WO2025048047A1 - Composition for diagnosis of radioresistance of colorectal cancer or for prognosis of colorectal cancer - Google Patents

Abstract

In the present invention, the mRNA of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4, and SYTL4 genes, or the proteins encoded by these genes, exhibit differential expression levels between radioresistant and radiosensitive colorectal cancer cells, and are closely correlated with reduced survival rates in colorectal cancer patients. Therefore, an agent for measuring the expression levels of the mRNA of the genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4, and SYTL4 or the proteins encoded by the genes according to the present invention effectively diagnoses radio-resistance in colorectal cancer or prognoses radiotherapy for colorectal cancer. This enables the determination of whether to proceed with radiotherapy and the optimal radiation dose, thereby improving the efficiency and outcomes of radiotherapy for colorectal cancer.

Description

Composition for diagnosing radiation resistance of colon cancer or predicting prognosis of colon cancer

[Cross-reference with related applications]

This application claims the benefit of priority from Korean Patent Application No. 10-2023-0112012, filed August 25, 2023, the entire contents of which are incorporated herein by reference.

[Technical Field]

The present invention relates to a composition for diagnosing radiation resistance of colon cancer and applications thereof.

Cancer treatment methods include surgery, chemotherapy, and radiation therapy, and the importance of radiation therapy has increased recently. In cases where surgery is difficult, cases where tumors were effectively cured with radiation therapy alone have been reported, and tumor treatment methods using radiation are also developing year after year, so radiation therapy has become established as a method that can effectively treat tumors in the body without any particular pain or discomfort to the patient. Statistically, about 30-50% of cancer patients receive radiation therapy at some point during the treatment period. As the number of cancer patients receiving radiation therapy is increasing every year, the importance of radiation therapy in cancer treatment is also increasing.

Radiation therapy is commonly used to treat various types of cancer, either alone or in combination with chemotherapy. However, the effectiveness of radiation therapy varies depending on the nature of the cancer, the patient, and whether radiation is combined with other treatments. Cancers for which radiation therapy is commonly performed include uterine cancer, pharyngeal cancer, lung cancer, brain cancer, and breast cancer. Although these cancers are the main targets of radiation therapy, they often do not respond well to radiation therapy, so strategies to increase treatment efficiency are necessary. In addition, even if radiation therapy is effective and the initial response is good, relapses often occur, so it is important to establish countermeasures for recurrent cancer to increase the effectiveness of radiation therapy.

Furthermore, since the acquisition of radiation resistance by cancer cells and damage to normal tissue during high-dose radiation therapy are pointed out as problems that reduce the effectiveness of radiation therapy, research is needed to improve the effectiveness of radiation therapy. Currently, radiation therapy is used as an effective cancer treatment method along with surgical operation or chemical drug therapy, and is used to kill cancer cells by treating the cancerous tissue of the patient with radiation.

Although there are differences in the sensitivity of cancer cells to radiation therapy, if we develop a technology that can predict individual radiation sensitivity before radiation therapy, we will be able to provide the most suitable radiation therapy for each individual by adjusting the radiation dose to treat cancer.

Meanwhile, research on companion diagnostics, which refers to approved diagnoses that can select appropriate targeted anticancer drugs and treatment methods based on systematic analysis results of recent patient personal factors, is actively being conducted. Companion diagnostics can provide clear clinical grounds for prescriptions based on a doctor's diagnosis and can suggest appropriate treatment methods to patients, which can not only increase the effectiveness of cancer treatment but also reduce the misuse of targeted anticancer drugs and contribute to the soundness of national health insurance finances. Currently, the companion diagnostics market is growing in treatment fields such as breast cancer, lung cancer, colon cancer, stomach cancer, and melanoma, and breast cancer and lung cancer fields are expected to lead market growth in particular.

Korean Patent Application No. 10-2018-0143493 discloses a technique for diagnosing radioresistant lung cancer or pancreatic cancer by measuring the expression level of PMVK protein or mRNA, but no data is provided regarding colorectal cancer cell specificity.

