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WO2018026190A1 - Biomarker for predicting cancer prognosis - Google Patents

Biomarker for predicting cancer prognosis Download PDF

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Publication number
WO2018026190A1
WO2018026190A1 PCT/KR2017/008336 KR2017008336W WO2018026190A1 WO 2018026190 A1 WO2018026190 A1 WO 2018026190A1 KR 2017008336 W KR2017008336 W KR 2017008336W WO 2018026190 A1 WO2018026190 A1 WO 2018026190A1
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cancer
biomarker
expression
prognosis
pkr
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French (fr)
Korean (ko)
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이연수
김인후
이용선
이은경
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National Cancer Center Japan
National Cancer Center Korea
University of Texas System
University of Texas at Austin
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National Cancer Center Japan
National Cancer Center Korea
University of Texas System
University of Texas at Austin
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    • 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
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • G01N33/575—
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a composition for predicting cancer prognosis comprising a gene biomarker for predicting the prognosis of cancer, and an agent for measuring the expression level of the gene biomarker.
  • nc886 The role of nc886 in cancer is known because of its expression pattern in many cancer cell lines and patient samples. Previous studies in the inventors and other laboratories have highlighted its putative tumor suppressor role, based on its epigenetic silence in some tumors. However, several data suggest that increased nc886 expression is a more common phenomenon, especially during the early stages of oncogenesis. The nc886 level is much higher in proliferating cells than in non-proliferating tissue. For example, most immortalized cells express abundantly nc886. In many cases, high levels of nc886 are maintained or even higher as immortalized cells progress into transformed cells. Increased expression of nc886 in cancer is also shown in miRNA profiling data with probes that detect nc886. All these observations are consistent with higher PolIII activity in cancer cells than in normal cells.
  • nc886 plays an important role in determining cell death or proliferation through its inhibitory role in PKR.
  • PKR was originally known as a viral sensor that, when activated for viral infection, eIF2 ⁇ phosphorylation leads to cell death and consequently shut down cellular protein synthesis.
  • PKR is involved in various cellular signaling pathways and in cancer.
  • pro-apoptotic role suggested by PKR as its original tumor suppressor, its important role in cancer is still controversial.
  • Epigenetic silencing of nc886 occurs in subgroups of cancer cells, nc886 knockdown (KD) activates PKR, resulting in apoptosis, as in viral infection.
  • KD nc886 knockdown
  • nc886 in relation to PKR in tumor palpation or inhibition is not yet known. Increased expression of nc886 in proliferating cells is consistent with its oncogenic role corresponding to the putative tumor suppressor function of PKR: but this idea has not been demonstrated. Of course, the nc886 will play a PKR-independent role. Studies on nc886 have been limited due to the depletion of acute cell death through PKR activation. For this reason, the challenge for obtaining an nc886 ⁇ cell line from a homologous nc886 + control cell line. Comparison between the two cell lines is essential to measure the exact role of nc886 in cancer. In this study, we solved this problem by continuously producing PKR and nc886 knockout (KO) cells, and for the first time clearly determined the role in cancer, in particular thyroid and liver cancer.
  • PKR and nc886 knockout (KO) cells we solved this problem by continuously producing PKR and nc886 knock
  • the present invention is to provide a biomarker for predicting the prognosis of cancer comprising the nc886 gene.
  • the present invention provides a biomarker for predicting the prognosis of cancer comprising the nc886 gene.
  • the cancer may be thyroid cancer or the liver.
  • One embodiment of the present invention provides a composition for predicting cancer prognosis comprising an agent for measuring the expression level of the biomarker of the present invention.
  • the agent for measuring the expression level of the biomarker may comprise primer pairs, probes or antisense nucleotides that specifically bind to the biomarker.
  • Another embodiment of the present invention provides a kit for predicting cancer prognosis comprising the composition of the present invention.
  • the kit can be an RT-PCR kit, a competitive RP-PCR kit, a real time RT-PCR kit, a quantitative RT-PCR kit or a DNA chip kit.
  • step (a) obtaining the expression level, or expression pattern of the biomarker of claim 1 from a biological sample isolated from a cancer patient; And (b) comparing the expression level or expression pattern obtained in step (a) with a biomarker expression level or expression pattern of a corresponding gene of a cancer patient whose prognosis is known. to provide.
  • the method of measuring the biomarker level is reverse transcriptase polymerase (RT-PCR), competitive reverse transcriptase polymerase (competitive RT-PCR), real time quantitative RT-PCR, quantitative polymerase Quantitative RT-PCR, RNase protection method, Northern blotting or DNA chip technology, immunohistochemical staining, immunoprecipitation assay , Complementary fixation assay (complenent Fixation Assay) or immunofluorescence (immunofluorescence).
  • RT-PCR reverse transcriptase polymerase
  • competitive RT-PCR competitive reverse transcriptase polymerase
  • real time quantitative RT-PCR quantitative polymerase Quantitative RT-PCR
  • RNase protection method reverse transcriptase polymerase Quantitative RT-PCR
  • Northern blotting or DNA chip technology Western blotting or DNA chip technology
  • immunohistochemical staining immunoprecipitation assay
  • Complementary fixation assay Complementary fixation assay
  • immunofluorescence immunofluorescence
  • the method comprising the steps of: (a) treating the candidate material with a sample isolated from a cancer patient; (b) measuring the expression level of the biomarker according to claim 1 in a sample of the cancer patient treated with the candidate substance; And (c) if the biomarker expression level of step (b) is lower than before treatment of the candidate, determining the candidate as a material for preventing or treating cancer; It provides a cancer screening system for preventing or treating cancer.
  • a substance for inhibiting the expression or activity of the biomarker according to the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier.
  • the material may be an antisense oligonucleotide, aptamer, siRNA or shRNA to the biomarker.
  • the substance may be an antibody or an antigen-binding fragment thereof that inhibits the activity of the biomarker.
  • composition may inhibit the expression of one or more anti-apoptotic proteins of the group consisting of EGFR, HIPK2, HSPBL2 or TAXIBP1.
  • composition can increase the expression of CDKN2C or DKK1.
  • a substance for inhibiting the expression or activity of the biomarker according to the present invention comprising administering a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier.
  • a substance for inhibiting the expression or activity of the biomarker according to the present invention for the manufacture of a prophylactic or therapeutic agent for cancer for the manufacture of a prophylactic or therapeutic agent for cancer; And pharmaceutically acceptable carriers.
  • the present invention relates to a biomarker for predicting prognosis of thyroid cancer or liver cancer comprising the nc886 gene, and by measuring the expression level of the biomarker, the prognosis of the cancer patient can be predicted.
  • it is possible to determine the appropriate treatment direction according to the predicted prognosis it is possible to provide a treatment method for each patient, and to reduce the cancer recurrence and mortality of cancer patients with poor prognosis can more effectively treat breast cancer patients.
  • nc886 shows the expression of nc886 in tissue samples obtained from thyroid cancer patients and cell lines. Specifically, (a) shows the results of qRT-PCR measurements on nc886 of 37 pairs of thyroid tumors and adjacent normal tissues, and (b) and (c) show T stage (panel B) and lymph node metastasis (panel C). According to the subclassification of patients in the nc886 expression group, (d) shows the results of qRT-PCR measurement for nc886 thyroid cell line.
  • FIG. 2 shows nc886KD activating PKR that exacerbates cell proliferation in tissue samples obtained from thyroid cancer patients and cell lines. Specifically, (a) shows the results of Northern hybridization of nc886 and 5SrRNA by nc886 KD, loading control (top panel) and cell proliferation (MRS) analysis (bottom panel), and (b) shows panel N after panel nc86 KD. The displayed protein was confirmed by Western blot, (c) shows the nc886 KD data summary of panels A ⁇ B and the expected cell results of nc886 KO.
  • Figure 3 shows the results of cell proliferation analysis of PKR or nc886 KO cells in tissue samples obtained from thyroid cancer patients and cell lines. Specifically, (a) confirms KO cell lines by Western / Northern blots of PKR / nc886 with ⁇ -actin and 5S rRNA as loading controls, respectively, (b) shows MTT cell proliferation assay of the indicated KO cell line, (c) and (d) show colony formation assays.
  • FIG. 4 shows the results of cell migration ((a)-(b)) and invasion ((c)-(d)) of PKR or nc886 KO cells in tissue samples obtained from thyroid cancer patients and cell lines.
  • FIG. 5 shows the results of comparing gene expression profiles between PKR wt / nc886 wt , PKR KO / nc886 wt , and PKR KO / nc886 KO in tissue samples obtained from thyroid cancer patients and cell lines.
  • (a) represents a heat map showing the hierarchical clustering of 226 genes in which expression values were significantly changed (log2 (fc)> 1 or ⁇ -1) in PKR or nc886 KO
  • (b) is indicated Kaplan-Meier curves stratify the survival of 505 patients (TCGA cohort) according to gene expression.
  • FIG. 6 is a schematic diagram summarizing the role of nc886 in thyroid cancer.
  • FIG. 8 shows the results of (a) classifying patients with increased nc886 expression (39 patients) and patients with reduced nc886 expression (19 patients) in two subgroups of tumors of 58 liver cancer patients, and (b A Kaplan-Meier curve showing the probability of RFP and OS of the two subgroups, and (c) a heat map generated from mRNA microarray data of the 58 patient samples.
  • the inventors have completed the present invention by using the CRIPR / Cas-mediated gene KO to find out that nc886 plays an oncogene role in thyroid cancer or liver cancer.
  • the present invention provides a biomarker for predicting prognosis of cancer comprising the nc886 gene.
  • the cancer may be thyroid cancer or liver cancer.
  • nc886 actively proliferates thyroid cells and is essential for cell proliferation, migration and invasion, and high expression of nc886 is closely associated with tumor aggressiveness and lateral lymph node metastasis.
  • nc886 KO induced downregulation of a set of genes, some of which were found to be associated with good survival of patients in the TCGA cohort. That is, genes associated with nc886 may be a useful indicator in determining the extent of surgery before and after surgery and in determining the dose of radiation iodine therapy to improve the outcome after surgery.
  • the present invention provides a composition for predicting cancer prognosis comprising an agent for measuring the expression level of the biomarker according to the present invention. Details of the biomarker are as described above.
  • the agent for measuring the expression level of the biomarker may be a primer, probe, antisense oligonucleotide, aptamer or antibody specific for the biomarker.
  • the composition according to the present invention can perform thyroid cancer or liver cancer through the PCR amplification using the sense and antisense primer of the polynucleotide of nc886 to produce the desired product, PCR conditions, sense and antisense primer length is Modifications can be made based on what is known in.
  • the primers of the present invention can be chemically synthesized using phosphoramidite solid support methods or other well known methods, and such nucleic acid sequences can also be modified using many means known in the art. .
  • Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).
  • the present invention provides a kit for predicting cancer prognosis comprising a composition for predicting cancer prognosis according to the present invention. Details of the composition for predicting cancer prognosis are as described above.
  • the kit for measuring the expression level of the biomarker may be a kit containing the necessary elements necessary to perform RT-PCR.
  • the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC-water ( DEPC-water), sterile water, and the like.
  • the kit of the present invention may be a kit for detecting a gene for diagnosis of thyroid cancer or liver cancer, including the essential elements necessary to perform the DNA chip.
  • the DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached with a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.
  • the present invention comprises the steps of (a) obtaining the expression level, or expression pattern of the biomarker of claim 1 from a biological sample isolated from a cancer patient; And (b) comparing the expression level or expression pattern obtained in step (a) with a biomarker expression level or expression pattern of a corresponding gene of a cancer patient whose prognosis is known. to provide. Details of the biomarker are as described above.
  • the information providing method comprises the step [(a)] of obtaining the expression level, or expression pattern of the biomarker of claim 1 from a biological sample isolated from cancer patients.
  • the step of measuring the expression level of the biomarker from the biological sample separated from the individual to be diagnosed is preferably made through the step of contacting the diagnostic composition for thyroid cancer or liver cancer with the biological sample, but is not limited thereto.
  • the method for measuring the expression level is reverse transcriptase polymerase (RT-PCR), competitive reverse transcriptase polymerase (competitive RT-PCR), real time quantitative RT-PCR, quantitative polymerase Quantitative RT-PCR, RNase protection method, Northern blotting or DNA chip technology, immunohistochemical staining, immunoprecipitation assay Complement Fixation Assay, Immunofluorescence, but is not limited thereto.
  • the biological sample may be liver-derived tissue, cells, whole blood, blood serum, plasma, saliva, sputum or urine, but is not limited thereto.
  • the information providing method is a step of comparing the expression level or expression pattern obtained in step (a) with the biomarker expression level, or expression pattern of the corresponding gene of cancer patients with known prognosis [(b) step ] Is included. Specifically, comparing the expression level of the biomarker with the expression level of nc886 of the normal control sample is to confirm that the level of the biomarker in the sample is higher than the control.
  • the information providing method according to the present invention may further comprise the step of determining as thyroid cancer or liver cancer, if the expression level measured in step (a) is higher than the expression level of the biomarker of the normal control sample. Specifically, by comparing the expression level of nc886 in the normal control group and the expression level of nc886 in patients with suspected thyroid cancer or liver cancer, it is possible to diagnose whether the patient is actually a thyroid cancer or liver cancer, and further, the progression or prognosis of thyroid cancer or liver cancer. Can be predicted.
  • the information providing method according to the present invention it is possible to accurately develop thyroid cancer or liver cancer, and to predict the progression stage or the prognosis, and there is an advantage of making an appropriate treatment plan according to the predicted prognosis.
  • the present invention comprises the steps of (a) treating the candidate substance to a sample isolated from a cancer patient; (b) measuring the expression level of the biomarker according to claim 1 in a sample of the cancer patient treated with the candidate substance; And (c) if the biomarker expression level of step (b) is lower than before treatment of the candidate, determining the candidate as a material for preventing or treating cancer; It provides a cancer screening system for preventing or treating cancer. Details of the biomarker are as described above.
  • the screening system includes the step of treating the candidate material to the sample separated from the cancer patient [step (a)].
  • the sample may be thyroid or liver-derived tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine in patients with thyroid cancer or liver cancer, but is not limited thereto.
  • the candidate material may be individual nucleic acids, peptides, proteins, antibodies, other extracts or natural products, compounds, or the like, which are estimated to have potential as therapeutic agents for thyroid cancer or liver cancer according to a conventional selection method, or randomly selected. It is preferable that the compound inhibits the expression of the biomarker, but is not limited thereto.
  • the screening system comprises the step of (b) measuring the expression level of the biomarker according to claim 1 in the sample of cancer patients treated with the candidate. Specifically, by treating candidate cells for the treatment or prevention of thyroid cancer or liver cancer to liver cells or tissues or other biological samples, by measuring the expression level of the biomarker and the expression and phosphorylation of the signaling protein related to biomarker expression, thyroid cancer Or a substance for treating or preventing liver cancer.
  • step (c) determining the candidate as a substance for preventing or treating cancer.
  • a substance that inhibits the expression of the biomarker and the expression and phosphorylation of the biomarker expression-related signaling protein is determined as a substance for treating or preventing a disease of thyroid cancer or liver cancer.
  • Confirmation of the reaction between the substances, as a protein-protein, a protein-compound, or the above-mentioned candidates can use conventional methods used to confirm the reaction between protein-nucleic acid, peptides, antibodies, other extracts or natural products. .
  • the present invention is a substance for inhibiting the expression or activity of the biomarker according to claim 1; And it provides a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier. Details of the biomarker are as described above.
  • the substance may be an antisense oligonucleotide, aptamer, siRNA or shRNA to the biomarker, and may be an antibody or an antigen-binding fragment thereof that inhibits the activity of the protein encoded by the biomarker.
  • the antibody is not particularly limited thereto, but may be any antibody that can specifically bind to a protein encoded by the biomarker of the present invention, preferably a monoclonal antibody or a chimeric antibody. , Humanized antibodies, human antibodies, and the like, as well as functional fragments of the antibodies.
  • the antibody has the characteristic of binding to specifically recognize the protein encoded by the biomarker of the present invention, only the full form having the full length of two heavy chains and two light chains is provided. But includes functional fragments of antibody molecules.
  • the functional fragment of the molecule of an antibody means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.
  • composition can inhibit the proliferation and migration of thyroid cells by increasing the expression of CDKN2C or DKK1.
  • composition provided by the present invention may further include a therapeutic agent that exhibits therapeutic activity of thyroid cancer or liver cancer, in addition to an oligonucleotide or antibody that inhibits protein expression or activity encoded by the biomarker used as an active ingredient.
  • the composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent depending on the mode of administration. Specifically, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and any one or more of the above components may be mixed and used as necessary, antioxidants, Other additives commonly used, such as buffers, may additionally be included.
  • Diluents, dispersants, surfactants, binders, and lubricants may also be added to formulate into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions, etc., depending upon the purpose of administration, and may be specific to the target organ.
  • Target organ or tissue specific antibodies or other ligands can be used in combination with the carrier so as to facilitate the use.
  • Such types of carriers, excipients or additives include all conventional formulations in the art, and the types of carriers, excipients or additives usable by the above examples are not limited.
  • compositions or mixtures may be suitably administered to a subject according to conventional methods, routes of administration, and dosages used in the art, depending on the purpose or need.
  • routes of administration may be administered orally, orally, subcutaneously, intraperitoneally, intrapulmonally, and intranasally, and are administered by suitable methods, including intralesional administration if necessary for local immunosuppressive treatment.
  • Non-oral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • the appropriate dosage and frequency of administration may be selected according to methods known in the art, and the amount and frequency of administration of the composition comprising the antisense oligonucleotide, siRNA or shRNA of the present invention to be administered are symptoms to be prevented or treated. And various factors such as the type, route of administration, sex, health condition, diet, age and weight of the individual, and severity of the disease.
  • Nthy-ori 3-1 (Lemoine NR, Mayall ES, Jones T, Sheer D, McDermid S, Kendall-Taylor P et al. Characterization of human thyroid epithelial, SV-40 immortalized cell line derived from normal primary thyroid follicular epithelial cells cells immortalized in vitro by simian virus 40 DNA transfection.
  • BCPAP KTC-1, SNU-790 derived from papillary thyroid carcinoma; FTC133 derived from follicular thyroid carcinoma; C643, SW1736, and Cal-62 derived from anaplastic thyroid carcinoma [SNU-790, (Koh CS, Ku JL, Park SY, Kim KH, Choi JS, Kim IJ et al.
  • Nthy-ori 3-1 was purchased from Sigma-Aldrich (St. Louis, MO, USA) and BCPAP and Cal-62 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany); C643 and SW1736 from CLS (Cell Line service, Germany); KTC-1, SNU-790 and FTC133 lines were obtained from our laboratory stocks (National Cancer Center, Center for Thyroid Cancer, Korea). Cells were cultured in growth media rich in 10% fetal bovine serum (FBS) (GE Healthcare Life Sciences; Logan, UT, USA) and 1% antibiotic-antimyotic (Life Technologies; Carlsbad, CA, USA). All cell lines were incubated in a humidified incubator with 5% CO 2 at 7 ° C.
  • FBS fetal bovine serum
  • All cell lines were incubated in a humidified incubator with 5% CO 2 at 7 ° C.
  • Typical reagents used in this example are
  • Anti-oligos (“anti886 75-56” and “anti-vt 21-2”, which are designed as “anti-nc886” and “anti-control” in this study, respectively) are described as "Lee K, Kunkeaw N, Jeon SH, Lee I.” , Johnson BH, Kang GY et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA 2011; 17 (6): 1076-1089. ”And transfection. Total RNA of patient tissue samples and cell lines was isolated with Trizol reagent (Life Technologies; Carlsbad, Calif., USA).
  • hCas9 and "gRNA_Cloning Vector” were purchased from Addgene (plasmid # 41815 and # 41824, respectively).
  • PKR's sgRNA-expressing plasmid (“pCR sgPKR-1a") is a D. Found by Stacy Horne.
  • the sgRNA-expressing plasmids of nc886 were constructed according to the gDNA synthesis protocol [https://www.addgene.org/41824/and (33)]. Briefly, annealing two partially complementary oligos containing sgRNA sequences (FIG.
  • the Cas9-expressing plasmid (“hCas9"), combined with “pCR sgPKR-1a” (for PKR KO) or “pCR sg886-164" and “pCR sg886 + 15" (for nc886 KO), is a Lipofectamine 2000 (Life Technologies Transfection). Untransfected cells were treated in parallel with negative controls upon G418 selection. After 24 hours of transfection, the cells were transferred to a growth medium containing 1 mg / ml of G418. G418-resistant colonies were individually isolated and further cultured.
  • MTT 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) dye or its derivative MTT dye.
  • MTT and MTS dyes were purchased from Sigma-Aldrich and Promega (Madison, WI, USA) respectively and the assay was performed according to the manufacturer's instructions.
  • MTT and MTS dyes were purchased from Sigma-Aldrich and Promega (Madison, WI, USA) respectively and the assay was performed according to the manufacturer's instructions.
  • 100 cells were seeded in one of the 6-well plates and maintained for 7 days. Thereafter, the cells were stained and fixed with 1% crystal violet to count colonies.
  • Cell migration assays were performed using an 8- ⁇ m pore filter insert (BD Biosciences; San Jose, CA, USA). The cells were resuspended in serum-free RPMI-1640 medium, added to the upper chamber, and transferred to a lower chamber containing 1% PBS and RPMI-1640 for 24 hours. Migrated cells on the bottom surface of the insert were fixed, air dried for 20 minutes and stained with 1% crystal violet for 20 minutes. Thereafter, the retaining cells on the top surface were removed by wiping with a cotton swab. To quantify the migration rates, the insert was measured for absorbance at 564 nm with 10% acetic acid. Cell invasion assays were performed with matrigel-coated inserts (BD Biosciences). Cells were stained with Diff-Quik stain TM (Sysmex; Kobe, Japan) and cell number was measured.
  • PKR KO / nc886 KO cells grow more slowly than nc886 wt cells (PKR wt / nc886 wt and PKR KO / nc886 wt , FIGS. 3B-D and S7A-C). I could confirm it.
  • PKR KO itself which gives a growth advantage, can be seen as a larger colony size of PKR KO / nc886 wt cells than the colony size of PKR wt / nc886 wt .
  • MTT values and colony numbers did not increase significantly in the absence of PKR (FIGS. 3B-D and).
  • PKR KO / nc886 KO cells were clearly less migrating and invasive than PKR KO / nc886 wt cells, and PKR KO / nc886 wt cells were slightly more migrating and invasive than PKR wt / nc886 wt cells (FIGS. 4A-D). Overall, all of these data predicted that nc886 would play an oncogenic role.
  • Hierarchical clustering and generation of a heat map were performed using Cluster 3.0 and Java TreeView (version 1.1.6r4) softwares. Direct physical interactions and their pathways are described in the "GeneMANIA" Cytoscape plug-in function (http://www.cytoscape.org/) (Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P et al. . the GeneMANIA prediction server:.; : was estimated using the W214-220) biological network integration for gene prioritization and predicting gene function Nucleic Acids Res 2010 38 (Web Server issue)..
  • TCGA Cancer Genomic Altas
  • TCGA provides RNA-seq data in Expectation Minimization values (RSEM) at standardized expression levels.
  • RSEM Expectation Minimization values
  • fc fold-change
  • nc886 was measured in a pair of normal / tumor samples from 37 patients with thyroid cancer and divided into three groups (low, medium and high) according to the nc886 level (FIG. 1A). The proportions of each group were compared using? 2 and Fisher's exact test. Measurements were made for continuous variables, mean, and standard deviation analysis. The difference of the continuous variables was analyzed using Mann-Whitney U test, Student's unpaired two-sided t-test or one-way ANOVA. Statistical analysis was performed with STATA software (version 10, StataCorp., College Station, TX, USA). All p-values were two-sided, and p-values were considered statistically significant below 0.05.
  • nc886 in the thyroid cell line was measured, and it was confirmed that the expression was higher in most of the cancer cell line than the immortal cell line Nthy-ori-3-1 (FIG. 1D).
  • Nthy-ori 3-1 cells were not fully transformed but proliferated well and expressed significantly higher nc886 than non-proliferatong thyroid tissues.
  • Very low expression of nc886 was also observed in normal tissues of various organs, indicating that nc886 expression was proportional to cell proliferation. That is, by decreasing the serum concentration in the culture medium, when cell proliferation was slowed down, the expression of nc886 was decreased.
  • nc886 expression pattern is believed to play an oncogene role in thyroid cancer with respect to cell proliferation in vitro and tumor progression and aggression in patients.
  • nc886 is associated with a subset of thyroid cells, as seen in other types of cancer, including esophageal squamous cell carcinoma, gastric cancer, acute myeloid leukemia, and lung cancer. It was epigenetically silenced. Thus, the loss of phenotype was evaluated to clarify the role of nc886 in thyroid cancer since the possibility of a tumor suppressor role could not be ruled out.
  • nc886 suppresses PKR.
  • Nthy-ori 3-1, SW1736 and C643 thyroid cell lines were transfected with antisense oligonucleotides targeting nc886, the expression level of nc886 was reduced, as shown in the Northern blot of FIG. 2A.
  • nc886 KD induced PKR activation by increasing the active form of phospho-PKR (FIG. 2B).
  • Active PKR phosphorylated its best substrate eIF2® and consequently inhibited cell proliferation not only in immortalized Nthy-ori 3-1 cell line but also in thyroid cancer cell line SW1736 (FIGS. 2A-B).
  • eIF2 active phosphorylated its best substrate eIF2® and consequently inhibited cell proliferation not only in immortalized Nthy-ori 3-1 cell line but also in thyroid cancer cell line SW1736 (FIGS. 2A-B).
  • C643, eIF2? No phosphorylation or cell proliferation effect was observed
  • nc886 KD The damage of cell proliferation at nc886 KD appeared to be consistent with its putative oncogenic role. However, this phenomenon should be understood as a PKR dependent "tumor sensing model" rather than the role of nc886 in the etiology and / or progression of thyroid cancer. That is, nc886 KD immediately induced the PKR cell killing pathway before observing other phenotypes that could reflect the functional significance of elevated expression of nc886 in immortalized or transformed cells. In order to clarify this test the long-term cell phenotype and nc886-null (nc886 -) should be compared to the cells and isogenic nc886 + cells. Thus, the nc886 KO cell line was generated.
  • PKR KO cell line ahead of nc886 KO cell line was made.
  • wild type PSR wt / nc886 wt
  • PKR KO PSR KO / nc886 wt
  • double KO PSR KO / mc886 KO
  • Nthy-on 3-1 cells can be modified in either direction positive or below compared to nc886 / PKR KO. This is likely to be a significant advantage since nc886 and PKR KO are expected to show opposite phenotypes.
  • sgRNA small guide RNA
  • nc886 KO is a non-coding RNA
  • functional KO could not be guaranteed even if 1 ⁇ 2nt were deleted.
  • two sgRNAs adjacent to the nc886 transcript were designed to remove the entire DNA portion between them.
  • Two sgRNA-expressing plasmids were transfected with parental Nthy-ori 3-1 (PKR wt ) and Nthy-ori 3-1 (PKR KO ) cells.
  • PKR KO / nc886 KO clones confirmed that nc886 was not expressed by Northern hybridization, and these cell lines were used to investigate the role of nc886 in thyroid cancer. It was.
  • PKR KO / nc886 KO cells grew more slowly than nc886 wt cells.
  • PKR KO itself showed a fine growth advantage, as seen in PKR KO / nc886 wt cells of colony size larger than PKR wt / nc886 wt cells.
  • MTT value and colony number did not increase significantly.
  • Cell migration and invasive analysis also showed similar results. PKR KO / nc886 KO cells were found to be less cell migration and invasion than PKR KO / nc886 wt cells.
  • nc886 KO cells were in PKR KO -background, so all phenotypes represented by nc886 KO are PKR independent.
  • the proliferative role of nc886 cannot be attributed to the inhibition of pro-apoptotic function of PKR. If PKR inhibition is the only function of nc886, PKR KO / nc886 KO cells and PKR KO / nc886 wt cells will have the same phenotype.
  • Another important conclusion drawn from the data is when PKR appears to play an important role when contributing.
  • nc886 / PKR was further investigated.
  • Microarray experiments were performed on three cell lines (PKR wt / nc886 wt , PKR KO / nc886 wt and PKR KO / nc886 KO ) to obtain a list of 226 genes from expression changes between the cell lines.
  • FIG. 5A left panel
  • the expression of 201 genes and 25 genes were altered by PKR and nc886 KO, respectively.
  • Microarray data was confirmed by measuring some genes altered by qRT-PCR (FIG. 5A, right panel).
  • the inventors conducted genetic network analysis using the GeneMANIA plug-in tool in Cytoscape [http://www.cytoscape.org/] to identify direct physical interactions and pathway interconnections between the 201 genes.
  • p53 and the most renowned oncogene (MyC) are important hubs.
  • p53 and MYC are the most famous tumor suppressor and tumor genes, respectively, and are interestingly known to regulate Pol III transcription and thus nc886 expression. More research will be needed to determine the relationship between nc886 and p53, MYC and 201 genes.
  • TCGA Cancer Genome Atlas
  • nc886 normal and cancerous tissues were obtained from 58 liver cancer patients (from Dr. Lee's group at the MD Anderson Cancer Center, USA), RNA was isolated, and nc886 expression was measured by Northern blot. To properly calibrate the nc886 signal, the Northern experiment was performed once more using a probe for 5S rRNA in the same blot. After quantifying the northern result by a densitometer, the value of nc886 / 5S rRNA for each sample was obtained and used as the nc886 expression. By comparing nc886 expression levels of liver cancer samples and corresponding normal tissue samples, 58 patients were divided into two subgroups. As a result, nc886 was increased in 39 patients and nc886 was decreased in 19 patients. This tendency was similar to that in thyroid cancer.
  • MRNA array results are present in 58 pairs of samples (obtained from Dr. Lee's group at the MD Anderson Cancer Center, USA). As a result of clustering the mRNA expression patterns according to the two groups with high and low nc886 expression, the activity of the cancer-promoting transcription factor was increased in the high nc886 group, and the activity of the cancer suppressor microRNA was decreased.
  • nc886 acts as a cancer promoting factor in liver cancer.

