KR20210094356A - Composition For Predicting Radiation Resistance Of Cancer - Google Patents

Abstract

The present invention relates to a marker for diagnosing radiation resistance of cancer. By using the marker and composition for diagnosing radiation resistance of the present invention, it is possible to diagnose radiation resistance of cancer cells to be treated with radiation and provide customized radiation therapy, and it is possible to enhance a therapeutic effect and reduce side effects in cancer patients. In addition, it is possible to provide information necessary for predicting the prognosis of radiation therapy patients.

Description

Composition for diagnosing radiation resistance of cancer {Composition For Predicting Radiation Resistance Of Cancer}

The present invention relates to a marker for diagnosing radiation resistance of cancer. More specifically, the present invention provides a composition for diagnosing radiation resistance comprising an agent for measuring the presence or expression level of the marker, a kit for diagnosing radiation resistance comprising the composition, and providing information necessary for diagnosing radiation resistance using the same it's about how

As life expectancy increases, the probability that an individual will develop cancer over their lifetime increases. Cancer, which was considered an incurable disease in the past, is being recognized as a disease that can be cured if detected at an early stage and combined with appropriate treatment with advances in medical science.

However, since the prognosis may vary depending on the type of cancer, the patient's family history, or age, etc., interest in personalized medicine or precision medicine has recently been attracting attention. Customized medicine or precision medicine refers to predicting disease prognosis and treatment responsiveness based on genetic, environmental, and biological factors that are different for each patient and providing customized treatment optimized for each patient.

As an example, in common cancer types such as breast cancer, lung cancer, urological cancer and gastrointestinal cancer, radiation therapy is a treatment method that is generally combined with surgery, chemotherapy, or hormone therapy, but some patients are resistant to radiation therapy. decreased survival rate.

In addition, since melanoma, a malignant cancer type that occurs in melanocytes, exhibits strong radiation resistance, radiation therapy is rarely used to treat melanoma. Melanoma is also resistant to targeted anticancer drugs. For example, there is a report that recurrence occurred within 6 months of patients receiving long-term treatment with vemurafenib (Non-Patent Document 1). Because of these characteristics, melanoma has very limited treatment options and is considered one of the most dangerous types of skin cancer.

In order for this personalized medicine to become universal, the importance of diagnostic technology that can help establish individual treatment strategies is emphasized. The present inventors recognized the need to develop a diagnostic marker capable of diagnosing radiation-resistant cancer cells, and in particular, completed the present invention as a result of repeated research and experiments to discover a diagnostic marker capable of diagnosing radiation-resistant melanoma.

Obenauf AC, Zou Y, Ji AL, Vanharanta S, Shu W, Shi H, Kong X, Bosenberg MC, Wiesner T, Rosen N et al: Therapy-induced tumoursecretomes promote resistance and tumour progression. Nature 2015, 520(7547):368-372.

One object of the present invention is to provide a marker composition for diagnosing radiation resistance of cancer cells.

Another object of the present invention is to provide a composition for diagnosing radiation resistance of cancer cells.

Another object of the present invention is to provide a kit for diagnosing radiation resistance of cancer cells comprising the composition for diagnosing radiation resistance.

Another object of the present invention is to provide a method of providing information for diagnosing radioresistance in a cancer patient using the composition or a method of providing information necessary for predicting a radiotherapy prognosis of a cancer patient.

Another object of the present invention is to provide a composition for enhancing radiation sensitivity to cancer cells.

Another object of the present invention is to provide a method for screening a radiation sensitivity enhancer for cancer cells.

According to one aspect of the present invention, there is provided a marker composition for diagnosing radiation resistance of cancer cells comprising one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN.

According to another aspect of the present invention, the mRNA of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN or a protein thereof is measured by radiation of cancer cells comprising an agent for measuring the expression level A composition for diagnosing resistance is provided.

According to another aspect of the present invention, there is provided a kit for diagnosing radiation resistance of cancer cells, comprising the composition for diagnosing radiation resistance of cancer cells.

According to another aspect of the present invention, measuring the mRNA or protein expression level of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN from a biological sample; and comparing the measured expression level of mRNA or protein with the expression level of mRNA or protein measured in a control sample; it provides a method of providing information for diagnosing radiation resistance in cancer patients.

According to another aspect of the present invention, in cancer cells comprising an inhibitor of mRNA or protein expression or activity of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN as an active ingredient. To provide a composition for enhancing radiation sensitivity.

According to another aspect of the present invention, the step of contacting a test substance with cancer cells; Measuring the expression or activity level of mRNA or protein of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN in cancer cells in contact with the test substance; And compared to the control sample, the CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2, and selecting a test substance with a reduced level of mRNA or protein expression or activity of one or more genes selected from the group consisting of SRGN; Provided is a method for screening a radiation sensitivity enhancer for cancer cells, including cancer cells.

