Olfml2b biomarker for treatment of hepatocellular carcinoma and prediction of recurrence, and use thereof

Abstract

The present invention relates to an OLFML2B biomarker for treatment of hepatocellular carcinoma and prediction of recurrence, and use thereof. Specifically, OLFML2B exhibits a high AUC in hepatocellular carcinoma diagnostic performance and is more useful for predicting recurrence, and has been identified to have consistent performance in various public data such as MDACC and GSE series. In Cox regression analysis, OLFML2B has been identified to be a significant factor in survival and recurrence, and has shown a more distinct increase in expression in malignant hepatocytes, PVTT, and recurrent tumor tissue than in normal tissue or primary tumor. In addition, it has been identified that the invasiveness and proliferation of hepatocellular carcinoma cell lines are significantly reduced when OLFML2B is inhibited. Therefore, the present invention can ultimately promote an improvement in survival rate and treatment outcomes of hepatocellular carcinoma patients.

Description

OLFML2B Biomarker for Liver Cancer Treatment and Recurrence Prediction and Its Uses

[0001] The present invention relates to the OLFML2B biomarker for the treatment of liver cancer and the prediction of recurrence, and the use thereof.

[0002] Hepatocellular carcinoma (HCC) is a major type of cancer in Korea, ranking fourth in incidence and second in mortality. The 5-year relative survival rate for liver cancer patients is very low, at less than 40%, necessitating continuous efforts to improve survival rates. In particular, liver cancer accounts for the highest mortality rate among all cancers in the socioeconomically active age group of 40-50 year olds, posing a significant national problem. Premature death due to liver cancer negatively impacts not only an individual's quality of life but also economic productivity. Furthermore, liver cancer presents with almost no early symptoms, and existing biomarkers (e.g., AFP) have limitations in terms of sensitivity and specificity.

[0003] Meanwhile, for patients with recurrence, the prognosis worsens and precise prediction is essential for establishing treatment strategies; therefore, the development of markers to predict postoperative recurrence is necessary.

[0004] The object of the present invention is to provide a biomarker composition for predicting liver cancer metastasis or recurrence comprising the OLFML2B protein or a gene encoding the same.

[0005] In addition, another objective of the present invention is to provide a composition for predicting liver cancer metastasis or recurrence comprising, as an active ingredient, a preparation capable of measuring the expression level of the OLFML2B protein or the gene encoding it.

[0006] In addition, another objective of the present invention is to provide a kit for predicting liver cancer metastasis or recurrence comprising the above composition.

[0007] In addition, another objective of the present invention is to provide a method for providing information necessary for predicting liver cancer metastasis or recurrence, comprising the step of measuring the expression level of the OLFML2B protein or the gene encoding it.

[0008] In addition, another objective of the present invention is to provide a composition for preventing, improving, or treating liver cancer metastasis or recurrence comprising an inhibitor of the expression or activity of the OLFML2B protein or the gene encoding it as an active ingredient.

[0009] In addition, another objective of the present invention is to provide a screening method for therapeutic agents to inhibit liver cancer metastasis or recurrence, comprising the step of selecting a test substance in which the expression or activity level of the OLFML2B protein or the gene encoding it is reduced.

[0010] To achieve the above objective, the present invention provides a biomarker composition for predicting liver cancer metastasis or recurrence comprising the OLFML2B protein or a gene encoding the same.

[0011] In addition, the present invention provides a composition for predicting liver cancer metastasis or recurrence comprising, as an active ingredient, a preparation capable of measuring the expression level of the OLFML2B protein or the gene encoding it.

[0012] In addition, the present invention provides a kit for predicting liver cancer metastasis or recurrence comprising the above composition.

[0013] In addition, the present invention provides a method for providing information necessary for predicting liver cancer metastasis or recurrence, comprising: (1) measuring the expression level of an OLFML2B protein or a gene encoding it from a sample isolated from a liver cancer patient; (2) comparing the expression level of the OLFML2B protein or the gene encoding it with a control sample; and (3) determining that there is a high probability of liver cancer recurrence if the expression level of the OLFML2B protein or the gene encoding it is higher than that of the control sample.

[0014] In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer metastasis or recurrence, comprising as an active ingredient an inhibitor of the expression or activity of the OLFML2B protein or the gene encoding it.

[0015] In addition, the present invention provides a health functional food for preventing or improving liver cancer metastasis or recurrence, comprising as an active ingredient an inhibitor of the expression or activity of the OLFML2B protein or the gene encoding it.

[0016] In addition, the present invention provides a screening method for therapeutic agents to inhibit liver cancer metastasis or recurrence, comprising the steps of: (1) contacting isolated liver cancer cells with a test substance; (2) measuring the degree of expression or activity of the OLFML2B protein or the gene encoding it in the liver cancer cells contacted with the test substance; and (3) selecting a test substance in which the degree of expression or activity of the OLFML2B protein or the gene encoding it is reduced compared with a control sample.

[0017] The present invention relates to the OLFML2B biomarker for the treatment of liver cancer and the prediction of recurrence, and to its applications. Specifically, OLFML2B exhibits a high AUC in liver cancer diagnostic efficacy and is more useful for predicting recurrence; consistent performance has been confirmed across various public datasets, including the MDACC and GSE series. In Cox regression analysis, OLFML2B was identified as a significant factor in survival and recurrence, showing a distinct increase in expression in malignant hepatocytes, PVTT, and recurrent tumor tissue compared to normal tissue or primary tumor. Furthermore, it was confirmed that the invasiveness and proliferation of liver cancer cell lines significantly decrease upon inhibition of OLFML2B. Accordingly, the present invention can ultimately promote improved survival rates and treatment outcomes for liver cancer patients.

[0018] Figure 1 shows the results of recurrence prediction analysis using a protein chip.

[0019] Figure 2 shows the OS and DFS results of LAMP1, LAT, and CTRL in the TCGA data.

[0020] Figure 3 shows the OS and DFS results of OLFML2B in four public omics datasets.

[0021] Figure 4 shows the results of Cox regression analysis on the GSE114564 data.

[0022] Figure 5 shows the results of confirming OLFML2B expression in HCC through GepLiver DB analysis.