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Abstract

The present invention relates to a genetic biomarker for predicting prognosis of cancer, particularly, thyroid cancer or liver cancer, a cancer prognosis predicting composition comprising an agent for measuring an expression level of the genetic biomarker, a cancer prognosis predicting kit comprising the composition, an information providing method for predicting prognosis of cancer by use of the kit, a system for screening a material prophylactic or curative of cancer by use of the genetic biomarker, and a pharmaceutical composition targeting the genetic biomarker to prevent or treat cancer. More particularly, the prognosis of a cancer patient can be predicted by measuring an expression level of the genetic biomarker of the present invention in the cancer patient, and the expression of a corresponding genetic biomarker responsible for poor prognosis can be suppressed, depending on prognosis prediction results, so that cancer patients can be more effectively treated.

Description

암 예후 예측용 바이오마커Biomarkers for predicting cancer prognosis

본 출원은 2016년 08월 02일 출원된 대한민국 특허출원 제10-2016-0098635호 를 우선권으로 주장하고, 상기 명세서 전체는 본 출원의 참고문헌이다. This application claims the priority of Korean Patent Application No. 10-2016-0098635, filed August 02, 2016, the entirety of which is a reference of the present application.

본 발명은 암의 예후를 예측하기 위한 유전자 바이오마커, 상기 유전자 바이오마커의 발현수준을 측정하는 제제를 포함하는 암 예후 예측용 조성물에 관한 것이다.The present invention relates to a composition for predicting cancer prognosis comprising a gene biomarker for predicting the prognosis of cancer, and an agent for measuring the expression level of the gene biomarker.

암에서의 nc886의 역할은 다수의 암세포 주 및 환자 시료에서 이것의 발현 패턴 때문에 알려졌다. 본 발명자 및 다른 연구실에서의 이전의 연구들은 몇몇 종양들에서 이것의 후성 유적학적 침묵에 기반하여, 이것의 추정적인 종양 억제 역할을 강조했다. 그러나, 여러 데이터들은 nc886 발현의 증가가, 특히 종양발생의 초기 단계 동안에 좀 더 일반적인 현상이라는 것을 암시했다. 상기 nc886 레벨은 비-증식 조직보다 증식 세포에서 훨씬 더 높다. 예를 들면, 대부분 불멸화 세포(immortalized cell)들은 nc886을 풍부하게 발현한다. 많은 케이스에서, nc886의 높은 레벨은 불멸화 세포(immortalized cell)가 형질전환된 세포로 진행됨에 따라 유지되거나 심지어 더 높아진다. 암에서 nc886의 증가된 발현은 nc886을 검파하는 프로브가 있는 miRNA 프로파일링 데이터에서 또한 보여진다. 모든 이러한 관찰들은 정상 세포에서보다 암세포에서의 더 높은 PolⅢ 활성과 일치한다. The role of nc886 in cancer is known because of its expression pattern in many cancer cell lines and patient samples. Previous studies in the inventors and other laboratories have highlighted its putative tumor suppressor role, based on its epigenetic silence in some tumors. However, several data suggest that increased nc886 expression is a more common phenomenon, especially during the early stages of oncogenesis. The nc886 level is much higher in proliferating cells than in non-proliferating tissue. For example, most immortalized cells express abundantly nc886. In many cases, high levels of nc886 are maintained or even higher as immortalized cells progress into transformed cells. Increased expression of nc886 in cancer is also shown in miRNA profiling data with probes that detect nc886. All these observations are consistent with higher PolIII activity in cancer cells than in normal cells.

nc886은 PKR에서 이것의 억제 역할을 통해 세포 사멸 또는 증식을 결정하는데 있어서 중요한 역할을 한다. PKR은 원래 바이러스 감염에 활성화될 때 eIF2α 인산화함으로써 세포 사멸을 이끌고 결과적으로 세포성 단백질 합성을 shut down 되는 바이러스성 센서로 알려져 있다. 바이러스성 센싱 뿐만 아니라, PKR은 다양한 세포성 신호 통로에 관여하고 암에 관여한다. PKR의 본래 종양 억제자 역할로 제시된 pro-apoptotic 역할에도 불구하고 암에서 이것의 중요한 역할은 여전히 논쟁적이다. nc886의 후성유전학적 침묵은 암세포의 아군에서 발생하고, nc886 knockdown(KD)은 PKR을 활성화시키며, 결과적으로 바이러스 감염에서와 마찬가지로, 세포사멸을 유도한다. 이러한 결과는 제거 전-암성 세포에서 nc886/PKR의 중요성을 설명하기 위하여, PKR의 본래 바이러스 감지 역할과 비교된 "종양 감지 모델"을 제안하였다.nc886 plays an important role in determining cell death or proliferation through its inhibitory role in PKR. PKR was originally known as a viral sensor that, when activated for viral infection, eIF2α phosphorylation leads to cell death and consequently shut down cellular protein synthesis. In addition to viral sensing, PKR is involved in various cellular signaling pathways and in cancer. Despite the pro-apoptotic role suggested by PKR as its original tumor suppressor, its important role in cancer is still controversial. Epigenetic silencing of nc886 occurs in subgroups of cancer cells, nc886 knockdown (KD) activates PKR, resulting in apoptosis, as in viral infection. These results suggested a "tumor detection model" compared with PKR's original virus detection role to explain the importance of nc886 / PKR in pre-cancerous cells removed.

