WO2022019604A1 - Biomarker specific for liver cancer, and use thereof - Google Patents

Biomarker specific for liver cancer, and use thereof Download PDF

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Publication number
WO2022019604A1
WO2022019604A1 PCT/KR2021/009313 KR2021009313W WO2022019604A1 WO 2022019604 A1 WO2022019604 A1 WO 2022019604A1 KR 2021009313 W KR2021009313 W KR 2021009313W WO 2022019604 A1 WO2022019604 A1 WO 2022019604A1
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liver cancer
smarca4
irak1
gene
expression level
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PCT/KR2021/009313
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French (fr)
Korean (ko)
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남석우
김상연
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주식회사 네오나
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Priority to US18/006,075 priority Critical patent/US20230272478A1/en
Publication of WO2022019604A1 publication Critical patent/WO2022019604A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a liver cancer-specific biomarker and its use, and more particularly, using genes whose expression is specifically changed in hepatocellular carcinoma as biomarkers for hepatocellular carcinoma detection and diagnosis, and to treat liver cancer by targeting them It's about use.
  • Cancer is a representative disease that threatens human health and is the most representative cause of death as a single disease in industrialized countries. Although the cause of cancer is still unknown, it is considered that complex factors of genetic factors, which are internal factors, and carcinogenic chemicals acting as factors that cause cancer, which are external factors, continuous inflammation and damage, and cancer-causing virus infection are acting. . However, cancer is not so desperate that it can be concluded that it is an incurable disease, and it can be cured by early diagnosis and active treatment. Therefore, early detection, early diagnosis, and treatment are crucially important to increase the effectiveness of cancer treatment, and even in advanced cancer, if various and active methods are used, a cure or life can be prolonged and painful symptoms can be improved.
  • liver cancer is known as one of the most lethal cancers worldwide, and its mortality rate is the third most aggressive cancer (Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004; 50:556-573). It is possible only in % to 25% of patients, and most liver cancer patients die within a relatively short period of time due to locally advanced or metastatic diseases (Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets, Semin Liver Dis , 2005; 25:212-225) Hepatitis B virus, hepatitis C virus, and aflatoxin B1 are well known as major causes of liver cancer.
  • liver cancer can be broadly divided into primary liver cancer (hepatocellular carcinoma) arising from hepatocellular carcinoma and metastatic liver cancer in which cancers of other tissues have metastasized to the liver. More than 90% of liver cancers are primary liver cancer.
  • HCC Hepatocellular carcinoma
  • protooncogenes such as various growth factor genes are mutated into oncogenes for various reasons and overexpressed or overactive, or tumors such as Rb protein or p53 protein It has been reported that when a tumor suppressor gene is mutated by various causes and underexpressed or loses its function, it causes the onset and progression of various cancers including liver cancer.
  • Another object of the present invention is to provide a composition for diagnosing liver cancer.
  • Another object of the present invention is to provide a liver cancer diagnostic kit.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer.
  • Another object of the present invention is to provide a method for providing information necessary for diagnosing liver cancer.
  • Another object of the present invention is to provide a method for providing information for predicting the prognosis of liver cancer.
  • the present invention provides a biomarker for diagnosing liver cancer comprising one or more genes selected from the group consisting of SMARCA4, SMARCC1, SMARCA2 and IRAK1 or proteins expressed from the genes.
  • the present invention provides a composition for diagnosing liver cancer comprising an agent for measuring the expression level of one or more biomarker genes selected from the group consisting of SMARCA4, SMARCC1, SMARCA2 and IRAK1 at the mRNA or protein level.
  • the present invention provides a liver cancer diagnostic kit comprising the composition.
  • the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
  • the present invention provides a method for providing information necessary for diagnosing liver cancer.
  • the present invention provides a method of providing information for predicting the prognosis of liver cancer.
  • the present invention provides a method for predicting the responsiveness of a SMARCA4 inhibitor to the treatment of liver cancer.
  • the present invention provides a method for preventing or treating liver cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
  • the expression of SMARCA4 was increased in HCC, it was found that SMARCA4 directly increased the expression of IRAK1, and by confirming that the oncoproteins Gankyrin and AKR1B10 were induced through transcriptional activation of IRAK1, SMARCA4-IRAK1- Gankyrin and AKR1B10 can be used as specific markers for liver cancer, and they can be usefully used as targets for liver cancer treatment.
  • FIG. 1 is a diagram confirming the genetic variation and abnormal expression of the SWI / SNF subunit gene in HCC:
  • Figure 2 is a diagram analyzing the tumorigenicity (Tumorigenicity) of SMARCA4, SMARCC1 and SMARCA2 in HCC:
  • FIG. 3 is a diagram confirming the metastatic potential of SMARCA4 in a mouse HCC model:
  • A Pie charts, Venn diagrams and schematics of the enhancer gene and SMARCA4-disruption gene
  • FIG. 5 is a diagram confirming SMARCA4 and IRAK1 overexpression in the HCC patient cohort.
  • Figure 6 is a diagram confirming the anti-tumor effect in HCC by target-inhibition of IRAK1:
  • A differential gene expression of the IRAK1 gene
  • FIG. 7 is a diagram confirming the regulation of IRAK1 activity of SMARCA4 in HCC:
  • FIG. 8 is a diagram confirming that SMARCA4 regulates IRAK1 in HCC and activates tumor proteins Gankyrin and AKR1B10:
  • the term "subject” or “patient” means any single individual in need of treatment, including humans, apes, monkeys, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included in the subject are any subjects who participated in a clinical study trial without any clinical manifestations of any disease, or subjects who participated in epidemiological studies or subjects used as controls.
  • sample refers to a biological sample obtained from a subject or patient.
  • Sources of biological samples may include fresh, frozen and/or preserved organ or tissue samples or solid tissue from biopsies or aspirates; blood or any blood component; The cells may be at any point in the pregnancy or development of the subject.
  • the present invention is selected from the group consisting of SMARCA4 (SWI / SNF related, Matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), SMARCC1, SMARCA2 and IRAK1 (Interleukin-1 receptor-associated kinase 1) It relates to a biomarker for diagnosing liver cancer comprising one or more genes or proteins expressed from the genes.
  • SMARCA4 SWI / SNF related, Matrix associated, actin dependent regulator of chromatin, subfamily a, member 4
  • SMARCC1 SMARCA2
  • IRAK1 Interleukin-1 receptor-associated kinase 1
  • the biomarker may include genes of SMARCA4 and IRAK1 or proteins expressed from the genes together.
  • the liver cancer may be hepatocellular carcinoma (HCC), more preferably hepatocellular carcinoma overexpressing IRAK1.
  • HCC hepatocellular carcinoma
  • the hepatocellular carcinoma may be IRAK1 overexpression, but is not limited thereto.
  • it may further include a gene of Gankyrin or AKR1B10 (Aldo-keto reductase family 1 member B10) or a protein expressed from the gene.
  • AKR1B10 Aldo-keto reductase family 1 member B10
  • SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 may increase liver cancer-specific expression, and SMARCA2 may decrease liver cancer-specific expression.
  • the present invention relates to a composition for diagnosing liver cancer, comprising an agent for measuring the expression level of one or more biomarker genes selected from the group consisting of SMARCA4, SMARCC1, SMARCA2 and IRAK1 at the mRNA or protein level.
  • the composition may further comprise an agent for measuring the expression level of Gankyrin or AKR1B10 at the mRNA or protein level.
  • the liver cancer may be hepatocellular carcinoma, more preferably hepatocellular carcinoma overexpressing IRAK1.
  • the composition of the present invention may include an agent for measuring the expression levels of SMARCA4 and IRAK1 genes at the mRNA or protein level.
  • the agent for measuring the expression level of the biomarker gene at the mRNA level is a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence and the complementary sequence to specifically recognize It may include a primer pair, a probe, or a primer pair and a probe, and the measurement is a polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction ( Competitive RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or a method selected from the group consisting of a DNA chip.
  • the agent for measuring the expression level of a biomarker gene at the protein level is an antibody, antibody fragment, aptamer, avimer that specifically recognizes the full protein length of the marker or a fragment thereof ) or peptidomimetics
  • the measurement may be Western blot, ELISA (Enzyme linked immunosorbent assay), Radioimmunoassay (RIA), Radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining , immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or a method selected from the group consisting of protein microarray.
  • the term “detection” or “measurement” refers to quantifying the concentration of a detected or measured target.
  • primer is a nucleic acid sequence having a short free 3 hydroxyl group, which can form a complementary template and base pair, and serves as a starting point for template strand copying. It refers to a short nucleic acid sequence that functions.
  • the primers are capable of initiating DNA synthesis in the presence of the four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerate or reverse transcriptase) in an appropriate buffer and temperature.
  • probe refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to mRNA, and is labeled to determine the presence or absence of a specific mRNA.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. The selection of suitable probes and hybridization conditions may be modified based on those known in the art, and therefore, the present invention is not particularly limited thereto.
  • the primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods.
  • Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phossotriesters, phosphoro amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
  • suitable conditions for hybridizing a probe with a cDNA molecule can be determined in a series of processes by an optimization procedure. These procedures are carried out as a series of procedures by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999).
  • the high stringency conditions among the stringent conditions include hybridization in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and in 0.1 ⁇ SSC (Standard saline citrate)/0.1% SDS. It means washing at 68°C conditions.
  • high stringency conditions mean washing at 48° C. in 6 ⁇ SSC/0.05% sodium pyrophosphate.
  • Low stringency conditions mean washing at 42° C. in, for example, 0.2 ⁇ SSC/0.1% SDS.
  • the term “antibody” refers to a specific protein molecule directed to an antigenic site as a term known in the art. This also includes partial peptides that can be made from the protein.
  • the form of the antibody of the present invention is not particularly limited, and a part thereof is also included in the antibody of the present invention as long as it has a polyclonal antibody, a monoclonal antibody, or antigen-binding property, and all immunoglobulin antibodies are included.
  • the antibody of the present invention includes a special antibody such as a humanized antibody.
  • the present invention relates to a liver cancer diagnostic kit comprising the composition of the present invention.
  • the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient, as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA or protein from the sample. have.
  • it may include PCR primers for amplifying a relevant region of genomic DNA.
  • the kit may include probes of genetic factors useful for pharmacogenomic profiling.
  • the labeled oligonucleotide can be easily identified during analysis.
  • the term "diagnosis” as used herein refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, a specific disease or disorder Determining the prognosis of a subject with the disease (eg, identification of a pre-metastatic or metastatic cancer state, staging the cancer, or determining the responsiveness of the cancer to treatment), or Therametrics (eg, monitoring the condition of the subject to provide information on treatment efficacy).
  • the kit may further contain a labeling material such as a DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP), a fluorescent material, and the like.
  • a labeling material such as a DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP), a fluorescent material, and the like.
  • the present invention relates to a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
  • the SMARCA4 inhibitor may be siRNA, or siRNA of SEQ ID NO: 1.
  • SMARCA2 or an expression promoter thereof, a Gankyrin inhibitor, an AKR1B10 inhibitor, a SMARCC1 inhibitor, or an IRAK1 inhibitor may be further included.
  • the IRAK1 inhibitor may be siRNA, or siRNA of SEQ ID NO: 2.
  • the liver cancer may be hepatocellular carcinoma, more preferably hepatocellular carcinoma overexpressing IRAK1.
  • the pharmaceutical composition according to the present invention may include any substance capable of inhibiting the expression of SMARCA4, SMARCC1, or IRAK1 or promoting the expression of SMARCA2.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not normally cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness when administered to humans.
  • Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like, and carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. and the like, and may further include a stabilizer and a preservative.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • the pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and as a representative dosage form for parenteral administration, an isotonic aqueous solution or suspension is preferred as an injectable dosage form.
  • Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving it in saline or buffer.
  • formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.
  • the pharmaceutical composition formulated in the above manner may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the “administration” refers to introducing a predetermined substance into a patient by any suitable method. and the route of administration of the substance can be administered through any general route as long as it can reach the target tissue.
  • the term “effective amount” refers to an amount exhibiting a preventive or therapeutic effect when administered to a patient.
  • the dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the type and severity of the patient's disease, age, sex, weight, sensitivity to drugs, the type of current treatment, administration method, target cell, etc.
  • the pharmaceutical composition of the present invention may be administered in combination with a conventional therapeutic agent, may be administered sequentially or simultaneously with the conventional therapeutic agent, and may be administered single or multiple.
  • an amount that can obtain the maximum effect with a minimum amount without side effects can be administered, more preferably 1 to 10000 ⁇ g/weight kg/day, even more preferably 10 to 1000 It can be administered repeatedly several times a day at an effective dose of mg/kg body weight/day.
  • the SMARCA2 expression promoter preferably a chemical substance, a nucleotide, a vector containing the gene, a protein or fragment thereof into which the gene is translated, or a natural product extract may be included as an active ingredient.
  • promotion of expression means to cause enhancement of expression of target gene mRNA or translation into protein.
  • the expression inhibitor of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 may include a chemical substance, nucleotide, antisense, siRNA oligonucleotide or a natural product extract as an active ingredient, more preferably the gene An antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to the nucleotide sequence of may be included as an active ingredient.
  • siRNA small interference RNA
  • expression inhibition means to cause a decrease in target gene mRNA expression or translation into a protein, and preferably means that the target gene expression becomes undetectable or exists at an insignificant level. do.
  • siRNA Small interfering RNA
  • RNA interference or gene silencing International Patent Publication Nos. 00/44895, 01/36646, 99/32619, 01 see /29058, 99/07409 and 00/44914. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method.
  • the sense strand (sequence corresponding to the mRNA sequence of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10) and the antisense strand (sequence complementary to the mRNA sequence of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10) are mutually exclusive
  • the siRNA molecule of the present invention may have a structure that is positioned on the opposite side to form a double-stranded chain, and the siRNA molecule of the present invention may have a single-stranded structure having self-complementary sense and antisense strands.
  • siRNA is not limited to the complete pairing of double-stranded RNA parts that are paired with each other, but pairs are formed by mismatch (corresponding bases are not complementary), bulges (there is no base corresponding to one chain), etc. There may be parts that are not achieved.
  • siRNA end structure can inhibit the expression of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 genes by the RNAi effect, both blunt ends or cohesive ends are possible, and the cohesive end structure is 3'- Both a terminal overhang structure and a 5'-end overhang structure are possible.
  • the siRNA molecule of the present invention may have a form in which a short nucleotide sequence is inserted between self-complementary sense and antisense strands.
  • the siRNA molecule formed by expression of the nucleotide sequence is used for intramolecular hybridization.
  • a hairpin structure is formed, and as a whole, a stem-and-loop structure is formed (shRNA).
  • shRNA stem-and-loop structure
  • Methods for preparing siRNA include direct synthesis of siRNA in a test tube and introduction into cells through transformation, and transfection of an siRNA expression vector or PCR-derived siRNA expression cassette prepared so that siRNA is expressed in the cell. There is a way to convert or infect.
  • the composition of the present invention comprising a gene-specific siRNA may include an agent that promotes the influx of the siRNA into a cell.
  • an agent that promotes nucleic acid influx can be used for an agent that promotes siRNA entry into cells.
  • a liposome is used or a lipophilic one of a number of sterols including cholesterol, cholate and deoxycholic acid. It can be formulated with a carrier.
  • poly-L-lysine, spermine, polysilazane, polyethylenimine (PEI), polydihydroimidazolenium, polyallylamine ), a cationic polymer such as chitosan may be used, and succinylated PLL (Succinylated PLL), succinylated PEI (Succinylated PEI), polyglutamic acid, polyaspartic acid (Polyaspartic acid), polyacrylic acid (Polyacrylic acid), polymethacylic acid (Polymethacylic acid), dextran sulfate (Dextran sulfate), heparin (Heparin), anionic polymers such as hyaluronic acid (Hyaluronic acid) Available.
  • succinylated PLL succinylated PLL
  • succinylated PEI succinylated PEI
  • polyglutamic acid polyaspartic acid
  • Polyacrylic acid Polyacrylic acid
  • an antibody specific for the protein when used as a substance for reducing the expression and activity of the SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 protein, the antibody is directly or indirectly through a linker with an existing therapeutic agent. couple (eg, covalently).
  • antisense oligonucleotide refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and is bound to a complementary sequence in mRNA to form SMARCA4, SMARCC1, IRAK1 , inhibits the translation of Gankyrin or AKR1B10 into protein.
  • the antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 mRNA and is capable of binding to SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 mRNA, SMARCA4, SMARCC1, IRAK1, Gankyrin or It can inhibit AKR1B10 mRNA's essential activity for translation, translocation into the cytoplasm, maturation or all other overall biological functions.
  • the antisense nucleic acid may be modified at one or more bases, sugars, or positions of the backbone to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol ., 5, 3, 343-55, 1995). ).
  • the nucleic acid backbone may be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic intersaccharide linkages, and the like.
  • Antisense nucleic acids may also include one or more substituted sugar moieties.
  • Antisense nucleic acids may include modified bases.
  • Modified bases include hypoxanthine, 6-methyladenine, 5-methylpyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2-aminoadenine , 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diamino purines, etc.
  • the antisense nucleic acid of the present invention may be chemically bound to one or more moieties or conjugates that improve the activity and cell adsorption properties of the antisense nucleic acid.
  • the modified nucleic acid may increase the stability to nucleases and increase the binding affinity between the antisense nucleic acid and the target mRNA.
  • Antisense oligonucleotides can be synthesized in vitro by a conventional method and administered in vivo, or antisense oligonucleotides can be synthesized in vivo.
