Biomarker composition for diagnosis of liver disease and pharmaceutical composition for treatment comprising Lrg1 protein or gene encoding Lrg1 protein

Abstract

The present invention relates to a biomarker composition for diagnosing liver diseases, which is capable of confirming an increase in Lrg1 in the case of liver fibrosis and using this to apply Lrg1 to diagnosis of liver diseases including liver fibrosis, and which is also capable of confirming the possibility of Lrg1 as a therapeutic target for liver diseases and applying same to prevention or treatment of liver diseases.

Description

Technical Field

[0001] The present invention provides a biomarker composition for liver disease diagnosis and a pharmaceutical composition for treatment, comprising Lrg1 protein or a gene encoding Lrg1 protein. Background Art

[0002] Liver disease can be caused by a variety of factors, including alcohol, various drugs, toxic chemicals, hepatitis B and C viruses, cholestasis, and autoimmunity. It typically progresses through fatty liver disease, eventually developing into hepatitis, liver fibrosis, and cirrhosis. Fatty liver disease itself is not a disease, but rather a reversible condition that resolves naturally after the causative agent is removed. However, if excessive fat accumulates in liver tissue over a prolonged period, steatohepatitis develops, resulting in repeated liver cell necrosis and regeneration. During this process, the fibrous extracellular matrix (ECM) increases and accumulates, leading to liver fibrosis.

[0003] Liver fibrosis remains a significant disease worldwide. Liver fibrosis results from chronic liver damage, accompanied by the accumulation of extracellular matrix (ECM) proteins such as collagen, fibronectin, and laminin. Most chronic diseases are characterized by recurrent liver damage accompanied by persistent inflammation, wound tissue formation, tissue structural changes, and organ failure. Hepatic stellate cells (HSCs) have garnered attention as key cells in liver fibrosis. As the primary source of both fibrillary and non-microfibrillary matrix proteins, they are central to the fibrotic process. While resting HSCs produce fewer ECM proteins than activated HSCs, repeated injury-induced activation of resting HSCs leads to their proliferation and transformation into a myofibroblast-like phenotype, a process known as activation.

[0004] Activated HSC function leads to excessive accumulation of ECM, destroying the liver of normal structure, which causes damage to the liver in pathophysiology. Overexpression of ECM proteins eventually induces liver failure, fibrosis, cirrhosis or cancer caused by HSC activation. The abnormal state of accumulation and overexpression of ECM proteins in liver tissue leads to excessive liver matrix in late-stage liver disease or liver disorder. This state is the most important stage during progressive liver disease. Therefore, the prevention and suppression of liver fibrosis are very important for improving chronic liver disease. However, no FDA-approved liver fibrosis therapeutic agent has been developed so far, and the specific mechanism of liver cancer caused by liver fibrosis has not yet been found.

[0005] Therefore, in order to solve the above problems, there is a need to develop methods for diagnosing liver diseases including liver fibrosis and therapeutic agents. Summary of the Invention

[0006] Technical issues An object of the present invention is to provide a biomarker composition for diagnosing liver disease, comprising a leucine-rich alpha-2-glycoprotein 1 (Lrg1) protein or a gene encoding the Lrg1 protein.

[0007] Another object of the present invention is to provide a composition for diagnosing liver diseases, comprising as an active ingredient an agent capable of measuring the expression or activity level of Lrg1 protein or the expression level of a gene encoding the protein.

[0008] Another object of the present invention is to provide a kit for diagnosing liver disease comprising the composition.

[0009] Another object of the present invention is to provide a method for providing information required for liver disease diagnosis, comprising the following steps: (1) measuring the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein in a sample isolated from a patient with liver disease; (2) comparing the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein with that of a control group sample; and (3) determining that the patient has liver disease when the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein is higher than that of the sample in the control group.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver disease, comprising an inhibitor of Lrg1 expression or activity as an active ingredient.

[0011] Another object of the present invention is to provide a method for screening therapeutic agents for liver disease, comprising the following steps: (1) contacting a test substance with liver disease cells; (2) measuring the expression or activity level of Lrg1 protein in the liver disease cells contacted with the test substance; and (3) selecting a test substance in which the expression or activity level of Lrg1 protein is reduced compared to a control sample.