By using the composition for diagnosing radiation resistance of the present invention, it is possible to provide customized radiation therapy by determining whether or not radiation resistance of cancer cells to be treated with radiation. Specifically, it is possible to enhance the therapeutic effect and reduce the side effects by diversifying the applied treatment regimen depending on whether the cancer cells are radiation-resistant or sensitive. In addition, using the radiation-resistant diagnostic composition of the present invention can help predict the prognosis of radiation-treated cancer patients.

1 shows the results of measuring the viability of cells against gamma (γ)-irradiation through MTT analysis for various melanoma cell lines (panel AE) as an embodiment of the present invention. After the melanoma cell line was gamma (γ)-irradiated with O Gy or 8 Gy, the viability of the cells was measured by MTT assay. The experimental results of FIG. 1 show that the SK-Mel5 cell line has radiation-sensitive properties, and the remaining cell lines have radiation-resistance.
2 is an embodiment of the present invention, a heat map plot result (panel A) and protein-protein interaction (PPI, Protein-Protein Interaction Network) analysis result (panel B) for 139 genes selected by RNA sequencing. indicates
3 shows RT-PCR analysis results for 8 genes selected from PPI analysis as an embodiment of the present invention.
4 shows the results of cell cycle analysis performed on various melanoma cell lines (panel AE) as an embodiment of the present invention.
5 is an embodiment of the present invention, Western blot test results for cyclin D1 (CCND1) in various melanoma cell lines (panel A) and fluorescence activated cell sorting (FACS) analysis results in SK-Mel28 and SK-Mel2 cell lines. (Panel B) is shown.

In this specification, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.

According to one aspect of the present invention, the present invention is CASP1 (Caspase 1), CCND1 (Cyclin D1), ENO2 (Enolase 2), HSPA1A (Heat shock protein 70), NGFR (Nerve Growth Factor Receptor), OAS1 (Oligoadenylate synthetase 1) , OAS2 (Oligoadenylate synthetase 2) and SRGN (Serglycin) provides a marker composition for diagnosing radiation resistance of cancer cells comprising one or more genes selected from the group consisting of.

The radiation-resistance diagnostic marker composition of the present invention is composed of a gene having a differential expression level in radiation-resistant cancer cells compared to general cancer cells that do not exhibit radiation resistance, so that radiation resistance of an individual or cell can be determined. That is, an individual or cancer cell having a higher or lower expression level of each marker gene included in the radioresistance diagnostic marker composition of the present invention than a control group can be determined to have radiation resistance or sensitivity.

If the radiation resistance or sensitivity of the cancer to be treated with radiation is determined and the level thereof is determined, the radiation dose of radiation therapy can be determined by reflecting this, thereby improving the efficiency and treatment results of radiation therapy.

In the present invention, the term “marker” refers to a measurable index capable of predicting the responsiveness of cells to radiation.

More specifically, the marker is a molecule, such as a polypeptide, a nucleic acid (eg, mRNA, etc.), a lipid that exhibits a significant difference in expression or activity between a cell or individual exhibiting resistance to radiation and a cell or individual exhibiting sensitivity. , and organic biomolecules such as glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.). For the purposes of the present invention, the marker may include a nucleic acid molecule (DNA or mRNA) or an expression protein of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN. .

In one embodiment of the present invention, the marker may include one or more genes selected from the group consisting of OAS1, HSPA1A, and CCND1.

As an example, the marker composition for diagnosing radiation resistance of cancer cells of the present invention comprises (i) an OAS1 gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS2 and SRGN.

As an example, the marker composition for diagnosing radiation resistance of cancer cells of the present invention comprises (i) HSPA1A gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, NGFR, OAS1, OAS2 and SRGN.

As an example, the marker composition for diagnosing radiation resistance of cancer cells of the present invention includes (i) a CCND1 gene; and (ii) at least one gene selected from the group consisting of CASP1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN.

As an example, the marker composition for diagnosing radiation resistance of cancer cells of the present invention may include all of OAS1, HSPA1A, and CCND1 genes.

In the present invention, the term "radio-resistance" refers to a state in which cells are not easily affected by radiation, and there is little or no change in cell repair or proliferation when exposed to radiation.

In the present invention, the term "radio-sensitive" refers to a state in which cells are easily affected by radiation and show changes in cell repair or proliferation upon exposure to radiation.

As used herein, the term "cancer cell" refers to any cell that exhibits uncontrolled growth in a tissue or organ of a multicellular organism.