  • An example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I.
  • One example of allowing antisense RNA to be synthesized in vivo is to use a vector with the origin of the recognition site (MCS) in the opposite direction to allow the antisense RNA to be transcribed.
  • MCS origin of the recognition site
  • Such antisense RNA preferably has a translation stop codon in the sequence so that it is not translated into the peptide sequence.
  • the present invention comprises the steps of (a) measuring the gene expression level of SMARCA4 or IRAK1 in a biological sample isolated from a test subject; (b) comparing the corresponding gene of the normal control sample with the corresponding result; and (c) when the gene expression level in step (a) is higher than the expression level of the corresponding gene in step (b), determining that the test subject is liver cancer, providing information necessary for diagnosing liver cancer It's about how to do it.
  • the method comprises the steps of (a) measuring the gene or protein expression level of SMARCC1, SMARCA2, Gankyrin, or AKR1B10 in a biological sample isolated from a test subject; (b) comparing the corresponding gene or protein of the normal control sample with the corresponding result; And (c) when the expression level in step (a) of SMARCC1, Gankyrin or AKR1B10 is high compared to the expression level of the corresponding gene or protein in step (b), or the expression level in step (a) of SMARCA2 (b) When it is low compared to the expression level of the gene or protein in the step, the step of determining that the test subject is liver cancer may be further included.
  • the biological sample may include a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and the like.
  • the present invention comprises the steps of (a) measuring the gene expression level of SMARCA4 or IRAK1 in a biological sample isolated from a test subject; (b) comparing the corresponding gene of the normal control sample with the corresponding result; and (c) when the gene expression level in step (a) is higher than the expression level of the corresponding gene in step (b), determining that the prognosis of the test subject is bad, for predicting the prognosis of liver cancer How to provide information.
  • the present invention relates to a method for predicting liver cancer treatment responsiveness of a SMARCA4 inhibitor, comprising measuring the expression level of IRAK1.
  • the method may further comprise measuring the expression level of Gankyrin or AKR1B10.
  • it relates to a method for preventing or treating liver cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
  • TCGA_LIHC The Cancer Genome Atlas liver hepatocellular carcinoma project
  • ICGC_LIRI International Cancer Genome Consortium liver Cancer-RIKEN, JP
  • GEO Gene Expression Omnibus
  • SMARCA4 and SMARCC1 are HCC patients was significantly overexpressed, and at the same time, it could be confirmed that SMARCA2 was down-regulated ( ⁇ ⁇ 1.5 fold, P ⁇ 0.05).
  • HCC data TCGA_LIHC, ICGC_LIRI, GSE77314
  • SMARCA4 and SMARCC1 were overexpressed in noncancerous liver and HCC tissues of the same patient, and SMARCA2 was downregulated at the same time. ( ⁇ ⁇ 1.5 fold, P ⁇ 0.05) (Fig. 1c).
  • each gene was knocked down by RNA interference.
  • each gene was knocked down by RNA interference in Hep3B, Huh7 and SNU-449 liver cell lines with relatively high SMARCA4 and SMARCC1 expression and relatively low SMARCA2 expression than in normal hepatocytes, and cell viability and cell proliferation rates were analyzed by MTT analysis, BrdU It was confirmed by analysis and colonogenic assay analysis.
  • the migration and invasion of knockdown HCC cells of each gene were confirmed by Boyden chamber motility assay and wound healing assay.
  • the effect of knockdown of each gene on cell cycle regulation in HCC cells was confirmed by flow cytometric analysis, and cell cycle regulatory proteins were confirmed by Western blot analysis.
  • SMARCA4 knockdown selectively induces p27 Kip1 expression in HCC cells, and at the same time inhibits cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6, thereby hyperphosphorylating pRb (Hypophosphorylation) (p-pRb) was shown to induce (Fig. 3d).
  • p-pRb hyperphosphorylating pRb (Hypophosphorylation)
  • SMARCA4 To determine whether the regulation of SMARCA4, SMARCA2 and SMARCC1 induces tumor suppressive effects in vivo, SMARCA4 from 14 weeks of age in 8 Ras- Tg (H-ras12V homozygous transgenic) male mice, where HCC develops at about 15 to 18 weeks of age.
  • liver HCC of mice was confirmed by ultrasonography (Philips, Amsterdam, Nederland) from 14 weeks of age to 24 weeks of age to obtain livers, and the weight and tumor mass number of the obtained livers were confirmed and disrupted by Western blot analysis SMARCA4, SMARCA2 and SMARCC1 was confirmed.
  • HOMER FullPeaks
  • FDR-adjusted p-value cutoff 0.001
  • 9,198 genes out of 21,684 genes closest to the peak were identified as active enhancers (FIG. 4a).
  • SMARCA4 knockdown was performed in Hep3B to identify regulated gene elements in HCC cells, and 1,865 gene elements regulated by SMARCA4 were found (> ⁇ 1.5 fold, P ⁇ 0.05), of which 1,054 genes were shown to be downregulated in SMARCA4 knockdown HCC cells.
  • 563 genes were analyzed to be regulated by SMARCA4-SWI/SNF in HCC (Fig. 4a).
  • 563 SMARCA4-associated genes showed progressive increases or decreases in transcription levels across different stages of HCC, and consistently highest or lowest levels in advanced HCC (Fig. 4b).
  • genes related to carcinogenesis including angiogenesis, Wnt signaling, integrin signaling, PDGF signaling, and G-protein signaling, were searched for in the 563 gene sets (FIG. 4c). ).
  • ChIP analysis was performed using anti-SMARCA4 and anti-H3K27ac antibodies, respectively, and the active enhancer region of IRAK1 (H3K27ac) was Enrichment rates were confirmed.
  • Chromatin immunoprecipitation (ChIP) analysis was performed according to the manufacturer's (Pierce Agarose ChIP kit; Thermo Fisher Scientific, Waltham, MA) instructions, and DNA was RT- with primers for enhancer regions of each candidate gene regulated by SMARCA4. It was amplified by qPCR.
  • Invivofectamine 3.0 Invitrogen containing 25 mg/kg of si-SMARCA4 or si-IRAK1 was administered intravenously to 14-week-old Ras-Tg mice. Injections, and ultrasonography at 15, 17, 19, 21 and 23 weeks of age (Fig. 8c). As a result, in the case of the control group (si-Cont), HCC was detected at 19 weeks of age, and a large number of large tumors occurred in 4 out of 4 mice.

Abstract

The present invention relates to a use of using, as biomarkers for the detection and diagnosis of hepatocellular carcinoma, genes of which the expression changes specifically to hepatocellular carcinoma, and treating liver cancer by using same as targets. In the present invention, it has been discovered that the expression of SMARCA4 is increased in HCC and SMARCA4 directly increases the expression of IRAK1, and it has been ascertained that oncogenic proteins Gankyrin and AKR1B10 are induced through the transcriptional activation of IRAK1, and thus SMARCA4-IRAK1-Gankyrin and AKR1B10 can be used as markers specific for liver cancer and can be effectively used as targets for liver cancer treatment.

Description

간암 특이적 바이오 마커 및 이의 용도Liver cancer-specific biomarkers and uses thereof
본 발명은 간암 특이적 바이오 마커 및 이의 용도에 관한 것으로, 더욱 상세하게는, 간세포암에 특이적으로 발현이 변화하는 유전자들을 간세포암 검출 및 진단용 바이오 마커로 이용하고, 이들을 표적으로 간암을 치료하는 용도에 관한 것이다.The present invention relates to a liver cancer-specific biomarker and its use, and more particularly, using genes whose expression is specifically changed in hepatocellular carcinoma as biomarkers for hepatocellular carcinoma detection and diagnosis, and to treat liver cancer by targeting them It's about use.
암은 인류의 건강을 위협하는 대표적인 질병으로서 산업화된 국가에서 단일 질병으로는 가장 대표적인 사망 원인이다. 암의 발생원인은 아직도 규명되지 않고 있으나 내적 요인인 유전적 요소와 외적 요인인 암 발생 유발요소로 작용되는 발암화학물질, 계속적인 염증과 손상, 암 유발 바이러스 감염의 복합적 요소가 작용하는 것으로 간주된다. 그러나 암은 불치의 병으로 단정할 만큼 절망적인 것은 아니며 조기진단과 함께 적극적인 치료에 임하면 완치될 수 있다. 따라서 조기발견과 조기진단과 치료가 암치료의 효과를 높이는 데 결정적으로 중요하며, 진행된 암에서도 다각적이고 적극적인 방법들을 동원한다면 완치 내지 생명을 연장시키고 괴로운 증세를 호전시킬 수 있다. Cancer is a representative disease that threatens human health and is the most representative cause of death as a single disease in industrialized countries. Although the cause of cancer is still unknown, it is considered that complex factors of genetic factors, which are internal factors, and carcinogenic chemicals acting as factors that cause cancer, which are external factors, continuous inflammation and damage, and cancer-causing virus infection are acting. . However, cancer is not so desperate that it can be concluded that it is an incurable disease, and it can be cured by early diagnosis and active treatment. Therefore, early detection, early diagnosis, and treatment are crucially important to increase the effectiveness of cancer treatment, and even in advanced cancer, if various and active methods are used, a cure or life can be prolonged and painful symptoms can be improved.
암 중에서도 간암은 세계적으로 가장 치명적인 암의 하나로 알려져 있으며, 그로 인한 사망률은 3위에 해당하는 공격적인 암이다(Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004; 50:556-573) 치료목적의 수술은 단지 15%에서 25% 정도의 환자에게만 가능하고 대부분의 간암 환자들은 국부적으로 진행하거나 전이되는 질병들에 의해 비교적 단기간 내에 사망한다(Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets, Semin Liver Dis, 2005; 25:212-225) 간암의 주요 원인으로 B형 간염 바이러스(Hepatitis B virus), C형 간염 바이러스(Hepatitis C virus), 및 아플라톡신 B1(Aflatoxin B1) 등이 잘 알려져 있다. 하지만, 지난 20년간 간암 환자의 전체적인 생존율은 크게 증가하지 않았고, 간암의 발달(Development) 및 진전(Progression) 기작은 여전히 잘 알려져 있지 않은 상태이다(Bruix J, et al., Focus on hepatocellular carcinoma, Cancer Cell, 2004; 5:215-219) 지금까지, 분자표적치료(Molecular targeted therapy)가 성숙한 간암의 치료에 효과적인 것으로 나타났지만(Shen YC, Hsu C, Cheng AL Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives, J Gastroenterol; 45:794-807), 어떻게 이러한 유전적 변화가 간암 환자들 개개인에게 관찰되는 임상적 특징들을 야기하는지는 불명확하다. 간암은 크게 간세포 자체로부터 발생한 원발성 간암(간세포암; Hepatocellular carcinoma)과 다른 조직의 암이 간으로 전이되어 온 전이성 간암으로 구분할 수 있는데, 간암의 약 90% 이상은 원발성 간암이다. Among cancers, liver cancer is known as one of the most lethal cancers worldwide, and its mortality rate is the third most aggressive cancer (Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004; 50:556-573). It is possible only in % to 25% of patients, and most liver cancer patients die within a relatively short period of time due to locally advanced or metastatic diseases (Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets, Semin Liver Dis , 2005; 25:212-225) Hepatitis B virus, hepatitis C virus, and aflatoxin B1 are well known as major causes of liver cancer. However, the overall survival rate of liver cancer patients has not increased significantly over the past 20 years, and the mechanisms of development and progression of liver cancer are still not well known (Bruix J, et al., Focus on hepatocellular carcinoma, Cancer Cell , 2004; 5:215-219) so far, although molecular targeted therapy has been shown to be effective in the treatment of mature hepatocellular carcinoma (Shen YC, Hsu C, Cheng AL Molecular targeted therapy for advanced hepatocellular carcinoma: current status) and future perspectives, J Gastroenterol ; 45:794-807), it is unclear how these genetic changes cause the clinical features observed in individual liver cancer patients. Liver cancer can be broadly divided into primary liver cancer (hepatocellular carcinoma) arising from hepatocellular carcinoma and metastatic liver cancer in which cancers of other tissues have metastasized to the liver. More than 90% of liver cancers are primary liver cancer.
간세포암(Hepatocellular carcinoma, HCC)은 해마다 50 만명이 사망하는 세계에서 5번째로 일반적인 종양이다(Okuda 2000). HCC 환자의 생존률은 지난 20년에 걸쳐 개선되지 않았고 사망율과 거의 동일한 발병률을 지니고 있다(Marrero, Fontana et al., 2005). B형 간염 바이러스 또는 C형 간염 바이러스에 감염되어 발병하는 만성간염 및 아플라톡신 B1(Aflatoxin B1)과 같은 발암물질에의 노출은 HCC에 대한 주요 위험 인자로 알려져 있고(Thorgeirsson and Grisham, 2002), 세포주기 기작에서 G1 단계로 진행하는 세포주기 조절물질들의 변화가 간암 형성에 관련되어 있다는 보고도 있으나(Hui et al., Hepatogasteroenterology 45:1635-1642, 1998), 간암의 발병 및 진행에 관한 세포 내의 분자적 메커니즘은 아직도 명확히 규명되지 못한 상태이다. 종래의 연구에 따르면 각종 성장인자 유전자와 같은 전-종양형성유전자(Protooncogene)가 다양한 원인에 의하여 종양형성유전자(Oncogene)로 돌연변이되어 과발현하거나 과활성을 갖는 경우, 또는 Rb 단백질이나 p53 단백질과 같은 종양형성 억제 유전자(Tumor suppressor gene) 가 다양한 원인에 의하여 돌연변이 되어 저발현하거나 기능을 잃는 경우 간암을 비롯한 다양한 암의 발병과 진행을 유발하는 것으로 보고되어 있다. 이외에도 DNA 돌연변이와 유전자 발현의 유전적 변화(Genetic alteration)등이 간암 환자조직에서 확인된다고 보고되고 있다(Park et al., Cancer Res 59:307-310, 1999; Bjersing et al., J Intern Med, 234:339-340, 1993; Tsopanomichalou et al., Liver 19:305-311, 1999; Kusano et al., Hepatology 29:1858-1862, 1999; Keck et al., Cancer Genet Cytogenet 111:37-44, 1999). 최근에는 간암을 비롯한 대부분의 암의 발병 및 진행은 특정 몇몇 유전자들에 의하여 이루어지는 것이 아니라 세포주기, 신호전달 등과 관련된 다양한 각종 유전자들의 복합적인 상호작용에 발병하는 것으로 인식되고 있으며, 따라서 개별적인 유전자나 단백질의 발현이나 기능에만 중점을 두는 것에서 벗어나 다양한 유전자나 단백질에 대한 총체적인 연구의 필요성이 대두되고 있다. Hepatocellular carcinoma (HCC) is the fifth most common tumor in the world, with 500,000 deaths annually (Okuda 2000). The survival rate of HCC patients has not improved over the past 20 years and has an incidence rate that is almost equal to the mortality rate (Marrero, Fontana et al., 2005). Chronic hepatitis caused by infection with hepatitis B virus or hepatitis C virus and exposure to carcinogens such as Aflatoxin B1 are known as major risk factors for HCC (Thorgeirsson and Grisham, 2002), and the cell cycle There is also a report that changes in cell cycle regulators that progress from the mechanism to the G1 stage are involved in the formation of hepatocarcinoma (Hui et al., Hepatogasteroenterology 45:1635-1642, 1998). The mechanism is still not clearly elucidated. According to prior studies, protooncogenes such as various growth factor genes are mutated into oncogenes for various reasons and overexpressed or overactive, or tumors such as Rb protein or p53 protein It has been reported that when a tumor suppressor gene is mutated by various causes and underexpressed or loses its function, it causes the onset and progression of various cancers including liver cancer. In addition, it has been reported that DNA mutations and genetic alteration of gene expression are confirmed in liver cancer patient tissues (Park et al., Cancer Res 59:307-310, 1999; Bjersing et al., J Intern Med , 234:339-340, 1993; Tsopanomichalou et al., Liver 19:305-311, 1999; Kusano et al., Hepatology 29:1858-1862, 1999; Keck et al., Cancer Genet Cytogenet 111:37-44, 1999). Recently, it is recognized that the onset and progression of most cancers, including liver cancer, is caused by the complex interaction of various genes related to the cell cycle, signal transduction, etc., rather than by a few specific genes. Therefore, individual genes or proteins Rather than focusing only on the expression or function of
본 발명의 목적은 간암 진단용 바이오 마커를 제공하는 것이다.It is an object of the present invention to provide a biomarker for diagnosing liver cancer.
또한, 본 발명의 목적은 간암 진단용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for diagnosing liver cancer.
또한, 본 발명의 목적은 간암 진단 키트를 제공하는 것이다.Another object of the present invention is to provide a liver cancer diagnostic kit.
또한, 본 발명의 목적은 간암 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer.
또한, 본 발명의 목적은 간암 진단에 필요한 정보를 제공하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for providing information necessary for diagnosing liver cancer.
또한, 본 발명의 목적은 간암의 예후 예측을 위한 정보를 제공하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for providing information for predicting the prognosis of liver cancer.
아울러, 본 발명의 목적은 SMARCA4 억제제의 간암 치료 반응성 예측방법을 제공하는 것이다.In addition, it is an object of the present invention to provide a method for predicting liver cancer treatment responsiveness of a SMARCA4 inhibitor.