[0023] Figure 6 shows the results of confirming OLFML2B expression in HCC through spatial transcriptome analysis.

[0024] Figure 7 shows the results of confirming OLFML2B expression in GSE77509.

[0025] Figure 8 shows the results of confirming OLFML2B mRNA expression in the Ajou University Hospital cohort.

[0026] Figure 9 shows the results of confirming OLFML2B protein expression in the Ajou University Hospital cohort.

[0027] Figure 10 shows the results of confirming OLFML2B mRNA expression in a patient who relapsed after surgery.

[0028] Figure 11 shows the results of confirming OLFML2B mRNA expression in a patient who relapsed after surgery.

[0029] Figure 12 shows the results of the comparison of expression and diagnostic accuracy analysis of OLFML2B and AFP in liver cancer tissue derived from the same patient.

[0030] Figure 13 shows the results of confirming OLFML2B mRNA and protein expression in normal hepatocyte cell lines and liver cancer cell lines.

[0031] Figure 14 shows the results of confirming the KD efficiency of OLFML2B in two liver cancer cell lines.

[0032] Figure 15 shows the results of the wound healing effect on liver cancer cells when OLFML2B is inhibited.

[0033] Figure 16 shows the results of the inhibitory effect on liver cancer cell colony formation when OLFML2B is inhibited.

[0034] Figure 17 shows the results of inhibiting liver cancer cell migration by OLFML2B siRNA treatment.

[0035] Figure 18 shows the results of inhibiting liver cancer cell invasion by inhibiting OLFML2B expression.

[0036] Figure 19 shows the results of EMT / vascular infiltration-related protein expression regulation.

[0037] Figure 20 shows the results of activation of the apoptosis pathway in liver cancer cells through OLFML2B inhibition.

[0038] Figure 21 shows the results of increased OLFML2B expression in recurrent tumors in an orthotopic liver cancer mouse model.

[0039] Figure 22 shows the result graph following OLFML2B inhibition in a metastatic mouse model.

[0040] Figure 23 shows the results of reduced lung metastasis following OLFML2B inhibition in a metastasis model.

[0041] Figure 24 shows the results of the analysis of liver metastasis after intravenous infusion of NIH-3T3-ras cells.

[0042] The present invention identifies the role of OLFML2B, a novel factor involved in the recurrence and metastasis of hepatocellular carcinoma (HCC), and provides a diagnostic and therapeutic technology capable of predicting cancer recurrence and inhibiting metastasis by targeting this factor. In particular, the present invention is expected to have the following effects.

[0043]

[0044] 1. Clinical Value as a Biomarker Specific for Recurrent Liver Cancer

[0045] OLFML2B is specifically overexpressed in recurrent liver cancer tissues when comparing primary and recurrent liver cancer tissues taken from the same patient. Increased expression in recurrent tissues was also confirmed in an orthotopic liver cancer mouse model. Through this, the potential for clinical application as a molecular marker for identifying patients at high risk of recurrence and determining treatment strategies has been secured.

[0046]

[0047] 2. Potential for developing therapeutic agents as metastasis inhibition targets

[0048] When OLFML2B is knocked down or inhibited, migration, invasion, EMT, vascular invasion, and anti-apoptotic signaling of liver cancer cells are significantly suppressed. In an in vivo tail vein injection model, the number and area of ​​lung metastatic lesions were significantly reduced. This implies that OLFML2B performs a direct function as a regulator of metastasis and recurrence, and the development of therapeutic agents such as anti-OLFML2B antibodies, siRNA, ASOs, and small molecule inhibitors is possible.

[0049]

[0050] 3. Diagnostic accuracy overcoming the limitations of existing biomarkers (AFP)

[0051] When compared to AFP in tissues from the same patient, OLFML2B was highly expressed and detectable even in samples with low AFP expression. ROC analysis also showed that OLFML2B had a higher AUC (0.98 vs. 0.75) compared to AFP. This implies that it can serve as a diagnostic complement in the AFP-negative patient population and facilitates expansion into a multi-biomarker-based diagnostic panel.

[0052]

[0053] 4. Potential for Technology Transfer and Commercialization Linkage

[0054] This invention has potential applications in both diagnosis and treatment, such as identifying patients with recurrent liver cancer, predicting high-risk groups for metastasis, and evaluating anticancer drug candidates. In particular, the reproducible results based on patient tissues and the in vivo metastasis inhibitory effect allow for expansion into the preclinical candidate discovery and animal testing CRO stages. Among the liver cancer-related genes known to date, factors that simultaneously regulate both recurrence and metastasis and have been proven as therapeutic targets are rare.

[0055]

[0056] The present invention provides a biomarker composition for predicting liver cancer metastasis or recurrence comprising the OLFML2B protein or a gene encoding the same.

[0057]

[0058] In this specification, the term “prediction” is used to refer to the likelihood that a patient will respond favorably or unfavorably to a drug or a set of drugs. In one embodiment, the prediction relates to the degree of such response. For example, the prediction relates to whether and / or the probability that a patient will survive without cancer recurrence after treatment, for example, treatment with a specific therapeutic agent and / or surgical removal of the primary tumor and / or chemotherapy for a specific period. The prediction of the present invention can be clinically used to determine treatment by selecting the most appropriate treatment method for a liver cancer patient. The prediction of the present invention is a useful tool for predicting whether a patient will respond favorably to a therapeutic treatment, such as a given therapeutic treatment, for example, administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc., or whether long-term survival of the patient is possible after the therapeutic treatment.

[0059]

[0060] In addition, the present invention provides a composition for predicting liver cancer metastasis or recurrence comprising, as an active ingredient, a preparation capable of measuring the expression level of the OLFML2B protein or the gene encoding it.

[0061] More specifically, the agent capable of measuring the expression level of the OLFML2B protein or the gene encoding it may be a primer or probe that specifically binds to the OLFML2B gene, an antibody, peptide, aptamer, or compound that specifically binds to the OLFML2B protein, but is not limited thereto.

[0062]

[0063] In addition, the present invention provides a kit for predicting liver cancer metastasis or recurrence comprising the above composition.

