteins.

However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and additional problems not mentioned will be clearly understood by those skilled in the art from the description below.

To solve the above-mentioned problems, the present inventors have identified CLC (Galectin-10, UniProt Accession No. Q05315), TNFAIP8 (Tumor necrosis factor alpha-induced protein 8, UniProt Accession No. O95379), EVI5L (EVI5-like protein, UniProt Accession No. Q96CN4), ISG15 (Ubiquitin-like protein ISG15, UniProt Accession No. P05161), PIGS (GPI transaminase component PIG-S, UniProt Accession No. Q96S52), SLC4A1 (Band 3 anion transport protein, UniProt Accession No. P02730), MYH1 (Myosin-1, UniProt Accession No. P12882), CLTA (Clathrin light chain A, UniProt Accession No. P09496), CCAR2 (Cell cycle and apoptosis regulator protein) 2, UniProt Accession No. Q8N163), GRK6 (G protein-coupled receptor kinase 6, UniProt Accession No. P43250), ATP5F1D (ATP synthesis subunit delta, mitochondrial, UniProt Accession No. P30049), ASRGL1 (Isoaspartyl peptide/L-asparaginase, UniProt Accession No. Q7L266), BST1 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, UniProt Accession No. Q10588), CYBA (Cytochrome b-245 light chain, UniProt Accession No. P13498), ASAP1 (Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1, UniProt Accession No. Q9ULH1), MAP2K4 (Dual specificity mitogen-activated protein kinase kinase 4, UniProt Accession No. P45985), RNASE2 (Non-secretory ribonuclease, UniProt Accession No. P10153), NIPSNAP2 (Protein NipSnap homolog 2, UniProt Accession No. O75323), MMP1 (Interstitial collagenase, UniProt Accession No. P03956), EWSR1 (RNA-binding protein EWS, UniProt Accession No. Q01844), CCL28 (CC motif chemokine 28, UniProt Accession No Q9NRJ3), LEG1 (Protein LEG1 homolog, UniProt Accession No Q6P5S2), ACBP (Acyl-CoA-binding protein, UniProt Accession No. P07108), APOM (Apolipoprotein M, UniProt Accession No. O95445), TFF3 (Trefoil factor 3, UniProt Accession No. Q07654), FOLR1 (Folate receptor alpha, UniProt Accession No. P15328), WFDC2 (WAP four-disulfide core domain protein 2, UniProt Accession No. Q14508), SERPINA1 (Alpha-1-antitrypsin, UniProt Accession No P01009), SLURP1 (Secreted Ly-6/uPAR-related protein 1, UniProt Accession No. P55000), DEFB4A (Defensin beta 4A, UniProt Accession No. O15263), SLPI (Antileukoproteinase, UniProt Accession No P03973), IGFBP7 (Insulin-like growth factor-binding protein 7, UniProt Accession No. Q16270), IGLV1-51 (Immunoglobulin lambda variable 1-51, UniProt Accession No. P01701), SERPINI1 (Neuroserpin, UniProt Accession No. Q99574), IGLV3-9 (Immunoglobulin lambda variable 3-9, UniProt Accession No. A0A075B6K5), SERPINA3 (Alpha-1-antichymotrypsin, UniProt Accession No P01011), SPATS2L (SPATS2-like protein, UniProt Accession No Q9NUQ6), APOC1 (Apolipoprotein C-I, UniProt Accession No P02654), IGKV2-29 (Immunoglobulin Provided is a biomarker composition for diagnosing oral lichen planus, comprising one or more selected from the group consisting of kappa variable 2-29, UniProt Accession No. A2NJV5) and KALRN (Kalirin, UniProt Accession No. O60229).

According to a further embodiment of the present invention, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, S A diagnostic composition for oral lichen planus is provided, which includes a preparation capable of measuring the expression level of one or more proteins selected from the group consisting of ERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or genes encoding such proteins.

In one aspect, the preparation 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.

On the other hand, the preparation 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.

According to a further embodiment of the present invention, there is provided a diagnostic kit for oral lichen planus, comprising any one of the diagnostic compositions described above.

According to a further embodiment of the present invention, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WF are isolated from a biological sample isolated from a subject. A method for providing information for diagnosing oral lichen planus is provided, which includes measuring the expression level of one or more proteins selected from the group consisting of DC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or genes encoding the same.