[0012] Technical Solution To achieve the above-mentioned object, the present invention provides a biomarker composition for diagnosing liver disease, comprising a leucine-rich alpha-2-glycoprotein 1 (Lrg1) protein or a gene encoding the Lrg1 protein.

[0013] Furthermore, the present invention provides a composition for diagnosing liver diseases, comprising, as an active ingredient, an agent capable of measuring the expression or activity level of Lrg1 protein or the expression level of a gene encoding the protein.

[0014] Furthermore, the present invention provides a kit for diagnosing liver diseases comprising the composition.

[0015] Furthermore, the present invention provides a method for providing information required for liver disease diagnosis, comprising the following steps: (1) measuring the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein in a sample isolated from a patient with liver disease; (2) comparing the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein with that of a sample of a control group; and (3) determining that the patient has liver disease when the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein is higher than that of the sample of the control group.

[0016] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising an inhibitor of Lrg1 expression or activity as an active ingredient.

[0017] Furthermore, the present invention provides a method for screening therapeutic agents for liver disease, comprising the following steps: (1) contacting a test substance with liver disease cells; (2) measuring the expression or activity level of Lrg1 protein in the liver disease cells contacted with the test substance; and (3) selecting a test substance in which the expression or activity level of the Lrg1 protein is reduced compared to a control sample.

[0018] Technical Effects The present invention relates to a biomarker composition for diagnosing liver diseases. In the case of liver fibrosis, an increase in Lrg1 can be confirmed, and Lrg1 can be used to diagnose liver diseases including liver fibrosis. Furthermore, the possibility of Lrg1 as a therapeutic target for liver diseases can be confirmed and applied to the prevention or treatment of liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a result confirming increased inflammation in Runx3-deficient cells.

[0020] Figure 2 Shown is a method for preparing Runx3-deficient mice.

[0021] Figure 3 This is the result of confirming abnormal liver vascular endothelial cell function in Runx3-deficient mice.

[0022] Figure 4 This is the result of confirming liver fibrosis symptoms in Runx3-deficient mice.

[0023] Figure 5 This is a result confirming the increase of Lrg1 in Runx3-deficient cells.

[0024] Figure 6 The results confirmed that Lrg1 is secreted through the IL-6 / JAK / STAT3 pathway in Runx3-deficient cells.

[0025] Figure 7 The results confirmed that Lrg1 increases α-SMA and collagen I in HSCs through SMAD2 and SMAD3 signaling.

[0026] Figure 8 These results confirm that HSC activation by Lrg1 is mediated by TGF-βR signaling.

[0027] Figure 9 This result confirmed that Lrg1 increases α-SMA and collagen I independently of TGF-β.

[0028] Figure 10 It is confirmed that Lrg1 is increased in liver fibrosis and is expressed in Runx3 ΔEC Further additional results were obtained in mouse models. Best Mode for Carrying Out the Invention

[0029] Hereinafter, the present invention will be described in more detail.

[0030] The present invention provides a biomarker composition for diagnosing liver disease, comprising a leucine-rich alpha-2-glycoprotein 1 (Lrg1) protein or a gene encoding the Lrg1 protein.

[0031] The liver disease may be one selected from the group consisting of liver fibrosis, liver cancer, hepatitis, hepatotoxicity, alcoholic fatty liver disease and non-alcoholic fatty liver disease, but is not limited thereto.

[0032] "Diagnosis" of the present invention includes determining an individual's susceptibility to a particular disease or condition, determining whether an individual currently has a particular disease or condition, determining the prognosis of an individual with a particular disease or condition, or therametrics (e.g., monitoring an individual's status to provide information about the efficacy of a treatment).

[0033] The "biomarkers" of the present invention are substances that can distinguish tissues or cells of a subject with liver disease from tissues or cells of a normal control group for diagnosis, and include proteins or organic biomolecules such as nucleic acids, lipids, glycolipids, and glycoproteins that are increased or decreased in the tissues or cells of a subject with liver disease compared to a normal control group.

[0034] Furthermore, the present invention provides a composition for diagnosing liver diseases, comprising, as an active ingredient, an agent capable of measuring the expression or activity level of Lrg1 protein or the expression level of a gene encoding the protein.

