KR101793174B1 - Method for Diagnosing Recurrent Cancer Using GOLGB1 or SF3B3 and Composition for Treating Recurrent Cancer Containing Inhibitors of GOLGB1 or SF3B3 - Google Patents

Abstract

The present invention relates to a composition for diagnosis or prognosis prediction of recurrent cancer using GOLGB1 or SF3B3, a composition for preventing or treating recurrence cancer containing an expression or activity inhibitor of GOLGB1 or SF3B3 protein, and a composition for preventing or treating recurrent cancer using GOLGB1 or SF3B3 ≪ / RTI > Since GOLGB1 or SF3B3 of the present invention is suitable as a therapeutic target of recurrent cancer, it can be applied to the development of an agent capable of preventing or treating recurrent cancer by inhibiting GOLGB1 or SF3B3. It is very useful for management.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for diagnosing recurrent cancer using GOLGB1 or SF3B3, and a composition for treating recurrent cancer containing GOLGB1 or SF3B3 inhibitor,

The present invention relates to a composition for diagnosis or prognosis prediction of recurrent cancer using GOLGB1 or SF3B3, a composition for preventing or treating recurrence cancer containing an expression or activity inhibitor of GOLGB1 or SF3B3 protein, and a composition for preventing or treating recurrent cancer using GOLGB1 or SF3B3 ≪ / RTI >

Hepatocellular carcinoma (HCC) is the fifth most malignant cancer in the world, the second leading cause of cancer death (Jemal A et al., Cancer J Clin . 61: 69-90, 2011). In the present study, patients with hepatocellular carcinoma (HCC) were treated with surgical resection and liver transplantation (Aravalli RN et al., Hepatology . 48: 2047-2063, 2008) (Llovet JM et al., Semin Liver Dis . 25: 181-200, 2005). Recurrent tumors have a distinct genomic profile compared to primary tumors, suggesting intracellular reprogramming at tumor recurrence. In fact, recurrent tumors acquire aggressive phenotypes such as invasion, metastasis, EMT and chemical resistance, which aggravate the prognosis of tumor patients (Brodsky AS et al., PLoS One. 9: e94476, 2014; Woo HG et al., Clin Cancer Res . 14: 2056-2064, 2008; Kim JH et al., PLoS medicine. 11: e1001770, 2014). Therefore, in order to improve the clinical outcome of patients with hepatocellular carcinoma, it is essential to clarify the mechanism of hepatocellular carcinoma recurrence.

Recently, the development of high-throughput sequencing (HTS) technology has enabled us to analyze the genomic variation of genetic factors that play an important role in cancer progression. Recurrent mutations were found in TP53, CTNNB1, AXIN1, IRF2, CDKN1A, CDKN2A, ARID1A, ARID2 and TERT in hepatocellular carcinoma, leading to clinical results that promote the development and / or progression of hepatocellular carcinoma (Guichard C et al , Nat Genet. 44: 694-698, 2012; Woo HG et al., PLoS One. 9: e115152, 2014). Recurrent tumors showed a clear mutation and / or transcriptomic profile compared to primary tumors (Kim J et al., Cancer Cell 28: 318-328, 2015; Eleveld TF et al., Nat Genet. 47: 864-871, 2015 ). Therefore, comparing the genomic and / or transcriptomic profiles of recurrent or metastatic tumors to primary tumors of the same patient can reveal the mechanism for recurrent tumor progression.

On the other hand, the carcinogenic functions of GOLGB1 and SF3B3 are largely unknown. Golgi1, a giant cell membrane protein associated with Golgi, has been reported to promote in vivo formation and retention of small Golgi forms (Linstedt AD et al., Mol Biol Cell. 4: 679-693, 1993), but the oncological role has not yet been established. In addition, SF3B3 is a protein for spliceosome assembly, which is associated with cancer cell growth and has been implicated in the prognosis of breast cancer patients (Srihari S et al., Molecular bioSystems. 12: 963-972 , 2016).

Accordingly, the present inventors have made extensive efforts to predict the diagnosis or prognosis of cancer by biomarker discovery through the genome and / or transcriptomic profile of the tumor and to develop a therapeutic agent. As a result, it has been found that overexpression of GOLGB1 or SF3B3 gene It was confirmed that the mutation of the gene can aggressively progress the cancer and inhibit the progress of the cancer through its inhibition, thus completing the present invention.

It is an object of the present invention to provide a composition for predicting the diagnosis or prognosis of recurrent cancer, which comprises a substance capable of measuring mRNA of GOLGB1 or SF3B3 gene or a protein expression level encoded by the gene.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating recurrent cancer, which comprises as an active ingredient an agent for inhibiting the expression or activity of GOLGB1 or SF3B3 protein.

It is still another object of the present invention to provide a screening method for preventing or treating recurrent cancer using GOLGB1 or SF3B3.

In order to achieve the above object, the present invention provides a composition for predicting the diagnosis or prognosis of recurrent cancer, which comprises a substance capable of measuring mRNA of GOLGB1 or SF3B3 gene or a protein expression level encoded by the gene.

The present invention also provides a pharmaceutical composition for preventing or treating recurrent cancer comprising, as an active ingredient, an agent for inhibiting the expression or activity of GOLGB1 or SF3B3 protein.

The present invention also relates to a method for screening a test substance, comprising: (i) treating a test substance with an expression cell line of GOLGB1 or SF3B3 protein; (ii) measuring the level of GOLGB1 or SF3B3 protein expression or activity in the cell line; And (iii) selecting a test substance whose expression or activity level of the GOLGB1 or SF3B3 protein is lower than that of a control group not treated with the test substance.

Since GOLGB1 or SF3B3 of the present invention is suitable as a therapeutic target of recurrent cancer, it can be applied to develop a substance capable of preventing or treating recurrent cancer by inhibiting GOLGB1 or SF3B3, and is also useful as a cancer marker for recurrence. It is very useful for management.

FIG. 1 shows the number of mutations found in primary and recurrent tumors. FIG. 1 (B) shows the number of mutations found in primary and recurrent tumors, Right), and (C) shows the assembled sequence lead with the mutation by the Integrated Genome Viewer (IGV).
FIG. 2A shows a heat map showing the expression of a set of genes "RER (reprogrammed expression by recurrence)", which is a gene that is differentially regulated upward and downward between primary and recurrent hepatocellular carcinoma. FIG. 2B shows RER_UP (n = 60, red) and RER_DOWN (n = 70, blue) genes, and (C) is a bar graph showing the population concentration of the genes of MsigDB (red: tumor formation, green: tumor suppression) (D) shows the overall survival rate between the RER_high and RER_low groups in the TCGA cohort (left) and the survival rate (left) between the RER_high and RER_low groups in the TCGA cohort ) And recurrence-free survival rate (right), and (E) shows the number of genes overlaid between the relapse-related gene and the RER_UP gene in the previous study.
FIG. 3 is a functional evaluation of a recurrence-specific mutation in which (A) shows the expression level of a recurrence-specific mutant gene in an HCC sample, (B) shows the expression level of a recursive-specific mutant gene in recurrent HCC (C) is the result of confirming cell proliferation by 72 hours of knockdown of HepG2, Huh7, Hep3B, SNU423 and PLC liver cancer cells with siRNA against NT-CTL (non-target control), GOLGB1 or SF3B3 D) is a measure of cell migration using transwell chambers for cells transfected with siRNA against NT-CTL (non-target control), GOLGB1 or SF3B3.
Figure 4 (A) is the MTT measurement of cell proliferation of HepG2 and Huh7 transfected with a mock control, wild-type, or GOLGB1 or SF3B3 mutant for 72 h, and (B) shows the mutation of GOLGB1 or SF3B3 for 14 days (C) and (D) are measurements of cell migration and infiltration of HepG2 and Huh7 transfected with mock control, wild-type, or GOLGB1 or SF3B3 mutants.
Figure 5 shows the predicted protein structure of the wild type (left) and mutant (right) of GOLGB1 (A) and SF3B3 (B).
FIG. 6 (A) shows genes expressed in HepG2 cells knocked down by GOLGB1 (siGOLGB1, top) or SF3B3 (siSF3B3, bottom), and (B) shows siGOLGB1_DOWN (top) and siSF3B3_DOWN (top) between recurrent HCC and primary HCC (C) shows the population concentration scores of siGOLGB1_DOWN (top) and siSF3B3_DOWN (bottom) genes in each HCC sample, and (D) siSF3B3_DOWN (n = 53) (E) shows the network analysis of the siGOLGB1_DOWN (n = 126) and RER_UP (n = 60) genes in the HepG2 cells transformed with NT-CTL (non-target control), GOLGB1 or SF3B3 siRNA, The expression level of mRNA was measured by qRT-PCR.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, RNA-Seq profiling of primary and recurrent hepatocellular carcinoma (HCC) was performed to identify seven somatic mutations in recurrent HCC. Among the seven mutations, the well-known cancer gene was included in primary liver cancer such as TP53 mutation, ARID1A, and CCNL2. These results demonstrate that the sample size of the present invention is limited but the reliability of the data and analysis strategy is high. It was confirmed that a new mutation of GOLGB1 and SF3B3 among the seven mutants can promote the carcinogenic characteristics.

