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WO2018026190A1 - Biomarqueur pour prédire un pronostic d'un cancer - Google Patents

Biomarqueur pour prédire un pronostic d'un cancer Download PDF

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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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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

La présente invention concerne un biomarqueur génétique pour prédire le pronostic d'un cancer, en particulier le cancer de la thyroïde ou le cancer du foie, une composition de prédiction de pronostic d'un cancer comprenant un agent pour mesurer un niveau d'expression du biomarqueur génétique, un kit de prédiction de pronostic d'un cancer comprenant la composition, un procédé d'obtention d'informations pour prédire le pronostic d'un cancer à l'aide du kit, un système de criblage d'un matériau prophylactique ou curatif d'un cancer à l'aide du biomarqueur génétique et une composition pharmaceutique ciblant le biomarqueur génétique pour prévenir ou traiter un cancer. Plus particulièrement, le pronostic d'un patient souffrant d'un cancer peut être prédit par mesure d'un niveau d'expression du biomarqueur génétique de la présente invention chez le patient souffrant d'un cancer et l'expression d'un biomarqueur génétique correspondant responsable d'un mauvais pronostic peut être supprimée, en fonction des résultats de prédiction de pronostic, de manière telle que les patients atteints d'un cancer peuvent être traités plus efficacement.
PCT/KR2017/008336 2016-08-02 2017-08-02 Biomarqueur pour prédire un pronostic d'un cancer Ceased WO2018026190A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110456042A (zh) * 2019-08-09 2019-11-15 臻悦生物科技江苏有限公司 血清学生物标志物在制备试剂盒和/或芯片中的应用、试剂盒或芯片及其制备方法
KR20200069258A (ko) * 2018-12-06 2020-06-16 국립암센터 식도편평상피암의 예후를 예측하는 방법
CN113692443A (zh) * 2018-12-06 2021-11-23 国立癌症中心 包含nc886的用于增强溶瘤病毒活性或用于促进溶瘤病毒生产的组合物

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110580956B (zh) * 2019-09-19 2022-03-11 青岛市市立医院 一组肝癌预后标志物及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014148780A1 (fr) * 2013-03-20 2014-09-25 서울대학교산학협력단 Biomarqueur pour le diagnostic du cancer du foie
CN104887694A (zh) * 2015-05-27 2015-09-09 中国科学院微生物研究所 一种靶向非编码rna的反义寡核苷酸及其在制备抗流感病毒药物中的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014148780A1 (fr) * 2013-03-20 2014-09-25 서울대학교산학협력단 Biomarqueur pour le diagnostic du cancer du foie
CN104887694A (zh) * 2015-05-27 2015-09-09 中国科学院微生物研究所 一种靶向非编码rna的反义寡核苷酸及其在制备抗流感病毒药物中的应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEE, EUN KYUNG ET AL.: "Nc886, a Non-coding RNA and Suppressor of PKR, Exerts an Oncogenic Function in Thyroid Cancer", ONCOTARGET, vol. 7, no. 46, 6 September 2016 (2016-09-06), pages 75000 - 75012, XP055461434 *
LEE, KWANG-SOO ET AL.: "Nc886, a Non-coding RNA of Anti-proliferative Role, Is Suppressed by CpG DNA Methylation in Human Gastric Cancer", ONCOTARGET, vol. 5, no. 11, 2014, pages 3944 - 3955, XP055461431 *
LEE, YONG SUN: "A Novel Type of Non-coding RNA, Nc886, Implicated in Tumor Sensing and Suppression", GENOMICS & INFORMATICS, vol. 13, no. 2, 2015, pages 26 - 30, XP055461430 *
SILVER, MATT J. ET AL.: "Independent Genomewide Screens Identify the Tumor Suppressor VTRNA2-1 as a Human Epiallele Responsive to Periconceptional Environment", GENOME BIOLOGY, vol. 16, no. 118, 2015, pages 1 - 14, XP021224292 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200069258A (ko) * 2018-12-06 2020-06-16 국립암센터 식도편평상피암의 예후를 예측하는 방법
KR20210117238A (ko) * 2018-12-06 2021-09-28 국립암센터 식도편평상피암의 예후를 예측하는 방법
CN113692443A (zh) * 2018-12-06 2021-11-23 国立癌症中心 包含nc886的用于增强溶瘤病毒活性或用于促进溶瘤病毒生产的组合物
KR102348837B1 (ko) 2018-12-06 2022-01-11 국립암센터 식도편평상피암의 예후를 예측하는 방법
KR102359423B1 (ko) 2018-12-06 2022-02-09 국립암센터 식도편평상피암의 예후를 예측하는 방법
CN110456042A (zh) * 2019-08-09 2019-11-15 臻悦生物科技江苏有限公司 血清学生物标志物在制备试剂盒和/或芯片中的应用、试剂盒或芯片及其制备方法

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