그럼에도 불구하고, 종양 촉진 또는 억제에 있어 PKR과의 관계에서 nc886의 정확한 역할은 아직 밝혀지지 않았다. 증식 세포에서 nc886의 증가된 발현은 PKR의 추정 종양 억제 기능에 대응하는 그것의 oncogenic 역할과 일치한다: 그러나 이러한 아이디어는 입증되지 않았다. 물론, nc886은 PKR-독립적인 역할을 할 것이다. nc886에 관한 연구는 PKR 활성화를 통해 급성 세포 사멸에서의 고갈 결과로 인한 한계가 있었다. 이러한 이유로, 동종 nc886+대조군 세포주로부터 nc886- 세포주를 수득하는 것에 대한 도전이 되었다. 두 세포주 사이의 비교는 암에서 nc886의 정확한 역할을 측정하기 위해 필수적이다. 본 연구에서, 본 발명자들은 PKR 및 nc886 낙아웃(knockout, KO) 세포를 연속적으로 생산함으로써 상기와 같은 문제를 해결하였고, 처음으로, 암 특히 갑상선암 및 간암에서의 역할을 명백히 결정하였다.Nevertheless, the exact role of nc886 in relation to PKR in tumor palpation or inhibition is not yet known. Increased expression of nc886 in proliferating cells is consistent with its oncogenic role corresponding to the putative tumor suppressor function of PKR: but this idea has not been demonstrated. Of course, the nc886 will play a PKR-independent role. Studies on nc886 have been limited due to the depletion of acute cell death through PKR activation. For this reason, the challenge for obtaining an nc886 − cell line from a homologous nc886 + control cell line. Comparison between the two cell lines is essential to measure the exact role of nc886 in cancer. In this study, we solved this problem by continuously producing PKR and nc886 knockout (KO) cells, and for the first time clearly determined the role in cancer, in particular thyroid and liver cancer.

본 발명은 nc886 유전자를 포함하는 암의 예후 예측용 바이오마커 등을 제공하고자 한다. The present invention is to provide a biomarker for predicting the prognosis of cancer comprising the nc886 gene.

그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다. However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

본 발명은 nc886 유전자를 포함하는 암의 예후 예측용 바이오마커 를 제공한다.The present invention provides a biomarker for predicting the prognosis of cancer comprising the nc886 gene.

상기 암은 갑상선암 또는 간일 수 있다.The cancer may be thyroid cancer or the liver.

본 발명의 일구현예는 본 발명의 바이오마커의 발현 수준을 측정하는 제제를 포함하는 암 예후 예측용 조성물을 제공한다.One embodiment of the present invention provides a composition for predicting cancer prognosis comprising an agent for measuring the expression level of the biomarker of the present invention.

상기 바이오마커의 발현 수준을 측정하는 제제는 상기 바이오마커에 특이적으로 결합하는 프라이머 쌍, 프로브 또는 안티센스 뉴클레오티드를 포함할 수 있다.The agent for measuring the expression level of the biomarker may comprise primer pairs, probes or antisense nucleotides that specifically bind to the biomarker.

본 발명의 다른 일구현예는 본 발명의 조성물을 포함하는 암 예후 예측용 키트를 제공한다.Another embodiment of the present invention provides a kit for predicting cancer prognosis comprising the composition of the present invention.

상기 키트는 RT-PCR 키트, 경쟁적 RP-PCR 키트, 실시간 RT-PCR 키트, 정량적 RT-PCR 키트 또는 DNA 칩 키트 일 수 있다.The kit can be an RT-PCR kit, a competitive RP-PCR kit, a real time RT-PCR kit, a quantitative RT-PCR kit or a DNA chip kit.

본 발명의 또 다른 일구현예로, (a) 암 환자에서 분리된 생물학적 시료로부터 제1항의 바이오마커의 발현 수준, 또는 발현 패턴을 얻는 단계; 및 (b) 상기 (a) 단계에서 얻은 발현 수준 또는 발현 패턴을 예후가 알려진 암 환자의 해당 유전자의 바이오마커 발현 수준, 또는 발현 패턴과 비교하는 단계를 포함하는 암 예후 예측을 위한 정보제공방법을 제공한다.In another embodiment of the present invention, (a) obtaining the expression level, or expression pattern of the biomarker of claim 1 from a biological sample isolated from a cancer patient; And (b) comparing the expression level or expression pattern obtained in step (a) with a biomarker expression level or expression pattern of a corresponding gene of a cancer patient whose prognosis is known. to provide.

상기 바이오마커 수준을 측정하는 방법은 역전사효소 중합반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(competitive RT-PCR), 실시간 역전사 효소 중합효소반응(real time quantitative RT-PCR), 정량적 중합효소반응(quantitative RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블랏팅(Nothern blotting) 또는 DNA 칩 방법(DNA chip technology), 면역조직화학염색법(immunohistochemical staining), 면역침전분석법(immunoprecipitation assay), 보체 고정 분석법(complenent Fixation Assay) 또는 면역형광법(immunofluorescence)인 것일 수 있다.The method of measuring the biomarker level is reverse transcriptase polymerase (RT-PCR), competitive reverse transcriptase polymerase (competitive RT-PCR), real time quantitative RT-PCR, quantitative polymerase Quantitative RT-PCR, RNase protection method, Northern blotting or DNA chip technology, immunohistochemical staining, immunoprecipitation assay , Complementary fixation assay (complenent Fixation Assay) or immunofluorescence (immunofluorescence).

본 발명의 또 다른 일구현예로, (a) 암 환자에서 분리한 시료에 후보물질을 처리하는 단계; (b) 후보물질이 처리된 암 환자의 시료에서 제1항에 따른 바이오마커의 발현 수준을 측정하는 단계; 및 (c) 상기 (b) 단계의 바이오마커 발현 수준이 후보물질의 처리 전보다 낮은 경우, 후보물질을 암의 예방 또는 치료용 물질로 판단하는 단계; 를 포함하는 암 예방 또는 치료용 물질 스크리닝 시스템을 제공한다. In another embodiment of the present invention, the method comprising the steps of: (a) treating the candidate material with a sample isolated from a cancer patient; (b) measuring the expression level of the biomarker according to claim 1 in a sample of the cancer patient treated with the candidate substance; And (c) if the biomarker expression level of step (b) is lower than before treatment of the candidate, determining the candidate as a material for preventing or treating cancer; It provides a cancer screening system for preventing or treating cancer.

본 발명의 또 다른 일구현예로, 본 발명에 따른 바이오마커의 발현 또는 활성을 억제하는 물질; 및 약학적으로 허용가능한 담체를 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.In another embodiment of the present invention, a substance for inhibiting the expression or activity of the biomarker according to the present invention; And it provides a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier.

상기 물질은 바이오마커에 대한 안티센스 올리고 뉴클레오티드, 앱타머(aptamer), siRNA 또는 shRNA인 것일 수 있다.The material may be an antisense oligonucleotide, aptamer, siRNA or shRNA to the biomarker.

상기 물질은 상기 바이오마커의 활성을 억제하는 항체 또는 그의 항원 결합 단편인 것일 수 있다.The substance may be an antibody or an antigen-binding fragment thereof that inhibits the activity of the biomarker.

상기 조성물은 ANLN, C5orf13(=NREP), COL5A1, VGLL2, C15orf52, 또는 KIAA1644의 발현을 억제할 수 있다. The composition can inhibit the expression of ANLN, C5orf13 (= NREP), COL5A1, VGLL2, C15orf52, or KIAA1644.

상기 조성물은 EGFR, HIPK2, HSPBL2 또는 TAXIBP1으로 구성된 군 중 하나 이상의 항-세포사멸 단백질(anti-apoptotic protein)의 발현을 억제할 수 있다. The composition may inhibit the expression of one or more anti-apoptotic proteins of the group consisting of EGFR, HIPK2, HSPBL2 or TAXIBP1.

상기 조성물은 CDKN2C 또는 DKK1의 발현을 증가시킬 수 있다. The composition can increase the expression of CDKN2C or DKK1.

본 발명의 또 다른 일구현예로, 본 발명에 따른 바이오마커의 발현 또는 활성을 억제하는 물질; 및 약학적으로 허용가능한 담체를 포함하는 암 예방 또는 치료용 약학적 조성물을 투여하는 것을 포함하는 암 예방 또는 치료 방법을 제공하고자 한다. In another embodiment of the present invention, a substance for inhibiting the expression or activity of the biomarker according to the present invention; And to provide a method for preventing or treating cancer comprising administering a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier.

본 발명의 또 다른 일구현예로, 암 예방 또는 치료제의 제조를 위한 본 발명에 따른 바이오마커의 발현 또는 활성을 억제하는 물질; 및 약학적으로 허용가능한 담체를 포함하는 조성물의 용도를 제공하고자 한다. In another embodiment of the present invention, a substance for inhibiting the expression or activity of the biomarker according to the present invention for the manufacture of a prophylactic or therapeutic agent for cancer; And pharmaceutically acceptable carriers.

본 발명은 nc886 유전자를 포함하는 갑상선암 또는 간암의 예후 예측용 바이오마커에 관한 것으로서, 상기 바이오마커의 발현 수준을 측정함으로써, 암 환자의 예후를 예측할 수 있다. 또한, 예측된 예후에 따라 적절한 치료방향을 결정할 수 있어 환자 맞춤별 치료방법의 제공이 가능하고, 불량한 예후의 암 환자의 암 재발 및 사망률을 감소시켜 유방암 환자를 더욱 효과적으로 치료할 수 있다.The present invention relates to a biomarker for predicting prognosis of thyroid cancer or liver cancer comprising the nc886 gene, and by measuring the expression level of the biomarker, the prognosis of the cancer patient can be predicted. In addition, it is possible to determine the appropriate treatment direction according to the predicted prognosis, it is possible to provide a treatment method for each patient, and to reduce the cancer recurrence and mortality of cancer patients with poor prognosis can more effectively treat breast cancer patients.

도 1은 갑상선암 환자 및 세포주로부터 얻은 조직샘플에서의 nc886의 발현을 나타낸 것이다. 구체적으로, (a)는 갑상선 종양 및 인접한 정상조직의 37쌍의 nc886에 대한 qRT-PCR 측정 결과를 나타낸 것이고, (b) 및 (c)는 T 병기(패널B) 및 림프절 전이(패널 C)에 따라 nc886 발현 그룹에서 환자의 하위 분류를 나타낸 것이며, (d)는 갑상선 세포주의 nc886에 대한 qRT-PCR 측정 결과를 나타낸 것이다. 1 shows the expression of nc886 in tissue samples obtained from thyroid cancer patients and cell lines. Specifically, (a) shows the results of qRT-PCR measurements on nc886 of 37 pairs of thyroid tumors and adjacent normal tissues, and (b) and (c) show T stage (panel B) and lymph node metastasis (panel C). According to the subclassification of patients in the nc886 expression group, (d) shows the results of qRT-PCR measurement for nc886 thyroid cell line.

도 2는 갑상선암 환자 및 세포주로부터 얻은 조직샘플에서 세포 증식을 악화시키는 PKR를 활성화시키는 nc886KD를 나타낸 것이다. 구체적으로, (a)는 nc886 KD후, 로딩 컨트롤(상단 패널) 및 세포증식(MRS)분석(하단 패널)으로 nc886 및 5SrRNA의 Northern hybridization 결과를 나타낸 것이고, (b)는 nc86 KD 후 패널 A에서 표시된 단백질을 Western blot으로 확인한 결과이며, (c)는 패널 A~B의 nc886 KD 데이터 요약 및 nc886 KO의 예상 세포 결과를 나타낸 것이다. FIG. 2 shows nc886KD activating PKR that exacerbates cell proliferation in tissue samples obtained from thyroid cancer patients and cell lines. Specifically, (a) shows the results of Northern hybridization of nc886 and 5SrRNA by nc886 KD, loading control (top panel) and cell proliferation (MRS) analysis (bottom panel), and (b) shows panel N after panel nc86 KD. The displayed protein was confirmed by Western blot, (c) shows the nc886 KD data summary of panels A ~ B and the expected cell results of nc886 KO.

도 3은 갑상선암 환자 및 세포주로부터 얻은 조직샘플에서 PKR 또는 nc886 KO 세포의 세포 증식 분석 결과를 나타낸 것이다. 구체적으로, (a)는 PKR/nc886의 Western/Northern blots에 의한 KO 세포주를 각각 loading controls로서 β-actin 및 5S rRNA로 확인한 것이고, (b)는 표시된 KO 세포주의 MTT 세포 증식 분석을 나타낸 것이며, (c) 및 (d)는 콜로니 형성 분석을 나타낸 것이다. Figure 3 shows the results of cell proliferation analysis of PKR or nc886 KO cells in tissue samples obtained from thyroid cancer patients and cell lines. Specifically, (a) confirms KO cell lines by Western / Northern blots of PKR / nc886 with β-actin and 5S rRNA as loading controls, respectively, (b) shows MTT cell proliferation assay of the indicated KO cell line, (c) and (d) show colony formation assays.

도 4는 (갑상선암 환자 및 세포주로부터 얻은 조직샘플에서 PKR 또는 nc886 KO 세포의 세포 이동((a)~(b)) 및 침습((c)~(d)) 결과를 나타낸 것이다.4 shows the results of cell migration ((a)-(b)) and invasion ((c)-(d)) of PKR or nc886 KO cells in tissue samples obtained from thyroid cancer patients and cell lines.

도 5는 갑상선암 환자 및 세포주로부터 얻은 조직샘플에서 PKRwt/nc886wt, PKRKO/nc886wt, 및 PKRKO/nc886KO 사이의 유전자 발현 프로필을 비교한 결과를 나타낸 것이다. 구체적으로, (a)는 PKR 또는 nc886 KO에서 발현 값이 유의하게(log2 (fc)> 1 또는 <-1) 변화된 226개의 유전자의 계층적 클러스터링을 보여주는 히트 맵을 나타내는 것이고, (b)는 표시된 유전자의 발현에 따라 505명의 환자(TCGA 코호트)의 생존율을 계층화하는 Kaplan-Meier 곡선을 나타낸 것이다.FIG. 5 shows the results of comparing gene expression profiles between PKR wt / nc886 wt , PKR KO / nc886 wt , and PKR KO / nc886 KO in tissue samples obtained from thyroid cancer patients and cell lines. Specifically, (a) represents a heat map showing the hierarchical clustering of 226 genes in which expression values were significantly changed (log2 (fc)> 1 or <-1) in PKR or nc886 KO, and (b) is indicated Kaplan-Meier curves stratify the survival of 505 patients (TCGA cohort) according to gene expression.

도 6은 nc886의 갑상선암에서의 역할을 요약한 모식도이다. 6 is a schematic diagram summarizing the role of nc886 in thyroid cancer.

도 7(a)~(c)는 간세포 암종양(hepatocellular carcinoma tumor) 및 정상 간 조직에서의 nc886 발현 여부를 비교한 그래프이다. 7 (a) to (c) is a graph comparing the expression of nc886 in hepatocellular carcinoma tumor and normal liver tissue.

도 8은 (a) 58명의 간암 환자의 종양에서 nc886의 발현이 증가된 환자(39명) 및 nc886의 발현이 감소된 환자(19명)을 두 하위 그룹으로 분류한 결과를 나타낸 것이고, (b) 상기 두 하위그룹의 RFP 및 OS의 확률을 보여주는 Kaplan-Meier 곡선을 나타낸 것이며, (c) 상기 58개의 환자 표본의 mRNA 마이크로어레이 데이터로부터 생성된 히트맵(heat map)을 나타내는 것이다. FIG. 8 shows the results of (a) classifying patients with increased nc886 expression (39 patients) and patients with reduced nc886 expression (19 patients) in two subgroups of tumors of 58 liver cancer patients, and (b A Kaplan-Meier curve showing the probability of RFP and OS of the two subgroups, and (c) a heat map generated from mRNA microarray data of the 58 patient samples.