상기 목적의 달성을 위해, 본 발명은 SMARCA4, SMARCC1, SMARCA2 및 IRAK1으로 이루어진 군으로부터 선택되는 하나 이상의 유전자 또는 상기 유전자로부터 발현된 단백질을 포함하는 간암 진단용 바이오 마커를 제공한다.In order to achieve the above object, the present invention provides a biomarker for diagnosing liver cancer comprising one or more genes selected from the group consisting of SMARCA4, SMARCC1, SMARCA2 and IRAK1 or proteins expressed from the genes.
또한, 본 발명은 SMARCA4, SMARCC1, SMARCA2 및 IRAK1로 이루어진 군으로부터 선택되는 하나 이상의 바이오 마커 유전자의 발현량을 mRNA 또는 단백질 수준에서 측정하는 제제를 포함하는 간암 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing liver cancer comprising an agent for measuring the expression level of one or more biomarker genes selected from the group consisting of SMARCA4, SMARCC1, SMARCA2 and IRAK1 at the mRNA or protein level.
또한, 본 발명은 상기 조성물을 포함하는 간암 진단 키트를 제공한다.In addition, the present invention provides a liver cancer diagnostic kit comprising the composition.
또한, 본 발명은 SMARCA4 억제제를 유효성분으로 포함하는 간암 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
또한, 본 발명은 간암 진단에 필요한 정보를 제공하는 방법을 제공한다.In addition, the present invention provides a method for providing information necessary for diagnosing liver cancer.
또한, 본 발명은 간암의 예후 예측을 위한 정보를 제공하는 방법을 제공한다.In addition, the present invention provides a method of providing information for predicting the prognosis of liver cancer.
또한, 본 발명은 SMARCA4 억제제의 간암 치료 반응성 예측방법을 제공한다.In addition, the present invention provides a method for predicting the responsiveness of a SMARCA4 inhibitor to the treatment of liver cancer.
아울러, 본 발명은 SMARCA4 억제제를 유효성분으로 포함하는 간암 예방 또는 치료용 약학적 조성물을 개체에 투여하는 단계를 포함하는 간암 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating liver cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
본 발명에 따르면, HCC에서 SMARCA4의 발현이 증가되었고, SMARCA4가 IRAK1 발현을 직접적으로 증가시키는 것을 밝혔으며, IRAK1의 전사적 활성화를 통해 종양단백질인 Gankyrin 및 AKR1B10를 유발하는 것을 확인함으로써, SMARCA4-IRAK1-Gankyrin, AKR1B10을 간암에 특이적인 마커로서 이용할 수 있으며, 이들을 간암 치료의 표적으로서 유용하게 활용할 수 있다.According to the present invention, the expression of SMARCA4 was increased in HCC, it was found that SMARCA4 directly increased the expression of IRAK1, and by confirming that the oncoproteins Gankyrin and AKR1B10 were induced through transcriptional activation of IRAK1, SMARCA4-IRAK1- Gankyrin and AKR1B10 can be used as specific markers for liver cancer, and they can be usefully used as targets for liver cancer treatment.
도 1은 HCC에서 SWI/SNF 서브유닛 유전자의 유전적 변이 및 이상 발현을 확인한 도이다:1 is a diagram confirming the genetic variation and abnormal expression of the SWI / SNF subunit gene in HCC:
A: HCC에서 SWI/SNF 복합체 서브유닛 유전자의 돌연변이 비율;A: mutation rate of SWI/SNF complex subunit gene in HCC;
B: TCGA_LIHC, ICGC_LIRI 및 Catholic_mLIHC(GSE114654) 데이터 세트에서 건강한 정상 환자(Normal patients, NC)와 비교한 HCC 환자에서의 SMARCA4, SMARCC1 및 SMARCA2의 차등 유전자 발현(Differential gene expression); B: Differential gene expression of SMARCA4, SMARCC1 and SMARCA2 in HCC patients compared to healthy normal patients (NC) in the TCGA_LIHC, ICGC_LIRI and Catholic_mLIHC (GSE114654) data sets;
C: HCC 조직과 이에 상응하는 비암성 간 생검의 HCC의 조직 쌍에서의 SMARCA4, SMARCC1 및 SMARCA2의 차등 유전자 발현;C: Differential gene expression of SMARCA4, SMARCC1 and SMARCA2 in pairs of HCC tissues and corresponding tissues of HCC from noncancerous liver biopsies;
D: qRT-PCR로 분석한 HCC 조직과 이에 상응하는 비암성 간 생검의 HCC의 조직 쌍에서의 SMARCA4, SMARCC1 및 SMARCA2의 발현; 및D: Expression of SMARCA4, SMARCC1 and SMARCA2 in pairs of HCC tissues analyzed by qRT-PCR and corresponding tissues of HCC from noncancerous liver biopsies; and
E: 10쌍의 선택된 HCC 서브셋에서의 SMARCA4, SMARCC1 및 SMARCA2의 웨스턴 블롯 분석 결과.E: Western blot analysis results of SMARCA4, SMARCC1 and SMARCA2 in 10 pairs of selected HCC subsets.
도 2는 HCC에서 SMARCA4, SMARCC1 및 SMARCA2의 종양원성(Tumorigenicity)을 분석한 도이다:Figure 2 is a diagram analyzing the tumorigenicity (Tumorigenicity) of SMARCA4, SMARCC1 and SMARCA2 in HCC:
A: si-SMARCA4 처리한 HCC의 세포 생존율;A: Cell viability of si-SMARCA4-treated HCC;
B: si-SMARCA4 처리한 HCC의 세포 증식률;B: cell proliferation rate of si-SMARCA4-treated HCC;
C: si-SMARCC1 처리한 HCC의 세포 생존율;C: cell viability of si-SMARCC1-treated HCC;
D: si-SMARCC1 처리한 HCC의 세포 증식률; 및 D: cell proliferation rate of HCC treated with si-SMARCC1; and
E: si-SMARCA2를 처리한 SMARCA4 고발현 세포 및 SMARCA4 비-발현 세포의 세포 생존율.E: Cell viability of SMARCA4 high-expressing cells and SMARCA4 non-SMARCA4 non-expressing cells treated with si-SMARCA2.
도 3은 마우스 HCC 모델에서 SMARCA4의 전이 잠재력을 확인한 도이다:3 is a diagram confirming the metastatic potential of SMARCA4 in a mouse HCC model:
A: si-SMARCA4 처리한 HCC 세포의 이동 및 침윤;A: Migration and invasion of si-SMARCA4-treated HCC cells;
B: 이동 세포 이미지;B: image of migrating cells;
C: si-SMARCA4 처리한 HCC 세포의 세포 주기 프로파일;C: Cell cycle profile of si-SMARCA4-treated HCC cells;
D: HCC 세포주에서의 세포 주기 관련 단백질의 웨스턴 블롯 분석 결과;D: Western blot analysis of cell cycle-related proteins in HCC cell lines;
E: Ras-Tg 마우스 모델의 si-Cont, si-SMARCA4, si-SMARCC1 및 pBJ-SMARCA2투여 스캐줄, 초음파 촬영 이미지 및 종양 지량 및 질량수; 및E: si-Cont, si-SMARCA4, si-SMARCC1 and pBJ-SMARCA2 dosing schedules, ultrasonographic images, and tumor mass and mass number in the Ras-Tg mouse model; and
F: Ras-Tg 마우스 모델의 간 조직에서의 SMARCA4, SMARCC1 및 SMARCA2의 웨스턴 블롯 분석 결과.F: Western blot analysis results of SMARCA4, SMARCC1 and SMARCA2 in liver tissue of the Ras-Tg mouse model.
도 4는 HCC에서 SMARCA4에 의해 조절되는 특이적인 표적 유전자를 확인한 도이다:4 is a diagram confirming specific target genes regulated by SMARCA4 in HCC:
A: 인핸서 유전자 및 SMARCA4-고절 유전자의 파이 차트, 벤다이어그램 및 회로도;A: Pie charts, Venn diagrams and schematics of the enhancer gene and SMARCA4-disruption gene;
B: 다단계 HCC에서 563개의 SMARCA4-관련 유전자들의 Heatmap;B: Heatmap of 563 SMARCA4-related genes in multi-stage HCC;
C: 563개 유전자의 기능적 분석;C: functional analysis of 563 genes;
D: si-SMARCA4 처리한 HCC 세포주에서의 SMARCA4 및 IRAK1의 발현 분석 결과(qRT-PCR);D: Results of SMARCA4 and IRAK1 expression analysis in si-S MARCA4-treated HCC cell lines (qRT-PCR);
E: IRAK1 인핸서에 결합하는 SMARCA4를 평가하기 위한 ChIP-qPCR 분석 결과; 및E: ChIP-qPCR assay results to evaluate SMARCA4 binding to the IRAK1 enhancer; and
F: si-SMARCA4 처리 후 IRAK1 인핸서 영역을 발현하는 리포터 플라스미드를 발현시킨 HCC 세포주에서의 듀얼 루시퍼레이즈 분석 결과.F: Result of dual luciferase analysis in HCC cell line expressing reporter plasmid expressing IRAK1 enhancer region after si-SMARCA4 treatment.
도 5는 HCC 환자 코호트에서의 SMARCA4 및 IRAK1 과발현을 확인한 도이다.5 is a diagram confirming SMARCA4 and IRAK1 overexpression in the HCC patient cohort.
도 6은 IRAK1의 표적-억제에 의한 HCC에서의 항-종양 효과를 확인한 도이다:Figure 6 is a diagram confirming the anti-tumor effect in HCC by target-inhibition of IRAK1:
A: IRAK1 유전자의 차등 유전자 발현;A: differential gene expression of the IRAK1 gene;
B: SMARCA4 또는 IRAK1 유전자 낙다운 후 세포 성장(세포 생존율) 및 세포 증식률;B: Cell growth (cell viability) and cell proliferation rate after SMARCA4 or IRAK1 gene knockdown;
C: 세포 주기 프로파일;C: cell cycle profile;
D: 세포 주기 관련 단백질의 웨스턴 블롯 분석 결과;D: Western blot analysis of cell cycle-related proteins;
E: Boyden chamber motility 분석을 이용하여 확인한 세포 이미지 및 세포 수; 및E: Cell images and cell numbers confirmed using Boyden chamber motility analysis; and
F: Transwell invasion 분석을 이용하여 확인한 세포 이미지 및 세포 수.F: Cell image and cell number confirmed using transwell invasion assay.
도 7은 HCC에서 SMARCA4의 IRAK1 활성 조절을 확인한 도이다:7 is a diagram confirming the regulation of IRAK1 activity of SMARCA4 in HCC:
A: SMARCA4를 낙다운한 뒤 IRAK1(pcDNA3.1_IRAK1)에 의한 항-종양 효과 (웨스턴 블롯 분석, MTT 분석 및 BrdU 분석); A: Anti-tumor effect by IRAK1 (pcDNA3.1_IRAK1) after knockdown of SMARCA4 (Western blot analysis, MTT analysis and BrdU analysis);
B: 세포 주기 프로파일;B: cell cycle profile;
C: 세포 주기 조절 단백질을 웨스턴 블롯 분석으로 확인한 결과;C: The result of confirming the cell cycle regulatory protein by Western blot analysis;
D: Boyden chamber motility 분석을 이용하여 확인한 세포 이미지 및 세포 수; 및D: Cell images and cell numbers confirmed using Boyden chamber motility analysis; and
E: Transwell invasion 분석을 이용하여 확인한 세포 이미지 및 세포 수.E: Cell image and cell number confirmed using Transwell invasion assay.
도 8은 HCC에서 SMARCA4가 IRAK1을 조절하여 종양 단백질인 Gankyrin 및 AKR1B10을 활성화 시키는 것을 확인한 도이다:8 is a diagram confirming that SMARCA4 regulates IRAK1 in HCC and activates tumor proteins Gankyrin and AKR1B10:
A: IRAK1의 하류 조절 분자들의 웨스턴 블롯 분석 결과;A: Western blot analysis of downstream regulatory molecules of IRAK1;
B: SMARCA4 낙다운(si-SMARCA4) 및 IRAK1 회복(pcDNA3.1_IRAK1)에 의한 IRAK1의 하류 조절 분자들의 발현 변화를 웨스턴 블롯 분석으로 확인한 결과;B: Western blot analysis confirmed the expression change of IRAK1 downstream regulatory molecules by SMARCA4 knockdown (si-SMARCA4) and IRAK1 recovery (pcDNA3.1_IRAK1);
C: Ras-Tg 마우스 모델의 투여 스캐줄;C: dosing schedule of Ras -Tg mouse model;
D: 종양 질량수;D: tumor mass number;
E: Ras-Tg 마우스의 간 조직 웨스턴 블롯 분석 결과; 및E: Western blot analysis of liver tissue from Ras-Tg mice; and
F: SMARCA4-IRAK1-Gankyrin, AKR1B10의 기전 모식도.F: Schematic diagram of the mechanism of SMARCA4-IRAK1-Gankyrin, AKR1B10.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.
또한, 본 명세서에서 사용되는 용어(Terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terms (Terminology) used in this specification are terms used to properly express a preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.
본 발명에서 용어, "대상체" 또는 "환자"는 인간, 유인원, 원숭이, 소, 개, 기니아 피그, 토끼, 닭, 곤충 등을 포함하여 치료가 요구되는 임의의 단일 개체를 의미한다. 또한, 임의의 질병 임상 소견을 보이지 않는 임상 연구 시험에 참여한 임의의 대상 또는 역학 연구에 참여한 대상 또는 대조군으로 사용된 대상이 대상에 포함된다. As used herein, the term "subject" or "patient" means any single individual in need of treatment, including humans, apes, monkeys, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included in the subject are any subjects who participated in a clinical study trial without any clinical manifestations of any disease, or subjects who participated in epidemiological studies or subjects used as controls.
본 발명에서 용어, "시료(샘플)"는 대상 또는 환자로부터 얻은 생물학적 시료를 의미한다. 생물학적 시료의 공급원은 신선한, 동결된 및/또는 보존된 장기 또는 조직 샘플 또는 생검 또는 흡인물로부터의 고형 조직; 혈액 또는 임의의 혈액 구성분; 대상의 임신 또는 발생의 임의의 시점의 세포일 수 있다. As used herein, the term “sample (sample)” refers to a biological sample obtained from a subject or patient. Sources of biological samples may include fresh, frozen and/or preserved organ or tissue samples or solid tissue from biopsies or aspirates; blood or any blood component; The cells may be at any point in the pregnancy or development of the subject.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다.All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.
일 측면에서, 본 발명은 SMARCA4(SWI/SNF related, Matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), SMARCC1, SMARCA2 및 IRAK1(Interleukin-1 receptor-associated kinase 1)으로 이루어진 군으로부터 선택되는 하나 이상의 유전자 또는 상기 유전자로부터 발현된 단백질을 포함하는 간암 진단용 바이오 마커에 관한 것이다.In one aspect, the present invention is selected from the group consisting of SMARCA4 (SWI / SNF related, Matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), SMARCC1, SMARCA2 and IRAK1 (Interleukin-1 receptor-associated kinase 1) It relates to a biomarker for diagnosing liver cancer comprising one or more genes or proteins expressed from the genes.
일 구현예에서, 상기 바이오 마커는 SMARCA4 및 IRAK1의 유전자 또는 상기 유전자로부터 발현된 단백질을 함께 포함할 수 있다.In one embodiment, the biomarker may include genes of SMARCA4 and IRAK1 or proteins expressed from the genes together.
일 구현예에서, 간암은 간세포암(Hepatocellular carcinoma, HCC)일 수 있으며, IRAK1이 과발현되는 간세포암인 것이 더욱 바람직하다.In one embodiment, the liver cancer may be hepatocellular carcinoma (HCC), more preferably hepatocellular carcinoma overexpressing IRAK1.
일 구현예에서, 간세포암은 IRAK1이 과발현되는 것일 수 있으나 이에 한정되는 것은 아니다.In one embodiment, the hepatocellular carcinoma may be IRAK1 overexpression, but is not limited thereto.
일 구현예에서, Gankyrin 또는 AKR1B10(Aldo-keto reductase family 1 member B10)의 유전자 또는 상기 유전자로부터 발현된 단백질을 추가로 포함할 수 있다.In one embodiment, it may further include a gene of Gankyrin or AKR1B10 (Aldo-keto reductase family 1 member B10) or a protein expressed from the gene.
일 구현예에서, SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10는 간암 특이적으로 발현이 증가할 수 있고, SMARCA2는 간암 특이적으로 발현이 감소할 수 있다.In one embodiment, SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 may increase liver cancer-specific expression, and SMARCA2 may decrease liver cancer-specific expression.
일 측면에서, 본 발명은 SMARCA4, SMARCC1, SMARCA2 및 IRAK1로 이루어진 군으로부터 선택되는 하나 이상의 바이오 마커 유전자의 발현량을 mRNA 또는 단백질 수준에서 측정하는 제제를 포함하는, 간암 진단용 조성물에 관한 것이다.In one aspect, the present invention relates to a composition for diagnosing liver cancer, comprising an agent for measuring the expression level of one or more biomarker genes selected from the group consisting of SMARCA4, SMARCC1, SMARCA2 and IRAK1 at the mRNA or protein level.
일 구현예에서, 상기 조성물은 Gankyrin 또는 AKR1B10의 발현량을 mRNA 또는 단백질 수준에서 측정하는 제제를 추가로 포함할 수 있다.In one embodiment, the composition may further comprise an agent for measuring the expression level of Gankyrin or AKR1B10 at the mRNA or protein level.