According to one aspect, the method comprises extracting CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, SLU from a biological sample isolated from a control group. The method may further include measuring the expression level of one or more proteins or genes encoding the same selected from the group consisting of RP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, and comparing the expression levels of the proteins or genes encoding the same between the subject and the control group.

In one aspect, if the expression level of one or more proteins or genes encoding the same selected from the group consisting of CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, and EWSR1 in the subject is higher than that in the control group, the subject can be diagnosed as having oral lichen planus.

In one aspect, if the expression level of one or more proteins selected from the group consisting of CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN or genes encoding the same is higher in the subject than in the control group, the subject can be determined to be normal.

According to one aspect, the biological sample may be saliva.

According to the present invention, oral lichen planus can be diagnosed quickly and accurately using saliva, a sample that can be collected non-invasively and is highly accessible.

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.

1 is a schematic diagram showing a saliva sample analysis method according to the present invention.

The inventors intend to collect saliva samples from a group of patients with oral lichen planus, subject them to the same analysis as samples from a normal control group, and identify proteins that are significantly more or less expressed in samples from oral lichen planus patients compared to samples from the control group. They then aim to complete and provide these as inventions for biomarkers and compositions for diagnosing oral lichen planus.

The present inventor has obtained CLC (Galectin-10, UniProt Accession No. Q05315), TNFAIP8 (Tumor necrosis factor alpha-induced protein 8, UniProt Accession No. O95379), EVI5L (EVI5-like protein, UniProt Accession No. Q96CN4), ISG15 (Ubiquitin-like protein ISG15, UniProt Accession No. P05161), PIGS (GPI transamidase component PIG-S, UniProt Accession No. Q96S52), SLC4A1 (Band 3 anion transport protein, UniProt Accession No. P02730), MYH1 (Myosin-1, UniProt Accession No. P12882), CLTA (Clathrin light chain A, UniProt Accession No. P09496), CCAR2 (Cell cycle and apoptosis regulator protein) 2, UniProt Accession No. Q8N163), GRK6 (G protein-coupled receptor kinase 6, UniProt Accession No. P43250), ATP5F1D (ATP synthesis subunit delta, mitochondrial, UniProt Accession No. P30049), ASRGL1 (Isoaspartyl peptide/L-asparaginase, UniProt Accession No. Q7L266), BST1 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, UniProt Accession No. Q10588), CYBA (Cytochrome b-245 light chain, UniProt Accession No. P13498), ASAP1 (Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1, UniProt Accession No. Q9ULH1), MAP2K4 (Dual specificity mitogen-activated protein kinase kinase 4, UniProt Accession No. P45985), RNASE2 (Non-secretory ribonuclease, UniProt Accession No. P10153), NIPSNAP2 (Protein NipSnap homolog 2, UniProt Accession No. O75323), MMP1 (Interstitial collagenase, UniProt Accession No. P03956), EWSR1 (RNA-binding protein EWS, UniProt Accession No. Q01844), CCL28 (CC motif chemokine 28, UniProt Accession No Q9NRJ3), LEG1 (Protein LEG1 homolog, UniProt Accession No Q6P5S2), ACBP (Acyl-CoA-binding protein, UniProt Accession No. P07108), APOM (Apolipoprotein M, UniProt Accession No. O95445), TFF3 (Trefoil factor 3, UniProt Accession No. Q07654), FOLR1 (Folate receptor alpha, UniProt Accession No. P15328), WFDC2 (WAP four-disulfide core domain protein 2, UniProt Accession No. Q14508), SERPINA1 (Alpha-1-antitrypsin, UniProt Accession No P01009), SLURP1 (Secreted Ly-6/uPAR-related protein 1, UniProt Accession No. P55000), DEFB4A (Defensin beta 4A, UniProt Accession No. O15263), SLPI (Antileukoproteinase, UniProt Accession No P03973), IGFBP7 (Insulin-like growth factor-binding protein 7, UniProt Accession No. Q16270), IGLV1-51 (Immunoglobulin lambda variable 1-51, UniProt Accession No. P01701), SERPINI1 (Neuroserpin, UniProt Accession No. Q99574), IGLV3-9 (Immunoglobulin lambda variable 3-9, UniProt Accession No. A0A075B6K5), SERPINA3 (Alpha-1-antichymotrypsin, UniProt Accession No P01011), SPATS2L (SPATS2-like protein, UniProt Accession No Q9NUQ6), APOC1 (Apolipoprotein C-I, UniProt Accession No P02654), IGKV2-29 (Immunoglobulin Provided is a biomarker composition for diagnosing oral lichen planus, comprising one or more selected from the group consisting of kappa variable 2-29, UniProt Accession No. A2NJV5) and KALRN (Kalirin, UniProt Accession No. O60229).