본 발명자들은 CRIPR/Cas-매개(mediated) 유전자 KO를 사용하여, nc886이 갑상선 암 또는 간암에서 종양 유발 유전자(oncogene) 역할을 한다는 것을 밝혀냄으로써, 본 발명을 완성하였다. The inventors have completed the present invention by using the CRIPR / Cas-mediated gene KO to find out that nc886 plays an oncogene role in thyroid cancer or liver cancer.

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

암의 예후 예측용 바이오마커Biomarkers for predicting cancer prognosis

본 발명은 nc886 유전자를 포함하는 암의 예후 예측용 바이오마커를 제공한다. 구체적으로, 상기 암은 갑상선암 또는 간암일 수 있다. The present invention provides a biomarker for predicting prognosis of cancer comprising the nc886 gene. Specifically, the cancer may be thyroid cancer or liver cancer.

상기 nc886의 발현은 갑상선 세포를 활발하게 증식시키며, 세포 증식, 이동 및 침습에 필수적이며, nc886의 고발현은 종양의 공격성 및 측면 림프절 전이와 밀접하게 관련되어 있다. nc886 KO는 한 세트 유전자의 하향 조절을 유도하고, 이들 중 일부 저발현은 TCGA 코호트(cohort)에서 환자의 양호한 생존과 관련이 있음을 확인할 수 있었다. 즉, nc886과 관련된 유전자는 수술 전후 수술의 범위를 결정하고, 수술 후의 결과를 개선하기 위해 방사선 요오드 요법의 용량을 결정하는데 유익한 지표가 될 수 있다. The expression of nc886 actively proliferates thyroid cells and is essential for cell proliferation, migration and invasion, and high expression of nc886 is closely associated with tumor aggressiveness and lateral lymph node metastasis. nc886 KO induced downregulation of a set of genes, some of which were found to be associated with good survival of patients in the TCGA cohort. That is, genes associated with nc886 may be a useful indicator in determining the extent of surgery before and after surgery and in determining the dose of radiation iodine therapy to improve the outcome after surgery.

암 예후 예측용 조성물Composition for predicting cancer prognosis

본 발명은 본 발명에 따른 바이오마커의 발현 수준을 측정하는 제제를 포함하는 암 예후 예측용 조성물을 제공한다. 상기 바이오마커의 구체적인 내용은 전술한 바와 같다. The present invention provides a composition for predicting cancer prognosis comprising an agent for measuring the expression level of the biomarker according to the present invention. Details of the biomarker are as described above.

구체적으로, 상기 바이오마커의 발현 수준을 측정하는 제제는 상기 바이오마커에 특이적인 프라이머, 프로브, 안티센스 올리고뉴클레오티드, 앱타머(aptamer) 또는 항체일 수 있다. 본 발명에 따른 조성물은 nc886의 폴리뉴클레오티드의 센스 및 안티센스 프라이머를 이용하여 PCR 증폭을 실시하여 원하는 생성물의 생성 여부를 통해 갑상선암 또는 간암을 진단할 수 있고, PCR 조건, 센스 및 안티센스 프라이머 길이는 당업계에 공지된 것을 기초로 변형할 수 있다. 또한, 본 발명의 프라이머는 포스포르아미다이트 고체 지지체 방법 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있으며, 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.Specifically, the agent for measuring the expression level of the biomarker may be a primer, probe, antisense oligonucleotide, aptamer or antibody specific for the biomarker. The composition according to the present invention can perform thyroid cancer or liver cancer through the PCR amplification using the sense and antisense primer of the polynucleotide of nc886 to produce the desired product, PCR conditions, sense and antisense primer length is Modifications can be made based on what is known in. In addition, the primers of the present invention can be chemically synthesized using phosphoramidite solid support methods or other well known methods, and such nucleic acid sequences can also be modified using many means known in the art. . Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

암 예후 예측용 키트Cancer prognosis prediction kit

본 발명은 본 발명에 따른 암 예후 예측용 조성물을 포함하는 암 예후 예측용 키트를 제공한다. 상기 암 예후 예측용 조성물의 구체적인 내용은 전술한 바와 같다. The present invention provides a kit for predicting cancer prognosis comprising a composition for predicting cancer prognosis according to the present invention. Details of the composition for predicting cancer prognosis are as described above.

구체적으로, 상기 바이오마커의 발현수준을 측정하기 위한 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. RT-PCR 키트는 마커 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합효소 및 역전사효소, DNase, RNase 억제제, DEPC-물(DEPC-water), 멸균수 등을 포함할 수 있다.Specifically, the kit for measuring the expression level of the biomarker may be a kit containing the necessary elements necessary to perform RT-PCR. In addition to each primer pair specific for the marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC-water ( DEPC-water), sterile water, and the like.

또한, 본 발명의 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 갑상선암 또는 간암의 진단용 유전자를 검출하기 위한 키트일 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고 기판은 정량 대조군 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다.In addition, the kit of the present invention may be a kit for detecting a gene for diagnosis of thyroid cancer or liver cancer, including the essential elements necessary to perform the DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached with a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

암 예후 예측을 위한 정보제공방법Methods of providing information for predicting cancer prognosis

본 발명은 (a) 암 환자에서 분리된 생물학적 시료로부터 제1항의 바이오마커의 발현 수준, 또는 발현 패턴을 얻는 단계; 및 (b) 상기 (a) 단계에서 얻은 발현 수준 또는 발현 패턴을 예후가 알려진 암 환자의 해당 유전자의 바이오마커 발현 수준, 또는 발현 패턴과 비교하는 단계를 포함하는 암 예후 예측을 위한 정보제공방법을 제공한다. 상기 바이오마커의 구체적인 내용은 전술한 바와 같다. The present invention comprises the steps of (a) obtaining the expression level, or expression pattern of the biomarker of claim 1 from a biological sample isolated from a cancer patient; And (b) comparing the expression level or expression pattern obtained in step (a) with a biomarker expression level or expression pattern of a corresponding gene of a cancer patient whose prognosis is known. to provide. Details of the biomarker are as described above.

먼저, 본 발명에 따른 정보제공방법은 암 환자에서 분리된 생물학적 시료로부터 제1항의 바이오마커의 발현 수준, 또는 발현 패턴을 얻는 단계[(a) 단계]를 포함한다. 구체적으로, 진단하고자 하는 개체로부터 분리된 생물학적 시료로부터 상기 바이오마커의 발현 수준을 측정하는 단계는 갑상선암 또는 간암의 진단용 조성물을 생물학적 시료와 접촉시키는 단계를 통하여 이루어지는 것이 바람직하나, 이에 한정되지 않는다. 이때, 발현 수준을 측정하는 방법은 역전사효소 중합반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(competitive RT-PCR), 실시간 역전사 효소 중합효소반응(real time quantitative RT-PCR), 정량적 중합효소반응(quantitative RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블랏팅(Nothern blotting) 또는 DNA 칩 방법(DNA chip technology), 면역조직화학염색법(immunohistochemical staining), 면역침전분석법(immunoprecipitation assay), 보체 고정 분석법(complenent Fixation Assay), 면역형광법(immunofluorescence)인 것일 수 있으나, 이에 한정되지 않는다. 또한, 상기 생물학적 시료는 간 유래 조직, 세포, 전혈, 혈정, 혈장, 타액, 객담 또는 뇨일 수 있으나, 이에 한정되지 않는다. First, the information providing method according to the present invention comprises the step [(a)] of obtaining the expression level, or expression pattern of the biomarker of claim 1 from a biological sample isolated from cancer patients. Specifically, the step of measuring the expression level of the biomarker from the biological sample separated from the individual to be diagnosed is preferably made through the step of contacting the diagnostic composition for thyroid cancer or liver cancer with the biological sample, but is not limited thereto. At this time, the method for measuring the expression level is reverse transcriptase polymerase (RT-PCR), competitive reverse transcriptase polymerase (competitive RT-PCR), real time quantitative RT-PCR, quantitative polymerase Quantitative RT-PCR, RNase protection method, Northern blotting or DNA chip technology, immunohistochemical staining, immunoprecipitation assay Complement Fixation Assay, Immunofluorescence, but is not limited thereto. In addition, the biological sample may be liver-derived tissue, cells, whole blood, blood serum, plasma, saliva, sputum or urine, but is not limited thereto.

다음으로, 본 발명에 따른 정보제공방법은 상기 (a) 단계에서 얻은 발현 수준 또는 발현 패턴을 예후가 알려진 암 환자의 해당 유전자의 바이오마커 발현 수준, 또는 발현 패턴과 비교하는 단계[(b) 단계]를 포함한다. 구체적으로, 상기 바이오카커의 발현 수준을 정상 대조군 시료의 nc886의 발현 수준과 비교하는 단계는 상기 시료 내 상기 바이오마커의 수준이 대조군에 비해 높은 것을 확인하는 단계이다. Next, the information providing method according to the present invention is a step of comparing the expression level or expression pattern obtained in step (a) with the biomarker expression level, or expression pattern of the corresponding gene of cancer patients with known prognosis [(b) step ] Is included. Specifically, comparing the expression level of the biomarker with the expression level of nc886 of the normal control sample is to confirm that the level of the biomarker in the sample is higher than the control.

이후, 본 발명에 따른 정보제공방법은 상기 (a) 단계에서 측정한 발현 수준이 정상 대조군 시료의 해당 바이오마커의 발현 수준보다 높은 경우, 갑상선암 또는 간암으로 판단하는 단계를 추가로 포함할 수 있다. 구체적으로, 정상 대조군에서의 nc886의 발현 수준과 갑상선암 또는 간암 의심 환자에서의 nc886의 발현 수준을 비교함으로써, 갑상선암 또는 간암의 실제 환자 여부를 진단할 수 있고, 나아가서는 갑상선암 또는 간암의 진행단계 또는 예후를 예측할 수 있다. Then, the information providing method according to the present invention may further comprise the step of determining as thyroid cancer or liver cancer, if the expression level measured in step (a) is higher than the expression level of the biomarker of the normal control sample. Specifically, by comparing the expression level of nc886 in the normal control group and the expression level of nc886 in patients with suspected thyroid cancer or liver cancer, it is possible to diagnose whether the patient is actually a thyroid cancer or liver cancer, and further, the progression or prognosis of thyroid cancer or liver cancer. Can be predicted.

따라서, 본 발명에 따른 정보제공방법에 의하면, 갑상선암 또는 간암을 정확하게 진달할 수 있고, 진행단계 또는 예후를 예측할 수 있는바, 예측된 예후에 따라 적절한 치료계획을 세울 수 있는 이점이 있다. Therefore, according to the information providing method according to the present invention, it is possible to accurately develop thyroid cancer or liver cancer, and to predict the progression stage or the prognosis, and there is an advantage of making an appropriate treatment plan according to the predicted prognosis.

암 예방 또는 치료용 물질 스크리닝 시스템Cancer Screening System for Preventing or Treating Cancer

본 발명은 (a) 암 환자에서 분리한 시료에 후보물질을 처리하는 단계; (b) 후보물질이 처리된 암 환자의 시료에서 제1항에 따른 바이오마커의 발현 수준을 측정하는 단계; 및 (c) 상기 (b) 단계의 바이오마커 발현 수준이 후보물질의 처리 전보다 낮은 경우, 후보물질을 암의 예방 또는 치료용 물질로 판단하는 단계; 를 포함하는 암 예방 또는 치료용 물질 스크리닝 시스템을 제공한다. 상기 바이오마커의 구체적인 내용은 전술한 바와 같다. The present invention comprises the steps of (a) treating the candidate substance to a sample isolated from a cancer patient; (b) measuring the expression level of the biomarker according to claim 1 in a sample of the cancer patient treated with the candidate substance; And (c) if the biomarker expression level of step (b) is lower than before treatment of the candidate, determining the candidate as a material for preventing or treating cancer; It provides a cancer screening system for preventing or treating cancer. Details of the biomarker are as described above.

먼저, 본 발명에 따른 스크리닝 시스템은 암 환자에서 분리한 시료에 후보물질을 처리하는 단계[(a) 단계]를 포함한다. 구체적으로, 상기 시료는 갑상선암 또는 간암 환자의 갑상선 또는 간 유래의 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨일 수 있으며, 갑상선 또는 간 종양 조직인 것이 바람직하나, 이에 한정되지 않는다. 또한, 상기 후보물질은 통상적인 선정방식에 따라 갑상선암 또는 간암 치료제로서 가능성을 지닌 것으로 추정되거나 또는 무작위적으로 선정된 개별적인 핵산, 펩타이드, 단백질, 항체, 기타 추출물 또는 천연물, 화합물 등이 될 수 있으며, 상기 바이오마커의 발현을 저해하는 화합물인 것이 바람직하나, 이에 한정되지 않는다. First, the screening system according to the present invention includes the step of treating the candidate material to the sample separated from the cancer patient [step (a)]. Specifically, the sample may be thyroid or liver-derived tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine in patients with thyroid cancer or liver cancer, but is not limited thereto. In addition, the candidate material may be individual nucleic acids, peptides, proteins, antibodies, other extracts or natural products, compounds, or the like, which are estimated to have potential as therapeutic agents for thyroid cancer or liver cancer according to a conventional selection method, or randomly selected. It is preferable that the compound inhibits the expression of the biomarker, but is not limited thereto.

다음으로, 본 발명에 따른 스크리닝 시스템은 후보물질이 처리된 암 환자의 시료에서 제1항에 따른 바이오마커의 발현 수준을 측정하는 단계[(b) 단계]를 포함한다. 구체적으로, 갑상선암 또는 간암의 치료 또는 예방의 후보물질을 간 세포 또는 조직 또는 기타 생물학적 시료에 처리하여, 상기 바이오마커의 발현 수준 및 바이오마커 발현 관련 신호전달 단백질의 발현 및 인산화 수준을 측정함으로써, 갑상선암 또는 간암의 치료 또는 예방 물질을 선별할 수 있다.Next, the screening system according to the present invention comprises the step of (b) measuring the expression level of the biomarker according to claim 1 in the sample of cancer patients treated with the candidate. Specifically, by treating candidate cells for the treatment or prevention of thyroid cancer or liver cancer to liver cells or tissues or other biological samples, by measuring the expression level of the biomarker and the expression and phosphorylation of the signaling protein related to biomarker expression, thyroid cancer Or a substance for treating or preventing liver cancer.

마지막으로, 본 발명에 따른 스크리닝 시스템은 상기 (b) 단계의 바이오마커 발현 수준이 후보물질의 처리 전보다 낮은 경우, 후보물질을 암의 예방 또는 치료용 물질로 판단하는 단계[(c) 단계]를 포함한다. 구체적으로, 상기 후보물질을 처리하지 않은 경우와 비교하여, 상기 바이오마커의 발현 및 바이오마커 발현 관련 신호전달 단백질의 발현 및 인산화를 저해하는 물질을 갑상선암 또는 간암의 질환 치료 또는 예방 물질로서 결정하며, 상기 물질 간의 반응 확인은, 단백질-단백질, 단백질-화합물, 또는 상기 언급된 후보물질로서, 단백질-핵산, 펩타이드, 항체, 기타 추출물 또는 천연물 간의 반응 여부를 확인하는데 사용되는 통상적인 방법들을 사용할 수 있다. Finally, the screening system according to the present invention, if the biomarker expression level of step (b) is lower than before the treatment of the candidate, determining the candidate as a substance for preventing or treating cancer [step (c)] Include. Specifically, compared with the case where the candidate is not treated, a substance that inhibits the expression of the biomarker and the expression and phosphorylation of the biomarker expression-related signaling protein is determined as a substance for treating or preventing a disease of thyroid cancer or liver cancer, Confirmation of the reaction between the substances, as a protein-protein, a protein-compound, or the above-mentioned candidates, can use conventional methods used to confirm the reaction between protein-nucleic acid, peptides, antibodies, other extracts or natural products. .

암 예방 또는 치료용 약학적 조성물Pharmaceutical composition for preventing or treating cancer

본 발명은 제1항에 따른 바이오마커의 발현 또는 활성을 억제하는 물질; 및 약학적으로 허용 가능한 담체를 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다. 상기 바이오마커의 구체적인 내용은 전술한 바와 같다. The present invention is a substance for inhibiting the expression or activity of the biomarker according to claim 1; And it provides a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier. Details of the biomarker are as described above.