일 구현예에서, 간암은 간세포암일 수 있으며, IRAK1이 과발현되는 간세포암인 것이 더욱 바람직하다.In one embodiment, the liver cancer may be hepatocellular carcinoma, more preferably hepatocellular carcinoma overexpressing IRAK1.
일 구현예에서, 본 발명의 조성물은 SMARCA4 및 IRAK1 유전자의 발현량을 mRNA 또는 단백질 수준에서 측정하는 제제를 포함할 수 있다.In one embodiment, the composition of the present invention may include an agent for measuring the expression levels of SMARCA4 and IRAK1 genes at the mRNA or protein level.
일 구현예에서, 바이오 마커 유전자의 발현량을 mRNA 수준에서 측정하는 제제는, 상기 마커의 핵산서열, 상기 핵산서열에 상보적인 핵산서열, 상기 핵산서열 및 상보적인 서열의 단편을 특이적으로 인식하는 프라미어 쌍, 프로브, 또는 프라이머 쌍 및 프로브를 포함할 수 있으며, 상기 측정은 중합효소연쇄반응, 실시간 RT-PCR (Real-time RT-PCR), 역전사 중합효소연쇄반응, 경쟁적 중합효소연쇄반응(Competitive RT-PCR), Nuclease 보호 분석(RNase, S1 nuclease assay), in situ 교잡법, 핵산 마이크로어레이, 노던블랏 또는 DNA 칩으로 이루어진 군으로부터 선택되는 방법으로 수행될 수 있다.In one embodiment, the agent for measuring the expression level of the biomarker gene at the mRNA level is a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence and the complementary sequence to specifically recognize It may include a primer pair, a probe, or a primer pair and a probe, and the measurement is a polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction ( Competitive RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or a method selected from the group consisting of a DNA chip.
일 구현예에서, 바이오 마커 유전자의 발현량을 단백질 수준에서 측정하는 제제는, 상기 마커의 단백질 전장 또는 그 단편을 특이적으로 인식하는 항체, 항체단편, 앱타머(Aptamer), 아비머(Avidity multimer) 또는 펩티도모방체(Peptidomimetics)일 수 있으며, 상기 측정은 웨스턴블랏, ELISA(Enzyme linked immunosorbent assay), 방사선면역분석(RIA: Radioimmunoassay), 방사면역확산법(Radioimmunodiffusion), 면역 전기영동, 조직면역염색, 면역침전 분석법(Immunoprecipitation assay), 보체 고정 분석법(Complement Fixation Assay), FACS, 질량분석 또는 단백질 마이크로어레이로 이루어진 군으로부터 선택되는 방법으로 수행될 수 있다.In one embodiment, the agent for measuring the expression level of a biomarker gene at the protein level is an antibody, antibody fragment, aptamer, avimer that specifically recognizes the full protein length of the marker or a fragment thereof ) or peptidomimetics, the measurement may be Western blot, ELISA (Enzyme linked immunosorbent assay), Radioimmunoassay (RIA), Radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining , immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or a method selected from the group consisting of protein microarray.
본 발명에서 사용된 용어 "검출" 또는 "측정"은 검출 또는 측정된 대상의 농도를 정량하는 것을 의미한다.As used herein, the term “detection” or “measurement” refers to quantifying the concentration of a detected or measured target.
본 발명에서 용어, "프라이머"는 짧은 자유 3말단 수산화기(Free 3 hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(Template)와 염기쌍(Base pair)를 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 폴리머레이트 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시할 수 있다. As used herein, the term "primer" is a nucleic acid sequence having a short free 3 hydroxyl group, which can form a complementary template and base pair, and serves as a starting point for template strand copying. It refers to a short nucleic acid sequence that functions. The primers are capable of initiating DNA synthesis in the presence of the four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerate or reverse transcriptase) in an appropriate buffer and temperature.
본 발명에서 용어, "프로브"란 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링 되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 프로브는 올리고 뉴클레오타이드(Oligonucleotide) 프로브, 단쇄 DNA(Single stranded DNA) 프로브, 이중쇄 DNA(Double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 통상의 기술분야에 공지된 것을 기초로 변형할 수 있으므로 본 발명에서는 이에 대해 특별히 한정하지 않는다.As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to mRNA, and is labeled to determine the presence or absence of a specific mRNA. can The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. The selection of suitable probes and hybridization conditions may be modified based on those known in the art, and therefore, the present invention is not particularly limited thereto.
본 발명의 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오타이드 하나 이상의 동족체로의 치환및 뉴클레오타이드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스소트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phossotriesters, phosphoro amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
본 발명에서, 프로브를 cDNA 분자와 혼성화시키는 적합한 조건은 최적화 절차에 의하여 일련의 과정으로 결정될 수 있다. 이런 절차는 연구실에서 사용을 위한 프로토콜을 수립하기 위하여 당업자에 의하여 일련의 과정으로 실시된다. 예를 들어, 온도, 성분의 농도, 혼성화 및 세척 시간, 완충액 성분 및 이들의 pH 및 이온세기 등의 조건은 프로브의 길이 및 GC 양 및 타깃 뉴클레오타이드 서열 등의 다양한 인자에 의존한다. 혼성화를 위한 상세한 조건은 Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); 및 M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.(1999)에서 확인할 수 있다. 예를 들어, 상기 엄격조건 중에서 고 엄격조건은 0.5 M NaHPO4, 7% SDS(Sodium dodecyl sulfate), 1 mM EDTA에서 65℃ 조건으로 혼성화하고, 0.1×SSC(Standard saline citrate)/0.1% SDS에서 68℃ 조건으로 세척하는 것을 의미한다. 또는, 고 엄격조건은 6×SSC/0.05% 소듐 파이로포스페이트에서 48℃ 조건으로 세척하는 것을 의미한다. 저 엄격조건은 예를 들어, 0.2×SSC/0.1% SDS에서 42℃ 조건으로 세척하는 것을 의미한다.In the present invention, suitable conditions for hybridizing a probe with a cDNA molecule can be determined in a series of processes by an optimization procedure. These procedures are carried out as a series of procedures by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999). For example, the high stringency conditions among the stringent conditions include hybridization in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and in 0.1×SSC (Standard saline citrate)/0.1% SDS. It means washing at 68°C conditions. Alternatively, high stringency conditions mean washing at 48° C. in 6×SSC/0.05% sodium pyrophosphate. Low stringency conditions mean washing at 42° C. in, for example, 0.2×SSC/0.1% SDS.
본 발명에서 용어, "항체"란 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 여기에는 상기 단백질에서 만들어질 수 있는 부분 펩티드도 포함된다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체도 포함된다.As used herein, the term “antibody” refers to a specific protein molecule directed to an antigenic site as a term known in the art. This also includes partial peptides that can be made from the protein. The form of the antibody of the present invention is not particularly limited, and a part thereof is also included in the antibody of the present invention as long as it has a polyclonal antibody, a monoclonal antibody, or antigen-binding property, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody.
일 측면에서, 본 발명은 본 발명의 조성물을 포함하는, 간암 진단 키트에 관한 것이다.In one aspect, the present invention relates to a liver cancer diagnostic kit comprising the composition of the present invention.
일 구현예에서, 상기 키트는 대상체 또는 환자로부터 생체 시료를 수집하기 위한 도구 및/또는 시약 뿐 아니라 그 시료로부터 게놈 DNA, cDNA, RNA 또는 단백질을 준비하기 위한 도구 및/또는 시약을 더 포함할 수 있다. 예를 들면, 게놈 DNA의 관련 영역을 증폭하기 위한 PCR 프라이머를 포함할 수 있다. 상기 키트는 약리 게놈학적 프로파일링에 유용한 유전 인자의 프로브를 포함할 수 있다. 또한, 이러한 키트의 사용에 있어서, 표지화된 올리고뉴클레오티드를 사용하여 분석 중 용이하게 동정할 수 있다.In one embodiment, the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient, as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA or protein from the sample. have. For example, it may include PCR primers for amplifying a relevant region of genomic DNA. The kit may include probes of genetic factors useful for pharmacogenomic profiling. In addition, in the use of such a kit, the labeled oligonucleotide can be easily identified during analysis.
본 발명에서, 사용된 용어 "진단"은 특정 질병 또는 질환에 대한 한 객체의 감수성(Susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는 지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(Prognosis)(예컨대, 전-전이성 또는 전이성 암 상태의 동정, 암의 단계 결정 또는 치료에 대한 암의 반응성 결정)를 판정하는 것, 또는 테라메트릭스(Therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링 하는 것)를 포함한다.In the present invention, the term "diagnosis" as used herein refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, a specific disease or disorder Determining the prognosis of a subject with the disease (eg, identification of a pre-metastatic or metastatic cancer state, staging the cancer, or determining the responsiveness of the cancer to treatment), or Therametrics (eg, monitoring the condition of the subject to provide information on treatment efficacy).
일 구현예에서, 상기 키트는 DNA 중합효소 및 dNTP(dGTP, dCTP, dATP 및 dTTP), 형광물질 등의 표지 물질을 추가로 더 함유할 수 있다.In one embodiment, the kit may further contain a labeling material such as a DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP), a fluorescent material, and the like.
일 측면에서, 본 발명은 SMARCA4 억제제를 유효성분으로 포함하는 간암 예방 또는 치료용 약학적 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
일 구현예에서, 상기 SMARCA4 억제제는 siRNA일 수 있으며, 서열번호 1의 siRNA일 수 있다.In one embodiment, the SMARCA4 inhibitor may be siRNA, or siRNA of SEQ ID NO: 1.
일 구현예에서, SMARCA2 또는 이의 발현 촉진제, Gankyrin 억제제, AKR1B10 억제제, SMARCC1 억제제, 또는 IRAK1 억제제를 추가로 포함할 수 있다. In one embodiment, SMARCA2 or an expression promoter thereof, a Gankyrin inhibitor, an AKR1B10 inhibitor, a SMARCC1 inhibitor, or an IRAK1 inhibitor may be further included.
일 구현예에서, 상기 IRAK1 억제제는 siRNA일 수 있으며, 서열번호 2의 siRNA일 수 있다.In one embodiment, the IRAK1 inhibitor may be siRNA, or siRNA of SEQ ID NO: 2.
일 구현예에서, 간암은 간세포암일 수 있으며, IRAK1이 과발현되는 간세포암인 것이 더욱 바람직하다.In one embodiment, the liver cancer may be hepatocellular carcinoma, more preferably hepatocellular carcinoma overexpressing IRAK1.
본 발명에 따른 약학적 조성물에는 SMARCA4, SMARCC1 또는 IRAK1의 발현을 억제하거나, SMARCA2의 발현을 촉진할 수 있는 물질이라면 모두 포함할 수 있다.The pharmaceutical composition according to the present invention may include any substance capable of inhibiting the expression of SMARCA4, SMARCC1, or IRAK1 or promoting the expression of SMARCA2.
본 발명에서 상기 본 발명의 약학적 조성물은 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 상기에서 "약학적으로 허용되는"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등 과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 약학적으로 허용되는 담체로는 예를 들면, 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등과 같은 경구 투여용 담체 및 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). In the present invention, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like, and carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
본 발명에 따른 약학적 조성물은 상술한 바와 같은 약학적으로 허용되는 담체와 함께 당업계에 공지된 방법에 따라 적합한 형태로 제형화될 수 있다. 즉, 본 발명의 약학적 조성물은 공지의 방법에 따라 다양한 비경구 또는 경구 투여용 형태로 제조될 수 있으며, 비경구 투여용 제형의 대표적인 것으로는 주사용 제형으로 등장성 수용액 또는 현탁액이 바람직하다. 주사용 제형은 적합한 분산제 또는 습윤제 및 현탁화제를 사용하여 당업계에 공지된 기술에 따라 제조할 수 있다. 예를 들면, 각 성분을 식염수 또는 완충액에 용해시켜 주사용으로 제형화될 수 있다. 또한, 경구 투여용 제형으로는, 이에 한정되지는 않으나, 분말, 과립, 정제, 환약 및 캡슐 등이 있다. 상기와 같은 방법으로 제형화된 약학적 조성물은 유효량으로 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있는데, 상기 "투여"란 어떠한 적절한 방법으로 환자에게 소정의 물질을 도입하는 것을 의미하며 물질의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 상기에서 "유효량"이란 환자에게 투여하였을 때, 예방 또는 치료 효과를 나타내는 양을 말한다. 본 발명에 따른 약학적 조성물의 투여량은 환자의 질환 종류 및 중증도, 연령, 성별, 체중, 약물에 대한 민감도, 현재 치료법의 종류, 투여방법, 표적 세포 등 다양한 요인에 따라 달라질 수 있으며, 당 분야의 전문가들에 의해 용이하게 결정될 수 있다. 또한, 본 발명의 약학적 조성물은 종래의 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 바람직하게는 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여할 수 있으며, 더욱 바람직하게는 1~10000 ㎍/체중kg/day, 더욱 더 바람직하게는 10~1000 ㎎/체중kg /day의 유효용량으로 하루에 수회 반복 투여될 수 있다. The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and as a representative dosage form for parenteral administration, an isotonic aqueous solution or suspension is preferred as an injectable dosage form. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving it in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules. The pharmaceutical composition formulated in the above manner may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the “administration” refers to introducing a predetermined substance into a patient by any suitable method. and the route of administration of the substance can be administered through any general route as long as it can reach the target tissue. As used herein, the term “effective amount” refers to an amount exhibiting a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the type and severity of the patient's disease, age, sex, weight, sensitivity to drugs, the type of current treatment, administration method, target cell, etc. can be easily determined by experts in In addition, the pharmaceutical composition of the present invention may be administered in combination with a conventional therapeutic agent, may be administered sequentially or simultaneously with the conventional therapeutic agent, and may be administered single or multiple. Preferably, in consideration of all of the above factors, an amount that can obtain the maximum effect with a minimum amount without side effects can be administered, more preferably 1 to 10000 μg/weight kg/day, even more preferably 10 to 1000 It can be administered repeatedly several times a day at an effective dose of mg/kg body weight/day.
일 구현예에서, SMARCA2의 발현 촉진제는, 바람직하게는 화학물질, 뉴클레오티드, 상기 유전자를 포함하는 벡터, 상기 유전자가 번역된 단백질 또는 이의 단편, 또는 천연물 추출물을 유효성분으로 포함할 수 있다.In one embodiment, the SMARCA2 expression promoter, preferably a chemical substance, a nucleotide, a vector containing the gene, a protein or fragment thereof into which the gene is translated, or a natural product extract may be included as an active ingredient.
본 발명에서 사용되는 용어, "발현 촉진"이란 표적 유전자 mRNA의 발현 또는 단백질로의 번역 증진을 야기하는 것을 의미한다.As used herein, the term "promotion of expression" means to cause enhancement of expression of target gene mRNA or translation into protein.
일 구현예에서, SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10의 발현 억제제는, 바람직하게는 화학물질, 뉴클레오티드, 안티센스, siRNA 올리고뉴클레오타이드 또는 천연물 추출물을 유효성분으로 포함할 수 있으며, 더욱 바람직하게는 상기 유전자의 뉴클레오티드 서열에 상보적인 서열을 가지는 안티센스 또는 siRNA(Small interference RNA) 올리고뉴클레오티드를 유효성분으로 포함할 수 있다.In one embodiment, the expression inhibitor of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 may include a chemical substance, nucleotide, antisense, siRNA oligonucleotide or a natural product extract as an active ingredient, more preferably the gene An antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to the nucleotide sequence of may be included as an active ingredient.
본 발명에서 사용되는 용어, "발현 억제"란 표적 유전자 mRNA 발현 또는 단백질로의 번역 저하를 야기하는 것을 의미하며, 바람직하게는 이에 의해 표적 유전자 발현이 탐지 불가능해지거나 무의미한 수준으로 존재하게 되는 것을 의미한다.As used herein, the term "expression inhibition" means to cause a decrease in target gene mRNA expression or translation into a protein, and preferably means that the target gene expression becomes undetectable or exists at an insignificant level. do.