[0035] The agent capable of measuring the expression level of Lrg1 may be a primer or probe that specifically binds to the Lrg1 gene, or an antibody, peptide, aptamer or compound that specifically binds to the Lrg1 protein, but is not limited thereto.

[0036] The term "primer" as used herein refers to a short genetic sequence that serves as the starting point for DNA synthesis and refers to an oligonucleotide synthesized for purposes such as diagnosis and DNA sequencing. The primer is typically synthesized in lengths of 15 to 30 base pairs for use, but the length may vary depending on the intended use and may be modified by known methods such as methylation and capping.

[0037] As used herein, the term "probe" refers to a nucleic acid that specifically binds to mRNA ranging from a few bases to several hundred bases in length, prepared by enzymatic chemical separation and purification or synthesis. The presence of mRNA can be confirmed by labeling with radioactive isotopes or enzymes, and can be designed and modified using known methods.

[0038] The term "antibody" as used herein is a well-known term in the art and refers to an immunoglobulin specific for an antigenic site. The antibody herein refers to an antibody that specifically binds to Lrg1 of the present invention, and can be prepared according to conventional methods in the art. The antibody may be a polyclonal antibody or a monoclonal antibody, and encompasses all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. Furthermore, the antibody also includes specialized antibodies such as humanized antibodies.

[0039] The term "peptide," as used herein, has the advantage of high binding affinity to target substances and resistance to denaturation even when subjected to thermal or chemical treatment. Furthermore, due to their small molecular size, peptides can be attached to other proteins for use as fusion proteins. Specifically, peptides can be attached to high-molecular-weight protein chains, enabling their use as diagnostic kits and drug delivery materials.

[0040] The term "aptamer," as used in this invention, refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that possesses a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to antigenic substances similar to antibodies. Furthermore, aptamers are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easily synthesized. Therefore, they can be used in place of antibodies.

[0041] Furthermore, the present invention provides a kit for diagnosing liver diseases comprising the composition.

[0042] The "kit" of the present invention may include an antibody that specifically binds to a biomarker component, a secondary antibody conjugate to which a marker is conjugated to develop color by reaction with a substrate, a color development substrate solution to be subjected to a color development reaction with the marker, a washing solution, and an enzyme reaction termination solution, and may be prepared as a plurality of separate packages or compartments containing the reagent components used.

[0043] Furthermore, the present invention provides a method for providing information required for diagnosing liver disease, comprising the following steps: (1) measuring the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein in a sample isolated from a patient with liver disease; (2) comparing the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein with that of a sample of a control group; and (3) determining that the patient has liver disease when the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein is higher than that of the sample of the control group.

[0044] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising an inhibitor of Lrg1 expression or activity as an active ingredient.

[0045] The Lrg1 expression inhibitor may be one selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the Lrg1 gene, but is not limited thereto.

[0046] The Lrg1 activity inhibitor may be one selected from the group consisting of low molecular weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the Lrg1 protein, but is not limited thereto.

[0047] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, antibacterial agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the preparation of pharmaceutical compositions. Specifically, the carriers, excipients, and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may be prepared by mixing at least one excipient (e.g., starch, calcium carbonate, sucrose or lactose, gelatin, etc.) with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, and syrups. In addition to the commonly used simple diluents water and liquid paraffin, they can also contain various excipients (e.g., wetting agents, sweeteners, fragrances, preservatives, etc.). Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, as well as injectable esters such as ethyl oleate. Suppository bases include synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, and glycerol gelatin. According to one embodiment of the present invention, the pharmaceutical composition is administered to the subject in a conventional manner by intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The dosage of the active ingredient according to the present invention can be different according to the state and weight of the subject, the type and degree of the disease, the drug form, the route of administration and the period, and can be appropriately selected by those skilled in the art. The daily dosage can be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, and more preferably 0.1 mg / kg to 100 mg / kg. Administration can be once a day or divided into several doses, and the scope of the present invention is not limited thereto.

[0048] Furthermore, the present invention provides a method for screening therapeutic agents for liver disease, comprising the following steps: (1) contacting a test substance with liver disease cells; (2) measuring the expression or activity level of Lrg1 protein in the liver disease cells contacted with the test substance; and (3) selecting a test substance in which the expression or activity level of the Lrg1 protein is reduced compared to a control sample.