구체적으로, 상기 물질은 상기 바이오마커에 대한 안티센스 올리고뉴클레오티드, 앱타머(aptamer), siRNA 또는 shRNA일 수 있으며, 상기 바이오마커에 의해 코딩되는 단백질의 활성을 억제하는 항체 또는 그의 항원 결합 단편일 수 있다. 또한, 상기 항체는 특별히 이에 제한되지 않으나, 본 발명의 바이오마커에 의해 코딩되는 단백질에 특이적으로 결합할 수 있는 모든 항체가 될 수 있고, 바람직하게는 단일클론항체, 카이메릭 항체(chimeric antibody), 인간화 항체(humanized antibody), 인간 항체(human antibody) 등이 될 수 있을 뿐만 아니라, 상기 항체의 기능적인 단편이 될 수도 있다. 또한 상기 항체는 본 발명의 바이오마커에 의해 코딩되는 단백질을 특이적으로 인식하는 결합의 특성을 갖는 한, 2개의 중쇄(heavy chain)와 2개의 경쇄(light chain)의 전체 길이를 가지는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체의 분자의 기능적인 단편이란, 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab')2 및 Fv 등이 있다.Specifically, the substance may be an antisense oligonucleotide, aptamer, siRNA or shRNA to the biomarker, and may be an antibody or an antigen-binding fragment thereof that inhibits the activity of the protein encoded by the biomarker. . In addition, the antibody is not particularly limited thereto, but may be any antibody that can specifically bind to a protein encoded by the biomarker of the present invention, preferably a monoclonal antibody or a chimeric antibody. , Humanized antibodies, human antibodies, and the like, as well as functional fragments of the antibodies. In addition, as long as the antibody has the characteristic of binding to specifically recognize the protein encoded by the biomarker of the present invention, only the full form having the full length of two heavy chains and two light chains is provided. But includes functional fragments of antibody molecules. The functional fragment of the molecule of an antibody means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.

본 발명에 따른 암 예방 또는 치료용 약학적 조성물은 ANLN, C5orf13(= NREP), COL5A1, VGLL2, C15orf52, 또는 KIAA1644의 발현을 억제함으로써, 종양 세포 증식 및 이동을 억제할 수 있다. 또한, EGFR, HIPK2, HSPBL2 또는 TAXIBP1으로 구성된 군 중 하나 이상의 항-세포사멸 단백질(anti-apoptotic protein)의 발현을 억제할 수 있다. The pharmaceutical composition for preventing or treating cancer according to the present invention can inhibit tumor cell proliferation and migration by inhibiting the expression of ANLN, C5orf13 (= NREP), COL5A1, VGLL2, C15orf52, or KIAA1644. It is also possible to inhibit the expression of one or more anti-apoptotic proteins from the group consisting of EGFR, HIPK2, HSPBL2 or TAXIBP1.

더 나아가, 상기 조성물은 CDKN2C 또는 DKK1의 발현을 증가시킴으로써, 갑상선 세포의 증식 및 이동을 억제할 수 있다. Furthermore, the composition can inhibit the proliferation and migration of thyroid cells by increasing the expression of CDKN2C or DKK1.

본 발명에서 제공하는 조성물은 유효성분으로 사용되는 상기 바이오마커에 의해 코딩되는 단백질 발현 또는 활성을 억제하는 올리고 뉴클레오티드 또는 항체 이외에, 갑상선암 또는 간암의 치료 활성을 나타내는 치료제를 추가로 포함할 수 있다. 본 발명의 조성물은 투여 방식에 따라 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 구체적으로, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 상기 성분들 중 어느 하나의 이상의 성분을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액 등 통상적으로 사용되는 다른 첨가제를 추가적으로 포함할 수 있다. 또한 투여 목적에 따라 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있고, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 또는 조직 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 상기와 같은 담체, 부형제 또는 첨가제의 종류는 당업계의 통상적인 제제를 모두 포함하며, 상기 예에 의해 사용가능한 담체, 부형제 또는 첨가제의 종류가 제한되는 것은 아니다.The composition provided by the present invention may further include a therapeutic agent that exhibits therapeutic activity of thyroid cancer or liver cancer, in addition to an oligonucleotide or antibody that inhibits protein expression or activity encoded by the biomarker used as an active ingredient. The composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent depending on the mode of administration. Specifically, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and any one or more of the above components may be mixed and used as necessary, antioxidants, Other additives commonly used, such as buffers, may additionally be included. Diluents, dispersants, surfactants, binders, and lubricants may also be added to formulate into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions, etc., depending upon the purpose of administration, and may be specific to the target organ. Target organ or tissue specific antibodies or other ligands can be used in combination with the carrier so as to facilitate the use. Such types of carriers, excipients or additives include all conventional formulations in the art, and the types of carriers, excipients or additives usable by the above examples are not limited.

상기와 같은 조성물 또는 혼합물은 목적 또는 필요에 따라 당업계에서 사용되는 통상적인 방법, 투여 경로, 투여량에 따라 적절하게 개체에 투여될 수 있다. 투여 경로의 예로는 경구, 비 경구, 피하, 복강 내, 폐 내, 및 비강 내로 투여될 수 있고, 국부적 면역억제 치료를 위해, 필요하다면 병변 내 투여를 포함하는 적합한 방법에 의해 투여된다. 비 경구 주입에는 근육 내, 정맥 내, 동맥 내, 복강 내 또는 피하투여가 포함된다. 또한 당업계에 공지된 방법에 따라 적절한 투여량 및 투여 횟수가 선택될 수 있으며, 실제로 투여되는 본 발명의 안티센스 올리고뉴클레오티드, siRNA 또는 shRNA를 포함하는 조성물의 양 및 투여 횟수는 예방 또는 치료하고자 하는 증상의 종류, 투여 경로, 성별, 건강 상태, 식이, 개체의 연령 및 체중, 및 질환의 중증도와 같은 다양한 인자에 의해 적절하게 결정될 수 있다.Such compositions or mixtures may be suitably administered to a subject according to conventional methods, routes of administration, and dosages used in the art, depending on the purpose or need. Examples of routes of administration may be administered orally, orally, subcutaneously, intraperitoneally, intrapulmonally, and intranasally, and are administered by suitable methods, including intralesional administration if necessary for local immunosuppressive treatment. Non-oral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the appropriate dosage and frequency of administration may be selected according to methods known in the art, and the amount and frequency of administration of the composition comprising the antisense oligonucleotide, siRNA or shRNA of the present invention to be administered are symptoms to be prevented or treated. And various factors such as the type, route of administration, sex, health condition, diet, age and weight of the individual, and severity of the disease.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[실험 방법][Experimental method]

1. 세포주 및 조직 시료(Cell lines and tissue samples)1. Cell lines and tissue samples

normal primary thyroid follicular epithelial cells에서 유래된 SV-40 immortalized cell line인 Nthy-ori 3-1(Lemoine NR, Mayall ES, Jones T, Sheer D, McDermid S, Kendall-Taylor P et al. Characterisation of human thyroid epithelial cells immortalized in vitro by simian virus 40 DNA transfection. Br J Cancer 1989; 60(6):897-903.); 유두 갑상선암종(papillary thyroid carcinoma)으로부터 유래된 BCPAP, KTC-1, SNU-790 ; 여포상 갑상선암종(follicular thyroid carcinoma)으로부터 유래된 FTC133; 역형성 갑상선암종(anaplastic thyroid carcinoma)로부터 유래된 C643, SW1736, 및 Cal-62 [SNU-790, (Koh CS, Ku JL, Park SY, Kim KH, Choi JS, Kim IJ et al. Establishment and characterization of cell lines from three human thyroid carcinomas: responses to all-transretinoic acid and mutations in the BRAF gene. Molecular and cellular endocrinology 2007;264(1-2):118-127.); 모든 다른 세포주(Pilli T, Prasad KV, Jayarama S, Pacini F, Prabhakar BS. Potential utility and limitations of thyroid cancer cell lines as models for studying thyroid cancer. Thyroid : official journal of the American Thyroid Association 2009; 19(12):1333-1342.) 및 그것의 참고문헌]) cell line이 본 연구를 위해 사용되었다.Nthy-ori 3-1 (Lemoine NR, Mayall ES, Jones T, Sheer D, McDermid S, Kendall-Taylor P et al. Characterization of human thyroid epithelial, SV-40 immortalized cell line derived from normal primary thyroid follicular epithelial cells cells immortalized in vitro by simian virus 40 DNA transfection. Br J Cancer 1989; 60 (6): 897-903.); BCPAP, KTC-1, SNU-790 derived from papillary thyroid carcinoma; FTC133 derived from follicular thyroid carcinoma; C643, SW1736, and Cal-62 derived from anaplastic thyroid carcinoma [SNU-790, (Koh CS, Ku JL, Park SY, Kim KH, Choi JS, Kim IJ et al. Establishment and characterization of cell lines from three human thyroid carcinomas: responses to all-transretinoic acid and mutations in the BRAF gene.Molecular and cellular endocrinology 2007; 264 (1-2): 118-127.); All other cell lines (Pilli T, Prasad KV, Jayarama S, Pacini F, Prabhakar BS.Potential utility and limitations of thyroid cancer cell lines as models for studying thyroid cancer.Thyroid: official journal of the American Thyroid Association 2009; 19 (12) : 1333-1342.) And its references]) Cell lines were used for this study.

Nthy-ori 3-1은 Sigma-Aldrich (St. Louis, MO, USA)에서 구매하였고 BCPAP 및 Cal-62은 DSMZ에서 구매하였으며(Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany); C643 및 SW1736은 CLS로부터(Cell Line service, Germany); KTC-1, SNU-790 및 FTC133 lines는 본 발명자들의 연구실 stocks에서 얻었다(National Cancer Center, Center for Thyroid Cancer, Korea). Cell을 10% fetal bovine serum (FBS)(GE Healthcare Life Sciences; Logan, UT, USA) 및 1% antibiotic-antimyotic(Life Technologies; Carlsbad,CA, USA)가 풍부한성장배지에서 배양하였다. 모든 cell line은 7℃에서 5% CO2의 humidified incubator에서 배양하였다.Nthy-ori 3-1 was purchased from Sigma-Aldrich (St. Louis, MO, USA) and BCPAP and Cal-62 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany); C643 and SW1736 from CLS (Cell Line service, Germany); KTC-1, SNU-790 and FTC133 lines were obtained from our laboratory stocks (National Cancer Center, Center for Thyroid Cancer, Korea). Cells were cultured in growth media rich in 10% fetal bovine serum (FBS) (GE Healthcare Life Sciences; Logan, UT, USA) and 1% antibiotic-antimyotic (Life Technologies; Carlsbad, CA, USA). All cell lines were incubated in a humidified incubator with 5% CO 2 at 7 ° C.

Normal 및 tumor tissues는 the National Cancer Center (NCC) Hospital에서 갑상선 수술을 받은 갑상선암 환자로부터 얻었다. 모든 fresh tissue는 액체 질소에서 수술 직후에 냉동하였고 Declaration of Helsinki의 원칙에 따른 NCC (NCC2014-0003)의 인간 대상 가이드라인에 대한 NCC, 기관검토위원회에 의해 승인된 프로토콜에 따라 -70℃에서 보관하였다. Normal and tumor tissues were obtained from thyroid cancer patients who underwent thyroid surgery at the National Cancer Center (NCC) Hospital. All fresh tissues were frozen immediately after surgery in liquid nitrogen and stored at −70 ° C. in accordance with the protocol approved by the NCC, Institutional Review Board for NCC (NCC2014-0003) human subject guidelines in accordance with Declaration of Helsinki's principle. .

병원 의료 기록 및 수술을 시행하고 병원에서 유두 갑상선 암으로 진단받은 37명의 환자의 병리보고서를 검토하였다. Pathologic staging는 International Union against Cancer and the American Joint Committee on Cancer, 7th edition에 따라 정의하였다. 환자의 임상적 및 병리학적 특징을 하기 표 1에 나타냈다.Hospital medical records and surgery were performed, and pathology reports of 37 patients diagnosed with papillary thyroid cancer were reviewed at the hospital. Pathologic staging was defined according to the International Union against Cancer and the American Joint Committee on Cancer, 7th edition. The clinical and pathological characteristics of the patients are shown in Table 1 below.

총 n=37Total n = 37 nc886의 발현 수준expression level of nc886 P-valueP-value 저(n=18)Low (n = 18) 중(n=8)(N = 8) 고(n=11)High (n = 11) 진단시 연령(세)Age at diagnosis 47.5±11.247.5 ± 11.2 50.2±10.850.2 ± 10.8 42.0±10.242.0 ± 10.2 47.1±12.047.1 ± 12.0 0.2280.228 종양의 크기(cm)Tumor size (cm) 1.7±1.01.7 ± 1.0 1.7±1.11.7 ± 1.1 1.9±1.31.9 ± 1.3 1.6±0.81.6 ± 0.8 0.7770.777 첫번째 종양의 수Number of first tumors 1.5±0.81.5 ± 0.8 1.3±0.61.3 ± 0.6 2.1±1.02.1 ± 1.0 1.5±0.71.5 ± 0.7 0.0280.028 과갑상선신장(extrathyroidal extension)Extrathyroidal extension 26(70.3%)26 (70.3%) 10(55.6%)10 (55.6%) 7(87.5%)7 (87.5%) 9(81.8%)9 (81.8%) 0.1570.157 후기 T 단계(T3-4)Late stage T (T3-4) 28(75.7%)28 (75.7%) 11(61.1%)11 (61.1%) 7(87.5%)7 (87.5%) 10(90.9%)10 (90.9%) 0.1310.131 림프절 전이(n,%)Lymph node metastasis (n,%) 22(59.5%)22 (59.5%) 5(27.8%)5 (27.8%) 8(100%)8 (100%) 9(81.8%)9 (81.8%) 0.00050.0005 N0N0 15(40.5%)15 (40.5%) 13(72.2%)13 (72.2%) 0(0%)0 (0%) 2(18.2%)2 (18.2%) 0.0280.028 N1aN1a 15(40.5%)15 (40.5%) 4(22.2%)4 (22.2%) 4(50.0%)4 (50.0%) 7(63.6%)7 (63.6%) N1bN1b 7(18.9%)7 (18.9%) 1(5.6%)1 (5.6%) 4(50.0%)4 (50.0%) 2(18.2%)2 (18.2%) 하시모토병(Hashimoto's thyroiditis)Hashimoto's thyroiditis 16(43.2%)16 (43.2%) 6(33.3%)6 (33.3%) 4(50.5%)4 (50.5%) 6(54.5%)6 (54.5%) 0.4860.486

평균±표준편차 Mean ± Standard Deviation

P 경향성=0.050P tendency = 0.050

2. 형질주입(2. Transfection TransfectionTransfection ), anti-), anti- oligosoligos , RNA/단백질 분리 및 측정, RNA / protein isolation and measurement

본 실시예에서 사용된 일반적인 시약은 Typical reagents used in this example are

· 문헌[Lee K, Kunkeaw N, Jeon SH, Lee I, Johnson BH, Kang GY et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA 2011; 17(6):1076-1089.] Lee K, Kunkeaw N, Jeon SH, Lee I, Johnson BH, Kang GY et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA 2011; 17 (6): 1076-1089.]

· 문헌[Kunkeaw N, Jeon SH, Lee K, Johnson BH, Tanasanvimon S, Javle M et al. Cell death/proliferation roles for nc886, a non-coding RNA, in the protein kinase R pathway in cholangiocarcinoma. Oncogene 2013; 32(32):3722-3731.]Kunkeaw N, Jeon SH, Lee K, Johnson BH, Tanasanvimon S, Javle M et al. Cell death / proliferation roles for nc886, a non-coding RNA, in the protein kinase R pathway in cholangiocarcinoma. Oncogene 2013; 32 (32): 3722-3731.]

· 문헌[Lee KS, Park JL, Lee K, Richardson LE, Johnson BH, Lee HS et al. nc886, a noncoding RNA of anti-proliferative role, is suppressed by CpG DNA methylation in human gastric cancer. Oncotarget 2014; 5(11):3944-3955.]에 충분히 기재되었다.Lee KS, Park JL, Lee K, Richardson LE, Johnson BH, Lee HS et al. nc886, a noncoding RNA of anti-proliferative role, is suppressed by CpG DNA methylation in human gastric cancer. Oncotarget 2014; 5 (11): 3944-3955.].

Anti-oligos(본 연구에서 각각 "anti-nc886" 및 "anti-control"으로 설계된 "anti886 75-56" 및 "anti-vt 21-2")는 「Lee K, Kunkeaw N, Jeon SH, Lee I, Johnson BH, Kang GY et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA 2011; 17(6):1076-1089.」에서 기술된 바와 같이 준비하였고 transfection 하였다. 환자 tissue sample 및 cell line의 total RNA는 Trizol reagent (Life Technologies; Carlsbad, CA, USA)로 isolation 하였다. nc886 및 control genes의 Northern hybridization, qRT-PCR measurement, PKR 및 다른 proteins의 Western blot을 「Lee K, Kunkeaw N, Jeon SH, Lee I, Johnson BH, Kang GY et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA 2011; 17(6):1076-1089.」에서 기술된 바와 같이 수행하였으며, qRT-PCR primers의 서열 정보는 요청시 이용 가능하다.Anti-oligos ("anti886 75-56" and "anti-vt 21-2", which are designed as "anti-nc886" and "anti-control" in this study, respectively) are described as "Lee K, Kunkeaw N, Jeon SH, Lee I." , Johnson BH, Kang GY et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA 2011; 17 (6): 1076-1089. ”And transfection. Total RNA of patient tissue samples and cell lines was isolated with Trizol reagent (Life Technologies; Carlsbad, Calif., USA). Northern hybridization of nc886 and control genes, qRT-PCR measurement, Western blot of PKR and other proteins were described in Lee K, Kunkeaw N, Jeon SH, Lee I, Johnson BH, Kang GY et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. RNA 2011; 17 (6): 1076-1089. ”, Sequence information of qRT-PCR primers is available upon request.