본 발명에서 사용되는 용어, "siRNA(Small interfering RNA)"란 RNA 방해 또는 유전자 사일런싱을 매개할 수 있는 핵산 분자를 의미한다(국제 공개특허 번호 00/44895, 01/36646, 99/32619, 01/29058, 99/07409 및 00/44914 참조). siRNA는 표적 유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 넉다운(Knock-down) 방법으로서 또는 유전자 치료 방법으로 제공된다. 본 발명의 siRNA 분자는, 센스 가닥(SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10의 mRNA 서열에 상응하는 서열)과 안티센스 가닥(SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10의 mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조를 가질 수 있으며, 또한, 본 발명의 siRNA 분자는 자기-상보성(Self-complementary) 센스 및 안티센스 가닥을 가지는 단일쇄 구조를 가질 수 있다. 나아가 siRNA는 RNA끼리 짝을 이루는 이중사슬 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 미스매치(대응하는 염기가 상보적이지 않음), 벌지(일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. 또한, siRNA 말단 구조는 SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10 유전자의 발현을 RNAi 효과에 의하여 억제할 수 있는 것이면 평활(Blunt) 말단 혹은 점착(Cohesive) 말단 모두 가능하고, 점착 말단 구조는 3'-말단 돌출 구조와 5'-말단 돌출 구조 모두 가능하다. 또한, 본 발명의 siRNA 분자는 자기-상보성(Self-complementary) 센스 및 안티센스 가닥 사이에 짧은 뉴클레오티드 서열이 삽입된 형태를 가질 수 있으며, 이 경우 뉴클레오타이드 서열의 발현에 의해 형성된 siRNA 분자는 분자내 혼성화에 의하여 헤어핀 구조를 형성하게 되며, 전체적으로는 스템-앤드-루프 구조를 형성하게 된다(shRNA). 이 스템-앤드-루프 구조는 인 비트로 또는 인 비보에서 프로세싱되어 RNAi를 매개할 수 있는 활성의 siRNA 분자를 생성한다. siRNA를 제조하는 방법은 시험관에서 siRNA를 직접 합성한 뒤, 형질전환 과정을 거쳐 세포 안으로 도입시키는 방법과 siRNA가 세포 안에서 발현되도록 제조된 siRNA 발현 벡터 또는 PCR-유래의 siRNA 발현 카세트 등을 세포 안으로 형질전환 또는 감염(Infection)시키는 방법이 있다.As used herein, the term "siRNA (Small interfering RNA)" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Patent Publication Nos. 00/44895, 01/36646, 99/32619, 01 see /29058, 99/07409 and 00/44914). Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method. In the siRNA molecule of the present invention, the sense strand (sequence corresponding to the mRNA sequence of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10) and the antisense strand (sequence complementary to the mRNA sequence of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10) are mutually exclusive The siRNA molecule of the present invention may have a structure that is positioned on the opposite side to form a double-stranded chain, and the siRNA molecule of the present invention may have a single-stranded structure having self-complementary sense and antisense strands. Furthermore, siRNA is not limited to the complete pairing of double-stranded RNA parts that are paired with each other, but pairs are formed by mismatch (corresponding bases are not complementary), bulges (there is no base corresponding to one chain), etc. There may be parts that are not achieved. In addition, as long as the siRNA end structure can inhibit the expression of SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 genes by the RNAi effect, both blunt ends or cohesive ends are possible, and the cohesive end structure is 3'- Both a terminal overhang structure and a 5'-end overhang structure are possible. In addition, the siRNA molecule of the present invention may have a form in which a short nucleotide sequence is inserted between self-complementary sense and antisense strands. In this case, the siRNA molecule formed by expression of the nucleotide sequence is used for intramolecular hybridization. Thus, a hairpin structure is formed, and as a whole, a stem-and-loop structure is formed (shRNA). This stem-and-loop structure can be processed in vitro or in vivo to generate active siRNA molecules capable of mediating RNAi. Methods for preparing siRNA include direct synthesis of siRNA in a test tube and introduction into cells through transformation, and transfection of an siRNA expression vector or PCR-derived siRNA expression cassette prepared so that siRNA is expressed in the cell. There is a way to convert or infect.
일 구현예에서, 유전자 특이적인 siRNA를 포함하는 본 발명의 조성물은 siRNA의 세포 내 유입을 촉진시키는 제제를 포함할 수 있다. siRNA의 세포 내 유입을 촉진시키는 제제에는 일반적으로 핵산 유입을 촉진하는 제제를 사용할 수 있으며, 이러한 예로는, 리포좀을 이용하거나 콜레스테롤, 콜레이트 및 데옥시콜산을 비롯한 다수의 스테롤류 중 1종의 친유성 담체와 함께 배합할 수 있다. 또한 폴리-L-라이신(Poly-L-lysine), 스퍼민(Spermine), 폴리실아잔(Polysilazane), 폴리에틸레민(PEI: polyethylenimine), 폴리디하이드로이미다졸레늄(Polydihydroimidazolenium), 폴리알리라민(Polyallylamine), 키토산(Chitosan) 등의 양이온성 고분자(Cationic polymer)를 이용할 수도 있고, 숙실화된 PLL(Succinylated PLL), 숙실화된 PEI(Succinylated PEI), 폴리글루타믹산(Polyglutamic acid), 폴리아스파틱산(Polyaspartic acid), 폴리아크릴산(Polyacrylic acid), 폴리메타아크릴산(Polymethacylic acid), 덱스트란 설페이트(Dextran sulfate), 헤파린(Heparin), 히아루릭산(Hyaluronic acid) 등의 음이온성 고분자(Anionic polymer)를 이용할 수 있다. In one embodiment, the composition of the present invention comprising a gene-specific siRNA may include an agent that promotes the influx of the siRNA into a cell. In general, an agent that promotes nucleic acid influx can be used for an agent that promotes siRNA entry into cells. For example, a liposome is used or a lipophilic one of a number of sterols including cholesterol, cholate and deoxycholic acid. It can be formulated with a carrier. Also, poly-L-lysine, spermine, polysilazane, polyethylenimine (PEI), polydihydroimidazolenium, polyallylamine ), a cationic polymer such as chitosan may be used, and succinylated PLL (Succinylated PLL), succinylated PEI (Succinylated PEI), polyglutamic acid, polyaspartic acid (Polyaspartic acid), polyacrylic acid (Polyacrylic acid), polymethacylic acid (Polymethacylic acid), dextran sulfate (Dextran sulfate), heparin (Heparin), anionic polymers such as hyaluronic acid (Hyaluronic acid) Available.
일 구현예에서, 상기 SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10 단백질의 발현 및 활성을 감소시키는 물질로서 상기 단백질에 특이적인 항체를 사용할 경우, 상기 항체는 기존의 치료제와 직접 또는 링커 등을 통하여 간접적으로 커플링(예를 들어, 공유결합)시킬 수 있다. In one embodiment, when an antibody specific for the protein is used as a substance for reducing the expression and activity of the SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 protein, the antibody is directly or indirectly through a linker with an existing therapeutic agent. couple (eg, covalently).
본 발명에서 사용되는 용어, "안티센스 올리고뉴클레오타이드"란 특정 mRNA의 서열에 상보적인 핵산 서열을 함유 하고 있는 DNA 또는 RNA 또는 이들의 유도체를 의미하고, mRNA 내의 상보적인 서열에 결합하여 SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10의 단백질로의 번역을 저해하는 작용을 한다. 본 발명의 안티센스 서열은 SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10 mRNA에 상보적이고 SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10 mRNA에 결합할 수 있는 DNA 또는 RNA 서열을 의미하며, SMARCA4, SMARCC1, IRAK1, Gankyrin 또는 AKR1B10 mRNA의 번역, 세포질 내로의 전위(Translocation), 성숙(Maturation) 또는 다른 모든 전체적인 생물학적 기능에 대한 필수적인 활성을 저해할 수 있다. 또한, 상기 안티센스 핵산은 효능을 증진시키기 위하여 하나 이상의 염기, 당 또는 골격(Backbone)의 위치에서 변형될 수 있다(De Mesmaeker et al., Curr Opin Struct Biol., 5, 3, 343-55, 1995). 핵산 골격은 포스포로티 오에이트, 포스포트리에스테르, 메틸 포스포네이트, 단쇄 알킬, 시클로알킬, 단쇄 헤테로아토믹, 헤테로시클릭 당 간 결합 등으로 변형될 수 있다. 또한, 안티센스 핵산은 하나 이상의 치환된 당 모이어티(Sugar moiety)를 포함할 수 있다. 안티센스 핵산은 변형된 염기를 포함할 수 있다. 변형된 염기에는 하이포크잔틴, 6-메틸아데닌, 5-메틸피리미딘(특히, 5-메틸시토신), 5-하이드록시메틸시토신(HMC), 글리코실 HMC, 젠토비오실 HMC, 2-아미노아 데닌, 2-티오우라실, 2-티오티민, 5-브로모우라실, 5-하이드록시메틸우라실, 8-아자구아닌, 7-데아자구아닌, N6(6-아미노헥실)아데닌, 2,6-디아미노퓨린 등이 있다. 또한, 본 발명의 안티센스 핵산은 상기 안티센스 핵산의 활성 및 세포 흡착성을 향상시키는 하나 이상의 모이어티(Moiety) 또는 컨쥬게이트(Conjugate)와 화학적으로 결합될 수 있다. 콜레스테롤 모이어티, 콜레스테릴 모이어티, 콜릭산, 티오에테르, 티오콜레스테롤, 지방성 사슬, 인지질, 폴리아민, 폴리에틸렌 글리콜 사슬, 아다맨탄 아세트산, 팔미틸 모이어티, 옥타데실아민, 헥실아미노카르보닐-옥시콜에스테롤 모이어티 등의 지용성 모이어티 등이 있고 이에 제한되지는 않는다. 지용성 모이어티를 포함하는 올리고뉴클레오티드와 제조 방법은 본 발명의 기술 분야에서 이미 잘 알려져 있다(미국특허 제5,138,045호, 제5,218,105호 및 제5,459,255호). 상기 변형된 핵산은 뉴클레아제에 대한 안정성을 증가시키고 안티센스 핵산과 표적 mRNA와의 결합 친화력을 증가시킬 수 있다. 안티센스 올리고뉴클레오타이드는 통상의 방법으로 시험관에서 합성되어 생체 내로 투여하거나 생체 내에서 안티센스 올리고뉴클레오타이드가 합성되도록 할 수 있다. 시험관에서 안티센스 올리고뉴클레오타이드를 합성하는 일예는 RNA 중합효소 I를 이용하는 것이다. 생체 내에서 안티센스 RNA가 합성되도록 하는 한 가지 예는 인식부 위(MCS)의 기원이 반대 방향에 있는 벡터를 사용하여 안티센스 RNA가 전사되도록 하는 것이다. 이런 안티센스 RNA는 서열 내에 번역 중지 코돈이 존재하도록 하여 펩타이드 서열로 번역되지 않도록 하는 것이 바람직하다.As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and is bound to a complementary sequence in mRNA to form SMARCA4, SMARCC1, IRAK1 , inhibits the translation of Gankyrin or AKR1B10 into protein. The antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 mRNA and is capable of binding to SMARCA4, SMARCC1, IRAK1, Gankyrin or AKR1B10 mRNA, SMARCA4, SMARCC1, IRAK1, Gankyrin or It can inhibit AKR1B10 mRNA's essential activity for translation, translocation into the cytoplasm, maturation or all other overall biological functions. In addition, the antisense nucleic acid may be modified at one or more bases, sugars, or positions of the backbone to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol ., 5, 3, 343-55, 1995). ). The nucleic acid backbone may be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic intersaccharide linkages, and the like. Antisense nucleic acids may also include one or more substituted sugar moieties. Antisense nucleic acids may include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-methylpyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2-aminoadenine , 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diamino purines, etc. In addition, the antisense nucleic acid of the present invention may be chemically bound to one or more moieties or conjugates that improve the activity and cell adsorption properties of the antisense nucleic acid. Cholesterol moiety, cholesteryl moiety, cholic acid, thioether, thiocholesterol, fatty chain, phospholipid, polyamine, polyethylene glycol chain, adamantane acetic acid, palmityl moiety, octadecylamine, hexylaminocarbonyl-oxychol fat soluble moieties such as esterol moieties, and the like. Oligonucleotides comprising a fat-soluble moiety and methods of preparation are well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid may increase the stability to nucleases and increase the binding affinity between the antisense nucleic acid and the target mRNA. Antisense oligonucleotides can be synthesized in vitro by a conventional method and administered in vivo, or antisense oligonucleotides can be synthesized in vivo. An example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to use a vector with the origin of the recognition site (MCS) in the opposite direction to allow the antisense RNA to be transcribed. Such antisense RNA preferably has a translation stop codon in the sequence so that it is not translated into the peptide sequence.
일 측면에서, 본 발명은 (a) 검사 대상체로부터 분리한 생물학적 시료에서 SMARCA4 또는 IRAK1의 유전자 발현량을 측정하는 단계; (b) 정상 대조군 시료의 해당 유전자의 상응하는 결과와 비교하는 단계; 및 (c) (a) 단계에서의 유전자 발현량이 (b) 단계에서의 해당 유전자의 발현 수준에 비해 높은 경우, 상기 검사 대상체가 간암일 것으로 판단하는 단계를 포함하는, 간암 진단에 필요한 정보를 제공하는 방법에 관한 것이다.In one aspect, the present invention comprises the steps of (a) measuring the gene expression level of SMARCA4 or IRAK1 in a biological sample isolated from a test subject; (b) comparing the corresponding gene of the normal control sample with the corresponding result; and (c) when the gene expression level in step (a) is higher than the expression level of the corresponding gene in step (b), determining that the test subject is liver cancer, providing information necessary for diagnosing liver cancer It's about how to do it.
일 구현예에서, 상기 방법은 (a) 검사 대상체로부터 분리한 생물학적 시료에서 SMARCC1, SMARCA2, Gankyrin 또는 AKR1B10의 유전자 또는 단백질 발현량을 측정하는 단계; (b) 정상 대조군 시료의 해당 유전자 또는 단백질의 상응하는 결과와 비교하는 단계; 및 (c) SMARCC1, Gankyrin 또는 AKR1B10의 (a) 단계에서의 발현량이 (b) 단계에서의 해당 유전자 또는 단백질의 발현 수준에 비해 높은 경우, 또는 SMARCA2의 (a) 단계에서의 발현량이 (b) 단계에서의 해당 유전자 또는 단백질의 발현 수준에 비해 낮은 경우, 상기 검사 대상체가 간암일 것으로 판단하는 단계를 추가로 포함할 수 있다.In one embodiment, the method comprises the steps of (a) measuring the gene or protein expression level of SMARCC1, SMARCA2, Gankyrin, or AKR1B10 in a biological sample isolated from a test subject; (b) comparing the corresponding gene or protein of the normal control sample with the corresponding result; And (c) when the expression level in step (a) of SMARCC1, Gankyrin or AKR1B10 is high compared to the expression level of the corresponding gene or protein in step (b), or the expression level in step (a) of SMARCA2 (b) When it is low compared to the expression level of the gene or protein in the step, the step of determining that the test subject is liver cancer may be further included.
일 구현예에서, 생물학적 시료는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨와 같은 시료 등을 포함할 수 있다.In one embodiment, the biological sample may include a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and the like.
일 측면에서, 본 발명은 (a) 검사 대상체로부터 분리한 생물학적 시료에서 SMARCA4 또는 IRAK1의 유전자 발현량을 측정하는 단계; (b) 정상 대조군 시료의 해당 유전자의 상응하는 결과와 비교하는 단계; 및 (c) (a) 단계에서의 유전자 발현량이 (b) 단계에서의 해당 유전자의 발현 수준에 비해 높은 경우, 상기 검사 대상체의 예후가 나쁜 것으로 판단하는 단계를 포함하는, 간암의 예후 예측을 위한 정보를 제공하는 방법에 관한 것이다.In one aspect, the present invention comprises the steps of (a) measuring the gene expression level of SMARCA4 or IRAK1 in a biological sample isolated from a test subject; (b) comparing the corresponding gene of the normal control sample with the corresponding result; and (c) when the gene expression level in step (a) is higher than the expression level of the corresponding gene in step (b), determining that the prognosis of the test subject is bad, for predicting the prognosis of liver cancer How to provide information.
일 측면에서, 본 발명은 IRAK1의 발현량을 측정하는 단계를 포함하는, SMARCA4 억제제의 간암 치료 반응성 예측방법에 관한 것이다.In one aspect, the present invention relates to a method for predicting liver cancer treatment responsiveness of a SMARCA4 inhibitor, comprising measuring the expression level of IRAK1.
일 구현예에서, 상기 방법은 Gankyrin 또는 AKR1B10의 발현량을 측정하는 단계를 추가로 포함할 수 있다.In one embodiment, the method may further comprise measuring the expression level of Gankyrin or AKR1B10.
일 측면에서, SMARCA4 억제제를 유효성분으로 포함하는 간암 예방 또는 치료용 약학적 조성물을 개체에 투여하는 단계를 포함하는 간암 예방 또는 치료 방법에 관한 것이다.In one aspect, it relates to a method for preventing or treating liver cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.