Although the carcinogenic functions of GOLGB1 and SF3B3 are largely unknown, the present invention demonstrated that overexpression of GOLGB1 or SF3B3 promotes aggressive cancer progression, such as cell proliferation, invasion and migration, reflecting aggressive behavior of recurrent HCC (Fig. 4) . In addition, somatic mutations in these genes have aggravated the aggressive phenotype of HCC cells, suggesting their action as activating mutants. Structural modifications of mutant proteins were also predicted to support their operability (Figure 5). That is, mutations in GOLGB1 or SF3B3 promote aggressive progression of HCC.

Next, CXCL8 and SOX4 were identified as common downstream targets of GOLGB1 and SF3B3 (Fig. 6D). CXCL8 (IL-8 encoding) activates the EGF and MAPK signaling pathways leading to cancer progression and metastasis in various cancers (Inoue K et al., Cancer Res. 60: 2290-2299, 2000; Luppi F et al. , Lung Cancer. 56: 25-33, 2007), SOX4 is an important modulator of EMT function in cancer progression (Tiwari N et al., Cancer Cell. 23: 768-783, Migration, invasion and metastasis (Liao YL et al., Oncogene . 27: 5578-5589, 2008). In fact, it has been established that CXCL8 (IL-8) and SOX4 can promote development and progression of cancer, confirming that CXCL8 and SOX4 are key downstream genes for aggressive progression by mutant GOLGB1 or SF3B3.

In conclusion, by performing a comprehensive analysis of mutation and transcriptome profiles and experimental evaluation, we successfully identified new driver mutations and their potential target genes. GOLGB1 or SF3B3 is very useful in the management of HCC patients, either therapeutically or genuinely.

Accordingly, the present invention relates to a composition for predicting the diagnosis or prognosis of recurrent cancer, which comprises a substance capable of measuring mRNA of GOLGB1 or SF3B3 gene or a protein expression level encoded by the gene.

In the present invention, the GOLGB1 gene (NM_001256488.1) is preferably a nucleotide sequence of SEQ ID NO: 1, and the SF3B3 gene (NM_012426.4) is a nucleotide sequence of SEQ ID NO: 2, But is not limited thereto.

The term "diagnosis" as used herein means to identify the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the recurrence of cancer. Preferably, it is confirmed whether or not the liver cancer recurs.

In the present invention, the term "prognosis" means a prediction for a medical cause (e.g., long-term viability, disease-free survival rate, etc.) and includes a positive prognosis (positive prognosis) or a negative prognosis Prognosis includes progression or mortality of recurrence, tumor growth, metastasis, drug resistance, etc., and positive prognosis may include disease progression such as absence of disease, improvement or stabilization of disease such as tumor regression stabilization.

In the present invention, the term "prediction" refers to predicting medical consequences, and for the purpose of the present invention, the course of disease in a patient diagnosed with cancer (progression, improvement of disease, recurrence of gastric cancer, Drug resistance).

In the present invention, the term "substance for measuring the expression level of the gene mRNA" means a molecule that can be used for confirming the expression level of GOLGB1 or SF3B3 gene of the present invention or a protein encoded by these genes, May be an antibody, an extramammer, a primer, or a probe specific to the GOLGB1 or SF3B3 gene.

The term "primer" in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group and can form a base pair with a complementary template, and a starting point for copying the template Quot; refers to a short nucleic acid sequence that functions as a < / RTI > In the present invention, PCR amplification using the sense and antisense primers of the marker polynucleotide of the present invention can predict the prognosis of gastric cancer through the production of desired products. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

In the present invention, "primer" is a PCR primer that specifically binds to the mRNA of the GOLGB1 or SF3B3 gene. By performing RT-PCR or quantitative RT-PCR using the primer, the expression level of the gene is compared with the normal group, And the prognosis can be predicted.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to mRNA, and the presence or absence of a specific mRNA can be confirmed have. The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe.

In the present invention, a concept including a nucleic acid probe capable of specifically recognizing and binding to the GOLGB1 or SF3B3 gene is included. By performing Northern blot analysis or the like, the expression level of the gene is compared with a normal group The diagnosis or prognosis of recurrent cancer can be predicted.

In the present invention, the term "measurement of protein expression level" is a process for confirming the presence and expression level of a protein expressed in a GOLGB1 or SF3B3 gene in a biological sample in order to predict cancer prognosis. The amount of the protein can be confirmed by using an antibody that specifically binds to the protein. Methods for this analysis include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, Immunoprecipitation assays, complete fixation assays, FACS, protein chips, and the like, but are not limited thereto.

The term "antibody" as used herein in the present invention means a specific protein molecule indicated for an antigenic site as a term known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker of the present invention, which clones each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene , And can be prepared from the obtained protein by a conventional method. It also includes partial peptides that can be made from the protein.

"Antibody" of the present invention includes both a polyclonal antibody, a monoclonal antibody and a recombinant antibody, and an antibody refers to a specific protein molecule directed against an antigenic site.

Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injection of the antigen into the animal and blood collection from the animal. Such polyclonal antibodies can be prepared from any animal species host such as goats, rabbits, sheep, monkeys, horses, pigs, rats, rats, cows, dogs and the like. Monoclonal antibodies may be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 624 Biol., 222: 58, 1-597, 1991) techniques. The antibody prepared by the above method can be separated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

That is, the present invention may include a monoclonal antibody or polyclonal antibody that specifically binds to an epitope of a protein encoded by the GOLGB1 or SF3B3 gene.

Such antibodies are generally quantitatively analyzed by color reaction using secondary antibodies conjugated with enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and their substrates, The protein monoclonal antibody directly conjugated with AP or HRP enzyme or the like may be quantitatively analyzed.

In the present invention, the GOLGB1 protein preferably has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, more preferably a nucleotide sequence of nucleotides 8445 (adenine) of SEQ ID NO: 1 But it is not limited thereto.

In the present invention, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 is GOLGB1 (E2721V) in which the 2721st amino acid of the wild type GOLGB1 protein is substituted with valine in glutamic acid, but is not limited thereto.

In the present invention, it is preferable that the SF3B3 protein has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, more preferably a nucleotide sequence of nucleotide 2621 of C (cytosine) of SEQ ID NO: 2 But it is not limited thereto.

In the present invention, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 is SF3B3 (H804Y) in which the 804th amino acid of wild-type SF3B3 protein is substituted with tyrosine at histidine, but is not limited thereto.

In the present invention, the recurrent cancer is preferably selected from the group consisting of liver cancer, pancreatic cancer, gastric cancer, colon cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, uterine cancer and lung cancer.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating recurrence cancer, which comprises an expression or activity inhibitor of GOLGB1 or SF3B3 protein as an active ingredient.

In the present invention, the GOLGB1 protein preferably has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, more preferably GOLGB1 (E2721V) in which the 2721st amino acid of wild-type GOLGB1 protein is substituted with valine in glutamic acid, But is not limited thereto.

In the present invention, the SF3B3 protein preferably has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, more preferably SF3B3 (H804Y) in which the 804th amino acid of wild-type SF3B3 protein is substituted with tyrosine at histidine, But is not limited thereto.

In the present invention, the expression inhibitor of the GOLGB1 or SF3B3 protein is selected from the group consisting of an antisense nucleotide complementary to mRNA of GOLGB1 or SF3B3 gene, a short interfering RNA, and a short hairpin RNA But it is not limited thereto.

In the present invention, the GOLGB1 or SF3B3 protein activity inhibitor may be any one selected from the group consisting of compounds, peptides, aptamers, and antibodies that complementarily bind to the GOLGB1 or SF3B3 protein, but the present invention is not limited thereto.

The term "substance capable of inhibiting expression" of the present invention means a substance capable of inhibiting the production of a transcript or protein expressed and produced in a gene. Such agents include, but are not limited to, transcription factors that bind to the gene and inhibit at the transcription level; Interfering RNAs such as miRNA, siRNA, and shRNA that bind to the transcribed and synthesized transcript and degrade the transcript; An antibody capable of binding to the expressed GOLGB1 or SF3B protein, and the like.