[0064]

[0065] In this specification, the term “primer” refers to a short nucleic acid sequence having a short free 3’ hydroxyl group, capable of forming base pairs with a complementary template, and acting as a starting point for template strand replication. 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 at an appropriate buffer solution and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0066] In this specification, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA. It is labeled to confirm the presence or absence and expression level of a specific mRNA. Probes may be constructed in the form of oligonucleotide probes, single-strand DNA probes, double-strand DNA probes, RNA probes, etc. The selection of appropriate probes and hybridization conditions may be appropriately selected according to techniques known in the art.

[0067] In this specification, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed toward an antigenic site. An antibody in the present invention refers to an antibody that specifically binds to the biomarker of the present invention, and the antibody may be prepared according to conventional methods in the art. The forms of the antibody include polyclonal antibodies or monoclonal antibodies, and include all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

[0068] In this specification, the term "peptide" has the advantage of high binding affinity to target substances and does not undergo denaturation even during heat or chemical treatment. Additionally, due to its small molecular size, it can be attached to other proteins to be used as a fusion protein. Specifically, since it can be attached to high-molecular-weight protein chains, it can be used as a diagnostic kit and a drug delivery material.

[0069] In this specification, the term "aptamer" refers to a type of polynucleotide composed of a special kind of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and can bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to antigenic substances just like antibodies, but because they are composed of polynucleotides that are more stable than proteins, have a simple structure, and are easy to synthesize, they can be used as a substitute for antibodies.

[0070]

[0071] In addition, the present invention provides a method for providing information necessary for predicting liver cancer metastasis or recurrence, comprising: (1) measuring the expression level of an OLFML2B protein or a gene encoding it from a sample isolated from a liver cancer patient; (2) comparing the expression level of the OLFML2B protein or the gene encoding it with a control sample; and (3) determining that there is a high probability of liver cancer recurrence if the expression level of the OLFML2B protein or the gene encoding it is higher than that of the control sample.

[0072] Preferably, the sample isolated from the liver cancer patient may be a sample isolated after the liver resection of the liver cancer patient, but is not limited thereto.

[0073] Specifically, the method for measuring the gene expression level described above uses RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chips, but is not limited thereto.

[0074] Specifically, the method for measuring the protein expression level described above uses Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chips, but is not limited thereto.

[0075]

[0076] In this specification, the term “sample isolated from a patient” includes, but is not limited to, samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differ from the control group in the expression level of the OLFML2B gene or OLFML2B protein, which is the biomarker of the present invention.

[0077]

[0078] In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer metastasis or recurrence, comprising as an active ingredient an inhibitor of the expression or activity of the OLFML2B protein or the gene encoding it.

[0079] More specifically, the inhibitor of expression or activity of the OLFML2B protein or the gene encoding it may be any one selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA) and short hairpin RNA (shRNA), compounds, peptides, aptamers, and antibodies that specifically bind to the OLFML2B protein or the gene encoding it, but is not limited thereto.

[0080] Preferably, the above pharmaceutical composition may be metastasized to the lungs, but is not limited thereto.

[0081] The pharmaceutical composition of the present invention may include chemical substances, nucleotides, antisense, siRNA oligonucleotides, and natural product extracts as active ingredients. The pharmaceutical composition or complex formulation of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients, and said adjuvants may include solubilizing agents such as excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, or flavoring agents. The pharmaceutical composition of the present invention may preferably be formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients for administration. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0082] The pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, or injectable liquids, as well as sustained-release formulations of the active compound. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intra-arterial, intraperitoneal, intramuscular, intra-arterial, intraperitoneal, intrasternal, transdermal, nasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes. The effective amount of the active ingredient in the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Accordingly, it may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health status, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and concurrently used drugs. Although not limited thereto, for example, in the case of an adult, when administered once or several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg in the case of a compound, 0.1 ng / kg to 10 g / kg in the case of a polypeptide, protein or antibody, and 0.01 ng / kg to 10 g / kg in the case of an antisense nucleotide, siRNA, shRNAi, or miRNA.

[0083]

[0084] In addition, the present invention provides a health functional food composition for preventing or improving liver cancer metastasis or recurrence, comprising an inhibitor of the expression or activity of the OLFML2B protein or the gene encoding it as an active ingredient.

[0085] The health functional food composition of the present invention may be provided in the form of powder, granules, tablets, capsules, syrup, or beverage, and the health functional food composition may be used in combination with other foods or food additives in addition to the active ingredient, and may be used appropriately according to conventional methods. The amount of the mixture of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health, or therapeutic treatment.

[0086] The effective dose of the active ingredient contained in the above health functional food composition may be used in accordance with the effective dose of the above pharmaceutical composition, but in the case of long-term consumption for the purpose of health and hygiene or health control, it may be less than the above range, and it is certain that the active ingredient may be used in an amount greater than the above range because there is no problem in terms of safety.

[0087] There are no special restrictions on the types of health foods mentioned above, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0088]

[0089] In addition, the present invention provides a screening method for therapeutic agents to inhibit liver cancer metastasis or recurrence, comprising the steps of: (1) contacting isolated liver cancer cells with a test substance; (2) measuring the degree of expression or activity of the OLFML2B protein or the gene encoding it in the liver cancer cells contacted with the test substance; and (3) selecting a test substance in which the degree of expression or activity of the OLFML2B protein or the gene encoding it is reduced compared with a control sample.

[0090] The term "test substance" used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. The sample includes, but is not limited to, chemical substances, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0091] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0092]

[0093] <Experimental Example>

[0094] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0095]

[0096] 1. Autoantibody Analysis

[0097] Subjects: Plasma samples from patients with recurrence (10) and non-recurrence (10) after surgery at Ajou University Hospital.

[0098] Method: Evaluation of autoantibody levels using a 22K protein chip.

[0099] Objective: To identify proteins that show differences between groups.

[0100]

[0101] 2. Candidate Marker Selection and Performance Evaluation

[0102] Analysis: Evaluation of recurrence prediction performance using ROC curves.

[0103] Selected candidates: 4 proteins: LAMP1, OLFML2B, LAT, and CTRL.

[0104]

[0105] 3. TCGA Data Analysis

[0106] Subjects: TCGA dataset patient group.

[0107] Analysis: OS (Overall Survival) and DFS (Disease-Free Survival) evaluation.