실시예 1. HCC에서 SWI/SNF 복합체의 서브유닛 유전자 발현 수준 평가Example 1. Evaluation of subunit gene expression level of SWI/SNF complex in HCC
HCC에서 서브유닛 유전자의 발현 수준을 확인하기 위해, TCGA_LIHC(The Cancer Genome Atlas liver hepatocellular carcinoma project), ICGC_LIRI(International Cancer Genome Consortium liver Cancer-RIKEN, JP) 및 NCBI의 GEO(Gene Expression Omnibus) 데이터베이스(Accession Numbers: GSE6764, GSE22058, GSE25097, GSE54238, GSE62043, GSE77314, GSE89377 및 GSE114564)에서 데이터를 얻었다. TCGA-LIHC HTSeq-FPKM의 레벨 3 mRNA 발현 데이터를 log2 변환시키고[log2(fpkm+1)] 유전자 발현 수준을 평가하는데 사용하였다. 그 결과, 인간 HCC(cBioPortal; n=1,019)는 SWI/SNF 서브유닛 유전자의 돌연변이 비율이 매우 낮은 것으로 나타났다(도 1a). 이와 같은 유전자 변형(Genetic alteration)의 낮은 발생율 및 비균질성(Heterogeneity)은 특정 서브유닛 유전자의 우세한 이상 발현이 SWI/SNF 복합체의 시스템 장애(Systemic disturbances)를 야기할 수 있음을 시사한다. 이에, HCC 데이터(Catholic_mLIHC; GSE114564)와 TCGA_LIHC, ICGC_LIRI 세트를 이용하여 SWI/SNF 서브유닛 유전자의 발현을 검토한 결과(도 1b), 10개의 SWI/SNF 서브유닛 유전자 중에, SMARCA4 및 SMARCC1가 HCC 환자에서 현저히 과발현되어 있었으며, 동시에 SMARCA2가 하향 조절되어 있음을 확인할 수 있었다(≥ ± 1.5 fold, P<0.05). 또한, 동일한 환자 정보를 포함하고 있는 HCC 데이터(TCGA_LIHC, ICGC_LIRI, GSE77314)에서 동일한 환자의 비암성 간 조직과 HCC조직에서 SMARCA4 및 SMARCC1이 과발현되어 있었으며, 동시에 SMARCA2가 하향 조절되어 있음을 확인 할 수 있었다(≥ ± 1.5 fold, P<0.05) (도 1c). 또한, 34개의 HCC 조직과 이에 상응하는 비암성 간 생검의 HCC의 조직 쌍을 한국의 National Biobank에서 획득하여 이에 대해 qRT-PCR로 분석한 결과, SMARCA4, SMARCA2 및 SMARCC1의 이상 조절이 TCGA_LIHC, ICGC_LIRI 및 GSE77314에서 일치하는 HCC의 조직 쌍에서도 확인되었다(도 1c). 또한, 무작위로 선택된 34개의 HCC 조직의 추가 세트에서 qRT-PCR로도 추가 검증한 결과, SMARCA4(67.6%) 및 SMARCC1(52.9%) 과발현과 SMARCA2(61.8%) 억제가 이 세트에서 일관되게 관찰되었다(도 1d). 아울러, 무작위로 선별한 10개 조직 쌍을 웨스턴 블롯으로 분석한 결과에서도 SMARCA4 및 SMARCC1의 현저한 과발현과 동시에 SMARCA2의 억제가 HCC에서 관찰되었다(도 1e).To confirm the expression level of subunit genes in HCC, TCGA_LIHC (The Cancer Genome Atlas liver hepatocellular carcinoma project), ICGC_LIRI (International Cancer Genome Consortium liver Cancer-RIKEN, JP) and NCBI's Gene Expression Omnibus (GEO) database (Accession) Numbers: GSE6764, GSE22058, GSE25097, GSE54238, GSE62043, GSE77314, GSE89377 and GSE114564). Level 3 mRNA expression data of TCGA-LIHC HTSeq-FPKM were log2 transformed [log2(fpkm+1)] and used to evaluate gene expression levels. As a result, it was found that human HCC (cBioPortal; n=1,019) had a very low mutation rate of the SWI/SNF subunit gene ( FIG. 1A ). The low incidence and heterogeneity of such genetic alterations suggests that the predominantly aberrant expression of specific subunit genes may cause systemic disturbances of the SWI/SNF complex. Accordingly, as a result of examining the expression of SWI/SNF subunit genes using HCC data (Catholic_mLIHC; GSE114564), TCGA_LIHC, and ICGC_LIRI sets (FIG. 1b), among 10 SWI/SNF subunit genes, SMARCA4 and SMARCC1 are HCC patients was significantly overexpressed, and at the same time, it could be confirmed that SMARCA2 was down-regulated (≥ ± 1.5 fold, P<0.05). Also, in the HCC data (TCGA_LIHC, ICGC_LIRI, GSE77314) containing the same patient information, it was confirmed that SMARCA4 and SMARCC1 were overexpressed in noncancerous liver and HCC tissues of the same patient, and SMARCA2 was downregulated at the same time. (≥ ± 1.5 fold, P<0.05) (Fig. 1c). In addition, as a result of qRT-PCR analysis of 34 HCC tissues and corresponding HCC tissue pairs from noncancerous liver biopsies obtained from the National Biobank of Korea, abnormal regulation of SMARCA4, SMARCA2 and SMARCC1 showed that TCGA_LIHC, ICGC_LIRI and A tissue pair of matched HCC in GSE77314 was also identified (Fig. 1c). In addition, further validation with qRT-PCR in an additional set of 34 randomly selected HCC tissues, SMARCA4 (67.6%) and SMARCC1 (52.9%) overexpression and SMARCA2 (61.8%) inhibition were consistently observed in this set ( Fig. 1d). In addition, as a result of Western blot analysis of 10 randomly selected tissue pairs, significant overexpression of SMARCA4 and SMARCC1 and inhibition of SMARCA2 were observed in HCC at the same time (FIG. 1e).
실시예 2. SMARCA4, SMARCA2 및 SMARCC1의 발현 조절에 의한 항종양 효과Example 2. Anti-tumor effect by regulation of expression of SMARCA4, SMARCA2 and SMARCC1
2-1. in vitro 분석2-1. in vitro analysis
상기 실시예에서 HCC 특이적으로 발현이 변화한 SMARCA4, SMARCA2 및 SMARCC1가 간암 발생(Hepatocarcinogenesis)에서 어떤 역할을 하는지 확인하기 위해, 각 유전자를 RNA 간섭으로 낙다운하였다. 이를 위해, 정상 간세포보다 SMARCA4 및 SMARCC1 발현이 비교적 높고, SMARCA2 발현이 비교적 낮은 간 세포주인 Hep3B, Huh7 및 SNU-449에서 각 유전자를 RNA 간섭으로 낙다운하고, 세포생존율, 세포 증식률을 MTT 분석, BrdU 분석 및 집락형성 분석(Clonogenic assay) 분석으로 확인하였다. 또한, 각 유전자의 낙다운 HCC 세포의 이동 및 침습을 보이든 챔버 운동성 분석(Boyden chamber motility assay) 및 상처 치유 분석(Wound healing assay)으로 확인하였다. 아울러, 각 유전자의 낙다운이 HCC 세포에서 세포 주기 조절에 미치는 영향을 유세포 분석(Flow cytometric analysis)으로 확인하고, 세포 주기 조절 단백질을 웨스턴 블롯 분석으로 확인하였다.In order to confirm the role of SMARCA4, SMARCA2, and SMARCC1 whose HCC-specific expression is changed in the above example, in hepatocarcinogenesis, each gene was knocked down by RNA interference. To this end, each gene was knocked down by RNA interference in Hep3B, Huh7 and SNU-449 liver cell lines with relatively high SMARCA4 and SMARCC1 expression and relatively low SMARCA2 expression than in normal hepatocytes, and cell viability and cell proliferation rates were analyzed by MTT analysis, BrdU It was confirmed by analysis and colonogenic assay analysis. In addition, the migration and invasion of knockdown HCC cells of each gene were confirmed by Boyden chamber motility assay and wound healing assay. In addition, the effect of knockdown of each gene on cell cycle regulation in HCC cells was confirmed by flow cytometric analysis, and cell cycle regulatory proteins were confirmed by Western blot analysis.
그 결과, SMARCA4 및 SMARCC1의 낙다운이 HCC 세포의 세포 성장 및 증식 비율을 현저히 억제하는 것으로 나타났다(도 2a 내지 d). 또한, SMARCA4-null HCC 세포주인 PLC/PRF/5 및 SK-Hep-1 모두에서 SMARCA2 낙다운은 HCC 세포의 종합적인 치사율을 나타냈으나, SMARCA4를 고발현하는 Hep3B 세포 및 Huh7 세포는 성장률에 변화가 없는 것으로 나타났다(도 2e). 이 결과는 간세포암 발생에서 SMARCA4-SWI/SNF 복합체의 중추적인 역할을 나타낸다. 또한, 보이든 챔버 운동성 분석 결과에서 SMARCA4 낙다운이 HCC 세포의 혈청-자극된 이동 및 침습 반응을 현저히 억제하는 것으로 나타났으며(도 3a), 상처 치유 분석 결과에서도 이와 유사하게 HCC 세포의 상처 치유 효과를 SMARCA4 낙다운이 감소시키는 것으로 나타났다(도 3b). 아울러, 유세포 분석으로 SMARCA4 낙다운이 HCC 세포의 세포 주기 조절에 미치는 영향을 확인한 결과, SMARCA4 낙다운이 HCC 세포에서 G1기의 세포 수를 증가시키는 것으로 나타났고(도 3c), 웨스턴 블롯으로 세포 주기 조절 단백질들의 발현을 확인한 결과, SMARCA4 낙다운이 HCC 세포에서 p27 Kip1 발현을 선택적으로 유도하고, 동시에 사이클린 D1, 사이클린 E, 사이클린-의존성 카이네이즈 2(CDK2), CDK4 및 CDK6를 억제함으로써 pRb의 과인산화(Hypophosphorylation) (p-pRb)를 유도하는 것으로 나타났다(도 3d). 이를 통해, HCC에서 SMARCA4 과활성이 세포 주기 단백질의 전자를 조절하여 G1기에서 S기로의 전환을 촉진하는 것을 알 수 있었다.As a result, it was found that the knockdown of SMARCA4 and SMARCC1 significantly inhibited the cell growth and proliferation rate of HCC cells ( FIGS. 2A to 2D ). In addition, in both SMARCA4-null HCC cell lines PLC/PRF/5 and SK-Hep-1, SMARCA2 knockdown resulted in overall HCC cell lethality, whereas SMARCA4 high-expressing Hep3B cells and Huh7 cells showed a change in growth rate. was found to be absent (Fig. 2e). These results indicate a pivotal role of the SMARCA4-SWI/SNF complex in the development of hepatocellular carcinoma. In addition, the results of Boyden chamber motility analysis showed that SMARCA4 knockdown significantly inhibited the serum-stimulated migration and invasion response of HCC cells (FIG. 3a), and similarly, wound healing of HCC cells was shown in the wound healing analysis results. The effect was shown to be reduced by SMARCA4 knockdown (Fig. 3b). In addition, as a result of confirming the effect of SMARCA4 knockdown on the cell cycle regulation of HCC cells by flow cytometry, it was found that SMARCA4 knockdown increases the number of cells in G1 phase in HCC cells ( FIG. 3c ), and cell cycle by Western blot As a result of confirming the expression of regulatory proteins, SMARCA4 knockdown selectively induces p27 Kip1 expression in HCC cells, and at the same time inhibits cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6, thereby hyperphosphorylating pRb (Hypophosphorylation) (p-pRb) was shown to induce (Fig. 3d). Through this, it was found that SMARCA4 hyperactivity in HCC regulates the electrons of cell cycle proteins and promotes the conversion from G1 to S phase.
2-2. in vivo 분석2-2. in vivo analysis
SMARCA4, SMARCA2 및 SMARCC1의 조절이 생체 내에서 종양 억제 효과를 유발하는지 확인하기 위해, HCC가 약 15 내지 18주령에 발달되는 Ras-Tg(H-ras12V homozygous transgenic) 수컷 마우스 8마리에 14주령부터 SMARCA4-표적 마우스 siRNA(si-SMARCA4) 또는 SMARCC1 siRNA(si-SMARCC1)와 혼합한 간 특이적 전달 시약 Invivofectamine, 또는 마우스SMARCA2 발현 플라스미드(pBJ-SMARCA2)와 혼합한 Turbofect를 정맥주사하였다(도 3e). 마우스의 간 HCC는 14주령부터 간을 수득하는 24주령까지 초음파 촬영(Philips, Amsterdam, Nederland)으로 확인되었고, 수득한 간의 중량 및 종양 질량수를 확인하고 이를 파쇄하여 웨스턴 블롯 분석으로 SMARCA4, SMARCA2 및 SMARCC1의 발현을 확인하였다.To determine whether the regulation of SMARCA4, SMARCA2 and SMARCC1 induces tumor suppressive effects in vivo, SMARCA4 from 14 weeks of age in 8 Ras- Tg (H-ras12V homozygous transgenic) male mice, where HCC develops at about 15 to 18 weeks of age. - Liver specific delivery reagent Invivofectamine mixed with target mouse siRNA (si-SMARCA4) or SMARCC1 siRNA (si-SMARCC1), or Turbofect mixed with mouse SMARCA2 expression plasmid (pBJ-SMARCA2) was injected intravenously (Fig. 3e). Liver HCC of mice was confirmed by ultrasonography (Philips, Amsterdam, Nederland) from 14 weeks of age to 24 weeks of age to obtain livers, and the weight and tumor mass number of the obtained livers were confirmed and disrupted by Western blot analysis SMARCA4, SMARCA2 and SMARCC1 was confirmed.
그 결과, 대조군 마우스(si-Cont)에서는 18주령부터 HCC가 검출되었으며, 4마리 중 3마리는 다수의 큰 HCC가 발생하였다. 반면, si-SMARCA4 처리군 마우스에서는 24주령까지 HCC가 전혀 검출되지 않았다. 그러나, si-SMARCC1 처리군 마우스 및 pBJ-SMARCA2 처리군 마우스에서는 20주령에 HCC가 검출되었고, 4마리 중 3마리는 비교적 작은 HCC가 발생되었다. 간의 총 중량 변화 및 종양 질량수(Mass number)는 SMARCA4의 표적-억제가 종양 부하를 감소시키는데 현저하게 효과적이라는 것을 나타냈다. 아울러, 웨스턴 블롯 분석 결과, 각 군의 마우스 간 조직에서 SMARCA4 및 SMARCC1의 억제와 SMARCA2의 증가된 발현이 확인되었다(도 3f). As a result, in control mice (si-Cont), HCC was detected from 18 weeks of age, and 3 out of 4 mice developed many large HCCs. On the other hand, in the mice of the si-SMARCA4 treatment group, no HCC was detected until 24 weeks of age. However, HCC was detected at 20 weeks of age in si-SMARCC1 treated mice and pBJ-SMARCA2 treated mice, and relatively small HCC was developed in 3 out of 4 mice. Changes in total weight of liver and tumor mass number indicated that target-inhibition of SMARCA4 was remarkably effective in reducing tumor burden. In addition, as a result of Western blot analysis, inhibition of SMARCA4 and SMARCC1 and increased expression of SMARCA2 were confirmed in the mouse liver tissues of each group ( FIG. 3f ).
실시예 3. HCC에서 SMARCA4-조절 유전자 확인Example 3. Identification of SMARCA4-regulated genes in HCC
3-1. SMARCA4-조절 유전자 선발3-1. SMARCA4-regulated gene selection
SMARCA4-SWI/SNF 조절 유전자를 확인하기 위해, ENCODE(Encyclopedia of DNA Elements)(https://www.encodeproject.org/)로부터 입수 가능한 HepG2 세포의 히스톤 ChIP-seq를 이용하여 HCC 세포에서 SMARCA4에 의해 특이적으로 인식되는 마커인 H3K27ac 및 H3K27me3가 풍부한 위치를 포괄적으로 맵핑하였다. 여기에서 Bowtie 2(http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) 맵퍼를 사용하여 트리밍된 판독값을 인간 게놈에 정렬시켰고, PCR 편향(Bias)을 제거하기 위해, 중복 판독값을 Sambamba 툴(https://lomereiter.github.io/sambamba/)을 이용하여 제거하였다. HOMER(FindPeaks)를 이용하여 H3K27ac 또는 H3K27me3가 풍부한 위치를 확인하였다(FDR-조정된 p값 컷오프=0.001). 상기 분석을 통해, 피크에 가장 가까운 21,684 유전자 중 9,198개의 유전자가 활성 인핸서로 확인되었다(도 4a). 그 후, HCC 세포에서 조절된 유전자 요소(Elements)를 확인하기 위해 Hep3B에서 SMARCA4 낙다운을 수행하여 SMARCA4에 의해 조절되는 1,865개의 유전자 요소를 찾았으며(> ± 1.5 fold, P<0.05), 이 중 1,054개의 유전자가 SMARCA4가 낙다운된 HCC 세포에서 하향 조절되는 것으로 나타났다. 이들 SMARCA4-조절 유전자 1,054개를 9,198개의 활성 인핸서와 함께 벤다이어그램 분석으로 분석한 결과, 563개의 유전자가 HCC에서 SMARCA4-SWI/SNF에 의해 조절되는 것으로 분석되었다(도 4a). 563개의 SMARCA4-관련 유전자는 여러 단계의 HCC에 걸쳐 전사 수준이 점진적인 증가 또는 감소하고, 진행된 HCC에서 일관되게 최고 또는 최저 수준인 것으로 나타났다(도 4b). Gene Ontology terms를 사용하여, 상기 563개의 유전자 세트에서 신생혈관, Wnt 신호전달, 인테그린 신호전달, PDGF 신호전달 및 G-단백질 신호전달을 포함하는 발암(Carcinogenesis) 기능과 관련된 유전자를 탐색하였다(도 4c). HCC에서 SMARCA4에 의해 조절되는 유전자를 선발하기 위해, 563개의 유전자를 TCGA_LIHC 및 ICGC_LIRI 데이터 세트 모두에서 과발현되는 84개의 유전자로 줄이고(≥ 1.5 fold, P<0.05), 그 후, SMARCA4 발현과 유의한 상관관계가 있는 37개의 유전자 중 상향조절된 상위 5개의 유전자를 검사하고 검증하였다(도 4a). SMARCA4가 이들 유전자를 직접적으로 조절하는지 확인하기 위해, HCC 세포를 SMARCA4 siRNA(si-SMARCA4)로 낙다운하고 qRT-PCR로 유전자 발현 변화를 확인하였다. 그 결과, 모든 HCC 세포에서 SMARCA4 낙다운에 의해 IRAK1가 일관되게 억제된 것을 확인하였다(도 4d).To identify SMARCA4-SWI/SNF regulatory genes, by SMARCA4 in HCC cells using histone ChIP-seq of HepG2 cells available from ENCODE (Encyclopedia of DNA Elements) (https://www.encodeproject.org/) Locations rich in specifically recognized markers, H3K27ac and H3K27me3, were comprehensively mapped. Trimmed reads were aligned to the human genome using the Bowtie 2 ( http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) mapper here, and duplicate reads were used to eliminate PCR bias. The value was removed using the Sambamba tool ( https://lomereiter.github.io/sambamba/ ). H3K27ac or H3K27me3-rich sites were identified using HOMER (FindPeaks) (FDR-adjusted p-value cutoff=0.001). Through the above analysis, 9,198 genes out of 21,684 genes closest to the peak were identified as active enhancers (FIG. 4a). After that, SMARCA4 knockdown was performed in Hep3B to identify regulated gene elements in HCC cells, and 1,865 gene elements regulated by SMARCA4 were found (> ± 1.5 fold, P<0.05), of which 1,054 genes were shown to be downregulated in SMARCA4 knockdown HCC cells. As a result of analyzing 1,054 of these SMARCA4-regulated genes together with 9,198 active enhancers by Venn diagram analysis, 563 genes were analyzed to be regulated by SMARCA4-SWI/SNF in HCC (Fig. 4a). 563 SMARCA4-associated genes showed progressive increases or decreases in transcription levels across different stages of HCC, and consistently highest or lowest levels in advanced HCC (Fig. 4b). Using Gene Ontology terms, genes related to carcinogenesis, including angiogenesis, Wnt signaling, integrin signaling, PDGF signaling, and G-protein signaling, were searched for in the 563 gene sets (FIG. 4c). ). To select for genes regulated by SMARCA4 in HCC, 563 genes were reduced to 84 genes overexpressed in both TCGA_LIHC and ICGC_LIRI data sets (≥ 1.5 fold, P<0.05), and then significantly correlated with SMARCA4 expression Among the 37 genes that were related, the top 5 genes that were upregulated were tested and verified ( FIG. 4A ). To confirm whether SMARCA4 directly regulates these genes, HCC cells were knocked down with SMARCA4 siRNA (si-SMARCA4) and gene expression changes were confirmed by qRT-PCR. As a result, it was confirmed that IRAK1 was consistently inhibited by SMARCA4 knockdown in all HCC cells ( FIG. 4D ).