The term "short interfering RNA " of the present invention means a double stranded RNA capable of inducing RNAi which inhibits the activity of a gene. In the present invention, the interfering RNA may be miRNA, siRNA, shRNA, or the like capable of inhibiting the expression of GOLGB1 or SF3B. The interfering RNA may be any form of inducing mRNA of GOLGB1 or SF3B, For example, siRNAs obtained by chemical synthesis or biochemical synthesis or in vivo synthesis, or double-stranded RNA of 10 base pairs or more in which a double-stranded RNA of about 40 bases or more is degraded in the body, etc. can be used.

The interference RNA is at least about 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, of the GOLGB1 or SF3B RNA nucleic acid sequence. Or more, particularly preferably 95% or more, and most preferably 100%. The RNA or double-stranded variant thereof including the double-stranded portion may be used. The sequence portion having homology is usually at least 15 nucleotides, preferably at least about 19 nucleotides, more preferably at least 20 nucleotides, and even more preferably at least 21 nucleotides.

In the present invention, the antibody may be an antibody capable of specifically binding to GOLGB1 or SF3B protein, preferably a polyclonal antibody capable of specifically binding to GOLGB1 or SF3B protein, a monoclonal antibody Or a part thereof.

In the present invention, the aptamer is a single-stranded DNA or RNA molecule, which can be detected by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment) (Tuerand L. Gold, Science 249, 505-510, 2005; AD Ellington and JW Szostak, Nature 346, 818-822, 1990), which can be obtained by separating oligomers that bind with high affinity and selectivity to molecules. M. Wilson and Szostak, Annu Rev. Biochem. 68, 611-647, 1999). Aptamers can specifically bind to a target and modulate the activity of the target, for example, by blocking the ability of the target to function through binding.

In the present invention, the recurrent cancer is preferably selected from the group consisting of liver cancer, pancreatic cancer, gastric cancer, colon cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, uterine cancer and lung cancer.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions. Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The content of the formulation in the pharmaceutical composition according to an embodiment of the present invention is not particularly limited, but may be in the range of 0.0001 to 50% by weight, more preferably 0.01 to 10% by weight, based on the total weight of the final composition .

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means a therapeutic or prophylactic treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the level of the disease to be treated, the severity of the disease, the activity of the drug, the age, body weight, health, sex, sensitivity of the patient to the drug, Duration, duration of administration, factors involved in combination with or contemporaneously with the composition of the present invention, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the age, body weight, sex, history, or kind of the substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered to a mammal including a human at a dose of 10 to 100 mg / kg, more preferably 10 to 30 mg / kg, and the administration frequency of the composition of the present invention It may be administered once to three times a day, or may be administered several times in divided doses.

The term "treatment" in the present invention means any action that improves or alters the radiation or drug resistant cancer by administering the pharmaceutical composition of the present invention.

The term "administration" of the present invention means the introduction of the pharmaceutical composition of the present invention to a subject by any appropriate method, and the administration route can be administered through various routes of oral or parenteral administration, have.

The route of administration of the pharmaceutical composition of the present invention can be administered through any conventional route so long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intracorporally, or rectally, as desired, though not particularly limited thereto. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

In another aspect, the present invention relates to a method for screening a test substance, comprising: (i) treating a test substance to an expression cell line of GOLGB1 or SF3B3 protein; (ii) measuring the level of GOLGB1 or SF3B3 protein expression or activity in the cell line; And (iii) selecting a test substance whose expression or activity level of the GOLGB1 or SF3B3 protein is lower than that of a control not treated with the test substance.

In the present invention, the GOLGB1 protein preferably has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, more preferably GOLGB1 (E2721V) in which the 2721st amino acid of wild-type GOLGB1 protein is substituted with valine in glutamic acid, But is not limited thereto.

In the present invention, the SF3B3 protein preferably has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, more preferably SF3B3 (H804Y) in which the 804th amino acid of wild-type SF3B3 protein is substituted with tyrosine at histidine, But is not limited thereto.

In the present invention, the degree of expression or activity of the protein in step (ii) may be determined by immunofluorescence, enzyme immunoassay (ELISA), Western blot, flow cytometry (FACS), immunohistochemistry, immunoprecipitation ), Radioimmunoassay (RIA), and RT-PCR, but the present invention is not limited thereto.

In the present invention, the recurrent cancer is preferably selected from the group consisting of liver cancer, pancreatic cancer, gastric cancer, colon cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, uterine cancer and lung cancer.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Patient and sample

Surgically resected primary and recurrent tumor specimens from two HCC patients were obtained from the AHBB, a human - derived life resource bank of the National Biological Bank of Korea, sponsored by the Ministry of Health and Welfare. Recurrent tumors occurred 14 months after surgery. The Institutional Review Board of Ajou University approved the study with consent from the donor.

RNA- Seq  Profiling and Data Processing

Total RNA was isolated using TRIzol® RNA (Life technologies, Carlsbad, Calif.) And confirmed using an Agilent RNA 6000 pico kit (Agilent, Santa Clara, Calif.). Sequencing libraries for mRNA were prepared using the TruSeq RNA sample preparation kit (Illumina, San Diego, Calif.) according to the manufacturer's instructions. Sequencing reactions were performed on an Illumina HiSeq2000, and the image data was converted and stored in FASTQ format. A low-quality sequence of less than 20 PHRED points is set to 'N' using the 'fastq_masker' command of the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and Tophat (Trapnell C et al. , Bioinformatics. 25: 1105-1111, 2009) as a basic parameter to the human reference genome (hg19). PCR replicas were removed by 'picard' MarkDuplicates (http://picard.sourceforge.net) and gene expression levels of RNASeq data were determined by Cufflinks (Trapnell C et al., Nat Biotechnol. 28: 511-515, 2010) And estimated by log2-transformed FPKM values.

RNA- On Seq  Mutation call by

RNA-Seq data were processed for mutation invocation and location repositioning and score normalization were performed using GATK IndelReligner and Recalibrator (DePristo MA et al., Nat Genet. 43: 491-498, 2011) . Sequence mutations were filtered using GATK Unitied Genotyper (DePristo MA et al., Nat Genet. 43: 491-498, 2011) according to the following conditions. (i) MQ0 <4 and MQ0 / (1.0 * DP)> 0.1, (ii) DP <5, (iii) QUAL <50, (iv) QD <1.5 (MQ0 is the mapping quality, DP is the depth , QUAL is the default call quality, and QD is the variation reliability depending on the depth). In addition, sequence variants with no more than 10 mutant read depths, known SNPs (dbSNP138) or non-exonic variants were further removed and the mutations were processed using ANNOVAR software. Identified mutations were verified by Sanger sequencing.

Example  1: RNA- Seq  Through profiling HCC's  Identification of mutations acquired by recurrence

As a result of HCC RNA-Seq profiling on primary and recurrent tumor specimens of 2 HCC patients, patient 1 showed 98 mutations in primary tumor (P1), 85 mutations in recurrent tumor (R1), 41 (31.65%), respectively. On the other hand, Patient 2 had 76 mutations in the primary tumor (P2) and 78 mutations in the recurrent tumor (R2), with 14 new mutations in the recurrence-specific mutation (71.11%) 1A). This suggests a mutation spectrum similar to previous studies (Guichard C et al., Nat Genet. 44: 694-698, 2012; Totoki Y et al., Nat Genet. 43: 464-469, 2011) There was no significant difference in mutation spectrum between recurrent tumors (Fig. 1B). Overall, mutations in C> T / G> A (38.79%, n = 64) and T> C / A> G (22.42%, n = 37) were frequent in tumors. The ratio of synonymous SNV ranged from 2.1 to 2.71 (Fig. 1B, right).

Of the mutations (R1; n = 41, R2; n = 14) obtained by recurrence of HCC, nine recurring specific mutations were found in recurrent tumors but not in primary and non-tumor surrounding tissues. These recurrence-specific mutations were verified by Sanger sequencing and found to be present in the genes for TP53 (P278R), CCNL2 (R499R), ARID1A (G2012D), GOLGB1 (E2721V), EXOC3 (V202I), SF3B3 (H804Y) and C18orf32 Seven recurrence specific mutations were identified (Table 1), but verification of VHL (E148fs) and BAAT (R297H) failed. These mutations included cancer-associated genes such as TP53, CCNL2 and ARID1A, which are known to be functionally related to HCC recurrence.

Example  2: Recurrence HCC's transcriptomic Reprogramming

To investigate the transcriptomic changes associated with the recurrence of hepatocellular carcinoma, we compared the gene expression levels of primary and recurrent hepatocellular carcinoma and found 130 differentially expressed genes with more than 2-fold differences, (RER) contains 60 upregulated genes (RER_UP) and 70 downregulated genes (RER_DOWN) (Figure 2A).