[0108] Objective: Search for prognosis-related proteins among four candidate proteins.

[0109]

[0110] 4. Cox Regression Analysis

[0111] Data: GSE114564 data used.

[0112] Analysis: Performed Cox regression analysis.

[0113] Objective: Evaluate whether OLFML2B is a key variable related to survival prediction.

[0114]

[0115] 5. Analysis of Expression Specificity

[0116] Data: Comparison with normal and various liver diseases in the GepLiver DB.

[0117] Objective: Confirmation of liver cancer-specific expression of OLFML2B.

[0118]

[0119] 6. ST Analysis and Comparison of Expression by Tissue Type

[0120] Data: Mendeley Data (skrx2fz79n) and GSE77509 utilized.

[0121] Analysis: Comparison of expression between non-malignant hepatocytes and malignant hepatocytes.

[0122] Objective: To identify changes in OLFML2B expression during the progression of liver cancer.

[0123]

[0124] 7. Evaluation of Liver Cancer Diagnostic History in Ajou University Hospital Patient Group

[0125] Subjects: 132 patients who underwent hepatectomy.

[0126] Experiment: Comparison of expression in liver cancer tissue (T) and surrounding normal tissue (NT) using qRT-PCR and WB

[0127] After passing the IRB review at Ajou University Hospital, the sample was secured as a test cohort through the Human Bioresource Bank. Clinical data was obtained from the Human Bioresource Center at Ajou University Hospital, including liver cancer recurrence, metastasis, surgery date, time of recurrence, last estimated date of death, and survival status.

[0128]

[0129] (1) RNA extraction from tissue

[0130] RNA from the tissue was extracted using QIAzol Lysis Reagent (Cat#79306).

[0131]

[0132] (2) Measurement of OLFML2B expression levels in tissues via qRT-PCR analysis

[0133] cDNA synthesis was performed using the PrimeScript™ RT Master Mix (Perfect Real Time)(Cat #RR036A)(TaKaRa) kit.

[0134] cDNA synthesis was carried out by combining tissue RNA and RNA Free Water to make 8 µl, adding 2 µl of PrimeScript™ RT Master Mix for a total of 10 µl.

[0135] The cDNA conditions were set as follows.

[0136] A. Stage 1: 37℃, 15 minutes

[0137] B. Stage 2: 85℃, 4℃ after 5 seconds, -ing

[0138]

[0139] qRT-PCR was performed using primers with the sequences below. The primers used were purchased from M.biotech (Hanam, Korea). 5 µl of qPCR Master Mix (2X, High ROX) (Gendepot, Cat #Q5602) was used, and qRT-PCR was performed with a total volume of 10 µl.

[0140] GeneAccession No. Forward sequence Reverse sequence OLFML2BNM_015441.25'- AACAGACTCGCTGGGGAAAG-3'(Sequence No. 1)5'-CCCCGTGATTGTGGAGAGAGT-3'(Sequence No. 2)HMBSNM_001024382.25'-GGAGGGCAGAAGGAAGAAAACAG-3'(Sequence No. 3)5'-CACTGTCCGTCTGTATGCGAG-3'(Sequence No. 4)

[0141]

[0142] The qRT-PCR conditions were set as follows.

[0143] A. Stage 1: 95℃, 2 minutes

[0144] B. Stage 2: 95℃, 15 seconds

[0145] C. : 58℃, 34 seconds

[0146] D. : 72℃, 30 seconds

[0147] E. Repeat Stage 2 for 50 cycles

[0148]

[0149] (3) Measurement of OLFML2B expression levels in tissues through WB analysis

[0150] Proteins were separated by treating cells with protein lysis buffer and then subjected to SDS-PAGE electrophoresis, followed by a Western blot using the OLFML2B antibody (Atlas Antibodies, Cat HPA062739).

[0151]

[0152] 8. Evaluation of Recurrence Predictability through qRT-PCR and WB Analysis

[0153] Subjects: For qRT-PCR samples, we evaluated the liver tumor tissue (PT) and surrounding tissue (NT) at the time of the initial surgery, as well as the liver tumor tissue (RT) at the time of recurrence, from patients who underwent surgery at Ajou University Hospital and subsequently experienced recurrence. For WB samples, we collected the liver tumor tissue (primary tumor, PT) and surrounding normal tissue (P-NT) at the time of the initial surgery, the liver tumor tissue (recurrent tumor, RT) at the time of recurrence, and the surrounding normal tissue (R-NT) at that time from patients who underwent surgery and subsequently experienced recurrence.

[0154]

[0155] 9. Cell Culture

[0156] Normal hepatocytes (LO-2) were obtained from ATCC (VA, USA), and human liver cancer cell lines (Hep3B, Huh-7, PLC / PRF / 5, SNU368, SNU398, SNU423, SNU449, SNU475) were obtained from the Korean Cell Line Bank (Seoul, Korea). Hep3B, Huh-7, PLC / PRF / 5, SNU368, SNU398, and SNU423 liver cancer cell lines were cultured in DMEM medium containing 10% FBS and 100 units / mL penicillin-streptomycin (Invitrogen, CA, USA). SNU449 and SNU475 liver cancer cell lines were cultured in RPMI 1640 medium containing 10% FBS and 100 units / mL penicillin-streptomycin (Invitrogen, CA, USA). All cultures were cultured under conditions where 5% carbon dioxide and a temperature of 37°C were maintained.

[0157]

[0158] 10. Transfection

[0159] For the siRNAs used for transfection, the control siRNA was synthesized by Genolution (Seoul, Korea), and the OLFML2B siRNA was AccuTarget™ Predesigned siRNA, a product manufactured and sold by Bioneer (Daejeon, Korea). The nucleotide sequences of the control siRNA and OLFML2B siRNA are shown in Table 2 below.

[0160] RNA duplexesSense sequenceControllsiRNAUUC UCC GAA CGU GUC ACG UUU (Sequence No. 5)OLFML2BsiRNAGAG AAC GUU UUA UCU CAG U (Sequence No. 6)

[0161] Transfection was performed using Lipofectamine 2000 (Invitrogen) reagent.