3. 3. CRISPRCRISPR /Of CasCas KO 세포주의 생성(generation) Generation of KO Cell Lines

"hCas9" 및 "gRNA_Cloning Vector"는 Addgene (plasmid #41815 및 #41824 각각)에서 구매하였다. PKR의 sgRNA-expressing plasmid ("pCR sgPKR-1a")는 Duke University의 Dr. Stacy Horne에 의해 발견되었다. nc886의 sgRNA-expressing plasmids는 gDNA synthesis protocol [https://www.addgene.org/41824/and (33)]에 따라 구성되었다. 간략하게, sgRNA sequences 를 포함하는 두 개의 partially complementary oligo를 annealing하고(도 5A) Phusion™ High-Fidelity DNA Polymerase 「New England Biolabs; Ipswich, MA, USA」에 의해 fully double-stranded DNA fragment를 전환함으로써 insert를 만들었다. 상기 insert는 "pCR sg886-164" 및 "pCR sg886+15"을 얻기 위해, Gibson assembly kit (New England Biolabs)를 사용함으로써 AflII-linearized "gRNA_Cloning Vector"로 통합하였다."hCas9" and "gRNA_Cloning Vector" were purchased from Addgene (plasmid # 41815 and # 41824, respectively). PKR's sgRNA-expressing plasmid ("pCR sgPKR-1a") is a D. Found by Stacy Horne. The sgRNA-expressing plasmids of nc886 were constructed according to the gDNA synthesis protocol [https://www.addgene.org/41824/and (33)]. Briefly, annealing two partially complementary oligos containing sgRNA sequences (FIG. 5A) and Phusion ™ High-Fidelity DNA Polymerase [New England Biolabs; Ipswich, Mass., USA, by inserting a fully double-stranded DNA fragment. The insert was integrated into an Afl II-linearized "gRNA_Cloning Vector" by using the Gibson assembly kit (New England Biolabs) to obtain "pCR sg886-164" and "pCR sg886 + 15".

"pCR sgPKR-1a" (for PKR KO) 또는 "pCR sg886-164" 및 "pCR sg886+15" (for nc886 KO)와 결합된, The Cas9-expressing plasmid ("hCas9")는 Lipofectamine 2000 (Life Technologies)를 이용함으로써 transfection 하였다. Untransfected cells은 G418 selection시, 음성대조군으로 병렬 처리하였다. 24시간 transfection한 후, cell을 G418의 1㎎/㎖을 포함하는 성장 배지에 옮겼다. G418-resistant colonies를 개별적으로 분리하고 추가 배양하였다. The Cas9-expressing plasmid ("hCas9"), combined with "pCR sgPKR-1a" (for PKR KO) or "pCR sg886-164" and "pCR sg886 + 15" (for nc886 KO), is a Lipofectamine 2000 (Life Technologies Transfection). Untransfected cells were treated in parallel with negative controls upon G418 selection. After 24 hours of transfection, the cells were transferred to a growth medium containing 1 mg / ml of G418. G418-resistant colonies were individually isolated and further cultured.

4. 세포 증식(cell proliferation), 이동(migration) 및 침습(invasion) 분석4. Cell proliferation, migration and invasion analysis

세포 증식은 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye의 변환 또는 이것의 유도체 MTT dye에 기반하여 측정하였다. MTT 및 MTS dyes를 Sigma-Aldrich 및 Promega (Madison, WI, USA)에서 각각 구매하였고 assay는 제조사의 지시에 따라 수행하였다. colony formation assay에서, 100 cells을 6-well plate 중 한 플레이트에 접종하였고 7일 동안 유지시켰다. 이후, 콜로니 수를 세기 위하여 1% 크리스탈 바이올렛으로 염색하고 고정하였다. Cell proliferation was determined based on the conversion of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) dye or its derivative MTT dye. MTT and MTS dyes were purchased from Sigma-Aldrich and Promega (Madison, WI, USA) respectively and the assay was performed according to the manufacturer's instructions. In the colony formation assay, 100 cells were seeded in one of the 6-well plates and maintained for 7 days. Thereafter, the cells were stained and fixed with 1% crystal violet to count colonies.

세포 이동 분석은 8-㎛ pore filter insert (BD Biosciences; San Jose, CA, USA)를 사용하여 수행하였다. Cell을 serum-free RPMI-1640 medium에 재부유(resuspention)시키고, upper chamber에 첨가하였으며, 24시간 동안 1% PBS와 RPMI-1640을 포함하는 lower chamber로 이동시켰다. 상기 insert의 바닥 표면에 있는 Migrated cells을 고정시키고 20분 동안 공기-건조(air dried) 시켰으며, 20분 동안 1% 크리스탈 바이올렛으로 염색하였다. 이후, top surface에 있는 Remaining cells을 cotton swab으로 wiping함으로써 제거하였다. migration rates을 정량하기 위해, insert는 10% acetic acid을 넣고 564nm에서 흡광도를 측정하였다. Cell invasion assays는 matrigel-coated insert (BD Biosciences)로 수행하였다. 세포를 Diff-Quik stain™ (Sysmex; Kobe, Japan)로 염색하고 세포수를 측정하였다. Cell migration assays were performed using an 8-μm pore filter insert (BD Biosciences; San Jose, CA, USA). The cells were resuspended in serum-free RPMI-1640 medium, added to the upper chamber, and transferred to a lower chamber containing 1% PBS and RPMI-1640 for 24 hours. Migrated cells on the bottom surface of the insert were fixed, air dried for 20 minutes and stained with 1% crystal violet for 20 minutes. Thereafter, the retaining cells on the top surface were removed by wiping with a cotton swab. To quantify the migration rates, the insert was measured for absorbance at 564 nm with 10% acetic acid. Cell invasion assays were performed with matrigel-coated inserts (BD Biosciences). Cells were stained with Diff-Quik stain ™ (Sysmex; Kobe, Japan) and cell number was measured.

본 실시예에서 MTT 및 콜로니 형성 분석을 통해 PKRKO/nc886KO cells이 nc886wt cells (PKRwt/nc886wt 및 PKRKO/nc886wt, 도 3B-D 및 도 S7A-C)에 비해 더 느리게 성장함을 확인할 수 있었다. 성장 이점을 부여하는, PKR KO 자체는 PKRwt/nc886wt의 콜로니 크기보다 PKRKO/nc886wt cells의 더 큰 콜로니 크기로 보여질 수 있다. 그러나, MTT 값 및 콜로니 수는 PKR의 부재로 현저하게 증가하지는 않았다(도 3B-D 및). 세포 이동 및 침습 분석도 비슷한 결과를 얻었다. PKRKO/nc886KO cells은 명백히 PKRKO/nc886wt cells에 비해 덜 migrating 하고 invasive했으며, PKRKO/nc886wt cells은 PKRwt/nc886wt cells에 비해 약간 더 migrating 하고 invasive했다(도 4A-D). 종합적으로 이러한 모든 데이터에서, nc886은 oncogenic한 역할을 할 것으로 예측하였다.In this example, MTT and colony formation analysis showed that PKR KO / nc886 KO cells grow more slowly than nc886 wt cells (PKR wt / nc886 wt and PKR KO / nc886 wt , FIGS. 3B-D and S7A-C). I could confirm it. PKR KO itself, which gives a growth advantage, can be seen as a larger colony size of PKR KO / nc886 wt cells than the colony size of PKR wt / nc886 wt . However, MTT values and colony numbers did not increase significantly in the absence of PKR (FIGS. 3B-D and). Similar results were obtained for cell migration and invasive analysis. PKR KO / nc886 KO cells were clearly less migrating and invasive than PKR KO / nc886 wt cells, and PKR KO / nc886 wt cells were slightly more migrating and invasive than PKR wt / nc886 wt cells (FIGS. 4A-D). Overall, all of these data predicted that nc886 would play an oncogenic role.

5. 5. mRNAmRNA 마이크로어레이Microarray 및 경로 분석 And path analysis

mRNA 발현 profiling 및 molecular signatures의 analysis를 mRNA expression profiling and analysis of molecular signatures

· 문헌[Lee HS, Lee K, Jang HJ, Lee GK, Park JL, Kim SY et al. Epigenetic silencing of the non-coding RNA nc886 provokes oncogenes during human esophageal tumorigenesis. Oncotarget 2014; 5(11):3472-3481.]Lee HS, Lee K, Jang HJ, Lee GK, Park JL, Kim SY et al. Epigenetic silencing of the non-coding RNA nc886 provokes oncogenes during human esophageal tumorigenesis. Oncotarget 2014; 5 (11): 3472-3481.]

· 문헌[Lee KS, Park JL, Lee K, Richardson LE, Johnson BH, Lee HS et al. nc886, a noncoding RNA of anti-proliferative role, is suppressed by CpG DNA methylation in human gastric cancer. Oncotarget 2014; 5(11): 3944-3955.]에 기술된 바와 같이 수행하였다. Lee KS, Park JL, Lee K, Richardson LE, Johnson BH, Lee HS et al. nc886, a noncoding RNA of anti-proliferative role, is suppressed by CpG DNA methylation in human gastric cancer. Oncotarget 2014; 5 (11): 3944-3955.].

Hierarchical clustering 및 generation of a heat map을 Cluster 3.0 및 Java TreeView (version 1.1.6r4) softwares를 이용하여 수행하였다. 직접 물리적 상호작용 및 해당 경로는 "GeneMANIA" Cytoscape plug-in function (http://www.cytoscape.org/) (Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P et al. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 2010; 38(Web Server issue):W214-220.)를 이용하여 추정하였다.Hierarchical clustering and generation of a heat map were performed using Cluster 3.0 and Java TreeView (version 1.1.6r4) softwares. Direct physical interactions and their pathways are described in the "GeneMANIA" Cytoscape plug-in function (http://www.cytoscape.org/) (Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P et al. . the GeneMANIA prediction server:.; : was estimated using the W214-220) biological network integration for gene prioritization and predicting gene function Nucleic Acids Res 2010 38 (Web Server issue)..

6. 6. TCGATCGA 데이터 분석 Data analysis

본 실시예에서는 nc886의 임상적 의의를 더 심문하기 위해 The Cancer Genomic Altas(TCGA)에서 데이터 검사를 하였다. ncc886은 mRNA도 miRNA에도 속하지 않기 때문에 상기 TCGA는 nc886 자체의 발현 데이터를 포함하지 않았고 따라서 대부분의 배열 또는 sequencing platforms에서 제외되었다. 대신, 본 발명자들은 환자 생존율에서 그 의미를 분석하기 위해 갑상선암 환자 505명의 TCGA RNA-seq data에서 201개 nc886-regulated genes의 발현을 조사하였다. 갑상선암 환자 505명의 RNA-seq data 및 임상 데이터를 TCGA database (https://tcga data.nci.nih.gov/tcga/tcgaHome2.jsp)에서 다운로드 하였다. 상기 TCGA RNA-seq data에서 이용 가능한 총 20,132 gene을 추가 분석을 위해 수집하였다. TCGA는 표준화 발현 수준으로 Expectation Minimization values (RSEM)에서 RNA-seq data를 제공한다. 유전자의 발현 배-변화 (fold-change, fc)는 Gene의 RSEM을 총 505개의환자 샘플로부터 측정된 RSEM의 평균으로 나누어 측정하였고 log2-transform하였다. 이전에 보고된 절차에 따라(34), 0~0.1의 RSEM 값은 상기 발현 fc를 계산하는데 있어 무한한 문제를 피하기 위해 0.1로 모두 대체하였다. In this example, data was examined in The Cancer Genomic Altas (TCGA) to further interrogate the clinical significance of nc886. Since ncc886 does not belong to either mRNA or miRNA, the TCGA did not include expression data of nc886 itself and thus was excluded from most alignment or sequencing platforms. Instead, we examined the expression of 201 nc886-regulated genes in TCGA RNA-seq data of 505 thyroid cancer patients to analyze their meaning in patient survival. RNA-seq data and clinical data of 505 patients with thyroid cancer were downloaded from the TCGA database (https: // tcga data.nci.nih.gov/tcga/tcgaHome2.jsp). A total of 20,132 genes available in the TCGA RNA-seq data were collected for further analysis. TCGA provides RNA-seq data in Expectation Minimization values (RSEM) at standardized expression levels. The expression fold-change (fc) of the gene was measured by dividing the RSEM of Gene by the mean of the RSEM measured from a total of 505 patient samples and log2-transformed. Following the previously reported procedure (34), RSEM values of 0-0.1 were all replaced with 0.1 to avoid infinite problems in calculating the expression fc.

선택된 유전자의 발현 수준에 따라, 환자들을 이전에 기술된 바와 같이, "high-expression group" (if a log2(fc) value is > 0)" 및 "low-expression group" (< 0)으로 분류하였다. 201개의 nc886-regulated gene(도 5) 중 189개의 gene을 상기 TCGA RNA-seq data의 리스트에서 찾았고 survival analysis를 위해 사용하였다. R studio는 모든 통계적 테스트 및 분석을 위해 사용되었다(http://www.rstudio.com/). 상기 '생존' 패키지는 Kaplan-Meier curve를 그리고 Cox proportional hazard ratio를 계산하기 위해 사용하였다. Depending on the expression level of the selected gene, patients were classified into "high-expression group" (if a log2 (fc) value is> 0) "and" low-expression group "(<0), as previously described. 189 of the 201 nc886-regulated genes (Figure 5) were found in the list of TCGA RNA-seq data and used for survival analysis R studio was used for all statistical tests and analysis (http: // www.rstudio.com/) The 'survival' package was used to calculate the Kaplan-Meier curve and calculate the Cox proportional hazard ratio.

그 결과, 본 발명자들은 ANLN, C5orf13(= NREP), COL5A1, VGLL2, C15orf52, 및 KIAA1644의 더 낮아진 발현이, nc886 KO에 따라 낮아진 발현 수준과 일치한다는, 환자의 이상 생존 (도 5A)과 크게(p<0.05) 관련이 있다는 것을 관찰하였다. As a result, we found that the lower expression of ANLN, C5orf13 (= NREP), COL5A1, VGLL2, C15orf52, and KIAA1644 is consistent with the abnormal survival of patients (FIG. 5A), consistent with lower expression levels according to nc886 KO (FIG. 5A). p <0.05).

7. 통계적 분석7. Statistical Analysis

환자 인구 통계를 식별하기 위해 기술적 분석을 수행하였다. 범주형 변수 분석을 위해, 주파수 및 백분율을 산출하였다. 갑상선암 환자 37명의 한 쌍의 정상/종양 시료에서 nc886의 발현을 측정하고 nc886 레벨에 따라 세 그룹(저, 중 및 고)으로 나누었다(도 1A). 각 그룹의 비율은 ?2 및 Fisher's exact test를 사용하여 비교하였다. 연속 변수, 평균, 및 표준편차 분석을 위해 측정하였다. 상기 연속 변수의 차이는 Mann-Whitney U test, Student's unpaired two-sided t-test 또는 one-way ANOVA를 이용하여 분석하였다. 통계적 분석은 STATA software (version 10, StataCorp., College Station, TX, USA)로 수행되었다. 모든 p-values는 two-sided했고, p-values는 0.05 미만이 통계적으로 유의한 것으로 간주하였다. Technical analysis was performed to identify patient demographics. For categorical variable analysis, frequencies and percentages were calculated. Expression of nc886 was measured in a pair of normal / tumor samples from 37 patients with thyroid cancer and divided into three groups (low, medium and high) according to the nc886 level (FIG. 1A). The proportions of each group were compared using? 2 and Fisher's exact test. Measurements were made for continuous variables, mean, and standard deviation analysis. The difference of the continuous variables was analyzed using Mann-Whitney U test, Student's unpaired two-sided t-test or one-way ANOVA. Statistical analysis was performed with STATA software (version 10, StataCorp., College Station, TX, USA). All p-values were two-sided, and p-values were considered statistically significant below 0.05.

[[ 실시예Example ]]

실시예Example 1. 환자 및 갑상선 세포주에서  1. In patients and thyroid cell lines nc886의nc886 발현 분석 Expression analysis

갑상선암 환자 37명의 정상 및 종양 시료에서 nc886의 발현을 측정하고 nc886의 발현 수준에 따라 세 그룹(저, 중, 고)으로 분류하였다(도 1A). 종양에서의 증가된 nc886의 발현은 원발성 종양(primary tumor)의 수(p=0.028), 종양의 공격성(aggressiveness) (도 1B에서 T 병기, P 경향성=0.050) 및 림프절로의 전이와 상관관계가 있었다(p=0.028). 그러나, 진단 당시의 나이, 원발성 종양의 크기 및 만성 염증으로 이어지는 자가 면역 질환인 하시모토 갑상선염(Hashimoto's thyroiditis)과 같은 임상적 변수와 nc886의 유의한 연관성은 없었다. The expression of nc886 in normal and tumor samples from 37 thyroid cancer patients was measured and classified into three groups (low, medium, and high) according to the expression level of nc886 (FIG. 1A). Increased expression of nc886 in tumors correlated with the number of primary tumors (p = 0.028), tumor aggressiveness (T stage in FIG. 1B, P trend = 0.050) and metastasis to lymph nodes. (P = 0.028). However, there was no significant association of nc886 with clinical variables such as age at diagnosis, primary tumor size and autoimmune disease Hashimoto's thyroiditis leading to chronic inflammation.