As used herein, the term “cancer” refers to a disease caused by the cancer cells.

In the present invention, "cancer" or "cancer cell" that can be distinguished by the expression difference of the marker gene is included without limitation, but preferably non-melanoma skin cancer (NMSC) or melanoma or cancer cells of these cancers, and more Preferably, it may be melanoma or a cancer cell thereof.

As used herein, the term "irradiation" includes exposure to, for example, X-rays, ultraviolet radiation, electromagnetic radiation, including alpha particles and gamma rays, or the action of alpha or beta particles.

As used herein, the term "gray (Gy)" is the amount of radiation emitted by 1 J in 1 kg of representative biological tissue.

Information on the marker genes may be obtained from a known database, for example, NCBI GenBank, but is not limited thereto.

According to another aspect of the present invention, the present invention provides an agent for measuring the mRNA or protein expression level of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN. Provided is a composition for diagnosing radiation resistance of cancer cells.

In one embodiment of the present invention, the composition for diagnosis of radiation resistance of the present invention may include an agent for measuring the expression level of mRNA of one or more genes selected from the group consisting of OAS1, HSPA1A, and CCND1 or a protein thereof.

As an example, the composition for diagnosis of radiation resistance of the present invention comprises (i) an OAS1 gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS2 and SRGN; an agent for measuring the expression level of mRNA or protein thereof.

As an example, the composition for diagnosing radiation resistance of the present invention comprises (i) HSPA1A gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, NGFR, OAS1, OAS2 and SRGN; an agent for measuring the expression level of mRNA or protein thereof.

As an example, the composition for diagnosis of radiation resistance of the present invention comprises (i) a CCND1 gene; and (ii) at least one gene selected from the group consisting of CASP1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN; an agent for measuring the expression level of mRNA or protein thereof.

As an example, the composition for diagnosing radiation resistance of the present invention may include an agent for measuring the expression level of mRNA or protein of OAS1, HSPA1A, and CCND1 genes.

In one embodiment, the expression level of the mRNA or protein of the marker gene may be the expression level of mRNA or protein derived from a cell obtained from a biological sample. The biological sample is a sample suspected of containing cancer cells, and may include, for example, tissue, whole blood, serum, plasma, urine, cell lysate, or cell culture, but is not limited thereto.

As used herein, the term "measurement of mRNA expression level" refers to a process of determining the presence and expression level of mRNA of the marker gene in a biological sample for diagnosing radiation resistance, and measuring the amount of mRNA. Analytical methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection) assay), Northern blotting, and a DNA chip, but is not limited thereto.

In the present invention, "measurement of protein expression level" refers to a process of confirming the presence and expression level of a protein expressed from the marker gene in a biological sample in order to diagnose radiation resistance, preferably, specific for the protein of the gene. The amount of protein can be confirmed by using an antibody that binds positively. Analysis methods for this include western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, and rocket. Immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited thereto. .

The agent for measuring the mRNA level of the gene of the present invention may include one or more selected from the group consisting of a probe, primer, antisense nucleotide, and PNA (peptide nucleic acid) that specifically binds to the mRNA of the gene. And, for example, a primer that specifically binds to the gene can be prepared by those skilled in the art using various programs based on the sequence of the gene known in an accessible database.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' terminal hydroxyl group, capable of base pairing with a complementary template, and serving as a starting point for template strand copying. It refers to a short nucleic acid sequence. do. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. The primers of the present invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific to the nucleotide sequence of each marker gene. Primers may incorporate additional features that do not change the basic properties of the primer to serve as the starting point for DNA synthesis. The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates. , carbamates, etc.) or charged linkages (eg phosphorothioate, phosphorodithioate, etc.). Nucleic acids may contain one or more additional covalently linked residues, e.g., proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.) ), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. The nucleic acid sequences of the present invention may also be modified using labels capable of providing a detectable signal, either directly or indirectly. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

As used herein, the term "peptide nucleic acid (PNA)" refers to a nucleic acid-like polymer having a structure in which the phosphate-ribose backbone of DNA is substituted with an amide backbone (N-(2-aminoethyl)glycine) similar to a peptide. PNA has greatly increased binding strength and stability to DNA or RNA, and is capable of complementary binding to a target nucleotide sequence like DNA despite structural changes in the backbone. Therefore, various fields in which oligonucleotides or nucleic acid molecules are used can be used in the same way.