3-2. SMARCA4 및 IRAK1의 상호작용 확인3-2. Confirm the interaction of SMARCA4 and IRAK1
상기 실시예에서 선발한 두 유전자 IRAK1의 인핸서 영역에 SMARCA4가 특이적으로 결합하는지 확인하기 위해, 항-SMARCA4 및 항-H3K27ac 항체를 각각 이용하여 ChIP 분석을 수행하고 IRAK1의 활성 인핸서 영역(H3K27ac)에서의 농축률(Enrichment rates)을 확인하였다. ChIP(Chromatin immunoprecipitation) 분석은 제조사(Pierce Agarose ChIP kit; Thermo Fisher Scientific, Waltham, MA)의 지시에 따라 수행하였고, DNA를 SMARCA4에 의해 조절되는 각 후보 유전자의 인핸서 영역에 대한 프라이머를 이용하여 RT-qPCR로 증폭하였다. 가교된 DNA(Cross-linked DNA)를 SMARCA4 항체(항-SMARCA4) 및 H3K27ac(Acetylated histone H3 at lysine 27) 항체(Abcam, Cambridge, UK) (항-H3K27ac)를 이용하여 침전시켰다. 상기 실험은 3회 반복되었으며 IgG으로 정규화하여 평균을 구하였다. 아울러, 내인성 SMARCA4가 IRAK1 인핸서 영역에 직접적으로 결합하는지 확인하기 위해, IRAK1 인핸서를 pGL4.23 리포터 벡터에 클로닝한 후, HCC 세포에서 SMARCA4의 존재 또는 부재하에 pGL4.23_IRAK1 인핸서의 듀얼 루시퍼레이즈 리포터 분석을 수행하였다. To confirm that SMARCA4 specifically binds to the enhancer region of the two genes IRAK1 selected in the above Example, ChIP analysis was performed using anti-SMARCA4 and anti-H3K27ac antibodies, respectively, and the active enhancer region of IRAK1 (H3K27ac) was Enrichment rates were confirmed. Chromatin immunoprecipitation (ChIP) analysis was performed according to the manufacturer's (Pierce Agarose ChIP kit; Thermo Fisher Scientific, Waltham, MA) instructions, and DNA was RT- with primers for enhancer regions of each candidate gene regulated by SMARCA4. It was amplified by qPCR. Cross-linked DNA was precipitated using SMARCA4 antibody (anti-SMARCA4) and H3K27ac (Acetylated histone H3 at lysine 27) antibody (Abcam, Cambridge, UK) (anti-H3K27ac). The experiment was repeated three times and the average was obtained by normalizing to IgG. In addition, to confirm that endogenous SMARCA4 directly binds to the IRAK1 enhancer region, after cloning the IRAK1 enhancer into the pGL4.23 reporter vector, a dual luciferase reporter assay of the pGL4.23_IRAK1 enhancer in the presence or absence of SMARCA4 in HCC cells was performed. carried out.
그 결과, 모든 HCC 세포에서 SMARCA4 낙다운에 의해 IRAK1 활성 인핸서 영역의 농축률이 음성 대조군(무작위로 선발된 비-SMARCA4 조절 유전자 GNLS, EIF4G2 및 CCT2)에 비해 현저히 감소하였으나, 동일 세포에서 CKAP4 활성 인핸서 영역은 SMARCA4 낙다운에 일관적으로 영향받지 않았다(도 4e). 아울러, 루시퍼레이즈 리포터 분석 결과, 모든 HCC 세포에서 SMARCA4 낙다운이 상대적인 루시퍼레이즈 활성을 현저하게 억제하는 것으로 나타나, SMARCA4 및 IRAK1 인핸서 간의 상호작용을 확인할 수 있었다(도 4f).As a result, the enrichment rate of the IRAK1 activity enhancer region by SMARCA4 knockdown in all HCC cells was significantly reduced compared to the negative control (randomly selected non-SMARCA4 regulatory genes GNLS, EIF4G2 and CCT2), but the CKAP4 activity enhancer in the same cells Regions were not consistently affected by SMARCA4 knockdown ( FIG. 4E ). In addition, as a result of the luciferase reporter analysis, SMARCA4 knockdown in all HCC cells significantly inhibited the relative luciferase activity, confirming the interaction between SMARCA4 and the IRAK1 enhancer ( FIG. 4f ).
실시예 4. HCC 환자 코호트에서의 SMARCA4 및 IRAK1 과발현 확인Example 4. Confirmation of SMARCA4 and IRAK1 overexpression in HCC patient cohort
TCGA_LIHC 및 ICGC_LIRI 데이터 세트에서 SMARCA4과 IRAK1의 과발현을 확인한 결과, TCGA_LIHC에서, 총 371명의 HCC 환자 중에서 정상인 50명의 평균 SMARCA4 또는 IRAK1 유전자 발현값의 1.5배 이상으로 SMARCA4의 발현이 증가되어 있는 환자는 318명(86%)로 나타났으며, IRAK1 발현이 증가되어 있는 환자는 335명(90%)으로 나타났고, 이 중, SMARCA4과 IRAK1이 동시에 모두 과발현된 환자는 318명중 304명(96%)으로 나타났다(도 5). 또한, ICGC_LIRI에서, 총 238명의 HCC 환자 중에서 정상인 202명의 평균 SMARCA4 또는 IRAK1 유전자 발현값의 1.5배 이상으로 SMARCA4의 발현이 증가되어 있는 환자는 172명(72%)이고, IRAK1 발현이 증가되어 있는 환자는 198명(83%)으로 나타났으며, 이 중, SMARCA4과 IRAK1이 동시에 모두 과발현된 환자는 172명중 152명(88%)으로 나타났다(도 5). 이를 통해, SMARCA4에 의해 IRAK1의 발현이 증가된 것을 유추할 수 있다.As a result of confirming the overexpression of SMARCA4 and IRAK1 in the TCGA_LIHC and ICGC_LIRI data sets, in TCGA_LIHC, among a total of 371 HCC patients, 318 patients with increased SMARCA4 expression at least 1.5 times the average SMARCA4 or IRAK1 gene expression value of 50 normal patients. (86%), and 335 patients (90%) had increased IRAK1 expression, of which 304 out of 318 patients (96%) had both SMARCA4 and IRAK1 overexpression. (Fig. 5). In addition, in ICGC_LIRI, among a total of 238 HCC patients, 172 patients (72%) with increased SMARCA4 expression by 1.5 times or more of the average SMARCA4 or IRAK1 gene expression value of 202 normal patients, and patients with increased IRAK1 expression was found in 198 (83%), among which, 152 (88%) of 172 patients with both SMARCA4 and IRAK1 overexpression were found (FIG. 5). Through this, it can be inferred that the expression of IRAK1 is increased by SMARCA4.
실시예 5. HCC에서 IRAK1의 종양 형성 역할 확인 Example 5. Identification of the tumorigenic role of IRAK1 in HCC
상기 실시예들을 통해 HCC 세포에서 SMARCA4가 IRAK1을 활성화시켰으므로, 간세포암 발생(Hepatocellular carcinogenesis)에서 IRAK1의 역할을 확인하기 위해, TCGA_LIHC 및 ICGC_LIRI에서 IRAK1의 발현 변화를 확인하였고, 그 결과, IRAK1의 SMARCA4와의 강한 양의 상관관계가 관찰되었다(도 6a). 또한, HCC 환자들의 Kaplan-Meier 생존 곡선은 IRAK1이 고발현된 환자들의 5년 전체 생존율이 IRAK1이 저발현된 환자들보다 현저히 낮은 것으로 나타났다(도 6a). 이에, 간세포암 발생에서 IRAK1의 종양 형성 기능을 확인하기 위해, HCC 세포에서 MTT 분석을 이용한 세포 성장 및 BrdU 분석을 이용한 세포 증식을 확인하였다. 그 결과, SMARCA4 및 IRAK1의 개별적인 낙다운이 HCC 세포에서 종양 세포 성장 및 증식률을 현저하게 억제하였다(도 6b). 또한, 세포 주기 조절을 확인하기 위해 PI-염색된 세포들을 유세포 분석으로 확인한 결과, SMARCA4 및 IRAK1의 낙다운 각각이 HCC 세포의 세포 주기 정지(Cell cycle arrest)를 유발하는 것으로 나타났다. 특히, SMARCA4에 의해 조절된 세포 주기 성분(Components)이 IRAK1과 함께 조절되어, HCC에서 IRAK1이 SMARCA4에 의해 조절되는 하류 분자임을 확인할 수 있었다(도 6c 및 d). 아울러, SMARCA4 및 IRAK1의 낙다운이 화학주성인자-자극된 HCC 세포의 이동 및 침윤 반응을 감소시켰다(도 6e 및 f).Since SMARCA4 activated IRAK1 in HCC cells through the above examples, in order to confirm the role of IRAK1 in hepatocellular carcinogenesis, the expression change of IRAK1 in TCGA_LIHC and ICGC_LIRI was confirmed. As a result, SMARCA4 of IRAK1 A strong positive correlation was observed with (Fig. 6a). In addition, the Kaplan-Meier survival curve of HCC patients showed that the 5-year overall survival rate of patients with high IRAK1 expression was significantly lower than that of patients with low IRAK1 expression (Fig. 6a). Therefore, in order to confirm the tumorigenic function of IRAK1 in the development of hepatocellular carcinoma, cell growth using MTT assay and cell proliferation using BrdU assay were confirmed in HCC cells. As a result, individual knockdown of SMARCA4 and IRAK1 significantly inhibited tumor cell growth and proliferation in HCC cells ( FIG. 6b ). In addition, as a result of confirming the PI-stained cells by flow cytometry to confirm the cell cycle regulation, it was shown that each knockdown of SMARCA4 and IRAK1 induces cell cycle arrest of HCC cells. In particular, cell cycle components regulated by SMARCA4 were regulated together with IRAK1, confirming that IRAK1 is a downstream molecule regulated by SMARCA4 in HCC ( FIGS. 6c and d ). In addition, the knockdown of SMARCA4 and IRAK1 decreased the migration and invasion response of chemoattractant-stimulated HCC cells ( FIGS. 6e and f ).
실시예 6. SMARCA4에 의한 IRAK1 활성 조절Example 6. Regulation of IRAK1 activity by SMARCA4
SMARCA4이 IRAK1 활성을 직접적으로 조절하는지 확인하기 위해, HCC 세포들을 SMARCA4 siRNA로 낙다운한 뒤, IRAK1 발현 플라스미드(pcDNA3.1_IRAK1)로 회복 실험을 수행하였다. 그 결과, SMARCA4 낙다운이 HCC 세포의 종양 세포 성장 및 증식을 둘 다 현저히 억제하였으나, 이와 같은 항-종양 효과는 IRAK1 플라스미드의 공동-형질전환에 의해 사라졌다(도 7a). 또한, PI-염색된 세포의 유세포 분석 결과, HCC 세포에서 SMARCA4 낙다운에 의해 G1기 정지가 현저히 유발되어 항-성장 효과를 나타낸 반면, 동일 세포에 IRAK1 플라스미드를 공동-형질전환하자 HCC 세포 주기가 정지되지 않아 종양 성장이 다시 재개되었다(도 7b). 이와 같은 결과는 세포주기 관련 단백질들을 웨스턴 블롯 분석으로 확인하여 추가 검증하였는데, 그 결과, HCC에서 SMARCA4 낙다운에 의해 선택적으로 유도된 p27 Kip1은 IRAK1 플라스미드 발현에 의해 억제되었으며, 동시에 SMARCA4 낙다운에 의해 발현이 억제된 사이클린 D1, 사이클린 E, CDK2, CDK4 및 CDK6와 pRb의 인산화는 회복되었다(도 7c). 아울러, HCC 세포에서 SMARCA4 낙다운에 의해 감소된 화학주성물질-자극된 이동 및 침윤 반응도 IRAK1 플라스미드의 발현으로 인해 회복되었다(도 7d 및 e).To confirm whether SMARCA4 directly regulates IRAK1 activity, HCC cells were knocked down with SMARCA4 siRNA, and then a recovery experiment was performed with an IRAK1 expression plasmid (pcDNA3.1_IRAK1). As a result, SMARCA4 knockdown significantly inhibited both tumor cell growth and proliferation of HCC cells, but this anti-tumor effect was abolished by co-transformation of the IRAK1 plasmid ( FIG. 7A ). In addition, as a result of flow cytometry analysis of PI-stained cells, SMARCA4 knockdown in HCC cells significantly induced G1 phase arrest to exhibit anti-growth effects, whereas co-transformation of the same cells with IRAK1 plasmid resulted in an abortion of the HCC cell cycle. It was not stopped and tumor growth resumed again ( FIG. 7B ). These results were further verified by confirming cell cycle-related proteins by Western blot analysis. As a result, p27 Kip1 selectively induced by SMARCA4 knockdown in HCC was inhibited by IRAK1 plasmid expression, and at the same time by SMARCA4 knockdown. Phosphorylation of the suppressed cyclin D1, cyclin E, CDK2, CDK4 and CDK6 and pRb was restored (Fig. 7c). In addition, the chemotactic-stimulated migration and invasion responses reduced by SMARCA4 knockdown in HCC cells were also restored due to the expression of the IRAK1 plasmid ( FIGS. 7d and e ).
실시예 7. IRAK1의 종양 단백질(Oncoprotein) 조절Example 7. Oncoprotein regulation of IRAK1
SMARCA4-IRAK1에 의해 종양 단백질들이 조절되는지 확인하기 위해, SMARCA4 및 IRAK1를 HCC 세포에서 낙다운하고 IRAK1 플라스미드로 IRAK1의 발현을 회복시켜 종양 단백질들의 조절 정도를 확인하였다. 그 결과, HCC에서 SMARCA4 및 IRAK1를 모두 낙다운한 결과, JNK-의존적 Gankyrin 및 AKR1B10가 현저히 억제되었다(도 8a). 또한, 상기 억제 효과가 IRAK1 플라스미드를 이용한 IRAK1의 공동 발현에 의해 회복되어, 간세포암 발생에서 SMARCA4-IRAK1-Gankyrin/AKR1B10의 조절 축을 확인하였다(도 8b). 아울러, 생체 내 SMARCA4-IRAK1 조절 축을 암 예방 표적으로 이용할 수 있을지 확인하기 위해, 14주령의 Ras-Tg 마우스에 25 mg/kg의 si-SMARCA4 또는 si-IRAK1가 함유된 Invivofectamine 3.0(Invitrogen)을 정맥주사하고, 15, 17, 19, 21 및 23주령에 초음파 촬영하였다(도 8c). 그 결과, 대조군의 경우(si-Cont), 19주령에 HCC 가 검출되었으며, 4마리 중 4마리에서 다수의 큰 종양이 발생하였다. 반면, si-IRAK1 처리군에서는 21주령에서 HCC가 관찰되었고, 4마리 중 1마리에서만 상대적으로 작은 HCC만이 발생하였고, si-SMARCA4 처리군에서는 24주 동안 HCC가 전혀 검출되지 않았다. SMARCA4 및 IRAK1를 모두 표적화한 경우 종양 부하가 감소되어 총 간 중량 및 종양 질량수 또한 현저하게 억제되었다(도 8d). 아울러, 웨스턴 블롯 분석에서도 si-SMARCA4 및 si-IRAK1 처리군의 경우 마우스 간에서 Gankyrin 및 AKR1B10의 발현이 억제되어, 간세포암 발생에서 SMARCA4-IRAK1-Gankyrin, AKR1B10를 통한 생체 내 조절 기전을 입증하였다(도 8e 및 f).To confirm whether tumor proteins are regulated by SMARCA4-IRAK1, SMARCA4 and IRAK1 were knocked down in HCC cells, and IRAK1 expression was restored with IRAK1 plasmid to confirm the degree of regulation of tumor proteins. As a result, as a result of knockdown of both SMARCA4 and IRAK1 in HCC, JNK-dependent Gankyrin and AKR1B10 were significantly inhibited (FIG. 8a). In addition, the inhibitory effect was restored by co-expression of IRAK1 using the IRAK1 plasmid, confirming the regulatory axis of SMARCA4-IRAK1-Gankyrin/AKR1B10 in the development of hepatocellular carcinoma ( FIG. 8b ). In addition, to determine whether the in vivo SMARCA4-IRAK1 regulatory axis can be used as a cancer prevention target, Invivofectamine 3.0 (Invitrogen) containing 25 mg/kg of si-SMARCA4 or si-IRAK1 was administered intravenously to 14-week-old Ras-Tg mice. Injections, and ultrasonography at 15, 17, 19, 21 and 23 weeks of age (Fig. 8c). As a result, in the case of the control group (si-Cont), HCC was detected at 19 weeks of age, and a large number of large tumors occurred in 4 out of 4 mice. On the other hand, in the si-IRAK1-treated group, HCC was observed at 21 weeks of age, and only 1 out of 4 had relatively small HCC, and in the si-SMARCA4 treated group, no HCC was detected for 24 weeks. When both SMARCA4 and IRAK1 were targeted, tumor burden was reduced, and total liver weight and tumor mass number were also significantly suppressed ( FIG. 8d ). In addition, in Western blot analysis, the expression of Gankyrin and AKR1B10 in the mouse liver was suppressed in the si-SMARCA4 and si-IRAK1 treatment groups, demonstrating an in vivo regulatory mechanism through SMARCA4-IRAK1-Gankyrin and AKR1B10 in the development of hepatocellular carcinoma ( 8e and f).