[0162]

[0163] 11. Wound healing assay

[0164] Experimental Subjects: SNU449 and SNU475 liver cancer cell lines used. Comparison of OLFML2B KD (Knockdown) group and Control group.

[0165]

[0166] (1) Cell culture

[0167] SNU449 and SNU475 cells were cultured in 6-well plates, respectively, to a density of 80–90%.

[0168] The medium used was RPMI 1640 + 10% FBS + 1% penicillin / streptomycin.

[0169]

[0170] (2) OLFML2B expression inhibition (KD) treatment

[0171] OLFML2B KD was performed using siRNA.

[0172] The control group was treated with non-targeting siRNA.

[0173]

[0174] (3) Wound creation

[0175] After cell culture was completed, a straight wound was created in the cultured cell layer using a pipette tip (200 μL).

[0176] The culture dish was washed with PBS to remove detached cells, and then replaced with serum-free media.

[0177]

[0178] (4) Taking an image

[0179] The wound was photographed with an optical microscope immediately after creation (0 hours), and after 24 and 48 hours, respectively.

[0180]

[0181] (5) Data Analysis

[0182] The wound closure area over time was calculated using ImageJ software.

[0183] The wound closure rate between the OLFML2B KD group and the Control group was compared.

[0184]

[0185] 12. Colony formation assay

[0186] Experimental Subjects: SNU449 and SNU475 liver cancer cell lines were used. The OLFML2B KD (Knockdown) group and the Control group were compared.

[0187]

[0188] (1) Cell culture and KD treatment

[0189] SNU449 and SNU475 cells were treated with OLFML2B siRNA or non-targeting siRNA, respectively.

[0190] Cells were collected after completing siRNA treatment for 24 hours.

[0191]

[0192] (2) Colony formation

[0193] Cells were seeded into a 6-well plate at a density of 4,000 cells / well.

[0194] Cultures were incubated for 8 days at 37°C and 5% CO₂, and the culture medium (RPMI1640 + 10% FBS + 1% penicillin / streptomycin) was replaced every 2 to 3 days.

[0195]

[0196] (3) Fixation and staining

[0197] After incubation, it was washed with PBS and fixed with 4% paraformaldehyde for 15 minutes.

[0198] Fixed cells were stained with 0.5% crystal violet (20 min).

[0199]

[0200] (4) Image capture and analysis

[0201] The number of colonies was counted by observing the stained plate under an optical microscope.

[0202] The number of colonies in the OLFML2B KD group and the Control group was compared.

[0203]

[0204] 13. Cell Migration and Invasion Analysis (Migration & invasion assay)

[0205] Cell migration analysis was performed using a modified Boyden chamber (BD Bioscience), and cell infiltration analysis was performed using Matrigel (BD Bioscience).

[0206] For infiltration analysis, Matrigel was diluted to a concentration of 0.3 mg / mL, 100 µl was dispensed onto the insert, and the samples were incubated at 37°C for 2 hours. Then, cells transfected with siNC and siOLFML2B were placed on the insert and cultured using a medium containing 2.5% FBS as a chemical attractant.

[0207] After 24 and 48 hours, the infiltrated and migrated cells were stained with the Diff Quick staining kit (Sysmex Corporation) and observed under a microscope.

[0208]

[0209] 14. Analysis of Protein Expression Related to EMT / Vascular Infiltration / Apootosis

[0210] Antibodies for EMT-related markers were E-cadherin (Cell Signaling Technology, 3195S), Occludin (Santa Cruz Biotechnology, 3195S), Vimentin (Cell Signaling Technology, 5741S), and Snail (Cell Signaling Technology, 3879S), and antibodies for vascular invasion-related markers were CD31 (Abcam, sc7269) and VEFG (Santa Cruz Biotechnology, sc7269).

[0211] Antibodies for apoptosis-related markers were obtained using the Apoptosis Antibody sampler kit (Cell Signaling Technology, #9915).

[0212]

[0213] 15. Analysis of OLFML2B Expression in Animal Models

[0214] (1) Analysis of OLFML2B expression in recurrent tumors in an orthotopic liver cancer mouse model

[0215] Using an orthotopic liver cancer mouse model in which liver cancer cell lines were directly injected into the liver, after tumor development in the liver, the tumor (primary tumor prior to resection) was removed and collected through surgical resection, and subsequently, the recurrent tumor tissue (recurrent tumor) was collected.

[0216] Immunohistochemical staining (IHC) was performed to determine the expression levels of OLFML2B protein in the primary tumor tissue prior to resection and the recurrent tumor tissue.

[0217]

[0218] (2) Tail vein mouse model

[0219] 3×10 NIH-3T3-ras cells into which the oncogene ras was introduced for a tail vein injection model to confirm the metastatic potential of liver cancer cells 5 Transfected cells were mixed with serum-free media, and the cell suspension was injected into the tail vein of 6-week-old Athymic mice.

[0220] The body weight of the mice was measured twice a week, and 14 days after the first injection, the mice were sacrificed and lung and liver tissues were collected to check the extent of metastasis through gross pathology and histopathological analysis (H&E).

[0221]

[0222] 16. Statistical Analysis of Results

[0223] Statistical analysis was conducted using MedCalc statistical software, GraphPad, and SPSS v22 analysis programs.

[0224]

[0225] <Example 1> Autoantibody Analysis Results

[0226] As a result of autoantibody analysis using a 22K protein chip, significant differences in expression were confirmed in 28 proteins in the relapsed group compared to the non-relapsed group.

[0227] LAMP1, OLFML2B, LAT, and CTRL showed high AUCs and were selected as candidate markers for recurrence prediction (Fig. 1).

[0228]

[0229] <Example 2> Survival Analysis Results in TCGA Data

[0230] To select markers for the diagnosis and recurrence prediction of liver cancer, four candidates from the TCGA dataset were evaluated by analyzing OS and DFS. No valid results were observed in OS and DFS for the three candidates, excluding OLFML2B (Fig. 2).

[0231]

[0232] <Example 3> Results of OLFM2B survival analysis in four public omics datasets

[0233] In addition to TCGA data, MDACC, GSE14520, and GSE16757 data, which can evaluate OS and DFS, were obtained to perform a survival analysis based on OLFM2B expression.