The agent for measuring the expression level of the protein of the marker gene may include one or more selected from the group consisting of an antibody, an interacting protein, a ligand, an oligopeptide, a nanoparticle, and an aptamer specific for the protein, for example, and antibodies specific for the protein.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The generation of an antibody using the radiation resistance marker protein as described above may be according to a technique well known in the art. The polyclonal antibody can be produced by a method well known in the art in which the above-described radioresistance marker protein antigen is injected into an animal and blood is collected from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be prepared from hosts of any animal species, such as goats, rabbits, sheep, monkeys, horses, pigs, bovine dogs, and the like. Monoclonal antibodies can be prepared using the hybridoma method well known in the art (see Kohler and Milstein (1976) European Jounral of Immunology 6:511-519), or a phage antibody library (Clackson et al, Nature, 352:624). -628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). The antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv.

As used herein, the term “ligand” refers to a substance that binds to a target protein and forms a complex with the target protein. More specifically, the ligand that can be used in the present invention may include a molecule capable of indicating the presence or absence of a target protein by generating a signal when it binds to a target protein.

As used herein, the term “aptamer” refers to a substance capable of binding to a target protein, and may be, for example, an oligonucleic acid or a peptide molecule. For a general description of aptamers, see Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl AcadSci USA. 95(24):142727 (1998).

According to another aspect of the present invention, there is provided a kit for diagnosing radiation resistance of cancer cells comprising the composition for diagnosing radiation resistance.

As used in the present invention, the term "kit" refers to the mRNA expression level or protein expression level of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN. It refers to a tool that can diagnose whether cancer cells are radiation-resistant.

The kit may be an RT-PCR kit, a DNA chip kit, a microarray or a protein chip kit. The diagnostic kit according to the present invention can be prepared according to a conventional method used in the art using the radiation-resistant diagnostic composition. The expression change of the radiation resistance gene may be measured using the radiation resistance diagnostic kit. In the kit for diagnosing radiation resistance of cancer cells of the present invention, primers and probes for measuring the expression level of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2, and SRGN as radioresistance diagnostic markers , or optionally an antibody recognizing the marker, as well as one or more other component compositions, solutions, or devices suitable for the assay method. For example, when the kit of the present invention is provided in the form of an ELISA kit, in addition to an antibody specific for a marker protein, an antibody specific for a control protein, a reagent capable of detecting bound antibody (eg, a labeled secondary antibody, chromophores, enzymes (eg, conjugated to an antibody) and their substrates or other substances capable of binding the antibody, etc.). In addition, when the kit of the present invention is provided in the form of a PCR kit, it may additionally include a thermostable DNA polymerase, a buffer, and dNTPs in addition to the primers and labeled probes that specifically bind to the nucleotide sequence of the above-described marker gene. can

The kit of the present invention may additionally include instructions for components/articles constituting the kit and instructions for use.

According to another aspect of the present invention, measuring the mRNA or protein expression level of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN from a biological sample ; and comparing the measured expression level of mRNA or protein with the expression level of mRNA or protein measured in a control sample, respectively; it provides a method of providing information for diagnosing radiation resistance in cancer patients, including.

In one embodiment of the present invention, the measuring step may be a step of measuring the expression level of the mRNA of one or more genes selected from the group consisting of OAS1, HSPA1A and CCND1 or a protein thereof.

As an example, the measuring step includes (i) OAS1 gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS2 and SRGN; measuring the expression level of mRNA or protein thereof.

As an example, the measuring step includes (i) HSPA1A gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, NGFR, OAS1, OAS2 and SRGN; measuring the expression level of mRNA or protein thereof.

As an example, the measuring step may include (i) a CCND1 gene; and (ii) at least one gene selected from the group consisting of CASP1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN; measuring the expression level of mRNA or protein thereof.

As an example, the measuring step may include measuring the expression level of the mRNA or protein thereof of OAS1, HSPA1A, and CCND1 genes.

For the purpose of the present invention, the expression level of mRNA or protein of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2, and SRGN measured in cancer cells is the level of mRNA or protein measured in a control sample. If it is higher than the expression level, it can be judged as a radiation-resistant cancer cell. Optionally, if the expression level of the gene in the cancer cell is lower than that measured in the control sample, it may be determined as a radiation-sensitive cancer cell.

For the measurement of the mRNA or protein expression level of the gene, the above-described agents may be used, respectively, and detailed descriptions are omitted to avoid repetitive description.

According to another aspect of the present invention, the present invention provides mRNA or protein expression of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN from an isolated sample of a cancer patient. comparing the level to the mRNA or protein expression level in normal cells, respectively; And when the mRNA or protein expression level of the isolated sample of the cancer patient is higher than that of normal cells, it provides a method of providing information necessary for predicting a radiotherapy prognosis of a cancer patient, comprising the step of diagnosing radiation resistance.

As used herein, the term "prediction of prognosis" means, by confirming the expression level of a gene showing a difference in expression in a radiation-resistant cancer cell line as compared to a radiation-sensitive cell line, the treatment effect, for example, survival potential, after radiation to a cancer patient; It is to predict the possibility of cure or recurrence.