The gene ontology (GO) analysis of the gene set was performed using the g: Profiler R package (Reimand J et al., Nucleic Acids Res. 44: W83-89, 2016) and the statistical significance was determined with a cutoff of P <0.01 . (ES1, nanog, sox2, oct4, NOS, etc.) to evaluate aggressive phenotypes of relapsed HCC, and mSigDB (Gerlinger M et al., N Engl J Med. 366: 883-892, myc1, myc2), EMT, and cell cycle related genes. Functional enhancement scores for gene features were determined by applying the Kolmogorov-Smirnov (KS) test, and for each sample, the directional P-values for the estimates D + and D- were calculated by KS-test, The score was calculated by -log10 (P value) (Woo HG et al., Cancer research. 70: 3034-3041, 2010). Also, GSEA software (Subramanian A et al., Proc Natl Acad Sci USA . 102: 15545-15550, 2005) was used to analyze gene cluster enrichment among sample groups and all statistical calculations were performed using R software (http://www.r-project.org).

As a result of the gene ontology (GO) analysis, the RER_UP gene was enriched with the gene related to movement (concentration score, ES = 4.12) and the inflammatory reaction (ES = 3.10) and the RER_DOWN gene was enriched with metabolic process related gene (ES = 7.12) (Fig. 2B). This reflects the aggressive phenotype of recurrent HCC and suggests a regulatory role for transcriptomic reprogramming in tumor recurrence.

Assessment of the aggressive behavior of recurrent HCCs by expression of cancer gene signals in the RNA-seq profile revealed that recurrent HCCs showed a significant concentration of carcinogenic signatures (eg, EGFR and MEK) as compared to primary HCC (Figure 2C) . In the recurrent HCC, downregulation of tumor suppressor gene signals such as TP53_DN.V1_UP, RPS14_DN.V1_UP, and MEL18_DN.V1_UP was observed, and recurrent HCC was observed in the cell cycle and stem cell (ES1, nanog, sox2, oct4, NOS, myc1 and myc2 ) And a set of genes known to be associated with cancer aggressiveness, including EMT-associated genes, have been enriched.

We also evaluated the prognostic relevance of the RER gene in independent HCC data from TCGA (http://cancergenome.nih.gov). HCC patients (n = 371) were divided into two groups based on the calculated concentration score (ES) of the RER gene. As a result, the prognosis of the overall survival rate (risk ratio HR = 2.07, P = 9.01x10-5) was not good and the RER expression was low (RER_high, ES> 0, n = 100) , ES <0, n = 271) showed a tumor recurrence-free survival rate (HR = 1.87, P = 2.16x10-4) (Fig. 2D). In addition, the RER gene signal was compared with previously reported recurrence related genes of hepatocellular carcinoma (Woo HG et al., Clin Cancer Res . 14: 2056-2064, 2008; Kim JH et al., PLoS medicine. , 2014), no significant overlap between these genes was found. However, 15 recurring genes of the previous study (Woo HG et al., Clin Cancer Res . 14: 2056-2064, 2008) were overlapped with the RER gene, and the recurrence of hepatocellular carcinoma such as CD24, TGFB1, CXCL6, CXCL8, (Fig. 2E). These results indicate that recurrent hepatocellular carcinoma expresses a more aggressive phenotype at the transcriptional level, thereby promoting genomic reprogramming upon recurrence of the liver cancer.

Example  3: GOLGB1  And SF3B3  Acquisition of aggressive phenotype by mutation

After confirming the aggressive phenotype of recurrent hepatocellular carcinoma, we investigated whether the mutation obtained by recurrence in recurrent HCC was involved in aggressive phenotype acquisition. First, we examined whether the mutation obtained by recurrence affects gene expression level.

As a result, the expression of 45 recurrence-specific mutant genes was significantly higher in recurrent HCC than in primary HCC (ES = 0.46, P-value = 0.03, FIGS. 3A-3B). However, other mutations except recurrent specific mutations did not show any significant change between primary and recurrent hepatocellular carcinoma. Since mutations can activate or deactivate other mutations, these results suggest that recurrence-specific mutations may be more likely to act as activating mutations causing overexpression of the mutated gene.

3-1: GOLGB1  And Of SF3B3 siRNA  Mediated knockdown

Next, siRNA mediated knockdown experiments using liver cancer cell lines were performed to confirm the possibility of mutation of recurrent specific mutations. HCC cell lines (HepG2, Huh7, Hep3B, SNU423 and PLC) obtained from the American Type Culture Collection (ATCC, Manassas, Va.) Were cultured in DMEM medium (Gibco BRL, Grand Island, NY) supplemented with 10% FBS and 100 U penicillin / At 37 &lt; 0 &gt; C, 5% CO2. Non-targeting control siRNA and siRNA for human GOLGB1 and SF3B3 were purchased from Dharmacon Inc. (Lafayette, CO) and cells were transfected with each siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, Calif.) According to the manufacturer's instructions.

One of seven proven recurring mutations (Figure 1B) is expected to have a deleterious structural change with a Polyphen2 score of greater than 0.9 and focused on GOLGB1 (H804Y) and SF3B3 (E2721V) mutations for which the functional role of the gene in HCC progression is not known Table 1). GOLGB1 mutations were frequently observed in lung cancer (9%) and SF3B3 mutations were frequently observed in bladder cancer (5.4%), but mutations in HCC were relatively low (GOLGB1, 2.7%, SF3B3, 1.9% .

SiRNA mediated knockdown of GOLGB1 and SF3B3 at transcriptional and protein levels were confirmed by quantitative RT-PCR and Western blotting analysis, respectively. For quantitative RT-PCR, total RNA was extracted from the cells using RNAiso plus (Takara, Tokyo, Japan), and cDNA was extracted with 2 μg of total RNA using a PrimeScript RT kit (Perfect Real Time; Takara, Tokyo, Japan) . Quantitation of gene expression levels was performed by the CF96 TM optical module using IQ SYBR Super Mix (Bio-Rad, Richmond, CA) and specific primers (Supplement Table S3). Western blotting analysis was performed by loading 20 μg of total protein onto 8% SDS-PAGE and transferring to nitrocellulose membrane (Bio-Rad), and the membrane was incubated in TBST containing 0.2% I-Block ™ (Applied Biosysms) Blocked and incubated with primary antibody diluted in TBST containing 0.2% I-Block ™ at room temperature or 4 ° C for 1 hour. primary antibodies of anti-GOLGB1 (R & D Systems, Minneapolis, MN) and anti-SF3B3 (Santa Cruz Biotechnology, Dallas, Tex.) were used. Immunoblots were visualized by EzWestLumi (Atto, Tokyo, Japan).

3-2: GOLGB1  And SF3B3  Proliferation of knockdown cells

GOLGB1 and SF3B3 constructs were cloned using an in-fusion cloning method (Clontech, Mountain View, Calif.). In summary, wild-type and mutant clones for GOLGB1 or SF3B3 were constructed by specific primers (Supplement Table S3) and CloneAmp HiFi PCR premix (TAKARA, Tokyo, Japan). The GOLGB1 is large in size, with two heads (1 ~ 5029 bp) and a tail (5030 ~ 9810 bp). The cDNA fragment was inserted into pcDNA3 vector using EcoRI and XbaI, and the PCR product of the mutant was confirmed by Sanger DNA sequencing method.

To measure cell proliferation, cells were inoculated into 96-well plates at 2 × 10 ³ cells per well and cultured overnight at 37 ° C. in a 5% CO ₂ incubator to transfect each construct. The 5 mg / mL MTT solution (Amresco, Cleveland, OH) was then added to each well and incubated for 2 hours, after which the blue crystal precipitate of each well was dissolved in 150 μl of DMSO per well and analyzed using a microplate reader The visible absorbance at 550 nm was quantified and cell proliferation was measured.

As a result, knockdown against GOLGB1 or SF3B3 inhibited cell proliferative activity in various hepatocarcinoma cell lines of HepG2, Huh7, Hep3B, SNU423 and PLC (Fig. 3C). In addition, the knockdown of these genes markedly reduced the migration activity of HepG2 cells, indicating increased transduction potential (FIG. 3D).

3-3: GOLGB1  And SF3B3  Cell proliferation, migration and invasion of mutant clones

To evaluate the carcinogenic function of mutants of GOLGB1 and SF3B3, a mutant clone prepared by performing site-specific mutagenesis was prepared. Expression clones were transfected into liver cancer cells (HepG2 and Huh7), and overexpression of each form was confirmed at the mRNA and protein levels.

Cells were then transfected with mock control and wild-type or mutant GOLGB1 or SF3B3 constructs for 48 hours, respectively, and then 500 cells / well seeded in 6-well plates for 14 days . The colonies were washed twice with PBS, fixed with 3.7% paraformaldehyde, and stained with a 1% crystal violet solution.