[0234] OLFML2B was found to be associated with poorer patient prognosis in OS and DFS with higher expression in a total of four datasets, including the TCGA dataset, MDACC, GSE14520, and GSE16757 (Fig. 3).

[0235]

[0236] <Example 4> Results of Cox regression analysis on GSE114564 data

[0237] As a result of performing Cox regression analysis on the GSE114564 data, OLFML2B was observed as a major variable with an HR of 1.706 (95% CI = 1.084-2.685, P = 0.020936) (Fig. 4).

[0238]

[0239] <Example 5> Evaluation of the Specificity of OLFML2B in Liver Cancer Using Public Omics Data

[0240] The expression of OLFML2B at various stages of HCC was further investigated through spatial transcriptome analysis using the GepLiver DB dataset.

[0241] Analysis of the heatmap generated from the GepLiver DB dataset revealed that OLFML2B expression was significantly increased in liver cancer compared to normal liver, viral hepatitis, and NAFLD (P< 0.05) (Fig. 5).

[0242] In addition, spatial transcriptome analysis showed that the expression of OLFML2B was significantly increased in malignant hepatocytes (Fig. 6).

[0243]

[0244] The GSE77509 dataset consists of transcriptome analysis performed on Normal, Tumor, and PVTT tissues from the same patient. When OLFML2B expression was confirmed, expression gradually increased in the Normal, Tumor, and PVTT tissues of the same patient (Fig. 7). The classification accuracy for each is as follows.

[0245] HCC vs. Normal: AUC = 0.95 (P < 0.0001).

[0246] PVTT vs. Normal & Tumor: AUC = 0.84 (P < 0.0001).

[0247]

[0248] <Example 6> Evaluation of the specificity of OLFML2B in liver cancer in a verification experiment

[0249] In public data, it was observed that the expression of OLFML2B is significantly increased in liver cancer, and is expressed more distinctly in malignant hepatocytes and PVTT tissues than in early liver cancer.

[0250] Accordingly, a verification analysis was performed based on tissue samples from liver cancer patients who underwent hepatectomy at Ajou University Hospital.

[0251]

[0252] 1. Results of Diagnostic History Evaluation for 132 Patients at Ajou University Hospital

[0253] qRT-PCR: OLFML2B expression was increased in liver cancer tissue in 118 out of 132 patients (89.4%) (Fig. 8).

[0254] Diagnostic power: AUC = 0.89 (P < 0.0001).

[0255]

[0256] WB: 90% increase in expression in T compared to NT in 10 patients (Fig. 9).

[0257] Diagnostic power: AUC = 0.97 (P < 0.0001).

[0258]

[0259] 2. Results of Recurrence Prediction Evaluation by qRT-PCR and WB Analysis

[0260] Using qRT-PCR, the liver cancer tissue (PT) and surrounding tissue (NT) at the time of the initial surgery, as well as the liver cancer tissue (RT) at the time of recurrence in patients who underwent surgery and subsequently experienced recurrence, were evaluated. Expression was higher in PT compared to NT and in RT compared to PT, with an AUC of 1.00 (P=0.0002) for total T compared to NT and an AUC of 0.92 (P=0.01) for RT compared to NT and PT (Fig. 10).

[0261] In the results of a WB study conducted on patients who underwent surgery and subsequently experienced recurrence, the liver cancer tissue at the time of the first surgery (primary tumor, PT), the normal tissue surrounding the liver cancer (P-NT), the liver cancer tissue at the time of recurrence (recurrent tumor, RT), and the normal tissue surrounding the liver cancer (R-NT) were collected and showed almost no expression in P-NT or R-NT, and it was found to be more increased in RT than in PT, with an AUC of 0.98 (P=0.001) for T relative to NT (Fig. 11).

[0262] In the expression analysis of AFP protein using the same samples, it was expressed in some samples at PT and RT, but overall, the expression intensity was weak and the expression pattern was inconsistent. In particular, in some RT samples, OLFML2B was strongly expressed despite almost no AFP being expressed.

[0263] Based on these qualitative results, quantification and AUC analysis were performed, and OLFML2B showed very high diagnostic accuracy with an AUC of 0.98 and a P of 0.001 in the ROC analysis of T (tumor) compared to NT (Fig. 12).

[0264] In contrast, the AUC of AFP was 0.84, indicating relatively low diagnostic performance.

[0265] In particular, OLFML2B is expressed stably and strongly even upon recurrence and is detectable in AFP-negative tumors, making it a candidate that can complement the limitations of the existing diagnostic biomarker AFP.

[0266]

[0267] <Example 7> Results of OLFML2B expression inhibition experiment

[0268] To determine the efficacy of inhibiting the overexpression of OLFML2B in liver cancer cell lines, we first checked the expression of OLFML2B in normal hepatocyte cell line LO2 and eight liver cancer cell lines using qRT-PCR and WB, and confirmed that SNU449 showed the highest expression (Fig. 13).

[0269] Additionally, the KD efficiency was confirmed by KDing OLFML2B in two cell lines with SNU475 (Fig. 14).

[0270] As a result of conducting a wound healing assay, it was observed that wound closure progressed less in both cell lines at 48 hours after KD in the group that KDed OLFML2B (Fig. 15).

[0271] Additionally, when viewed through a colony formation assay, it was confirmed that the colony number in the group that KD OLFML2B was significantly reduced (Fig. 16).

[0272]

[0273] After treating liver cancer cell lines SNU449 and SNU475 with OLFML2B-specific siRNA (siOLFML2B) and negative control siRNA (siNC), respectively, a Transwell migration assay was performed to analyze the migration ability of the cells.

[0274] As a result, in the siNC-treated group, a large number of cells that migrated to the lower chamber were observed, and the cell density remained high.

[0275] On the other hand, in the siOLFML2B treatment group, the number of cells that migrated downstream was significantly reduced, suggesting that inhibition of OLFML2B expression strongly inhibits the cell's migration ability.

[0276] Quantifying the number of migrated cells for each cell line, the number of migrated cells was significantly reduced in both SNU449 and SNU475 upon OLFML2B knockdown (P < 0.01) (Fig. 17).

[0277]

[0278] As a result of evaluating cell invasion using a transwell chamber coated with Matrigel, a large number of cells that had invaded to the bottom of the chamber were observed in the siNC-treated group.