According to another aspect of the present invention, the present invention provides a cancer cell comprising an inhibitor of the expression or activity of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN as an active ingredient To provide a composition for enhancing radiation sensitivity.

In one embodiment of the present invention, the protein expression or activity inhibitor may be a protein expression or activity inhibitor of one or more genes selected from the group consisting of OAS1, HSPA1A and CCND1.

As an example, the expression or activity inhibitor of the protein (i) OAS1 gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS2 and SRGN; may be an inhibitor of protein expression or activity.

As an example, the expression or activity inhibitor of the protein (i) HSPA1A gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, NGFR, OAS1, OAS2 and SRGN; It may be an inhibitor of protein expression or activity of

As an example, the expression or activity inhibitor of the protein is (i) CCND1 gene; and (ii) at least one gene selected from the group consisting of CASP1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN; may be an inhibitor of protein expression or activity.

As an example, the protein expression or activity inhibitor may be a protein expression or activity inhibitor of OAS1, HSPA1A and CCND1 genes.

In one embodiment of the present invention, the mRNA expression inhibitor of the gene is an antisense nucleotide complementary to the mRNA of the gene, small interfering RNA (siRNA) and short hairpin RNA (shRNA). It may include one or more selected from the group consisting of.

In another embodiment of the present invention, the protein activity inhibitor of the gene includes at least one selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the protein. can do.

According to another aspect of the present invention, the present invention comprises the steps of contacting a test substance with cancer cells; Measuring the expression or activity level of mRNA or protein of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN in cancer cells contacted with the test substance; And compared to the control sample, the CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and the expression or activity of one or more genes selected from the group consisting of SRGN mRNA or protein expression or activity of a reduced test substance; Provided is a method for screening a radiation sensitivity enhancer for cancer cells, including cancer cells.

In one embodiment of the present invention, the measuring step may be a step of measuring the expression or activity level of mRNA or protein of one or more genes selected from the group consisting of OAS1, HSPA1A, and CCND1.

As an example, the measuring step includes (i) OAS1 gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS2 and SRGN; measuring the expression or activity level of mRNA or protein.

As an example, the measuring step includes (i) HSPA1A gene; and (ii) at least one gene selected from the group consisting of CASP1, CCND1, ENO2, NGFR, OAS1, OAS2 and SRGN; It may be a step of measuring the level of expression or activity of mRNA or protein.

As an example, the measuring step may include (i) a CCND1 gene; and (ii) at least one gene selected from the group consisting of CASP1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN; measuring the expression or activity level of mRNA or protein.

As an example, the measuring step may be a step of measuring the expression or activity level of mRNA or protein of OAS1, HSPA1A and CCND1 genes.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Identification of radiation-sensitive or resistant cell lines

Cell culture and irradiation

SK-Mel5 and SK-Mel28 cells were cultured in the medium of a modified Eagle (DMEM) in Dulbecco, Malme-3M, UACC27 and SK-Mel2 cell lines are cultured in a humidified incubator containing 5% CO ₂ at 37 ℃ in RPMI1640 did. All media were supplemented with 10% fetal bovine serum (FBS) and Zellshield ^™ (Minerva Biolabs GmbH). If necessary, γ-irradiation of 8 Gy was performed with Gamma Cell 3000 (Atomic Energy) using ^{[ 137 Cs] in the cell line.}

Analysis of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

A 6-well plate was used for the experiments for each cell line. After irradiation (8 Gy), cells were cultured for 3 days. MTT solution (20 μl; Amresco, 5 mg/ml) was added and incubated for 4 hours. The growth medium was carefully removed, 200 μl of dimethylsulfoxide (DMSO) was added, and the optical density of each well was measured at 570 nm with a microplate reader. The results were measured as an average of three samples, and the results are shown in FIG. 1 . Data are presented as mean±SD of three independent experiments. A p-value of less than 0.05 was considered significant.

From the results in Figure 1, SK-Mel5 cell line (Panel A) was relatively sensitive to irradiation, whereas other cell lines SK-Mel28 (Panel B), Malme-3M (Panel C), UACC257 (Panel D) and SK-Mel2 (Panel E) was able to confirm that it was radiation-resistant.