Information on proteins or the genes encoding them using the UniProt Accession Number can be found at https://www.UniProt.org.

As used herein, "diagnosis" means predicting disease occurrence, determining risk or susceptibility, or confirming the presence or characteristics of a pathological condition. For purposes of this invention, diagnosis refers to the diagnosis of oral lichen planus.

In the present invention, the term "diagnostic (bio)marker, (bio)marker for diagnosing, or diagnostic marker" refers to a marker, including a preparation or composition contained therein, that can distinguish oral lichen planus from normal cells and diagnose the condition, and can measure the levels of organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, and sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increased or decreased pattern in cells with oral lichen planus compared to normal cells.

According to a further embodiment of the present invention, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, S A diagnostic composition for oral lichen planus is provided, which includes a preparation capable of measuring the expression level of one or more proteins selected from the group consisting of ERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or genes encoding such proteins.

As used herein, the term "measuring expression level" refers to measuring the presence or absence, expression, or degree of expression of a specific protein (peptide) or a gene encoding the protein, specifically CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, and MMP1. The expression level of one or more proteins selected from the group consisting of EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or the mRNA or gene encoding the same, can be measured.

In one aspect, the preparation 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.

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 may be directed against CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FO The antibody can specifically bind to one or more proteins or fragments thereof selected from the group consisting of LR1, WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN. The fragment refers to a protein fragment having one or more epitopes recognized by an antibody against the protein, and may be, for example, an immunogenic fragment. The antibody may be in the form of a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, including all immunoglobulin antibodies. The antibody also includes specialized antibodies such as humanized antibodies.

Using such antibodies, it is possible to confirm whether the protein is expressed in a biological sample using methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, polyacrylic gel Western blotting, or immunoblotting.

As used herein, the term "antibody fragment" 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. Such fragments include functional fragments of antibody molecules that are not intact antibodies having two light chains and two heavy chains. A functional fragment of an antibody molecule refers to a fragment that retains at least the antigen-binding function, and may be Fab, F(ab'), F(ab')2, or Fv. Such binding fragments may contain a minimum of 7 or more amino acids, e.g., 9 or more amino acids, or 12 or more amino acids.

Analytical methods for detecting the above proteins include Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoassay, immunohistochemistry assay, immunoprecipitation assay, complement fixation assay, and fluorescence-activated cell sorter. Examples include, but are not limited to, activated cell sorters (FACS), protein chips, etc.

In one aspect, the preparation 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. The analytical method may be, 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.

As used herein, the term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group that can form base pairs with a template complementary to a specific base sequence and act as a starting point for copying the template. A primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer solution and temperature. For example, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNAS E2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-5 The presence or absence of oral lichen planus in an individual can be confirmed by performing PCR amplification using sense and antisense primers having a sequence of 7 to 50 nucleotides as specific primers for a gene or mRNA encoding one or more proteins selected from the group consisting of SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN. The amount of the desired product produced can then be measured. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques well known in the art. The primers may have lengths of 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.

As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, that specifically binds to a target nucleic acid, e.g., mRNA, and may be labeled to enable the presence, content, and expression level of a specific mRNA to be confirmed. Probes may be prepared in the form of oligonucleotide probes, single-strand DNA probes, double-strand DNA probes, RNA probes, etc. For example, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2 K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, The presence or absence of oral lichen planus in an individual can be diagnosed by hybridizing 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 IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, and measuring the expression level of the mRNA through the degree of hybridization. Selection of an appropriate probe and hybridization conditions may be appropriately determined using techniques known in the art. The probe may have 10-100, 15-100, 10-80, 10-50, 10-30, 10-20, 15-80, 15-50, 15-30, 15-20, 20-100, 20-80, 20-50, or 20-30 nt.