[0279] On the other hand, the number of invading cells was significantly reduced in the siOLFML2B-treated group, and a significant inhibitory effect on invasion (P < 0.01) was observed in both SNU449 and SNU475 upon OLFML2B knockdown (Fig. 18).

[0280] This suggests that OLFML2B is involved in maintaining the invasiveness of liver cancer cells.

[0281]

[0282] Analysis of changes in EMT and vascular infiltration-related protein expression after OLFML2B knockdown treatment showed that E-cadherin (epithelial marker) significantly increased in siOLFML2B, while N-cadherin, Vimentin, Snail, ZEB1, and Slug (mesenchymal markers) all decreased.

[0283] This means that OLFML2B plays a role in promoting EMT, and that inhibition of expression leads to the recovery of the epithelial phenotype and inhibition of mesenchymal characteristics.

[0284] VEGF-A, VEGF-C, CD31, Angiopoietin-2, MMP2, and MMP9, which are markers related to vascular invasion, also showed decreased expression upon OLFML2B knockdown. This suggests that OLFML2B upregulates molecules associated with angiogenesis and vascular invasion, which may be associated with an increased risk of metastasis (Fig. 19).

[0285] These results demonstrate that OLFML2B is involved in regulating the invasiveness, EMT progression, and vascular invasion of liver cancer cells, suggesting that inhibiting the expression of this gene can effectively reduce the metastatic potential and malignancy of cancer cells.

[0286] Therefore, OLFML2B is evaluated as a candidate with high potential for use as a novel therapeutic target and prognostic marker to inhibit the recurrence and metastasis of liver cancer.

[0287]

[0288] After treating liver cancer cell lines SNU449 and SNU475 with OLFML2B-specific siRNA (siOLFML2B) and negative control siRNA (siNC), changes in the expression of apoptosis-related signaling proteins were analyzed using Western blot.

[0289] Analysis results showed that the expression of cleaved caspase-3 and cleaved PARP significantly increased in the OLFML2B knockdown group. This indicates that the apoptotic pathway was actively induced. On the other hand, the expression of cell survival-related proteins such as Bcl-2, Bcl-xL, and XIAP significantly decreased in the siOLFML2B treatment group, suggesting that OLFML2B is involved in maintaining anti-apoptotic signaling.

[0290] In the quantification results, the band intensities of cleaved caspase-3 and cleaved PARP were significantly increased compared to siNC, while Bcl-2 family proteins showed a decreasing pattern (P < 0.01 level) (Fig. 20).

[0291] These results suggest that OLFML2B is an important regulator contributing to the inhibition of apoptosis and the maintenance of survival signals in liver cancer cells, demonstrating that inhibiting this gene can effectively reduce the viability of cancer cells.

[0292] OLFML2B knockdown not only reduced the migration / invasion of liver cancer cells but also induced a decrease in tumor cell viability through the activation of apoptosis pathways.

[0293] Therefore, OLFML2B is highly promising as a therapeutic target for inhibiting recurrence and progression in liver cancer, and is a key factor capable of simultaneously regulating multiple mechanisms (EMT, vascular invasion, apoptosis, etc.).

[0294]

[0295] <Example 8> Results of OLFML2B expression inhibition experiment in vivo

[0296] After establishing an orthotopic HCC mouse model by directly injecting liver cancer cell lines into the liver, the tumor was removed via surgical resection after a certain period, and the recurrent tumor tissue was collected. At this time, immunohistochemical staining (IHC) was performed to determine the expression level of OLFML2B protein in the recurrent tumor tissue.

[0297] The IHC results in two animals in which recurrence was observed as a representative case are shown in Fig. 21.

[0298] Compared to the primary tumors prior to resection (PT1, PT2), the OLFML2B staining intensity in the recurrent tumors (RT1, RT2) was significantly increased.

[0299] In particular, a strong intracellular brown signal was observed spreading in the recurrent tissue, which suggests overexpression of the OLFML2B protein.

[0300] In contrast, OLFML2B expression was rarely observed in normal liver tissue.

[0301] In the quantitative image analysis results, it was visually confirmed that OLFML2B IHC intensity consistently increased in the relapse group, which is the same trend observed in actual patient samples (Fig. 21).

[0302]

[0303] A lung metastasis model was established by treating NIH-3T3-ras cells, into which the oncogene ras was introduced, with OLFML2B-specific siRNA (siOLFML2B) or negative control siRNA (siNC), respectively, and then injecting them intravenously into immunodeficient mice via tail vein injection. After a certain period, lung tissues were collected, and the extent of metastasis was analyzed quantitatively and histopathologically.

[0304] No significant difference was observed between the two groups in total weight change.

[0305] Gross examination of the dissected lung tissue revealed a large number of nodules in the siNC treatment group (Fig. 22).

[0306]

[0307] In contrast, clear masses were rarely observed on the lung surface in the siOLFML2B treatment group, and when the number of lung metastatic lesions was quantitatively analyzed, the number of metastatic lesions in the siOLFML2B treatment group was significantly reduced compared to siNC (P < 0.01).

[0308] In the quantification of the lesion area (%), it was statistically confirmed that the metastatic area was significantly reduced in the siOLFML2B treatment group.

[0309] In the siNC-treated group, typical cancer cell colonies were found to infiltrate multiplely within the lung parenchyma. In particular, aggregated tumor cells and infiltration were observed around blood vessels and airways.

[0310] In the siOLFML2B treatment group, almost no cancer cell infiltration lesions were observed in the lung parenchyma, and the normal lung tissue structure was well preserved overall (Fig. 23).

[0311]

[0312] After a certain period, liver tissue was collected and gross pathology and histopathological analysis (H&E) were performed.

[0313] Areas of high cell density were observed in the liver tissue of the siNC-treated group, suggesting the possibility of microinfiltration of cancer cells or reactive changes in the liver tissue (inflammation, cell proliferation). On the other hand, in the siOLFML2B-treated group, the liver parenchyma maintained a more uniform and normal arrangement of hepatocytes (Fig. 24).