또한, 갑상선 세포주에서 nc886의 발현을 측정하였고, 불멸화 세포주인 Nthy-ori-3-1보다 암 세포주의 대부분에서 더 높게 발현됨을 확인하였다(도 1D). Nthy-ori 3-1 세포는 완전히 형질 전환되지는 않았지만 잘 증식되었고 비 증식(non-proliferatong) 갑상선 조직보다 nc886이 현저하게 높게 발현되었다. nc886의 매우 낮은 발현은 여러 기관의 정상 조직에서도 나타났으며, nc886 발현이 세포 증식률에 비례함을 나타냈다. 즉, 배양 배지에서 혈청 농도를 감소시킴으로써, 세포 증식이 느려지면, nc886의 발현이 감소하였다. In addition, the expression of nc886 in the thyroid cell line was measured, and it was confirmed that the expression was higher in most of the cancer cell line than the immortal cell line Nthy-ori-3-1 (FIG. 1D). Nthy-ori 3-1 cells were not fully transformed but proliferated well and expressed significantly higher nc886 than non-proliferatong thyroid tissues. Very low expression of nc886 was also observed in normal tissues of various organs, indicating that nc886 expression was proportional to cell proliferation. That is, by decreasing the serum concentration in the culture medium, when cell proliferation was slowed down, the expression of nc886 was decreased.

상기 nc886 발현 패턴은 in vitro에서의 세포 증식 및 환자에서의 종양 진행 및 공격성과 관련하여 갑상선암에서 발암적(oncogene) 역할을 하는 것으로 추정된다. 그러나 nc886은 식도 편평 상피암(esophageal squamous cell carcinoma), 위암(gastric cancer), 급성 골수성 백혈병(acute myeloid leukemia) 및 폐암(lung cancer)을 포함한 다른 유형의 암에서 볼 수 있듯이 갑상선 세포의 서브 세트에서 후성적으로 침묵(epigenetically silenced)하였다. 따라서, 종양 억제 인자 역할의 가능성을 배제할 수 없으므로 갑상선암에서 nc886의 역할을 명확히 하기 위해, 기능 상실 표현형을 평가하였다.The nc886 expression pattern is believed to play an oncogene role in thyroid cancer with respect to cell proliferation in vitro and tumor progression and aggression in patients. However, nc886 is associated with a subset of thyroid cells, as seen in other types of cancer, including esophageal squamous cell carcinoma, gastric cancer, acute myeloid leukemia, and lung cancer. It was epigenetically silenced. Thus, the loss of phenotype was evaluated to clarify the role of nc886 in thyroid cancer since the possibility of a tumor suppressor role could not be ruled out.

실시예Example 2.  2. nc886nc886 침묵(silencing)에 의한 급성 세포 죽음(acute cell death) 및  Acute cell death by silencing and PKRPKR 활성화의 결과 The result of activation

nc886은 PKR을 억제한다. Nthy-ori 3-1, SW1736 및 C643 갑상선 세포주에 nc886을 표적으로 하는 안티센스 올리고 뉴클레오티드(anti-oligo)를 형질 감염시켰을 때, nc886의 발현 수준은 도 2A의 Northern blot에 나타난 바와 같이, 감소하였다. nc886 KD은 활성 형태인 phospho-PKR의 증가에 의해 PKR 활성화를 유도하였다(도 2B). 활성 PKR은 그것의 최상 기질인 eIF2?를 인산화시켰고, 결과적으로 불멸화 Nthy-ori 3-1 세포주뿐만 아니라 갑상선암 세포주 SW1736에서도 세포 증식을 억제하였다(도 2A-B). 반면, 다른 암 세포주인 C643에서는 eIF2? 인산화 또는 세포 증식 효과가 관찰되지 않았다.nc886 suppresses PKR. When Nthy-ori 3-1, SW1736 and C643 thyroid cell lines were transfected with antisense oligonucleotides targeting nc886, the expression level of nc886 was reduced, as shown in the Northern blot of FIG. 2A. nc886 KD induced PKR activation by increasing the active form of phospho-PKR (FIG. 2B). Active PKR phosphorylated its best substrate eIF2® and consequently inhibited cell proliferation not only in immortalized Nthy-ori 3-1 cell line but also in thyroid cancer cell line SW1736 (FIGS. 2A-B). On the other hand, in other cancer cell lines C643, eIF2? No phosphorylation or cell proliferation effect was observed.

nc886 KD에서 세포 증식의 손상은 그것의 추정 발암적 역할(putative oncogenic role)과 일치하는 것으로 보였다. 그러나, 이러한 현상은 병인(etiology) 및/또는 갑상선암의 진행에 있어서, nc886의 역할보다는 PKR 의존적 "종양 감지 모델"로 이해하여야 한다. 즉, nc886 KD은 불멸화 세포 또는 형질 전환된 세포에서 nc886의 상승된 발현의 기능적 중요성을 반영할 수 있는 다른 표현형을 관찰하기 전에, PKR 세포 사멸 경로를 즉시 유발하였다. 이것을 밝히기 위해서는 장기적인 세포 표현형을 검사하고 nc886-null(nc886-) 세포와 isogenic nc886+ 세포를 비교해야 한다. 따라서, nc886 KO 세포주를 생성하였다. The damage of cell proliferation at nc886 KD appeared to be consistent with its putative oncogenic role. However, this phenomenon should be understood as a PKR dependent "tumor sensing model" rather than the role of nc886 in the etiology and / or progression of thyroid cancer. That is, nc886 KD immediately induced the PKR cell killing pathway before observing other phenotypes that could reflect the functional significance of elevated expression of nc886 in immortalized or transformed cells. In order to clarify this test the long-term cell phenotype and nc886-null (nc886 -) should be compared to the cells and isogenic nc886 + cells. Thus, the nc886 KO cell line was generated.

실시예Example 3.  3. PKRPKR 및  And PKRPKR /Of nc886nc886 이중 KO 세포주의 순차적 생성 Sequential Generation of Double KO Cell Lines

antioligos는 자기 전파(self-propagation)를 하지 않아 장기적인 KD에는 부적합하기 때문에 "CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)"과 "CRISPR associated genes(Cas)"으로 구성된 박테리아 면역계로부터 새로운 유전자 편집 기술을 도입하였다. PKR은 nc886 KO 세포를 생성할 때도 nc886-가 PKR이 있는 상태에서 죽을 것으로 예상되기 때문에 문제가 되었으나(도 2C), 이러한 저항은 PKR 경로가 이미 잘못되었음을 나타냈다. 따라서, 상기 세포주의 어떤 데이터가 PKR과 함께 nc886의 역할을 갑상선 종양 형성에 적절하게 반영하는지 여부에 대한 의문이 들었다. Because antioligos do not self-propagation and are unsuitable for long-term KD, a new gene-editing technique has been introduced from the bacterial immune system consisting of "Clustered Regularly Interspaced Short Palindromic Repeats" and "CRISPR associated genes (Cas)". . PKR was problematic because nc886- was expected to die in the presence of PKR even when producing nc886 KO cells (FIG. 2C), but this resistance indicated that the PKR pathway was already wrong. Therefore, questions were asked as to which data of the cell line, along with PKR, adequately reflects the role of nc886 in thyroid tumor formation.

이러한 문제를 해결하기 위해, 본 발명자들은 PKR KO 세포주를 nc886 KO 세포주보다 앞서 만들었다. 이러한 방식으로. 야생형(PKRwt/nc886wt), PKR KO(PKRKO/nc886wt) 및 이중 KO(PKRKO/mc886KO)를 비교함으로써, 갑상선 종양 형성에 있어서, nc886 및/또는 PKR의 기여 여부를 확인할 수 있었다. 완전히 형질 전환된 세포주가 아닌 Nthy-ori 3-1을 선택하였고, 암세포주와 비교하여 더 자연적인 PKR 경로를 유지할 가능성이 높을 것으로 예상하였다. 같은 이유로, Nthy-on 3-1 세포는 nc886/PKR KO에 비해 종 양성 또는 이하의 어느 방향으로든 변형될 수 있다. 이것은 nc886과 PKR KO가 반대의 표현형을 나타낼 것으로 예상되었기 때문에 상당한 이점이 될 것으로 보인다. To solve this problem, we made PKR KO cell line ahead of nc886 KO cell line. In this way. By comparing wild type (PKR wt / nc886 wt ), PKR KO (PKR KO / nc886 wt ), and double KO (PKR KO / mc886 KO ), the contribution of nc886 and / or PKR to thyroid tumor formation was confirmed. . Nthy-ori 3-1 was chosen, not a fully transformed cell line, and was expected to maintain a more natural PKR pathway compared to cancer cell lines. For the same reason, Nthy-on 3-1 cells can be modified in either direction positive or below compared to nc886 / PKR KO. This is likely to be a significant advantage since nc886 and PKR KO are expected to show opposite phenotypes.

PKR 유전자의 첫 번째 엑손(exon)에서 아미노산 #15-21을 표적으로 하는 small guide RNA(sgRNA)를 디자인하고, DNA 시퀀싱과 웨스턴 블랏에 의한 PKR 단백질 발현에 따라, 1~2nt가 프레임시프트(frameshift)에 의해 결실된 PKRKO 클론을 선택하였다. Design a small guide RNA (sgRNA) targeting amino acids # 15-21 at the first exon of the PKR gene and frame shift 1 to 2 nt according to DNA sequencing and PKR protein expression by Western blot PKR KO clones deleted by) were selected.

이후, PKRKO 클론으로부터 nc886KO의 생성을 시도하였다. nc886은 non-coding RNA이기 때문에 1~2nt가 결실되었다 하더라도, 기능성 KO를 보장할 수 없었다. 따라서, nc886 전사체(transcript)와 인접한 두 개의 sgRNA를 설계하여 이들 사이의 전체 DNA 부분을 제거하였다. 2개의 sgRNA-발현 플라스미드를 parental Nthy-ori 3-1(PKRwt) 및 Nthy-ori 3-1(PKRKO) 세포로 형질 감염(transfection)시켰다. nc886KO 후보 클론을 초기 선택하는 동안, PKRwt Nthy-ori 3-1 세포에서는 콜로니가 거의 회수되지 않은 반면, 4개의 PKRKO 클론 모두에서 상당한 수의 콜로니가 회수되었음을 확인할 수 있었다. 이 결과는, nc886 결실(deletion)이 세포 증식에 해롭고, PKR 활성화가 그 원인 중 하나라는 생각을 확증하였다. 이후, 본 발명자들은 두 개의 sgRNA 사이의 nc886 DNA segment의 결실 및 시퀀싱(도 5C에 도시된 nc886KO 클론 #6의 대표 결과)을 나타내는 짧은 PCR 생성물을 확인함으로써, PKRK0/nc886KO 서브 클론을 얻었다. 또한, 3개의 PKRKO/nc886KO 클론(도 3A의 #6, #13 및 #17)에서 Northern hybridization에 의해 nc886이 발현되지 않음을 확인하고, 이들 세포주를 갑상선암에서의 nc886의 역할을 조사하는데 사용하였다. Thereafter, an attempt was made to generate nc886 KO from the PKR KO clone. Because nc886 is a non-coding RNA, functional KO could not be guaranteed even if 1 ~ 2nt were deleted. Thus, two sgRNAs adjacent to the nc886 transcript were designed to remove the entire DNA portion between them. Two sgRNA-expressing plasmids were transfected with parental Nthy-ori 3-1 (PKR wt ) and Nthy-ori 3-1 (PKR KO ) cells. During the initial selection of the nc886 KO candidate clone, few colonies were recovered in PKR wt Nthy-ori 3-1 cells, while a significant number of colonies were recovered in all four PKR KO clones. This result confirmed the idea that nc886 deletion is detrimental to cell proliferation and that PKR activation is one of the causes. We then obtained a PKR K0 / nc886 KO subclone by identifying a short PCR product showing deletion and sequencing of the nc886 DNA segment between two sgRNAs (representative results of nc886 KO clone # 6 shown in Figure 5C). . In addition, three PKR KO / nc886 KO clones (# 6, # 13, and # 17 of FIG. 3A) confirmed that nc886 was not expressed by Northern hybridization, and these cell lines were used to investigate the role of nc886 in thyroid cancer. It was.

실시예Example 4.  4. nc886nc886 KO 세포의 세포 배양(cell growth), 이동 및 침습의 지연 Retard cell growth, migration and invasion of KO cells

MTT 및 콜로니 형성 분석은 PKRKO/nc886KO 세포가 nc886wt 세포보다 천천히 성장하였다는 것을 입증하였다. PKR KO 자체는 PKRwt/nc886wt 세포보다 큰 콜로니 사이즈의 PKRKO/nc886wt 세포에서 볼 수 있듯이, 미세한 성장 이점을 나타냈다. 그러나, PKR KO의 경우 MTT 값과 콜로니 수가 유의적으로 증가하지는 않았다. 세포 이동 및 침습 분석 역시 비슷한 결과를 나타냈다. PKRKO/nc886KO 세포는 PKRKO/nc886wt 세포보다 세포 이동 및 침습이 덜 발생하는 것을 확인할 수 있었다. 즉, PKRKO/nc886wt 세포가 PKRwt/nc886wt 세포보다 약간 더 많은 것을 확인할 수 있었다(도 4A-D). 이러한 모든 데이터를 종합적으로 살펴보면, nc886이 oncogene으로서의 역할을 한다는 것을 확인할 수 있다. MTT and colony formation assays demonstrated that PKR KO / nc886 KO cells grew more slowly than nc886 wt cells. PKR KO itself showed a fine growth advantage, as seen in PKR KO / nc886 wt cells of colony size larger than PKR wt / nc886 wt cells. However, in the case of PKR KO, MTT value and colony number did not increase significantly. Cell migration and invasive analysis also showed similar results. PKR KO / nc886 KO cells were found to be less cell migration and invasion than PKR KO / nc886 wt cells. That is, it was confirmed that PKR KO / nc886 wt cells were slightly more than PKR wt / nc886 wt cells (FIGS. 4A-D). Comprehensive review of all these data shows that nc886 acts as an oncogene.

nc886KO 세포가 PKRKO-background에 있었고, 따라서 nc886 KO에 의해 나타나는 모든 표현형은 PKR 독립적인 것으로 주목해야 한다. 예를 들어, nc886의 증식적(proliferative) 역할은 PKR의 전-세포사멸(pro-apoptotic) 기능 억제에 기인할 수 없다. PKR 억제가 nc886의 유일한 기능이라면, PKRKO/nc886KO 세포와 PKRKO/nc886wt 세포는 동일한 표현형을 가질 것이다. 상기 데이터로부터 얻은 또 다른 중요한 결론은 PKR이 기여할 때, 중요한 역할을 하는 것처럼 보이는 경우이다. It should be noted that nc886 KO cells were in PKR KO -background, so all phenotypes represented by nc886 KO are PKR independent. For example, the proliferative role of nc886 cannot be attributed to the inhibition of pro-apoptotic function of PKR. If PKR inhibition is the only function of nc886, PKR KO / nc886 KO cells and PKR KO / nc886 wt cells will have the same phenotype. Another important conclusion drawn from the data is when PKR appears to play an important role when contributing.

실시예Example 5.  5. PKRPKR KO 및  KO and nc886 KO 세포의nc886 KO cells 유전자 발현 프로파일 Gene expression profile

다음으로 nc886/PKR의 역할에 대한 분자적 기초를 더 조사하였다. 3 가지 세포주(PKRwt/nc886wt, PKRKO/nc886wt 및 PKRKO/nc886KO)에서 마이크로어레이 실험을 수행하여 상기 세포주 사이의 표현 변화로부터 226개의 유전자 목록을 얻었다. 히트맵(heat map)에 나타난 바와 같이(도 5A, 좌측 패널), PKR 및 nc886 KO에 의해 201개 유전자 및 25개 유전자의 발현이 각각 변경되었다. 마이크로어레이 데이터는 qRT-PCR에 의해 변경된 일부 유전자를 측정함으로써 확인하였다(도 5A, 우측 패널). Next, the molecular basis for the role of nc886 / PKR was further investigated. Microarray experiments were performed on three cell lines (PKR wt / nc886 wt , PKR KO / nc886 wt and PKR KO / nc886 KO ) to obtain a list of 226 genes from expression changes between the cell lines. As shown in the heat map (FIG. 5A, left panel), the expression of 201 genes and 25 genes were altered by PKR and nc886 KO, respectively. Microarray data was confirmed by measuring some genes altered by qRT-PCR (FIG. 5A, right panel).