[0049] The Lrg1 expression level can be measured using one or more methods selected from the group consisting of reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, microarray, western blotting, and flow cytometry (FACS), but is not limited thereto.

[0050] To facilitate understanding of the present invention, the following detailed description is given with reference to examples and the like. However, the following examples and the like are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The examples and the like are provided to more fully explain the present invention to those skilled in the art. DETAILED DESCRIPTION

[0051] <Experimental Example 1> Confirmation of Cytokine Changes Induced by Runx3 Deficiency in Human Hepatic Endothelial Cells To confirm the cytokine changes caused by Runx3 deficiency in human liver sinusoidal endothelial cells (LSECs), siRunx3 and control siRNAs were transfected into TMNK-1 cells (human immortalized LSECs), and total RNA was isolated 36 hours later and then subjected to qRT-PCR.

[0052] <Experimental Example 2> Preparation of Runx3 Deficiency Mouse Model Endothelial cell (EC)-specific Runx3-deficient mice were generated using the tissue-specific Cre-loxP system. Figure 2As shown, mice in which Cre recombinase and Tie2 promoter were cloned to activate Cre specifically in Tie2-expressing ECs (Tie2-Cre mice) were cross-linked with Runx3 floxed mice (Runx3) expressing a gene generated by adding loxP sites before and after exon 4 of Runx3. f / f Mice lacking Runx3 exon 4 in EC were generated by mating with WT mice.

[0053] <Experimental Example 3> Immunohistochemistry method Mouse livers were fixed in formaldehyde, then paraffin blocks were prepared. These paraffin blocks were sectioned into 5μm sections and mounted on glass slides. Paraffin was removed from these sections, hydrated through various procedures, and then treated in a citrate solution (pH 6.0) at 95°C for 10 to 20 minutes before immunohistochemistry (IHC). After treatment in a 3% H₂O₂ solution for 10 minutes, the sections were blocked with 10% goat serum and incubated with a primary antibody solution overnight at 4°C. A biotinylated secondary antibody was diluted appropriately in a solution containing goat serum for one hour, followed by reaction with VECTASTAIN Elite ABC solution for 30 minutes. The sections were then treated and stained using a DAB substrate kit. Frozen tissue section slides were also used. Tissue sections were blocked with a 3% BSA / PBS solution and then incubated overnight at 4°C in a solution containing a diluted primary antibody (1% BSA, 0.3% Triton X-100). After treatment with a secondary fluorescent antibody solution for 1 hour, sections were mounted with a solution containing DAPI and observed using a fluorescence microscope or confocal microscope.

[0054] <Experimental Example 4> Western blotting After mixing liver tissue with the loading solution and boiling at 95°C for 10 minutes, proteins were separated by size using SDS-PAGE electrophoresis. The gel was then transferred to a fibrin membrane using a 0.45μm needle and blocked with 5% skim milk. After incubation with a primary antibody solution overnight at 4°C, the membrane was reacted with an HRP-conjugated secondary antibody for 1 hour, and protein bands were confirmed using an ECL substrate.

[0055] <Experimental Example 5> Measurement of serum ALT and AST The cells were coagulated in mouse serum for 1 hour, centrifuged at 1500 g for 15 minutes at 4°C, and assayed using AST and ALT kits (Bio-Vision).

[0056] <Experimental Example 6> Histological Staining Paraffin tissue slides were placed at 68°C for 1 hour, hydrated with various concentrations of ethanol solution, washed with water, and then stained according to standard protocols for H&E staining, Masson's trichrome staining, and Sirius red staining.

[0057] <Experimental Example 7> Single cell RNA-sequencing Hepatocytes and other cells were isolated from each mouse group, and 2 × 10 cells were analyzed per group at a ratio of 10% hepatocytes and 90% other cells. 7 GOBP analysis was performed on 10 cells. Protein expression in tissues was confirmed by IHC.

[0058] <Example 1> Confirmation of Cytokine Changes Caused by Runx3 Deficiency in Human Hepatic Endothelial Cells To confirm the changes in cytokines caused by Runx3 deficiency in human liver sinusoidal endothelial cells (LSEC), qRT-PCR was performed.