The primers or probes can be chemically synthesized using phosphoramidite solid support synthesis or a wide variety of other well-known methods. Furthermore, such nucleic acid sequences can be modified by a variety of methods known in the art. Examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with their analogs, or internucleotide modifications, such as uncharged linkers (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkers (e.g., phosphorothioates, phosphorodithioates, etc.). The primers or probes can also be modified with a label capable of directly or indirectly providing a detectable signal. Examples of such labels include radioisotopes, fluorescent molecules, or biotin.

According to a further embodiment of the present invention, there is provided a diagnostic kit for oral lichen planus comprising any one of the above diagnostic compositions.

For example, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, S By using a composition containing a protein detection agent for one or more proteins selected from the group consisting of ERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, the expression level of the corresponding protein in a sample can be confirmed, providing information for diagnosing oral lichen planus. The kit may be an immunoassay kit.

Another embodiment of the oral lichen planus diagnostic kit of the present invention includes CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR The present invention provides information useful for diagnosing oral lichen planus, including a preparation for detecting genes encoding any one or more proteins selected from the group consisting of: WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN. For example, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGFBP8, IGFBP9, IGFBP10, IGFBP11, IGFBP12, IGFBP13, IGFBP14, IGFBP15, IGFBP16, IGFBP17, IGFBP18, IGFBP19, IGFBP19, IGFBP16, IGFBP19 ... Nucleic acids encoding one or more proteins selected from the group consisting of GLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN are extracted, and a composition containing a preparation for detecting the extracted nucleic acids is used to confirm the level of expression of the nucleic acids encoding the corresponding proteins in the sample (when using a sample in which mRNA is reverse transcribed, cDNA and a preparation for detecting the cDNA are applied), thereby providing information for diagnosing oral lichen planus. The kit may be a DNA chip.

According to a further embodiment of the present invention, CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WF are isolated from a biological sample isolated from a subject. A method for providing information for diagnosing oral lichen planus is provided, which includes measuring the expression level of one or more proteins selected from the group consisting of DC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or genes encoding the same.

As used herein, the term "subject" refers to any living organism that develops or can develop oral lichen planus, and specific examples include, but are not limited to, mammals such as dogs, cats, mice, rats, monkeys, cows, pigs, minipigs, livestock, and humans.

As used herein, the term "sample" refers to a substance derived from the subject, and may specifically include tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, etc., and preferably includes, but is not limited to, saliva and oral mucosal tissues such as buccal smears and nasopharyngeal smears.

According to one aspect, the method comprises extracting CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, EWSR1, CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, S from a biological sample isolated from a control group. The method may further include measuring the expression level of one or more proteins or genes encoding the same selected from the group consisting of LURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, and comparing the expression levels of the proteins or genes encoding the same between the subject and the control group.

As used herein, the term "control group" preferably refers to a group that does not exhibit any pathological condition related to oral lichen planus.

In one aspect, if the expression level of one or more proteins selected from the group consisting of CLC, TNFAIP8, EVI5L, ISG15, PIGS, SLC4A1, MYH1, CLTA, CCAR2, GRK6, ATP5F1D, ASRGL1, BST1, CYBA, ASAP1, MAP2K4, RNASE2, NIPSNAP2, MMP1, and EWSR1 in the subject, or the gene encoding the same, is higher than that in the control group, preferably by two or more times., the subject can be determined to have oral lichen planus.

In one aspect, if the expression level of one or more proteins selected from the group consisting of CCL28, LEG1, ACBP, APOM, TFF3, FOLR1, WFDC2, SERPINA1, SLURP1, DEFB4A, SLPI, IGFBP7, IGLV1-51, SERPINI1, IGLV3-9, SERPINA3, SPATS2L, APOC1, IGKV2-29, and KALRN, or the gene encoding the same, in the subject is higher than that in the control group, preferably lower by 0.5 times or less, the subject can be determined to be normal.