본 발명자들은 추가적인 분석을 위하여, 201개의 유전자에 집중하였다. 유전자 온톨로지(GO, gene ontology) 분석 결과, 13개의 GO 세트가 nc886 KO에 비해 유의하게(p <0.05) 변경되었으며 대부분이 종양 형성에 직접 또는 간접적으로 관련되어 있음을 보여 주었으며, 그 결과를 하기 표 2에 나타냈다. 그 중에서도 세포 주기, 세포 성장 및 세포 사멸뿐만 아니라 세포 뼈대 구성(표 2에서 bold-highlighted GO 용어)은 세포 증식, 이동 및 침습에서 nc886 KO 표현형을 설명할 수 있다(그림 3-4). 예를 들어, EGFR, HIPK2, HSPBL2 및 TAX1BP1과 같은 항-세포사멸 단백질(anti-apoptotic protein)의 발현은 nc886 KO에 따라 감소되었고, 따라서, PKRKO/nc886KO 세포의 성장을 감소시키는 것으로 설명할 수 있을 것이다. We concentrated on 201 genes for further analysis. Gene ontology (GO) analysis showed that 13 GO sets were significantly altered (p <0.05) compared to nc886 KO, and most were directly or indirectly involved in tumor formation. 2 is shown. Among them, cell cycle, cell growth and cell death as well as cell skeletal composition (bold-highlighted GO term in Table 2) can explain the nc886 KO phenotype in cell proliferation, migration and invasion (Figure 3-4). For example, the expression of anti-apoptotic proteins such as EGFR, HIPK2, HSPBL2, and TAX1BP1 were reduced according to nc886 KO, and thus explained to reduce the growth of PKR KO / nc886 KO cells. Could be.

Figure PCTKR2017008336-appb-T000001
Figure PCTKR2017008336-appb-T000001

또한, 본 발명자들은 201개 유전자 사이의 직접적인 물리적 상호 작용과 경로의 상호 연결을 확인하기 위하여, GeneMANIA plug-in tool in Cytoscape [http://www.cytoscape.org/]을 사용하여 유전자 네트워크 분석을 수행하였다. 상기 유전자 네트워크에서, p53 및 MYC(the most renowned oncogene)는 중요한 허브(hub)이다. p53과 MYC는 각각 가장 유명한 종양 억제 인자(tumor suppressor)와 종양 유전자이며, 흥미롭게도 Pol III 전사와 따라서 nc886 발현을 조절하는 것으로 알려져 있다. nc886과 p53, MYC 및 201 개 유전자의 관계를 밝히기 위해서는 더 많은 연구가 필요할 것이다. In addition, the inventors conducted genetic network analysis using the GeneMANIA plug-in tool in Cytoscape [http://www.cytoscape.org/] to identify direct physical interactions and pathway interconnections between the 201 genes. Was performed. In this gene network, p53 and the most renowned oncogene (MyC) are important hubs. p53 and MYC are the most famous tumor suppressor and tumor genes, respectively, and are interestingly known to regulate Pol III transcription and thus nc886 expression. More research will be needed to determine the relationship between nc886 and p53, MYC and 201 genes.

다음으로, 본 발명자들은 TCGA(The Cancer Genome Atlas)의 데이터를 검토하여 nc886의 임상적 중요성에 대해 조사하였다. nc886은 mRNA 또는 miRNA에 속하지 않으므로 대부분의 어레이 또는 시퀀싱 플랫폼에서 제외되었기 때문에 TCGA는 nc886 자체의 발현 데이터를 포함하지 않는다. 대신, 환자의 생존에 미치는 영향을 분석하기 위해 갑상선암 환자 505명의 TCGA RNA-seq 데이터에서 201개의 nc886 조절 유전자 발현을 조사하였다. 그 결과, ANLN, C5orf13(= NREP), COL5A1, VGLL2, C15orf52 및 KIAA1644의 낮은 발현이 nc886 KO에 대한 발현 수준의 저하와 일치하여 환자의 장시간 생존과 유의미한(p <0.05) 관계가 있음을 확인할 수 있었다. 또한, 배열 데이터를 분자 표지 데이터베이스(MSigDB; http://software.broadinstitute.org/gsea/index.jsp)와 비교하여 어떤 전사 인자 (TF) 표적 유전자가 강화(enriched)되었는지 또는 감소(depletion)되었는지 분석하였다. 그 결과, nc886 KO에서 TF 표적의 일정한 평가값(evaluation) 또는 감소(depletion)를 발견할 수 없었으나, 예외적으로 nc886 KO에 풍부한 혈청 반응 인자(serum response factor) 시그니처(signature)를 발견할 수 있었다. 단, 상기 SRF의 증가된 활성이 nc886 KO의 직접적인 결과인지 여부를 명확히 할 필요성이 있다. Next, we examined the clinical significance of nc886 by examining the data of The Cancer Genome Atlas (TCGA). TCGA does not include the expression data of nc886 itself because nc886 does not belong to mRNA or miRNA and has therefore been excluded from most arrays or sequencing platforms. Instead, we examined the expression of 201 nc886 regulatory genes in the TCGA RNA-seq data of 505 patients with thyroid cancer to analyze their impact on patient survival. As a result, the low expression of ANLN, C5orf13 (= NREP), COL5A1, VGLL2, C15orf52, and KIAA1644 was in agreement with the decrease in expression level for nc886 KO, indicating a significant (p <0.05) relationship with long-term survival of patients. there was. In addition, the array data was compared with the molecular label database (MSigDB; http://software.broadinstitute.org/gsea/index.jsp) to determine which transcription factor (TF) target genes were enriched or depleted. Analyzed. As a result, a constant evaluation or depletion of the TF target could not be found in the nc886 KO, but the serum response factor signature abundant in the nc886 KO was found. . However, there is a need to clarify whether the increased activity of the SRF is a direct result of nc886 KO.

PKR KO가 한계 표현형과 유전자 발현의 중간 정도 변화를 보였음에도 불구하고, 많은 TF 표적 유전자가 현저하게 변화한다는 것을 발견하였다(Z-score cutoff = 3). PKRKO 세포에서 모두 고갈된 9개의 최상위 TF(Z-score cutoff = 4)는 NF-κB TF였다. 또한, Biocarta를 사용하여 NF-κB와 관련된 7개의 강화(enriched) 경로와 13 개의 감소(depletion) 경로 (Z-score cutoff = 4)를 확인하였다. 이러한 결과는 PKR이 NF-κB의 upstream에 있다는 것을 확증한다. Although PKR KO showed a moderate change in the marginal phenotype and gene expression, it was found that many TF target genes changed significantly (Z-score cutoff = 3). The nine highest TFs (Z-score cutoff = 4), all depleted in PKR KO cells, were NF-κB TF. Biocarta was also used to identify seven enriched and 13 depletion pathways (Z-score cutoff = 4) associated with NF-κB. These results confirm that PKR is upstream of NF-κB.

실시예Example 6. 환자 및 간암 세포주에서  6. In patients and liver cancer cell lines nc886의nc886 발현 분석 Expression analysis

간암에서 nc886의 발현양상을 보기 위해 58명의 간암환자로부터 정상조직 및 암조직을 얻고 (미국 MD Anderson Cancer Center의 이주석 박사 그룹으로부터 얻음), RNA를 분리한 후 nc886의 발현을 노던 블랏으로 측정하였다. nc886 시그날을 적절히 보정하기 위해서, 동일한 블랏에 5S rRNA에 대한 탐침을 사용하여 노던 실험을 한번 더 수행하였다. 노던 결과를 densitometer로 정량한 후, 각각의 샘플에 대해서 nc886 / 5S rRNA의 값을 구하여, 이를 nc886 발현량으로 사용하였다. 간암샘플과 그에 대응하는 정상조직 샘플의 nc886 발현량을 비교하여, 58환자를 두개의 소그룹으로 분류하였다. 그 결과 39 환자에서는 nc886이 증가하였고 19 환자에서는 nc886이 감소하였으며, 이러한 경향성은 갑상선암에서의 경향성과 유사함을 알 수 있었다. In order to see the expression of nc886 in liver cancer, normal and cancerous tissues were obtained from 58 liver cancer patients (from Dr. Lee's group at the MD Anderson Cancer Center, USA), RNA was isolated, and nc886 expression was measured by Northern blot. To properly calibrate the nc886 signal, the Northern experiment was performed once more using a probe for 5S rRNA in the same blot. After quantifying the northern result by a densitometer, the value of nc886 / 5S rRNA for each sample was obtained and used as the nc886 expression. By comparing nc886 expression levels of liver cancer samples and corresponding normal tissue samples, 58 patients were divided into two subgroups. As a result, nc886 was increased in 39 patients and nc886 was decreased in 19 patients. This tendency was similar to that in thyroid cancer.

위의 두 소그룹에서 환자의 생존을 Kaplan-Meier plot으로 본 결과, nc886이 증가한 환자들에서 RFS(recurrence-free survival)가 통계적으로 유의한 정도로 감소된 것을 확인할 수 있었다(p = 0.01). 비록 환자의 샘플수가 비교적 많지 않은 관계로 통계적 유의성을 볼 수는 없었으나(p = 0.19), 전체적인 생존율도 낮아진 경향을 보이는 것을 확인할 수 있었다. The Kaplan-Meier plot of the survival of the patients in the above two subgroups showed that the recurrence-free survival (RFS) was significantly reduced in patients with increased nc886 (p = 0.01). Although the number of patients was relatively small, statistical significance could not be seen (p = 0.19), but overall survival rate tended to decrease.

58쌍의 샘플에서 mRNA array 결과가 존재한다(미국 MD Anderson Cancer Center의 이주석 박사 그룹으로부터 얻음). nc886 발현이 높고 낮은 두 소그룹에 따라 mRNA 발현양상을 clustering한 결과, nc886이 높은 그룹에서 암촉진 전사인자의 활성이 증가되어 있었으며, 동시에 암억제 microRNA의 활성이 낮아져 있었다.MRNA array results are present in 58 pairs of samples (obtained from Dr. Lee's group at the MD Anderson Cancer Center, USA). As a result of clustering the mRNA expression patterns according to the two groups with high and low nc886 expression, the activity of the cancer-promoting transcription factor was increased in the high nc886 group, and the activity of the cancer suppressor microRNA was decreased.

이상의 결과를 정리하면, nc886 발현양이 암조직에서 전반적으로 증가되어 있었으며, nc886이 증가한 환자는 유전자 발현이 암을 유도하는 방향으로 변화하였고, 환자의 생존율이 낮은 것을 확인할 수 있다. 즉, 상기 결과는 nc886이 간암에서 암 촉진 인자로 작용함을 강하게 시사한다.In summary, the amount of nc886 expression was generally increased in cancer tissues, and the patients with increased nc886 showed a change in the direction of gene expression inducing cancer, and the survival rate of patients was low. That is, the results strongly suggest that nc886 acts as a cancer promoting factor in liver cancer.

전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (15)

nc886 유전자를 포함하는 암의 예후 예측용 바이오마커.Biomarker for predicting prognosis of cancer containing nc886 gene. 제1항에 있어서, The method of claim 1, 상기 암은 갑상선암 또는 간암인 것을 특징으로 하는 The cancer is characterized in that the thyroid cancer or liver cancer 예후 예측용 바이오마커. Biomarkers for prognostic prediction. 제1항의 바이오마커의 발현 수준을 측정하는 제제를 포함하는 암 예후 예측용 조성물.A composition for predicting cancer prognosis comprising an agent measuring the expression level of the biomarker of claim 1. 제2항에 있어서, The method of claim 2, 상기 바이오마커의 발현 수준을 측정하는 제제는 상기 바이오마커에 특이적으로 결합하는 프라이머 쌍, 프로브 또는 안티센스 뉴클레오티드를 포함하는 것을 특징으로 하는 The agent for measuring the expression level of the biomarker is characterized in that it comprises a primer pair, a probe or an antisense nucleotide that specifically binds to the biomarker 암 예후 예측용 조성물.Composition for predicting cancer prognosis. 제3항 또는 제4항의 조성물을 포함하는 암 예후 예측용 키트.A kit for predicting cancer prognosis comprising the composition of claim 3 or 4. 제5항에 있어서, The method of claim 5, 상기 키트는 RT-PCR 키트, 경쟁적 RP-PCR 키트, 실시간 RT_PCR 키트, 정량적 RT-PCR 키트 또는 DNA 칩 키트인 것을 특징으로 하는 The kit is characterized in that the RT-PCR kit, competitive RP-PCR kit, real-time RT_PCR kit, quantitative RT-PCR kit or DNA chip kit 암 예후 예측용 키트.Kit for predicting cancer prognosis. (a) 암 환자에서 분리된 생물학적 시료로부터 제1항의 바이오마커의 발현 수준, 또는 발현 패턴을 얻는 단계; 및(a) obtaining the expression level, or expression pattern, of the biomarker of claim 1 from a biological sample isolated from a cancer patient; And (b) 상기 (a) 단계에서 얻은 발현 수준 또는 발현 패턴을 예후가 알려진 암 환자의 해당 유전자의 바이오마커 발현 수준, 또는 발현 패턴과 비교하는 단계를 포함하는 (b) comparing the expression level or expression pattern obtained in step (a) with the biomarker expression level or expression pattern of the gene of the cancer patient of known prognosis. 암 예후 예측을 위한 정보제공방법.Methods of providing information for predicting cancer prognosis. 제7항에 있어서, The method of claim 7, wherein 상기 바이오마커 수준을 측정하는 방법은 역전사효소 중합반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(competitive RT-PCR), 실시간 역전사 효소 중합효소반응(real time quantitative RT-PCR), 정량적 중합효소반응(quantitative RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블랏팅(Nothern blotting) 또는 DNA 칩 방법(DNA chip technology), 노던 블랏팅(Nothern blotting), 면역조직화학염색법(immunohistochemical staining), 면역침전분석법(immunoprecipitation assay), 보체 고정 분석법(complenent Fixation Assay) 또는 면역형광법(immunofluorescence)인 것인 The method of measuring the biomarker level is reverse transcriptase polymerase (RT-PCR), competitive reverse transcriptase polymerase (competitive RT-PCR), real time quantitative RT-PCR, quantitative polymerase Quantitative RT-PCR, RNase protection method, Northern blotting or DNA chip technology, Northern blotting, immunohistochemical staining , Immunoprecipitation assay, complement fixation assay or immunofluorescence 암의 예후 예측을 위한 정보제공방법.Informational method for predicting the prognosis of cancer. (a) 암 환자에서 분리한 시료에 후보물질을 처리하는 단계;(a) treating the candidate material with a sample isolated from a cancer patient; (b) 후보물질이 처리된 암 환자의 시료에서 제1항에 따른 바이오마커의 발현 수준을 측정하는 단계; 및(b) measuring the expression level of the biomarker according to claim 1 in a sample of the cancer patient treated with the candidate substance; And (c) 상기 (b) 단계의 바이오마커 발현 수준이 후보물질의 처리 전보다 낮은 경우, 후보물질을 암의 예방 또는 치료용 물질로 판단하는 단계; 를 포함하는 암 예방 또는 치료용 물질 스크리닝 시스템.(c) if the biomarker expression level of step (b) is lower than before treatment of the candidate, determining the candidate as a material for preventing or treating cancer; Cancer screening system for preventing or treating cancer comprising a. 제1항에 따른 바이오마커의 발현 또는 활성을 억제하는 물질; 및 약학적으로 허용 가능한 담체를 포함하는 암 예방 또는 치료용 약학적 조성물.Substances that inhibit the expression or activity of the biomarker according to claim 1; And a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier. 제10항에 있어서, The method of claim 10, 상기 물질은 바이오마커에 대한 안티센스 올리고 뉴클레오티드, 앱타머(aptamer), siRNA 또는 shRNA인 것을 특징으로 하는 The substance is characterized in that the antisense oligonucleotide, aptamer, siRNA or shRNA to the biomarker 암 예방 또는 치료용 약학적 조성물.Pharmaceutical composition for preventing or treating cancer. 제11항에 있어서, The method of claim 11, 상기 물질은 상기 바이오마커의 활성을 억제하는 항체 또는 그의 항원 결합 단편인 것을 특징으로 하는 The substance is an antibody or antigen-binding fragment thereof that inhibits the activity of the biomarker. 암 예방 또는 치료용 약학적 조성물.Pharmaceutical composition for preventing or treating cancer. 제10항에 있어서, The method of claim 10, 상기 조성물은 ANLN, C5orf13(= NREP), COL5A1, VGLL2, C15orf52, 또는 KIAA1644의 발현을 억제하는 것을 특징으로 하는 The composition is characterized by inhibiting the expression of ANLN, C5orf13 (= NREP), COL5A1, VGLL2, C15orf52, or KIAA1644 암 예방 또는 치료용 약학적 조성물. Pharmaceutical composition for preventing or treating cancer. 제10항에 있어서, The method of claim 10, 상기 조성물은 EGFR, HIPK2, HSPBL2 또는 TAXIBP1으로 구성된 군 중 하나 이상의 항-세포사멸 단백질(anti-apoptotic protein)의 발현을 억제하는 것을 특징으로 하는 The composition is characterized by inhibiting the expression of one or more anti-apoptotic proteins of the group consisting of EGFR, HIPK2, HSPBL2 or TAXIBP1 암 예방 또는 치료용 약학적 조성물. Pharmaceutical composition for preventing or treating cancer. 제10항에 있어서, The method of claim 10, 상기 조성물은 CDKN2C 또는 DKK1의 발현을 증가시키는 것을 특징으로 하는 The composition is characterized in that to increase the expression of CDKN2C or DKK1 암 예방 또는 치료용 약학적 조성물. Pharmaceutical composition for preventing or treating cancer.
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