Example 2: Selection and verification of genes overexpressed in radiation-resistant cell lines

RNA isolation

Total RNA was extracted from the culture medium using TRI reagent (Molecular Research Center) according to the manufacturer's instructions. Briefly, cells were extracted with 500 μl TRI reagent, mixed with 100 μl chloroform, and centrifuged at 12,000 x g for 15 min at 4°C. The RNA containing aqueous phase was transferred to a new tube and mixed with 250 μl isopropyl alcohol to precipitate the RNA. An RNA pellet was obtained by centrifugation at 12,000 x g for 10 min at 4°C. The pellet was washed briefly with 200 μl of 75% ethanol, centrifuged at 7,500 x g for 5 minutes at 4° C., and then dried at room temperature for 5 minutes. Each resulting total RNA pellet was dissolved in RNase-free water and evaluated for quality and quantity.

RNA sequencing (Quant-seq) and protein-protein interaction (PPI) analysis

Quantitative sequencing of each cell line was performed (E-Biogen). The obtained data were analyzed using ExDEGA software, and 139 genes differentially expressed in each cell line with fold change>5.5 and p-value<0.01 were selected.

Subsequent analysis was performed on the selected 139 genes. The heat map was plotted using MultiExperiment Viewer (MeV) 4.9.0, and the results are shown in panel A of FIG. 2 .

In addition, in order to confirm the protein-protein interaction (PPI) for the selected gene, the 'Search Tool for the Retrieval of Interacting Genes (STRING)' application of Cytoscape 3.6.1 software was used, and the results are shown in panel B of FIG. shown in The interaction degree prediction score in the STRING application was applied as 0.35. Overall, 85 out of 139 genes were predicted to interact with each other. The 'yFields organic layout' was adopted with some modifications for visualization. Nodes represent selected genes and lines represent interactions between genes. From the results of panel B of FIG. 2 , it was confirmed that eight genes of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN exhibit strong intergene interactions.

Quantitative real-time PCR (qRT-PCR)

Quantitative RT-PCR was performed to verify the data obtained by RNA sequencing (Quant-seq). The expression levels of the 8 genes selected through STRING were analyzed for the 5 melanomas.

^{cDNA was prepared from total RNA through reverse transcription using 'SuperScript TM} VI First-Strand Synthesis System' (Invitrogen) according to the manufacturer's instructions. The resulting cDNA samples were diluted to a final concentration of 20 ng/μl. Real-time PCR reactions were performed using a BioRad CFX96 system (BioRad), and amplification was performed using AMPIGNE® qPCR Green Mix Lo-Rox (Enzo Life Sciences). The primer sequences used in the amplification reaction of each gene are shown in Table 1 below.

gene Accession No. gene ID primer SEQ ID NO: CASP1 NM_033294 834 F: 5'-TTT CTT GGA GAC ATC CCA C-3' One R: 5'-CAT CTG CGC TCT ACC ATC-3' 2 CCND1 NM_053056 595 F: 5'-TCA TTG AAC ACT TCC TCT CC-3' 3 R: 5'-GAG GGC GGA TTG GAA ATG-3' 4 ENO2 NM_001975 2026 F: 5'-CTC AAG GTC AAC CAG ATC G-3' 5 R: 5'-GTC TTG ATC TGG CCT GTG-3' 6 HSPA1A NM_005345 3303 F: 5'-GAG GCG GAC AAG AAG AAG-3' 7 R: 5'-GTA CAG TCC GCT GAT GAT G-3' 8 NGFR NM_002507 4804 F: 5'-CAA GAC CTC ATA GCC AGC-3' 9 R: 5'-CTT GCT TGT TCT GCT TGC-3' 10 OAS1 NM_001032409 4938 F: 5'-AGA AGA GGA CTG GAC CTG-3' 11 R: 5'-GAG CTT TGT CCT CTC TG-3' 12 OAS2 NM_016817 4939 F: 5'-TGG TGT TGG CAT CTT CTG-3' 13 R: 5'-GAT CCT AGC TCC ACA ACT TC-3' 14 SRGN NM_002727 5552 F: 5'-TCC TAA CGG AAA TGG AAC AG-3' 15 R: 5'-CTA ATC CAT GTT GAC CCA AG-3' 16

Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping genes are as follows: 5'-AGG GCT GCT TTT AAC TCT GGT-3' (forward) (SEQ ID NO: 17) and 5'-CCC CAC TTG ATT TTG GAG GGA-3' (reverse) (SEQ ID NO: 18). Thermal cycling conditions consisted of a denaturation step at 95° C. for 3 minutes followed by 40 cycles of 95° C. for 5 seconds, 58° C. for 30 seconds and 72° C. for 30 seconds. Experiments were performed three times for each data. The relative expression of the target gene ^{was quantified using the 2-ΔΔCt} method, and the results are shown in FIG. 3 .