[0059] As a result, according to Figure 1 , it was confirmed that in the absence of Runx3, the expression levels of IL-1β, IL-6, TNF-α, MCP-1, ICAM-1 and VCAM-1 were significantly increased compared with the control group.

[0060] <Example 2> Confirmation of abnormal liver vascular endothelial cell function and liver fibrosis symptoms in Runx3-deficient mice (1) Preparation of Runx3-deficient mouse model Endothelial cell (EC)-specific Runx3-deficient mice were generated using the tissue-specific Cre-loxP system. Figure 2 As shown, mice in which Cre recombinase and Tie2 promoter were cloned to activate Cre specifically in Tie2-expressing ECs (Tie2-Cre mice) and Runx3 floxed mice (Runx3) expressing a gene prepared by adding loxP sites before and after the Runx3 exon (exon4) were used. f / f Mice with Runx3 exon 4 deletion in EC were generated by mating with WT mice.

[0061] (2) Juvenile Runx3 ΔEC Mouse model situation In childhood Runx3 ΔEC In a mouse model, the genes for proteins whose expression changes due to Runx3 deficiency were identified using immunohistochemistry (IHC analysis).

[0062] As a result, according to Figure 3 , for Runx3 babies born 2 to 3 months ΔEC In the case of mouse models, the expression levels of CD34 and vWF were confirmed to be related to Runx3 f / f Compared with the increase in Runx3 ΔEC In the case of mouse models, the expression levels of CD31 and CD34 were confirmed to be related to Runx3. f / f Compared to increase.

[0063] (3) Elderly Runx3 ΔEC Mouse model situation In the elderly Runx3 ΔEC In mouse models, genes that are altered by Runx3 deficiency have been identified.

[0064] As a result, according to Figure 4 , for Runx3 of one year old ΔEC In the case of mouse models, the expression levels of CD31 and CD34 were confirmed to be related to Runx3. f / f Compared with the control group, the expression levels of α-SMA and collagen I were significantly increased in liver tissue, indicating the occurrence of liver fibrosis symptoms.

[0065] <Example 3> Confirmation of the increased expression and secretion pathway of Lrg1 caused by Runx3 deficiency To determine which genes were altered in expression in the absence of Runx3 in LSEC cells, single-cell RNA-sequencing and qRT-PCR were performed.

[0066] As a result, according to Figure 5 , it was confirmed that the expression level of Lrg1 was significantly increased compared with the case where Runx3 was not deficient, thereby confirming the possibility of Lrg1 as a therapeutic target for liver diseases such as liver fibrosis.

[0067] Furthermore, to find out the secretion pathway of Lrg1 in LSEC cells, immunoblotting was performed using cell lysates and conditioned medium.

[0068] As a result, according to Figure 5 and Figure 6 The researchers confirmed that Lrg1 expression increased in the absence of Runx3, while conversely, Lrg1 expression decreased in the absence of Runx3 when treated with the JAK inhibitor ruxolitinib or the IL-6 neutralizing antibody tocilizumab. This suggests that Lrg1 is secreted through the IL-6 / JAK / STAT3 pathway.

[0069] <Example 4> Confirmation of Increased Expression of α-SMA and Collagen I by Lrg1 Since hepatic stellate cells (HSCs) are the primary cells involved in liver fibrosis, the role of Lrg-1 in liver fibrosis was investigated using LX-2 cells, a HSC line. LX-2 cells were treated with recombinant Lrg-1 protein, and the expression of α-SMA and collagen I was confirmed by immunoblotting.

[0070] As a result, according to Figure 7 , it was confirmed that when HSCs were treated with Lrg1, the expression of α-SMA and collagen I increased. This suggests that Lrg1 exacerbates liver fibrosis.

[0071] In addition, it was confirmed that the expression of p-SMAD2 and p-SMAD3 increased after HSC treatment with Lrg1, indicating that Lrg1 can regulate the expression of p-SMAD2 and p-SMAD3 through the p-SMAD2 / p-SMAD3 pathway. Figure 8 When treated with galunisertib, a TGF-β receptor type 1 inhibitor, α-SMA, collagen I, p-SMAD2, and p-SMAD3, which were increased by Lrg1 treatment, were all reduced, confirming that Lrg1 utilizes the TGF-βR / p-SMAD2 / p-SMAD3 pathway.