[0314] Even if there are no clear metastatic lesions in high-magnification tissues, the relative increase in staining intensity and tissue heterogeneity observed in siNCs may indirectly support changes in the liver tissue microenvironment due to OLFML2B expression.

[0315] These results show that OLFML2B plays a very important role in the metastasis process of cancer cells.

[0316] In particular, inhibiting OLFML2B expression blocks the settlement, proliferation, and infiltration of tumor cells that have entered the bloodstream, which means that lung metastasis is significantly suppressed.

[0317] These results suggest the potential for OLFML2B to be utilized not only in liver cancer but also as a target for inhibiting metastasis in metastatic solid tumors.

[0318]

[0319] In conclusion, the present invention has the following technical features.

[0320]

[0321] 1. Superior diagnostic accuracy compared to existing liver cancer biomarkers (AFP, etc.)

[0322] OLFML2B exhibits high specificity in liver cancer tissue and demonstrates superior diagnostic performance compared to existing biomarkers, with an AUC of 0.98 and a P of 0.001 in ROC analysis. In particular, high expression was confirmed even in AFP-negative patient groups, distinguishing it as an auxiliary diagnostic marker capable of overcoming the limitations of existing biomarkers.

[0323]

[0324] 2. Specific high expression and prognostic predictive power in recurrent liver cancer tissue

[0325] In a comparison of primary and recurrent tumor tissues from the same patient, the expression of OLFML2B was significantly increased in the recurrent tissue. In an orthotopic liver cancer mouse model and patient-derived samples, expression was consistently and specifically elevated in the recurrent group. Cox regression analysis confirmed that OLFML2B is an independent prognostic factor significantly associated with survival and recurrence.

[0326]

[0327] 3. Verification of consistent performance across various public datasets

[0328] Analysis of various patient-derived public datasets, including MD Anderson Cancer Center (MDACC), GSE14520, and GSE199509, revealed that OLFML2B was consistently overexpressed in high-risk lesions—such as malignant hepatocytes, portal vein tumor thrombus (PVTT), and recurrent hepatitis C—compared to normal liver tissue. This data-driven evidence demonstrates that OLFML2B is a molecule closely associated with tumor malignancy and metastasis / recurrence.

[0329]

[0330] 4. Mechanistic functionality distinguishable from existing liver cancer targets

[0331] Inhibition of OLFML2B significantly reduced the expression of markers related to migration, invasion, EMT, and angiogenesis (MMPs, VEGF, etc.), induced apoptosis through increased cleaved caspase-3 and PARP, and decreased cell invasiveness and viability in liver cancer cell lines (SNU449, SNU475, etc.). In particular, inhibition of OLFML2B significantly reduced the number and area of ​​metastatic lesions in an in vivo lung metastasis model (tail vein injection). This suggests that, unlike existing diagnostic markers, it also functions as a target for inhibiting metastasis.

[0332]

[0333] 5. Potential to regulate complex biological phenomena rather than a single target

[0334] OLFML2B acts not merely as a marker for simple expression changes, but as a key gene that regulates complex cancer biological phenomena such as invasiveness, metastatic potential, vascular invasion, recurrence potential, and apoptosis. This makes it highly useful as a mechanism-based therapeutic target, differentiating it from existing liver cancer biomarkers that are primarily limited to diagnosis.

[0335]

[0336] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for predicting liver cancer metastasis or recurrence comprising the OLFML2B protein or a gene encoding it.

2. A composition for predicting liver cancer metastasis or recurrence comprising, as an active ingredient, a preparation capable of measuring the expression level of the OLFML2B protein or the gene encoding it.

3. A composition for predicting liver cancer metastasis or recurrence, characterized in that, in paragraph 2, the preparation capable of measuring the expression level of the OLFML2B protein or the gene encoding it is a primer or probe that specifically binds to the OLFML2B gene, or an antibody, peptide, aptamer, or compound that specifically binds to the OLFML2B protein.

4. A kit for predicting liver cancer metastasis or recurrence comprising the composition of paragraph 2 or 3.

5. (1) A step of measuring the expression level of the OLFML2B protein or the gene encoding it from a sample isolated from a liver cancer patient; (2) a step of comparing the expression level of the OLFML2B protein or the gene encoding it with a control sample; and (3) A method for providing information necessary for predicting liver cancer metastasis or recurrence, comprising the step of determining that the likelihood of liver cancer recurrence is high when the expression level of the OLFML2B protein or the gene encoding it is higher than that of a control sample.

6. A method for providing information necessary for predicting liver cancer metastasis or recurrence, characterized in that, in paragraph 5, the sample isolated from the liver cancer patient is a sample isolated after the liver resection of the liver cancer patient.

7. A pharmaceutical composition for preventing or treating liver cancer metastasis or recurrence, comprising as an active ingredient an inhibitor of the expression or activity of the OLFML2B protein or the gene encoding it.

8. A pharmaceutical composition for preventing or treating liver cancer metastasis or recurrence, wherein, in claim 7, the inhibitor of expression or activity of the OLFML2B protein or the gene encoding it is selected from the group consisting of an antisense nucleotide, a small interfering RNA (siRNA), and a short hairpin RNA (shRNA) that specifically binds to the OLFML2B protein or the gene encoding it, a compound, a peptide, an aptamer, and an antibody.

9. A pharmaceutical composition for preventing or treating liver cancer metastasis or recurrence, characterized in that, in claim 7 or 8, the liver cancer metastasis is metastasis to the lungs.

10. A health functional food composition for preventing or improving liver cancer metastasis or recurrence, comprising as an active ingredient an inhibitor of the expression or activity of the OLFML2B protein or the gene encoding it. 11.(1) A step of contacting isolated liver cancer cells with a test substance; (2) A step of measuring the degree of expression or activity of the OLFML2B protein or the gene encoding it in liver cancer cells contacted with the above test substance; and (3) A screening method for a therapeutic agent to inhibit metastasis or recurrence of liver cancer, comprising the step of selecting a test substance in which the expression or activity of the OLFML2B protein or the gene encoding it is reduced compared to a control sample.

12. A screening method for a therapeutic agent to inhibit liver cancer metastasis or recurrence, characterized in that, in claim 11, the isolated liver cancer cells are liver cancer cells isolated after liver resection of a liver cancer patient.

Images