Cell migration and invasion assays were performed with uncoupled uncoupled (for cell migration) or matrigel coated recoated (for cell invasion) transwell filters coated with Matrigel (100 μl, 1:10 dilution, BD Biosciences, Bedford, MA) 8-μm-pore transwell (6.5 mm, Costar, Corning, NY) for 1 hour. After washing the filter with PBS, cells were plated and incubated at 37 ° C, 5% CO ₂ . After infiltration of the upper chamber or non-migrating cells were removed with a cotton swab, infiltrated or migrated cells were washed with PBS through a filter, fixed with 3.7% formalin and stained with 0.1% crystal violet for 1 hour. The number of infiltrated or migrated cells was counted by light microscopy.

As a result, expression of wild type GOLGB1 or SF3B3 significantly increased cell proliferation and colony forming ability, respectively. In addition, the mutant type showed an effect of increasing the wild type, thereby promoting more aggressive cancer against cell proliferation, colonization, migration and invasion (Fig. 4). Indicating that the mutation acts as an activating mutation. In addition, the level of expression of the mutations was not significantly different between primary and recurrent hepatocellular carcinoma, indicating that the mutation effect is not due to an increase in gene expression levels.

Example  4: GOLGB1  And SF3B3  Structure of the mutant

The protein structure of the mutants was predicted to exhibit deleterious functional effects by PolyPhen2 software (Table 1).

The GOLGB1 (E2721V) mutation was located in the hinge region between the alpha helical modules and was predicted to change the rotation angle of the hinge region (Fig. 5A). The SF3B3 (H804Y) mutation is located at the border between the beta sheet and the loop and alters the wild type loop structure to the beta sheet (i.e., the 805-807 and 813-815 sites) and the alpha helix (i.e., the 820-823 site) (Fig. 5B).

These results may lead to structural changes in the protein that promote mutagenic aggressive behavior of cancer cells.

Example  5: GOLGB1  And Of SF3B3  Potential Downstream  Target CXCL8  And SOX4

5-1: GOLGB1 and SF3B3  Potential downstream target gene analysis due to mutation

In the above example, it was confirmed that mutations of GOLGB1 and SF3B3 can cause aggressive progression in HCC (FIGS. 3 and 4). In this example, a potential downstream target gene capable of causing a phenotype change due to mutation was analyzed.

Gene expression profiling was performed using the knockdown cells for GOLGB1 or SF3B3 to determine whether GOLGB1 knockdown (siGOLGB1_UP, n = 11 and siGOLGB1_DOWN, n = 126) and SF3B3 knockdown (siSF3B3_UP, n = 21, siSF3B3_DOWN, n = 53) And the differentially expressed gene (DEG) was confirmed (Fig. 6A). Since knockdown downregulated genes can be regarded as downstream genes, expression of siGOLB1_DOWN and siSF3B3_DOWN genes was analyzed.

As a result, it was found that siGOLB1_DOWN and siSF3B3_DOWN gene signatures were significantly rich in recurrent HCCs than primary HCC (FIG. 6B). The individual concentration score of this gene signature also showed a significant uptake in recurrent tumors (R1 and R2) compared to primary tumors (P1 and P2) (Fig. 6C). This indicates that the gene regulated by GOLGB1 or SF3B3 can play a crucial role in obtaining an aggressive phenotype in recurrent HCC by acting as a potential downstream apoptotic gene.

5-2: siGOLGB1 _DOWN and siSF3B3 _DOWN gene signature  analysis

Since siGOLGB1_DOWN and siSF3B3_DOWN signatures are considered potential targets regulated by GOLGB1 or SF3B3, network analysis was performed to predict the major regulator of gene expression changes. The network of siGOLGB1_DOWN, siSF3B3_DOWN and RER_UP gene signatures was constructed (Fig. 6D), and genetic, physical and pathway interactions between signatures were confirmed by GeneMANIA software (Montojo J et al., Bioinformatics. 26: 2927-2928, 2010) .

As a result, gene signals of siGOLGB1_DOWN and siSF3B3_DOWN share many genes (25.17%, 36 out of 143), indicating that simultaneous mutations may be synergistic for common downstream signaling pathways it means. Of these common target genes, CXCL8 and SOX4 were found to generally increase expression levels in recurrent HCC (RER_UP, Fig. 6D). In addition, down-regulation of CXCL8 and SOX4 expression was further confirmed by qRT-PCR in knockdown cells against GOLGB1 or SF3B3 (Fig. 6E).