[0072] Further, according to Figure 9 , increased expression of α-SMA and collagen I was confirmed when cells were treated with Lrg1 and TGF-β alone, and increased expression of α-SMA and collagen I was also confirmed when cells were treated with a combination of Lrg1 and TGF-β, thus confirming that Lrg1 increases α-SMA and collagen I independently of TGF-β.

[0073] <Example 5> Confirmation of Lrg1 as a therapeutic target for liver fibrosis To confirm that Lrg1 is a therapeutic target for liver fibrosis, IHC was performed on liver tissues isolated from animal models.

[0074] As a result, according to Figure 10, confirmed that the expression of Lrg1 increased in thioacetamide (TAA: Thioacetamide)-induced liver fibrosis model mice compared with the control group injected with normal saline, and also confirmed that Runx3 ΔEC The situation with Runx3 f / f Furthermore, Runx3 was also confirmed to be expressed in one-year-old mice showing symptoms of liver fibrosis. ΔEC The situation with Runx3 f / f Thus, it was confirmed that Lrg1 expression increases in the case of liver fibrosis, and it is known that Lrg1 can be a therapeutic target for liver fibrosis.

[0075] The above description of the present invention is for illustrative purposes only. Those skilled in the art will appreciate that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all aspects and are not restrictive.

[0076] The scope of the present invention is indicated by the appended claims, and all changes and modifications derived from the meaning and scope of the claims and their equivalents are to be construed as being included within the scope of the present invention.

Claims

A biomarker composition for diagnosing liver disease, comprising Lrg1 protein or a gene encoding the Lrg1 protein.

2. The biomarker composition according to claim 1, characterized in that The liver disease is one selected from the group consisting of liver fibrosis, liver cancer, hepatitis, hepatotoxicity, alcoholic fatty liver disease and non-alcoholic fatty liver disease.

3. A composition for diagnosing liver diseases, comprising as an active ingredient an agent capable of measuring the expression or activity level of Lrg1 protein or the expression level of a gene encoding the protein.

4. The composition for diagnosing liver disease according to claim 3, characterized in that The agent capable of measuring the expression level of Lrg1 is a primer or probe that specifically binds to the Lrg1 gene, or an antibody, peptide, aptamer or compound that specifically binds to the Lrg1 protein.

5. A kit for diagnosing liver disease comprising the composition according to claim 3 or claim 4.

6. A method for providing information required for diagnosing liver disease comprising the steps of: (1) Determine the expression level of Lrg1 gene mRNA or Lrg1 protein in samples isolated from patients with liver disease; (2) comparing the mRNA expression level of the Lrg1 gene or the expression level of the Lrg1 protein with that of a control group sample; and (3) When the expression level of the mRNA of the Lrg1 gene or the expression level of the Lrg1 protein is higher than that of the control sample, the patient is judged to have liver disease.

7. A pharmaceutical composition for preventing or treating liver disease, comprising an inhibitor of Lrg1 expression or activity as an active ingredient.

8. The pharmaceutical composition for preventing or treating liver disease according to claim 7, characterized in that The Lrg1 expression inhibitor is one selected from the group consisting of antisense nucleotides, small interfering RNAs, and short hairpin RNAs that complementarily bind to the mRNA of the Lrg1 gene.

9. The pharmaceutical composition for preventing or treating liver disease according to claim 7, characterized in that: The Lrg1 activity inhibitor is one selected from the group consisting of low molecular weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the Lrg1 protein.

10. A method for screening a liver disease therapeutic agent comprising the following steps: (1) Contacting the test substance with liver disease cells; (2) determining the expression or activity of Lrg1 protein in liver disease cells exposed to the test substance; and (3) Selecting a test substance that reduces the expression or activity of the Lrg1 protein compared to the control sample.

11. The method for screening therapeutic agents for liver diseases according to claim 10, wherein: The Lrg1 expression level is determined using one or more methods selected from the group consisting of reverse transcription-polymerase chain reaction, enzyme immunoassay, radioimmunoassay, immunohistochemistry, microarray, immunoblotting, and flow cytometry.

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