The terms used in the embodiments are for explanatory purposes only and should not be construed as limiting. The singular expressions include the plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" indicate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

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 present invention pertains. Commonly used, predefined terms should be interpreted to have a meaning consistent with the meaning they have in the context of the relevant art, and should not be interpreted as having an ideal or overly formal meaning unless expressly defined herein.

The present invention is susceptible to various modifications and can have various embodiments. Below, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but should be understood as including all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention. If a detailed description of related publicly known technology in the description of the present invention is deemed to obscure the gist of the present invention, that detailed description will be omitted.

Embodiment 1. Sample Collection

Saliva samples were collected from 11 patients with oral lichen planus at Seoul National University Bundang Hospital and 9 normal controls at Hanyang University Hospital (IRB No. B-2103-675-301). After spitting to remove mucus and foreign matter from the mouth, subjects gargled with 10 mL of distilled water for 1 minute, and the gargled fluid was used as the saliva sample. To prevent protein degradation or deformation in the saliva sample, cOmplete Mini EDTA-free protease inhibitor cocktail (Roche) was added as a protease inhibitor. 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 study.

Embodiment 2. Quantification of collected saliva samples

The collected saliva samples were quantified using the bicinchoninic acid (BCA) assay. The samples were diluted with water at a 1:2 ratio to measure protein concentration. Figure 1 shows a schematic diagram of the testing process for discovering protein biomarkers using saliva samples.

Embodiment 3. Sample preparation and in-solution digestion for mass spectrometry

For mass spectrometry, 200 μg of each of the 11 individual samples from the patient group collected in Example 1 above was taken based on the results of the protein concentration measurement described above, and 100 μg of each of the nine individual samples from the normal control group was taken and concentrated to 100 μL each. Then, 8 M urea and 2 M thiourea were added to the samples. Disulfide bonds were reduced at 37°C using 0.5 μmol of TCEP (tris(2-carboxyethyl)phosphine), followed by alkylation at 25°C using 1 μmol of IAA (2-iodoacetamide) in a dark environment. The 8 M urea was then diluted with 25 mM ammonium bicarbonate to 1 M urea. LysC (Endoproteinase Lys-C) was added at a ratio of 1:25 and digested at 30°C for 2 hours, followed by trypsin at a ratio of 1:50 and digestion overnight at 25°C. The peptide was then purified using a SepPak® tC18 cartridge (Sep-Pak tC18 1 cc Vac cartridge, 100 mg, Waters) and dried under vacuum. The dried peptide was dissolved in 100 mM tetraethylammonium bromide (TEAB) and quantified at a ratio of 1:25.

Embodiment 4. Tandem Mass Tag (TMT) labeling of peptide samples

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 (42 μg of peptide sample) prepared in Example 3. 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.

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 i.d. × 250 mm length; pore size 130 A; particle size 3.5 μm, Waters Corporation, Milford, MA, USA) using an Agilent 1290 Infinity liquid chromatography column, and high pH reversed-phase peptide fractionation was performed. The column was equilibrated with 100% Buffer A, 10 mM ammonium formate (pH 10). Buffer B was 90% acetonitrile in 10 mM ammonium formate (pH 10). The operating flow rate was 0.5 mL/min with the following gradient conditions:

0 min: 100% Buffer A and 0% Buffer B,
0 min to 10 min: 0% to 5% buffer B;
10 min to 48.5 min: 5% to 40% Buffer B;
48.5 min to 62.5 min: 40% to 70% Buffer B;
62.5 min to 72.5 min: 70% Buffer B,
72.5 min to 82.5 min: 70% to 5% Buffer B;
82.5 min to 92.5 min: 5% Buffer B

The sample was divided into 96 fractions containing peptides between 10 and 82.5 minutes. The 96 fractions were combined into 24 fractions, and then vacuum-dried. The dried peptide sample was resuspended and dissolved in 20.83 μL of 0.1% formic acid.