Thus, it was found that CXCL8 and SOX4 are downstream targets of GOLGB1 and SF3B3. In addition, we could observe a positive correlation between the expression level of GOLGB1 or SF3B3 and the expression level of CXCL8 and SOX4 in liver cancer data of TCGA, suggesting a potential linkage. That is, mutations in GOLGB1 and SF3B3 play a role in obtaining an aggressive phenotype in recurrent HCC, potentially resulting from the expression of CXCL8 and SOX4.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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ctgaaagaga agaagaatat 6480 gcaagagaaa ctggatgctt tgcgcagaga aaaagtccac ttggaagaga caattggaga 6540 gattcaggtt actttgaaca agaaagacaa ggaagttcag caacttcagg aaaacttgga 6600 cagtactgtg acccagcttg cagcctttac taagagcatg tcttccctcc aggatgatcg 6660 tgacagggtg atagatgaag ctaagaaatg ggagaggaag tttagtgatg cgattcaaag 6720 caaagaagaa gaaattagac tcaaagaaga taattgcagt gttctaaagg atcaacttag 6780 acagatgtcc atccatatgg aagaattaaa gattaacatt tccaggcttg aacatgacaa 6840 gcagatttgg gagtccaagg cccagacaga ggtccagctt cagcagaagg tctgtgatac 6900 tctacagggg gaaaacaaag aacttttgtc ccagctagaa gagacacgcc acctatacca 6960 cagttctcag aatgaattag ctaagttgga atcagaactt aagagtctca aagaccagtt 7020 gactgattta agtaactctt tagaaaaatg taaggaacaa aaaggaaact tggaagggat 7080 cataaggcag caagaggctg atattcaaaa ttctaagttc agttatgaac aactggagac 7140 tgatcttcag gcctccagag aactgaccag taggctgcat gaagaaataa atatgaaaga 7200 gcaaaagatt ataagcctgc tttctggcaa ggaagaggca atccaagtag ctattgctga 7260 actgcgtcag caacatgata aagaaattaa agagctggaa aacctgctgt cccaggagga 7320 agaggagaat attgttttag aagaggagaa caaaaaggct gttgataaaa ccaatcagct 7380 tatggaaaca ctgaaaacca tcaaaaagga aaacattcag caaaaggcac agttggattc 7440 ctttgttaaa tccatgtctt ctctccaaaa tgatcgagac cgcatagtgg gtgactatca 7500 acagctggaa gagcgacatc tctctataat cttggaaaaa gaccaactca tccaagaggc 7560 tgctgcagag aataataagc ttaaagaaga aatacgaggc ttgagaagtc atatggatga 7620 tctcaattct gagaatgcca agctagatgc agaactgatc caatatagag aagacctgaa 7680 ccaagtgata acaataaagg acagccaaca aaagcagctt cttgaagttc aacttcagca 7740 aaataaggag ctggaaaata aatatgctaa attagaagaa aagctgaagg aatctgagga 7800 agcaaatgag gatctgcgga ggtcctttaa tgccctacaa gaagagaaac aagatttatc 7860 taaagagatt gagagtttga aagtatctat atcccagcta acaagacaag taacagcctt 7920 gcaagaagaa ggtactttag gactctatca tgcccagtta aaagtaaaag aagaagaggt 7980 acacaggtta agtgctttgt tttcctcctc tcaaaagaga attgcagaac tggaagaaga 8040 attggtttgt gttcaaaagg aagctgccaa gaaggtaggt gaaattgaag ataaactgaa 8100 gaaagaatta aagcatcttc atcatgatgc agggataatg agaaatgaaa ctgaaacagc 8160 agaagagaga gtggcagagc tagcaagaga tttggtggag atggaacaga aattactcat 8220 ggtcaccaaa gaaaataaag gtctcacagc acaaattcag tcttttggaa ggtctatgag 8280 ttccttgcaa aatagtagag atcatgccaa tgaggaactt gatgaactga aaaggaaata 8340 tgatgccagt ctgaaggaat tggcacagtt gaaagaacag ggactcttaa acagagagag 8400 agatgctctt ctttctgaaa ccgccttttc aatgaactcc actgaggaga atagcttgtc 8460 tcaccttgag aaacttaacc aacagctcct atccaaagat gagcaattgc ttcacttgtc 8520 ctcacaacta gaagattctt ataaccaagt gcagtccttt tccaaggcta tggccagtct 8580 gcagaatgag agagatcacc tgtggaatga gctggagaaa tttcgaaagt cagaggaagg 8640 gaagcagagg tctgcagctc agccttccac cagcccagct gaagtacaga gtttaaaaaa 8700 agctatgtct tcactccaaa atgacagaga cagactactg aaggaattga agaatctgca 8760 gcagcaatac ttacagatta atcaagagat cactgagtta catccactga aggctcaact 8820 tcaggagtat caagataaga caaaagcatt tcagattatg caagaagagc tcaggcagga 8880 aaacctctcc tggcagcatg agctgcatca gctcaggatg gagaagagtt cctgggaaat 8940 acatgagagg agaatgaagg aacagtacct tatggctatc tcagataaag atcagcagct 9000 cagtcatctg cagaatctta taagggaatt gaggtcttct tcctcccaga ctcagcctct 9060 caaagtgcaa taccaaagac aggcatcccc agagacatca gcttccccag atgggtcaca 9120 aaatctggtt tatgagacag aacttctcag gacccagctc aatgacagct taaaggaaat 9180 tcaccaaaag gagttaagaa ttcagcaact gaacagcaac ttctctcagc tactggaaga 9240 gaaaaacacc ctttccattc agctctgcga taccagtcag agtcttcgtg agaaccagca 9300 gcactatggt gaccttttaa atcactgtgc agtcttggag aagcaggttc aagagctgca 9360 ggcggggcca ctaaatatag atgttgctcc aggagctccc caggaaaaga atggagttca 9420 cagaaagagt gaccctgagg aactaaggga accgcagcaa agcttttctg aagctcagca 9480 gcagctatgc aacaccagac aggaagtgaa tgaattaagg aagctgctgg aagaagaacg 9540 agaccaaaga gtggctgctg agaatgctct ctctgtggcc gaggagcaga tcagacggtt 9600 agagcacagt gaatgggact cttcccggac tcctatcatt ggctcctgtg gcactcagga 9660 gcaggcactg ttaatagatc ttacaagcaa cagttgtcga aggacccgga gtggcgttgg 9720 atggaagcga gtcctgcgtt cactctgtca ttcacggacc cgagtgccac ttctagcagc 9780 catctacttt ctaatgattc atgtcctgct cattctgtgt tttacgggcc atctatagac 9840 ttagttgtta ctctttggac cactcccctc aaaacttgga attctctcac ctctaacatc 9900 agaacatcaa ttccagtgga acagtcttcc catttacagg tcttctctcc aactcttcac 9960 ggaaagtgcc tgcaaaaaca gaggtggata cgaggacagg ttggagctgc agggactggc 10020 gagtctgctt tcttctactg ccctgagcct gaacgcttct gcttaatctg agaatcacat 10080 ttggtttgtt gagcctaata tttgttgaga ttttgcagga ccctgatctt ttgtggtcct 10140 gtaaaagata ctgaggaatg tctttcagcc aagccaagag gatggtttca ataaacctaa 10200 taatctgaag ttcagtatca ttttgattga taactttctt tgccttgttt gcctgtattt 10260 tctcctgttt cagggggaag gtggctctgc cataaacaga gttcagggaa atgaacatgt 10320 cacctcatag gacatctgat ttaggtctct tgcagaatag ggtggtgaaa gctgaaggaa 10380 ctccctaggg gttttagctg ttagatcaca tgggaggaca gcatcttctt ccctgccaat 10440 ttcagatata attggaggga gaatttcagg tcttagacta taaggataga atccctttgc 10500 ttttgccctt gatcacatgt atactaaggt attaagtggc ccaagtcttt cctttcacca 10560 aagggcaggg agaagtgtct atggacagca gggttccaat tcttgtcatt ccaggaatat 10620 ctgaatattc ctggaatgtg gaccctgaga cagtgctcag gggccactgg gagccaatat 10680 taggcattga ctctcagcca ggagtctgac tggtctaaca ccacactgca actcctgacc 10740 tcttgaagta ctaagctact ttgctgttag tgacagttat tacagtttct cagccccatt 10800 gtctctgccc tctgtggcaa tggaaggaga atatagagaa gacaaaatta aatatagata 10860 gacctgagaa ggacagccag gatagaattc catttgtgcc tattccctgc ctcccctccc 10920 ctcccccatc ctaccagttg gttattttct cattgcatac gtgatgtgtt cactgcctag 10980 cctctcccta aagaagagag aagacaagtg ggctgactga tctgctctaa atctactgtg 11040 gtgattaaat cttggttaca acatcctggg aagtttcctg aacaactgta aaataataa 11100 attattttca gaatgaagaa aaaaaaaaaa aaa 11133 <210> 2 <211> 9736 <212> DNA <213> Homo sapiens <400> 2 gcctttttcc gccgcgcgcc accagaatgt ccctgtcttg aggtctaatg gcggacgcca 60 gtatgttgga gttggtggtg gcttaagttt tgaagggagg tagcatccgt tggatatcca 120 caccatcctt ctcgctgcag gctttcttgg actccgtact gttggtgtaa ccaaggcctg 180 gaggtctggg tggctcaggt ttcctgcagc catgtttctg tacaacttaa ccttgcagag 240 agccactggc atcagctttg ccattcatgg aaacttttct ggaaccaaac aacaagaaat 300 tgttgtttcc cgtgggaaga tcttggagct gcttcgccca gaccccaaca ctggcaaagt 360 acatacccta ctcactgtgg aagtattcgg tgttatccgg tcactcatgg cctttaggct 420 gacaggtggc accaaagact acattgtagt tggcagtgac tctggtcgaa ttgttatttt 480 ggaataccag ccatctaaga atatgtttga gaagattcac caagaaacct ttggcaagag 540 tggatgccgt cgcatcgttc ctggccagtt cttagctgtg gatcccaaag ggcgagccgt 600 tatgattagt gccattgaga aacagaaatt ggtgtatatt ttgaacagag atgctgcagc 660 ccgacttacc atttcatctc ccctggaagc ccacaaagca aacactttag tgtatcatgt 720 agttggagta gatgtcggat ttgaaaatcc aatgtttgct tgtctggaaa tggattatga 780 ggaagcagac aatgatccaa caggggaagc agcagctaat acccagcaga cacttacttt 840 ctatgagcta gaccttggtt taaatcatgt ggtccgaaaa tacagtgaac ctttggagga 900 acacggcaac ttccttatta cagttccagg agggtcagat ggtccaagtg gagtactgat 960 ctgctctgaa aactatatta cttacaagaa ctttggtgac cagccagata tccgctgtcc 1020 aattcccagg aggcggaatg acctggatga ccctgaaaga ggaatgattt ttgtctgctc 1080 tgcaacccat aaaaccaaat cgatgttctt ctttttggct caaactgagc agggagatat 1140 ctttaagatc actttggaga cagatgaaga tatggttact gagatccggc tcaaatattt 1200 tgatactgta cccgttgctg ctgccatgtg tgtgcttaaa acagggttcc tttttgtagc 1260 atcagaattt ggaaaccatt acttatatca aattgcacat cttggagatg atgatgaaga 1320 acctgagttt tcatcagcca tgcctctgga agaaggagac acattctttt ttcagccaag 1380 accacttaaa aaccttgtgc tggttgatga gttggacagc ctctctccca ttctgttttg 1440 ccagatagct gatctggcca atgaagatac tccacagttg tatgtggcct gtggtagggg 1500 accccgatca tctctgagag tcctaagaca tggacttgag gtgtcagaaa tggctgtttc 1560 tgagctacct ggtaacccca acgctgtctg gacagtgcgt cgacacattg aagatgagtt 1620 tgatgcctac atcattgtgt ctttcgtgaa tgccacccta gtgttgtcca ttggagaaac 1680 tgtagaagaa gtgactgact ctgggttcct ggggaccacc ccgaccttgt cctgctcctt 1740 attaggagat gatgccttgg tgcaggtcta tccagatggc attcggcaca tacgagcaga 1800 caagagagtc aatgagtgga agacccctgg aaagaaaaca attgtgaagt gtgcagtgaa 1860 ccagcgacaa gtggtgattg ccctgacagg aggagagctg gtctatttcg agatggatcc 1920 ttcaggacag ctgaatgagt acacagaacg gaaggagatg tcagcagatg tggtgtgcat 1980 ggtctggcc aatgtacccc ctggagagca gcggtctcgc ttcctggctg tggggcttgt 2040 ggacaacact gtcagaatca tctccctgga tccctcagac tgtttgcaac ctctaagcat 2100 gcaggctctc ccagcccagc ctgagtcctt gtgtatcgtg gaaatgggtg ggactgagaa 2160 gcaggatgag ctgggtgaga ggggctcgat tggcttccta tacctgaata ttgggctaca 2220 gaacggtgtg ctgctgagga ctgtcttgga ccctgtcact ggggatttgt