From FIG. 3 , it was confirmed that the expression level of the eight genes selected through STRING was increased in the radiation-resistant cell line compared to the radiation-sensitive cell line. Among them, in the case of OAS1, HSPA1A and CCND1 genes, the expression increase rate was more than doubled in the radiation-resistant cell line compared to the radiation-sensitive cell line, and the expression gradually increased as the radiation resistance increased. Therefore, among the eight genes, the OAS1, HSPA1A and CCND1 genes are expected to be highly correlated with radiation resistance.

Fluorescence-activated cell sorting (FACS) analysis

For comparison and analysis of cell cycle distribution before and after irradiation, cells were harvested using a trypsin-EDTA solution 24 hours after irradiation with cells. Then, in order to inhibit the action of the trypsin-EDTA solution, the cells were suspended in a medium, washed with cold PBS, and fixed with cold 70% ethanol for 30 minutes. Cells were treated with RNase A (200 μg/ml) and propidium iodide (PI, 10 μg/ml) and incubated at room temperature for 15 minutes. CytoFLEX (BECKMAN COULTER) was used for cell cycle analysis, and flow cytometry software (CytExpert) was used for visualization, and the results are shown in FIG. 4 .

From the results of FIG. 4, it was confirmed that all melanomas exhibited a decrease in G0/G1 and S phases upon irradiation. However, in the radiation-resistant cell line, it was confirmed that the G2/M arrest after irradiation did not increase relatively significantly compared to the sensitive cell line. Specifically, in the radiation-sensitive cell line SK-Mel5, when gamma-irradiated, G1/G0 arrest decreased by 21.1% and S-phase by 15.1%, and G2/M arrest increased by 36.2%, whereas all radiation-resistant melanomas exhibited S-phase. Decrease width (SK-Mel28: -8.3%, Mamme-3M: -8.4%, UACC257: -5.4%, SK-Mel2: -0.5%) and G2/M arrester increase width (SK-Mel28: 17.6%, Mamme) -3M: 25.6%, UACC257: 24.5%, SK-Mel2: 23.5%) was reduced compared to the sensitive cell line. Therefore, it could be confirmed that the change pattern of the cell cycle is related to radiation resistance.

Example 3: Verification of correlation between CCND1 and radiation resistance

Western blot analysis

Western blot analysis was performed to verify the correlation between radiation resistance of melanoma and cyclin D1 expression. Western blot analysis was performed as follows: Antibodies to caspase-3 and cyclin D1 were purchased from Cell Signaling Technology, and antibodies to β-actin were purchased from Santa Cruz Biotechnology. Cells were extracted with a lysis solution (Cell signaling Technology), and the protein concentration of the lysate was measured using a BCA protein assay kit (Pierce) according to the manufacturer's instructions. The same amount of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10-12% gel and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) containing 0.2% Tween-20 for 1 hour and incubated with primary antibody overnight at 4°C. After washing 3 times for 1 hour with PBST, the blots were incubated with horseradish peroxide (HRP)-conjugated secondary antibody (Bethyl Laboratories) for 1 hour at room temperature. Protein bands were detected with enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotechnology) and X-ray film (AGFA).

The results of the Western blot experiment for cyclin D1 are shown in panel A of FIG. 5 . As can be seen from the experimental results of panel A of FIG. 5 , it was confirmed that the level of cyclin D1 protein was high in resistant melanoma.

Fluorescence-activated cell sorting (FACS) analysis

Fluorescence activated cell sorting (FACS) analysis was performed according to the experimental method described above, and the results are shown in panel B of FIG. 5 . In order to determine whether overexpressed cyclin D1 is involved in the formation of radiation resistance, SK-Mel28 and SK-Mel2 cell lines were pretreated with Arcyriaflavin A (indicated by Ar in FIG. 5), a CDK4-CCND1 inhibitor, for 6 hours, followed by FACS 24 hours after irradiation. was used to compare the cell cycle. As a result, it was confirmed that the G0/G1 and G2/M arrest levels were restored to their pre-irradiation levels by Arcyriaflavin A in both cell lines. This proves that cyclin D1 is involved in irradiation-induced cell cycle changes.

statistical analysis

All data are presented as mean±standard deviation (SD) and compared using Student's t-test. Analysis was performed using the statistical program Microsoft Office Excel (Microsoft Corp, USA), and a p-value <0.05 was considered statistically significant.

The specific embodiments described herein are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention as set forth in the claims herein.