Claims (24)

  1. SMARCA4(SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), SMARCC1, SMARCA2 및 IRAK1(Interleukin-1 receptor-associated kinase 1)으로 이루어진 군으로부터 선택되는 하나 이상의 유전자 또는 상기 유전자로부터 발현된 단백질을 포함하는 간암 진단용 바이오 마커.At least one gene or the gene selected from the group consisting of SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), SMARCC1, SMARCA2, and IRAK1 (Interleukin-1 receptor-associated kinase 1) A biomarker for diagnosing liver cancer comprising a protein expressed from
  2. 제1항에 있어서, According to claim 1,
    SMARCA4 및 IRAK1의 유전자 또는 상기 유전자로부터 발현된 단백질을 포함하는, 간암 진단용 바이오 마커.A biomarker for diagnosing liver cancer, comprising the genes of SMARCA4 and IRAK1 or proteins expressed from the genes.
  3. 제1항에 있어서, According to claim 1,
    간암은 간세포암(Hepatocellular carcinoma, HCC)인, 간암 진단용 바이오 마커.Liver cancer is a hepatocellular carcinoma (HCC), a biomarker for the diagnosis of liver cancer.
  4. 제3항에 있어서, 4. The method of claim 3,
    IRAK1이 과발현되는 간세포암인, 간암 진단용 바이오 마커.A biomarker for diagnosing liver cancer, which is a hepatocellular carcinoma in which IRAK1 is overexpressed.
  5. 제1항에 있어서, According to claim 1,
    Gankyrin 또는 AKR1B10의 유전자 또는 상기 유전자로부터 발현된 단백질을 추가로 포함하는 간암 진단용 바이오 마커.A biomarker for diagnosing liver cancer, further comprising a gene of Gankyrin or AKR1B10 or a protein expressed from the gene.
  6. SMARCA4, SMARCC1, SMARCA2 및 IRAK1로 이루어진 군으로부터 선택되는 하나 이상의 바이오 마커 유전자의 발현량을 mRNA 또는 단백질 수준에서 측정하는 제제를 포함하는, 간암 진단용 조성물.A composition for diagnosing liver cancer, comprising an agent for measuring the expression level of one or more biomarker genes selected from the group consisting of SMARCA4, SMARCC1, SMARCA2 and IRAK1 at the mRNA or protein level.
  7. 제6항에 있어서, 7. The method of claim 6,
    Gankyrin 또는 AKR1B10의 발현량을 mRNA 또는 단백질 수준에서 측정하는 제제를 추가로 포함하는, 간암 진단용 조성물. A composition for diagnosing liver cancer, further comprising an agent for measuring the expression level of Gankyrin or AKR1B10 at the mRNA or protein level.
  8. 제6항에 있어서, 7. The method of claim 6,
    간암은 간세포암인, 간암 진단용 조성물. The liver cancer is hepatocellular carcinoma, a composition for diagnosing liver cancer.
  9. 제6항에 있어서,7. The method of claim 6,
    SMARCA4 및 IRAK1 유전자의 발현량을 mRNA 또는 단백질 수준에서 측정하는 제제를 포함하는, 간암 진단용 조성물.A composition for diagnosing liver cancer, comprising an agent for measuring the expression levels of SMARCA4 and IRAK1 genes at the mRNA or protein level.
  10. 제6항에 있어서, 7. The method of claim 6,
    바이오 마커 유전자의 발현량을 mRNA 수준에서 측정하는 제제는, 상기 마커의 핵산서열, 상기 핵산서열에 상보적인 핵산서열, 상기 핵산서열 및 상보적인 서열의 단편을 특이적으로 인식하는 프라미어 쌍, 프로브, 또는 프라이머 쌍 및 프로브인, 간암 진단용 조성물.An agent for measuring the expression level of a biomarker gene at the mRNA level is a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair that specifically recognizes a fragment of the nucleic acid sequence and a complementary sequence, a probe , or a primer pair and a probe, a composition for diagnosing liver cancer.
  11. 제10항에 있어서, 11. The method of claim 10,
    측정은 중합효소연쇄반응, 실시간 RT-PCR(Real-time RT-PCR), 역전사 중합효소연쇄반응, 경쟁적 중합효소연쇄반응(Competitive RT-PCR), Nuclease 보호 분석(RNase, S1 nuclease assay), in situ 교잡법, 핵산 마이크로어레이, 노던블랏 또는 DNA 칩으로 이루어진 군으로부터 선택되는 방법으로 수행되는, 간암 진단용 조성물.Measurement is polymerase chain reaction, real-time RT-PCR (Real-time RT-PCR), reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in A composition for diagnosing liver cancer, which is performed by a method selected from the group consisting of in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip.
  12. 제6항에 있어서, 7. The method of claim 6,
    바이오 마커 유전자의 발현량을 단백질 수준에서 측정하는 제제는, 상기 마커의 단백질 전장 또는 그 단편을 특이적으로 인식하는 항체, 항체단편, 앱타머(Aptamer), 아비머(Avidity multimer) 또는 펩티도모방체(Peptidomimetics)인, 간암 진단용 조성물.An agent for measuring the expression level of a biomarker gene at the protein level is an antibody, antibody fragment, aptamer, avimer, or peptidomimetic that specifically recognizes the full-length protein of the marker or a fragment thereof. A body (Peptidomimetics), a composition for diagnosing liver cancer.
  13. 제12항에 있어서, 13. The method of claim 12,
    측정은 웨스턴블랏, ELISA(Enzyme linked immunosorbent assay), 방사선면역분석(RIA: Radioimmunoassay), 방사면역확산법(Radioimmunodiffusion), 면역 전기영동, 조직면역염색, 면역침전 분석법(Immunoprecipitation assay), 보체 고정 분석법(Complement Fixation Assay), FACS, 질량분석 또는 단백질 마이크로어레이로 이루어진 군으로부터 선택되는 방법으로 수행되는, 간암 진단용 조성물.Measurements are Western blot, ELISA (Enzyme linked immunosorbent assay), Radioimmunoassay (RIA), Radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, Immunoprecipitation assay, Complement fixation assay Fixation Assay), FACS, mass spectrometry, or a method selected from the group consisting of protein microarray, liver cancer diagnosis composition.
  14. 제6항의 조성물을 포함하는, 간암 진단 키트.A liver cancer diagnostic kit comprising the composition of claim 6 .
  15. SMARCA4 억제제를 유효성분으로 포함하는 간암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating liver cancer comprising a SMARCA4 inhibitor as an active ingredient.
  16. 제15항에 있어서, 16. The method of claim 15,
    SMARCA2 또는 이의 발현 촉진제, SMARCC1 억제제, 또는 IRAK1 억제제를 추가로 포함하는, 간암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating liver cancer, further comprising SMARCA2 or an expression promoter thereof, a SMARCC1 inhibitor, or an IRAK1 inhibitor.
  17. 제15항에 있어서, 16. The method of claim 15,
    간암은 IRAK1이 과발현되는 간암인, 간암 예방 또는 치료용 약학적 조성물.Liver cancer is a liver cancer in which IRAK1 is overexpressed, a pharmaceutical composition for preventing or treating liver cancer.
  18. 제15항에 있어서, 16. The method of claim 15,
    간암은 간세포암인, 간암 예방 또는 치료용 약학적 조성물.Liver cancer is hepatocellular carcinoma, a pharmaceutical composition for preventing or treating liver cancer.
  19. (a) 검사 대상체로부터 분리한 생물학적 시료에서 SMARCA4 또는 IRAK1의 유전자 발현량을 측정하는 단계;(a) measuring the gene expression level of SMARCA4 or IRAK1 in a biological sample isolated from the test subject;
    (b) 정상 대조군 시료의 해당 유전자의 상응하는 결과와 비교하는 단계; 및 (b) comparing the corresponding gene of the normal control sample with the corresponding result; and
    (c) (a) 단계에서의 유전자 발현량이 (b) 단계에서의 해당 유전자의 발현 수준에 비해 높은 경우, 상기 검사 대상체가 간암일 것으로 판단하는 단계를 포함하는, 간암 진단에 필요한 정보를 제공하는 방법. (c) when the gene expression level in step (a) is higher than the expression level of the corresponding gene in step (b), providing information necessary for diagnosing liver cancer, including determining that the test subject is liver cancer Way.
  20. 제19항에 있어서, 20. The method of claim 19,
    (a) 검사 대상체로부터 분리한 생물학적 시료에서 SMARCC1, SMARCA2, Gankyrin 또는 AKR1B10의 유전자 또는 단백질 발현량을 측정하는 단계;(a) measuring the gene or protein expression level of SMARCC1, SMARCA2, Gankyrin, or AKR1B10 in a biological sample isolated from the test subject;
    (b) 정상 대조군 시료의 해당 유전자 또는 단백질의 상응하는 결과와 비교하는 단계; 및 (b) comparing the corresponding gene or protein of the normal control sample with the corresponding result; and
    (c) SMARCC1, Gankyrin 또는 AKR1B10의 (a) 단계에서의 발현량이 (b) 단계에서의 해당 유전자 또는 단백질의 발현 수준에 비해 높은 경우, 또는 SMARCA2의 (a) 단계에서의 발현량이 (b) 단계에서의 해당 유전자 또는 단백질의 발현 수준에 비해 낮은 경우, 상기 검사 대상체가 간암일 것으로 판단하는 단계를 추가로 포함하는, 간암 진단에 필요한 정보를 제공하는 방법. (c) When the expression level of SMARCC1, Gankyrin or AKR1B10 in step (a) is high compared to the expression level of the corresponding gene or protein in step (b), or the expression level of SMARCA2 in step (a) in step (b) When the expression level of the corresponding gene or protein is low, the method of providing information necessary for diagnosing liver cancer, further comprising the step of determining that the test subject is liver cancer.
  21. (a) 검사 대상체로부터 분리한 생물학적 시료에서 SMARCA4 또는 IRAK1의 유전자 발현량을 측정하는 단계;(a) measuring the gene expression level of SMARCA4 or IRAK1 in a biological sample isolated from the test subject;
    (b) 정상 대조군 시료의 해당 유전자의 상응하는 결과와 비교하는 단계; 및 (b) comparing the corresponding gene of the normal control sample with the corresponding result; and
    (c) (a) 단계에서의 유전자 발현량이 (b) 단계에서의 해당 유전자의 발현 수준에 비해 높은 경우, 상기 검사 대상체의 예후가 나쁜 것으로 판단하는 단계를 포함하는, 간암의 예후 예측을 위한 정보를 제공하는 방법.(c) when the gene expression level in step (a) is higher than the expression level of the corresponding gene in step (b), information for predicting the prognosis of liver cancer, including the step of determining that the prognosis of the test subject is bad How to provide.
  22. IRAK1의 발현량을 측정하는 단계를 포함하는, SMARCA4 억제제의 간암 치료 반응성 예측방법.A method for predicting liver cancer treatment responsiveness of a SMARCA4 inhibitor, comprising the step of measuring the expression level of IRAK1.
  23. 제22항에 있어서, 23. The method of claim 22,
    Gankyrin 또는 AKR1B10의 발현량을 측정하는 단계를 추가로 포함하는, SMARCA4 억제제의 간암 치료 반응성 예측방법.A method for predicting liver cancer treatment responsiveness of a SMARCA4 inhibitor, further comprising the step of measuring the expression level of Gankyrin or AKR1B10.
  24. 제15항의 조성물을 개체에 투여하는 단계를 포함하는 간암 예방 또는 치료 방법.A method for preventing or treating liver cancer comprising administering the composition of claim 15 to a subject.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110124011A (en) * 2019-05-21 2019-08-16 上海市浦东新区公利医院(第二军医大学附属公利医院) Application of chromatin remodeling complex core catalytic subunit in preparing medicine for treating or preventing hepatitis B virus infection

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHEN ZHIAO, LU XINYUAN, JIA DESHUI, JING YING, CHEN DI, WANG QIFENG, ZHAO FANGYU, LI JINJUN, YAO MING, CONG WENMING, HE XIANGHUO: "Hepatic SMARCA4 predicts HCC recurrence and promotes tumour cell proliferation by regulating SMAD6 expression", CELL DEATH & DISEASE, NATURE PUBLISHING GROUP, GB, vol. 9, no. 2, 1 February 2018 (2018-02-01), GB , pages 1 - 12, XP055888979, ISSN: 2041-4889, DOI: 10.1038/s41419-017-0090-8 *
DISTEFANO JOHANNA, DAVIS BETHANY: "Diagnostic and Prognostic Potential of AKR1B10 in Human Hepatocellular Carcinoma", CANCERS, M D P I AG, CH, vol. 11, no. 4, 1 April 2019 (2019-04-01), CH , pages 486 - 13, XP055888999, ISSN: 2072-6694, DOI: 10.3390/cancers11040486 *
HU BO, LIN JIAN‐ZHEN, YANG XIAO‐BO, SANG XIN‐TING: "The roles of mutated SWI/SNF complexes in the initiation and development of hepatocellular carcinoma and its regulatory effect on the immune system: A review", CELL PROLIFERATION, OXFORD., GB, vol. 53, no. 4, 1 April 2020 (2020-04-01), GB , pages 12791 - 15, XP055888988, ISSN: 0960-7722, DOI: 10.1111/cpr.12791 *
JING HONGBIAO, ZHANG GUOMING, MENG LINGSHENG, MENG QINGDA, MO HAIYING, TAI YANHONG: "Gradually elevated expression of Gankyrin during human hepatocarcinogenesis and its clinicopathological significance", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, US, vol. 4, no. 1, 1 May 2015 (2015-05-01), US , pages 1 - 8, XP055888996, ISSN: 2045-2322, DOI: 10.1038/srep05503 *
KIM SANG YEAN; SHEN QINGYU; SON KEUNHONG; KIM HYUNG SEOK; YANG HEE DOO; NA MIN JEONG; SHIN EUNBI; YU SUJI; KANG KEUNSOO; YOU JUENG: "SMARCA4 oncogenic potential via IRAK1 enhancer to activate Gankyrin and AKR1B10 in liver cancer", ONCOGENE, NATURE PUBLISHING GROUP UK, LONDON, vol. 40, no. 28, 17 June 2021 (2021-06-17), London , pages 4652 - 4662, XP037509481, ISSN: 0950-9232, DOI: 10.1038/s41388-021-01875-6 *
YE ZHI-HUA, GAO LI, WEN DONG-YUE, HE YUN, PANG YU-YAN, CHEN GANG: "Diagnostic and prognostic roles of IRAK1 in hepatocellular carcinoma tissues: an analysis of immunohistochemistry and RNA-sequencing data from the cancer genome atlas", ONCOTARGETS AND THERAPY, vol. 10, 1 January 2017 (2017-01-01), pages 1711 - 1723, XP055888985, ISSN: 1178-6930, DOI: 10.2147/OTT.S132120 *
ZHANG FAN, DING LINDA, CUI LI, BARBER ROBERT, DENG BIN: "Identification of long non-coding RNA-related and –coexpressed mRNA biomarkers for hepatocellular carcinoma", BMC MEDICAL GENOMICS, BIOMED CENTRAL LTD, LONDON UK, vol. 12, no. S1, 1 January 2019 (2019-01-01), London UK , pages 1 - 12, XP055888992, ISSN: 1755-8794, DOI: 10.1186/s12920-019-0472-0 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110124011A (en) * 2019-05-21 2019-08-16 上海市浦东新区公利医院(第二军医大学附属公利医院) Application of chromatin remodeling complex core catalytic subunit in preparing medicine for treating or preventing hepatitis B virus infection

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