ctgatactcg 2280 cactcggtac ctggggtccc gtcctgtgaa gctcttccga gtccgaatgc aaggccagga 2340 ggcagtattg gccatgtcaa gccgctcatg gttgagctat tcttaccaat ctcgcttcca 2400 tctcacccca ctgtcttacg agacactgga atttgcatcg ggttttgcct cggaacagtg 2460 tcccgagggc attgtggcca tctccaccaa caccctacgg attttggcat tagagaagct 2520 cggtgctgtc ttcaatcaag tagccttccc actgcagtac acacccagga aatttgtcat 2580 ccaccctgag agtaacaacc ttattatcat tgaaacggac cacaatgcct acactgaggc 2640 caggaaagct cagagaaagc agcagatggc agaggaaatg gtggaagcag caggggagga 2700 tgagcgggag ctggccgcag agatggcagc agcattcctc aatgaaaacc tccctgaatc 2760 catctttgga gctcccaagg ctggcaatgg gcagtgggcc tctgtgatcc gagtgatgaa 2820 tcccattcaa gggaacacac tggaccttgt ccagctggaa cagaatgagg cagcttttag 2880 tgtggctgtg tgcaggtttt ccaacactgg tgaagactgg tatgtgctgg tgggtgtggc 2940 caaggacctg atactaaacc cccgatctgt ggcagggggc ttcgtctata cttacaagct 3000 tgtgaacaat ggggaaaaac tggagttttt gcacaagact cctgtggaag aggtccctgc 3060 tgctattgcc ccattccagg ggagggtgtt gattggtgtg gggaagctgt tgcgtgtcta 3120 tgacctggga aagaagaagt tactccgaaa atgtgagaat aagcatattg ccaattatat 3180 ctctgggatc cagactatcg gacatagggt aattgtatct gatgtccaag aaagtttcat 3240 ctgggttcgc tacaagcgta atgaaaacca gcttatcatc tttgctgatg atacctaccc 3300 ccgatgggtc actacagcca gcctcctgga ctatgacact gtggctgggg cagacaagtt 3360 tggcaacata tgtgtggtga ggctcccacc taacaccaat gatgaagtag atgaggatcc 3420 tacaggaaac aaagccctgt gggaccgtgg cttgctcaat ggggcctccc agaaggcaga 3480 ggtgatcatg aactaccatg tcggggagac ggtgctgtcc ttgcagaaga ccacgctgat 3540 ccctggaggc tcagaatcac ttgtctatac caccttgtct ggaggaattg gcatccttgt 3600 gccattcacg tcccatgagg accatgactt cttccagcat gtggaaatgc acctgcggtc 3660 tgaacatccc cctctctgtg ggcgggacca cctcagcttt cgctcctact acttccctgt 3720 gaagaatgtg attgatggag acctctgtga gcagttcaat tccatggaac ccaacaaaca 3780 aaagaacgtc tctgaagaac tggaccgaac cccacccgaa gtgtccaaga aactcgagga 3840 tatccggacc cgctacgcct tctgagccct cctttcccgg tggggcttgc cagagactgt 3900 gtgttttgtt tcccccacca ccatcactgc cacctggctt ctgccatgtg gcaggagggt 3960 gactggataa ttaagactgc attatgaaag tcaacagctc tttcccctca gctcttctcc 4020 tggaatgact ggcttcccct caaattggca ctgagatttg ctacacttct ccccacctgg 4080 tacatgatac atgaccccag gttccagtgt agaacctgag tcccccattc cccaaagcca 4140 tccctgcatt gatatgtctt gactctcctg tctacttttg cacacaccct taatttttaa 4200 ttggttttct tgtaaataca gttttgtaca atgttatctc tgtgggagga aggaggcagg 4260 ctgtggtggg actgggtagg gtatagtatc actcctgagt tccactgctc tagaatctaa 4320 ccagaaatag aaacctagtt tttaaggtga ctggcatcca tgtgtcttgt tctggagatg 4380 aggatgtagg tgggaggttt gaacccaagt tagagcagga agaactgagt agactccttc 4440 cttccagata ccgacttgga cttgcggcac tctgtggctc cccaccccca ggtctgtggt 4500 ggtttctctg ttttttcctg gttctttttg ctgtgctgat gaaacatgac ctcaataacc 4560 atgtgtatac ccacccctct tcccactggg tattgaggaa gggtggctga ttcttcctcc 4620 tcttctactc tgaggatgtt agtatgggga ttttagcatg aattctagct ggggagtctt 4680 aacagatgcc ccttttactg atacagcacc taaagcgatc tttggctcca taggaccata 4740 ggaagggtca gtacagaaga acctagatac tgccctgccc ctgagaactg tgtatatgtg 4800 gggcctgtct gcagcaccca tctcaggtgg gttccagagg gcctttaggg tataatgaga 4860 gcctgttagg tggaagaggc ccagttccag aaatgttcca gcccacccct gagaattcct 4920 cctgtttagt tgtgtgggaa gccctcgtct tccaggctgt ccttgcgcct tgaacctgga 4980 gaagtgagct cactgttctc aatacttcac aaatgtaaaa ctttctttcg tctgcatgtg 5040 ctcagccatc taaattgagc aaatgatctg gtgagcactg ggttagaatc aggaatggtg 5100 gaatacaatc tgaacctctc agagcccaga acagagggtt cctgacactg tgacactgtc 5160 tcctggaact aagtatctct tgaatcatga cttggtttta gatcagtcaa gagagaccca 5220 ggttttgcca ggaatcgaat ccctaaataa catgtttttt tctcacttag ctcatgaatt 5280 tgcatagtag acagtagttc tgaattagat tttgaaaacc taatttcagg gctcattttt 5340 tcctgtggcc ctaaatccat tctatcaaat tgtgtgatac tgacatgcag tcatctgagg 5400 aactcagcgt agatacttga gcagctcctc gcctcttttc taactcaagt ttgactaaaa 5460 tacatacact ccgtacagaa ggtagggggt tatgtaagaa aggaaaacct aatctatgga 5520 atcaggagtt gtcaccaccg agcttcctct ggaagtctgc ccatcagctt gcttgttctc 5580 tgttaagagg aagggctagg acaaggattt gggcttgaat atgtggaaag gaattttcat 5640 agttgttgct gcaggaccta caaaagttta aaattagatt ggatgtgact caatgacaag 5700 tcccatctgt gtaattgtta aggggacctg attgactcct gtggtttgat tgagcaacca 5760 ggtaaataga gacctctctc cagctttggc aaaacccatc agaggctgct gcagaactca 5820 gacagaggga tctgcccttg ggtttgcttc catcctgttc cattgctaag cccttgtgac 5880 ttggatccta ggactgaaaa gtttttagct gcctcagctt tcccctgacc ttactggcag 5940 aggttctgca gatgtttcct ttggaagatc tcttgccaag aatagcattc ctttggagga 6000 ggggggtttt agttggaatg ttgcttttct tggttagtgt aaatgtattg ctagtgagac 6060 agctgccggc gctggaaaag gctcgtctca cagggagagt gctggtcccc agaatgtgtg 6120 ctgttcccac gctgctgcct ttcttgagct tgttagagga aagccagaaa ggcattcaga 6180 tgggatcagt ctggctttca aatttttttt aattcctaag ttctgtttta ttttttaatt 6240 ttttaaaaaa aattttatta gagacagtct ctctctcttg cctagctggg agtgcagtgg 6300 agtgatcata gctcactgag gcttgaactc ctgggctcga gcaatccacc tcagcctcca 6360 gagtagggga gactacagat gtgtgccacc atactcagct aatttttaaa cttttgtaga 6420 gacagggtct ccctgtgttg cccaggctgg cctcgaactc ctgacctcaa aaaatcttcc 6480 tgccttggcc tcccaaagca ctgggattat aggtgtgagc cattgcgcct ggtcataaat 6540 tcttgttttt ttgtttgttt gtttattaga gatggaatct ctctctcttg accaggctag 6600 agggctgtgg tgcgatctca gcccactgca acctctatct cctgagctca agcgatcctc 6660 cttagcttcc caaatagctg gaactacagg catgtgccat cacgtccagc taatttttgt 6720 atttttagta gagaaggttt taccatgttg gacagggtgg tctcgaactc ctggcctaaa 6780 gtggtccacc tagctcagcc taccaaagtg ctgtgattac aggcgtgagc caccatgccc 6840 agcctctaaa ttctgttttc tattcaaagt aaaaatgaca tgtgtttgag tcatccactt 6900 gctcatttgc atatggaata ttgtatgtga atattttatt acacccttaa aattaatttt 6960 caagggctat atgtagagct cagaagaaac atctctgctg ctgaaatcaa acctggtcca 7020 aaaaacgtca caacggagga aaggaataga tggctctcga ggacaagata gggactctta 7080 aaacacctgc ttggccatgg ttgattgaca tttgagtgat ctggtagccc aacacaccct 7140 gtgaagttca ggtgaactga aatgggcagg tgtaggctac acaccagtct gaaccagcac 7200 aaccaagagt tgtttctctt ttttttcttt ttctttttct tttttttctt tttttttttt 7260 ttttttttga gatgaagtct tactctgttg cccaggctag agtacagagg cacaatctcg 7320 gctcaccaca gcctccgcct cctgggttca agcaattctc ctgcctcagc ctcccaagta 7380 gctgggacta caggcatgca ccaccatgcc tggctaattt ttgtattttt agtagagatg 7440 gggtttcacc atgttggcca ggctggtttc gaactcgtga cctcaggtga tccacccacc 7500 ttggtctccc aaagcgctgg gattacaggc gtgagccact gcacttggcc aggagctgtt 7560 taagccatca ggagacatgc tgacccacta gaagaggctg ctacgctcat ttcttccaca 7620 aacgtttact gggcacctac catgaaccac cacctcccag ctctgttctg cacccagagg 7680 ttggaggaga gtttataacc aggcagtctc tgtcgctgtg aggttggtgg tgaggtgagt 7740 ctgccagagg gcctgccacc agtttcagaa gagcctgcag cctgcatctg atttcctcag 7800 ccacaacctt gggacttcag gaaaaatcag aaggtcccaa gtctgaaaaa tgaagaggtg 7860 gggagtgggc agcaggggtt ttaggtcagg aaatggtagg aaacctgtag ttgtggctca 7920 taatcctagt gggatattag aaccctcttc atttgtcaga acagattcct ggggtctgct 7980 cccaattatt cacacggtgg ttcatgcctg gtaatcccag cactttgggc ggccagaggc 8040 aggcagattg cttgagccca gaagtttaag accagcctag gaaacagacc ccatctctac 8100 aaaaaattca aaaagccggg cgtgaagccc tgtttttttt tttttaaaaa aagagtctgc 8160 tggtgatgtc tgggcatgtg tctgcttcac agtccagtgt tatgatcagt agctgtgatg 8220 gacaatgaac attgctgctt gttcaaaatg aagaaaaaaa atatcaaaga aaaccaggtt 8280 tggaagtttc agatttggaa aaggagctgg gccagcccat ctggaatagc tgaggggccc 8340 tgttagcccc accctacccc accccacccc cgtatgcagc atctctggcg gtgcccagag 8400 gcaagccccc ttcagtttcc cacctctcac tctgccttcc agagcctagt caagttttaa 8460 attcccaggt ccactcagca gaacttgctc ccttctctga ctgcagtctc ataatgaaag 8520 gagaggtgct ttcagttggg tcatttggta gcgtttaggc ttagaagtta ctaatttgga 8580 gccgtgagta tatgtgtata agctagtaac acgtgatggt caagggaagt actaccattt 8640 cctgtgggtt attagggcct ccagctgtta tgaggacaca ctggggaatc tcagctttag 8700 ggagtcgatg atgtaactgg agaaaggcaa tgctgccctc ataaagcaat cagaaacgag 8760 attccacctg ccaaatgcca agaggcagca aagtccatga agagagcact gtatacagtc 8820 agatgacctg ggctcactag cctctaagca tagtcttggt ttcctgcctg taaattggta 8880 gaataagact gatttactgg gcctggcatg gtggctcaca cctgtaatcc cagcactttg 8940 agaggctgag gcaggaggat cccttgagcc caggagtttg agaccagcct gggcaacatg 9000 acaaaacccc atctctccaa aaatacaaaa attagccagg catggtggtg cacacttgta 9060 gtcccagtaa ttagggggct gagacaggag gatcacttca gcctatgagt ttgaggctgc 9120 agtgagctgt gattgcgcca ctacactcca gcctggatga caggacgaaa cctgtctcaa 9180 aaacaccaaa aaacaaaaac cggtctcctg gggtcatggt agcacaaacg cacatgactg 9240 agtgctcagg ggttctgagg cttgtccgct gacctggggc tctggccctg ggagatctgg 9300 gggacctgct gtcctatatg tgatgctttg aaagaaaggg gcatcattcc aagccaagag 9360 gccccagaga gggcaccgtg cggtgttcag gcttctgtga ggccccagtg agatcctgtg 9420 gctgtgcccc catcacctcc acccactctg ccctcccact agctgcccaa cggatgaatc 9480 aacgccttgg cagagttttc cagcagggcc ttgcagagag tgtgtgtgac ctgtgtggcc 9540 actgccttgg ggacgggtga ggagttagcc tggaacattc cagcgtgggc attattgtcc 9600 tgttgcaagt tcagggcaaa accaggaatc cagttttgtc gatccaattg agaaaacatt 9660 tcatgaacaa ctacttgtgg catgcattgg cactcggaat aaagcgcact attgtcacta 9720 aaaaaaaaaa aaaaaa 9736