<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> Composition For Predicting Radiation Resistance Of Cancer <130> 2019-DPA-3500 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> forward primer for CASP1 <400> 1 tttcttggag acaccccac 19 <210> 2 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for CASP1 <400> 2 catctgcgct ctaccat 18 <210> 3 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> forward primer for CCND1 <400> 3 tcattgaaca cttcctctcc 20 <210> 4 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for CCND1 <400> 4 gagggcggat tggaaatg 18 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> forward primer for ENO2 <400> 5 ctcaaggtca accagatcg 19 <210> 6 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for ENO2 <400> 6 gtcttgatct ggcctgtg 18 <210> 7 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> forward primer for HSPA1A <400> 7 gaggcggaca agaagaag 18 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for HSPA1A <400> 8 gtacagtccg ctgatgatg 19 <210> 9 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> forward primer for NGFR <400> 9 caagacctca tagccagc 18 <210> 10 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for NGFR <400> 10 cttgcttgtt ctgcttgc 18 <210> 11 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> forward primer for OAS1 <400> 11 agaagaggac tggacctg 18 <210> 12 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for OAS1 <400> 12 gagctttgtc ctctctg 17 <210> 13 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> forward primer for OAS2 <400> 13 tggtgttggc atcttctg 18 <210> 14 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for OAS2 <400> 14 gatcctagct ccacaacttc 20 <210> 15 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> forward primer for SRGN <400> 15 tcctaacgga aatggaacag 20 <210> 16 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> reverse primer for SRGN <400> 16 ctaatccatg ttgacccaag 20

Claims (16)

A marker composition for diagnosing radiation resistance of cancer cells comprising one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN.
The method according to claim 1,
Wherein the cancer cells are cancer cells of melanoma, a marker composition for diagnosing radiation resistance of cancer cells.
The method according to claim 1,
(i) the OAS1 gene; And (ii) CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS2 and a marker composition for diagnosing radiation resistance of cancer cells comprising one or more genes selected from the group consisting of SRGN.
CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and a composition for diagnosing radiation resistance of cancer cells comprising an agent for measuring the expression level of the mRNA of one or more genes selected from the group consisting of or a protein thereof.
5. The method according to claim 4,
Wherein the cancer cells are melanoma cancer cells, a composition for diagnosing radiation resistance of cancer cells.
5. The method according to claim 4,
(i) the OAS1 gene; And (ii) CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS2 and a composition for diagnosing radiation resistance of cancer cells comprising an agent for measuring the mRNA or protein expression level of one or more genes selected from the group consisting of SRGN.
5. The method according to claim 4,
The agent for measuring the expression level of the mRNA of the gene is a probe that specifically binds to the mRNA of the gene, a primer set, an antisense nucleotide and one or more selected from the group consisting of peptide nucleic acid (PNA) radiation of cancer cells A composition for diagnosis of resistance.
5. The method according to claim 4,
The agent for measuring the expression level of the protein of the gene is a composition for diagnosing radiation resistance of cancer cells comprising at least one selected from the group consisting of an antibody, an interacting protein, a ligand, an oligopeptide, a nanoparticle, and an aptamer specific for the protein.
A kit for diagnosing radiation resistance of cancer cells, comprising the composition for diagnosing radiation resistance according to any one of claims 4 to 8.
Measuring the expression level of the mRNA or protein of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN from the biological sample; and
Comparing the measured expression level of mRNA or protein with the expression level of mRNA or protein measured in a control sample; comprising, a method of providing information for diagnosing radiation resistance in cancer patients.
11. The method of claim 10,
When the expression level of the measured mRNA or protein is higher than the expression level of the mRNA or protein measured in the control sample, a method of providing information for diagnosing radiation resistance in a cancer patient diagnosed with radiation resistance.
11. The method of claim 10,
The method of providing information for diagnosing radiation resistance of a cancer patient, wherein the cancer is melanoma.
CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and a composition for enhancing radiation sensitivity to cancer cells comprising an inhibitor of the expression or activity of mRNA or protein of one or more genes selected from the group consisting of SRGN as an active ingredient.
14. The method of claim 13,
The mRNA expression inhibitor of the gene is one or more selected from the group consisting of antisense nucleotides complementary to the mRNA of the gene, small interfering RNA (siRNA) and short hairpin RNA (shRNA). A composition for enhancing radiation sensitivity to cancer cells, including.
14. The method of claim 13,
The protein activity inhibitor of the gene is a small molecule compound that specifically binds to the protein, a peptide, a peptide mimetics, an aptamer, an antibody and one or more selected from the group consisting of natural substances for enhancing radiation sensitivity to cancer cells. composition.
contacting the test substance to the cancer cells;
Measuring the expression or activity level of mRNA or protein of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN in cancer cells in contact with the test substance; and
Comparing with the control sample, the expression or activity level of the mRNA or protein of one or more genes selected from the group consisting of CASP1, CCND1, ENO2, HSPA1A, NGFR, OAS1, OAS2 and SRGN is reduced. A method for screening a radiation sensitivity enhancer for cancer cells.

Images