WO2022019604A1 - Biomarqueur spécifique du cancer du foie et utilisation associée - Google Patents

Biomarqueur spécifique du cancer du foie et utilisation associée Download PDF

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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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English (en)
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/fr

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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.

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Abstract

La présente invention concerne une utilisation , en tant que biomarqueurs pour la détection et le diagnostic d'un carcinome hépatocellulaire, de gènes dont l'expression change spécifiquement en cas de carcinome hépatocellulaire, et le traitement du cancer du foie les utilisant comme cibles. Dans la présente invention, il a été découvert que l'expression de SMARCA4 augmente en cas de HCC et que SMARCA4 augmente directement l'expression d'IRAK 1, et il a été confirmé que les protéines oncogènes gankyrines et AKR1B10 sont induites par l'activation transcriptionnelle d'IRAK 1, et ainsi SMARCA4-IRAK1-Gankyrine et AKR1B10 peuvent être utilisés en tant que marqueurs spécifiques du cancer du foie et peuvent être utilisées efficacement en tant que cibles pour le traitement du cancer du foie.
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CN110124011A (zh) * 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) 一种染色质重构复合物核心催化亚基在制备治疗或预防乙型肝炎病毒感染药物中的应用

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