Claims (16)

Wherein the protein encoded by the GOLGB1 gene comprises a substance capable of measuring mRNA of GOLGB1 or SF3B3 gene or a protein expression level encoded by the gene, wherein the protein encoded by the GOLGB1 gene is a protein encoded by the amino acid at position 2721 of glutamic acid Wherein the protein encoded by the SF3B3 gene is SF3B3 (H804Y) in which the 804th amino acid is substituted with tyrosine at histidine.
2. The method according to claim 1, wherein the substance for measuring the expression level of the mRNA is a primer capable of amplifying the GOLGB1 or SF3B3 gene or a probe specifically binding to the GOLGB1 or SF3B3 gene, A composition for predicting the diagnosis or prognosis of recurrent liver cancer, wherein the composition is an antibody or an umbilical artery specific for a protein encoded by the GOLGB1 or SF3B3 gene.
The composition according to claim 1, wherein the GOLGB1 gene has the nucleotide sequence of SEQ ID NO: 1, and the SF3B3 gene has the nucleotide sequence of SEQ ID NO: 2.
delete delete An inhibitor of the expression of GOLGB1 or SF3B3 protein selected from the group consisting of an antisense nucleotide complementary to mRNA of GOLGB1 or SF3B3 gene, a short interfering RNA (short interfering RNA) and a short hairpin RNA (short hairpin RNA), or an inhibitor of GOLGB1 or SF3B3 Wherein the GOLGB1 protein comprises the amino acid at position 2721 and the amino acid at position 2721 is selected from the group consisting of an aptamer that binds complementarily to a protein and an antibody that inhibits the activity of GOLGB1 or SF3B3 protein. Wherein the SF3B3 protein is SF3B3 (H804Y) in which the 804th amino acid is substituted with tyrosine at histidine. The pharmaceutical composition according to claim 1, wherein the SF3B3 protein is GOLGB1 (E2721V) substituted with valine in glutamic acid.
7. The method according to claim 6, wherein the GOLGB1 protein has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, and the SF3B3 protein has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: A pharmaceutical composition for preventing or treating liver cancer.
delete delete delete delete A method for screening a preventive or therapeutic agent for recurrent liver cancer comprising the steps of:
(i) treating the test substance with an expression cell line of GOLGB1 or SF3B3 protein;
(ii) measuring the level of GOLGB1 or SF3B3 protein expression or activity in the cell line; And
(iii) a step of selecting a test substance whose expression or activity level of the GOLGB1 or SF3B3 protein is decreased as compared with the control group not treated with the test substance,
Wherein the GOLGB1 protein is GOLGB1 (E2721V) in which the 2721st amino acid is substituted with valine in glutamic acid, and the SF3B3 protein is SF3B3 (H804Y) in which the 804th amino acid is substituted with tyrosine at histidine. &Lt; / RTI &gt;
13. The method according to claim 12, wherein the GOLGB1 protein has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, and the SF3B3 protein has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: A method for screening a preventive or therapeutic agent for liver cancer.
delete The method according to claim 12, wherein the level of expression or activity of the protein of step (ii) is determined by immunofluorescence, enzyme immunoassay (ELISA), Western blot, flow cytometry (FACS), immunohistochemistry, a method for screening a prophylactic or therapeutic agent for recurrent liver cancer, comprising the steps of: (a) detecting the presence or absence of a cancer cell;
delete

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