Embodiment 5. Peptide mass spectrum analysis

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 with the following gradient conditions:

0 min: 95% Buffer A and 5% Buffer B,
0 min to 4 min: 5% buffer B,
4 min to 13 min: 5% to 10% Buffer B;
13 min to 150 min: 10% to 25% Buffer B;
150-155 min: 25%-28% Buffer B;
155 min to 160 min: 28% to 40% Buffer B;
160-165 min: 40%-80% Buffer B;
165 min to 167 min: 80% buffer B;
167 min to 170 min: 80% to 5% Buffer B;
170-180 min: 5% Buffer B

The nanoLC system was installed on a Tribrid Orbitrap Eclipse. Full-scan MS spectra (300-16,000 m/z) were acquired with one microscan and a resolution of 120,000 m/z. Preview mode was used to select precursors and measure their charge states. MS/MS spectra for the 20 most intense ions from the preview illumination scan were acquired in the ion trap for the full scan with the following options: isolation window, 1.4 m/z; CID collision energy, 35%; and dynamic exclusion duration, 30 seconds. MS ³ scans were used for quantification: 10 synchronous precursor selection (SPS) ions; resolution of 50,000; higher-energy collisional dissociation (HCD) energy, 55%; scan range, 100-1000 m/z.

Embodiment 6. Data processing for protein identification and label-based quantitative analysis

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

The relative abundance of proteins present in two groups, the normal control group (nine samples) and the patient group (eleven samples), was calculated using Proteome Discoverer 2.4, using ^MS3 scans to calculate the signal-to-noise value of reporter ions labeled on peptides derived from each sample. To identify statistically significant proteins, a Student's t test analysis was performed on the abundance values based on the signal-to-noise value for the two groups (oral lichen planus patient group and normal control group) using Perseus software. A P value of less than 0.05 was considered statistically significant.

Embodiment 7. Identification of proteins differentially expressed in oral lichen planus

The results of identifying proteins present in the two groups of oral lichen planus patient and normal control samples confirmed that a total of 4,609 proteins were identified as one or more unique peptides. Of the identified proteins, 3,145 had signal-to-noise values that allowed for labeling-based quantitative analysis. To identify statistically significant proteins, a statistical analysis was performed on the abundance values of the 3,145 proteins based on the signal-to-noise values across the two groups (patient group and normal control group). 1,367 proteins were identified with a p-value of less than 0.05.

We attempted to select proteins that showed a statistically significant difference of 2-fold or more in expression in oral lichen planus patients from 1,367 statistically significant proteins, and identified the top 20 proteins. We also identified the top 20 proteins that showed a decrease of 0.5-fold or more in oral lichen planus patients, and the results are shown in Tables 1 and 2, respectively.

Although the embodiments of the present invention have been described in detail above with reference to the drawings, the present invention is not limited to the above-described embodiments, and those skilled in the art may apply various technical modifications and variations based on the above. For example, the described techniques may be performed in an order different from that described, and/or the components of the described systems, structures, devices, circuits, etc. may be combined or combined in a form different from that described, or may be substituted or replaced by other components or equivalents, and still achieve suitable results.

Accordingly, other embodiments, manufacturing examples, and equivalents to the claims are also within the scope of the following claims.

Claims (9)

A diagnostic biomarker composition for oral lichen planus, comprising CLC (Galectin-10, UniProt Accession No. Q05315 ) . A diagnostic composition for oral lichen planus, comprising a preparation capable of measuring the expression level of a CLC protein or a gene encoding the same. The diagnostic composition for oral lichen planus according to claim 2, wherein the preparation capable of measuring the expression level of the protein is an antibody or antigen-binding fragment thereof that specifically binds to the protein, or an aptamer that specifically binds to the protein. The diagnostic composition for oral lichen planus according to claim 2, wherein the preparation capable of measuring the expression level of the gene is a primer or probe that specifically binds to the nucleic acid molecule of the gene. 10. A method for providing information for diagnosing oral lichen planus, comprising the step of measuring the expression level of a CLC protein or a gene encoding the same from a biological sample isolated from a subject. The method for providing information according to claim 5, further comprising the steps of measuring the expression level of a CLC protein or a gene encoding the same from a biological sample isolated from a control group, and comparing the expression levels of the protein or the gene encoding the same between the subject and the control group. The information providing method according to claim 6, wherein the subject is diagnosed as having oral lichen planus when the expression level of the CLC protein or the gene encoding it in the subject is higher than that in the control group. The information providing method described in claim 5, wherein the biological sample is saliva. A diagnostic kit for oral lichen planus, comprising the diagnostic composition according to any one of claims 2 to 4.