In addition, Korean Patent Application No. 10-2018-0153659 discloses a technique for diagnosing the radiation resistance of cancer cells by measuring the expression level of the ROMO1 gene or protein, but does not present a technique for more accurately diagnosing radiation resistant colon cancer using multiple markers.

Under these circumstances, the inventors of the present invention conducted research on radiation resistance biomarkers and discovered a novel biomarker for diagnosing radiation resistant colon cancer. The biomarker can predict resistance to radiation therapy or the prognosis of radiation therapy, and is expected to be useful in the treatment of colon cancer.

[Prior art literature]

[Patent Document]

Republic of Korea Patent Application No. 10-2018-0143493

Republic of Korea Patent Application No. 10-2018-0153659

The purpose of the present invention is to provide a composition for diagnosing radiation resistance of colon cancer.

In addition, the present invention aims to provide a kit for diagnosing radiation resistance of colon cancer.

In addition, the present invention aims to provide a method for providing information for diagnosing radioresistance of colon cancer.

In addition, the present invention aims to provide a composition for predicting the prognosis of radiation therapy for colon cancer.

In addition, the present invention aims to provide a kit for predicting the prognosis of radiation therapy for colon cancer.

In addition, the present invention aims to provide a method for providing information for predicting the prognosis of radiation therapy for colon cancer.

To achieve the above object, one aspect of the present invention provides a composition for diagnosing radiation resistance of colon cancer, comprising an agent for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

In addition, another aspect of the present invention provides a kit for diagnosing radiation resistance of colon cancer, comprising the composition for diagnosing radiation resistance of colon cancer.

In addition, another aspect of the present invention provides a method for providing information for diagnosing radioresistance of colon cancer, comprising the steps of: (a) measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes in a sample isolated from an individual, or proteins encoded by the two or more genes; and (b) comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control.

In addition, another aspect of the present invention provides a composition for predicting the prognosis of radiation therapy for colon cancer, comprising an agent for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

In addition, another aspect of the present invention provides a kit for predicting the prognosis of radiation therapy for colon cancer, comprising a composition for predicting the prognosis of radiation therapy for colon cancer. Another aspect of the present invention provides a method for providing information for predicting the prognosis of radiation therapy for colon cancer, comprising the steps of: (a) measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes in a sample isolated from a subject; and (b) comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control.

Hereinafter, the present invention will be described in detail.

1. Composition for diagnosing radiation resistance of colon cancer or predicting prognosis of radiation therapy of colon cancer

One aspect of the present invention provides a composition for diagnosing radiation resistance of colon cancer.

In addition, another aspect of the present invention provides a composition for predicting the prognosis of radiation therapy for colon cancer.

The composition for diagnosing radiation resistance of colon cancer of the present invention contains, as an active ingredient, an agent for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

In addition, the composition for predicting the prognosis of radiation therapy for colon cancer of the present invention contains, as an active ingredient, an agent for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

CXCL5 (C-X-C motif chemokine ligand 5, NCBI gene ID: 6374) protein is a small cytokine belonging to the CXC chemokine family. It is produced by stimulation with the inflammatory cytokines interleukin-1 and tumor necrosis factor-alpha, and is known to regulate neutrophil homeostasis by inhibiting type II interferon-gamma.

In the present invention, the CXCL5 protein refers to not only the wild-type CXCL5 protein consisting of the amino acid sequence of SEQ ID NO: 1, but also an equivalent that has a different partial sequence but has the same or similar function or activity as the wild-type CXLC5 protein, and the equivalent may include an amino acid sequence having a sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 1.

CD68 (Cluster of Differentiation 68, NCBI gene ID: 968) protein is a protein expressed in monocyte lineage cells and macrophages, and is generally known as a scavenger receptor that plays a role in removing cellular debris, promoting phagocytosis, and mediating macrophage recruitment and activation.

In the present invention, the CD68 protein refers to not only the wild-type CD68 protein consisting of the amino acid sequence of SEQ ID NO: 2, but also an equivalent that has a different sequence from the wild-type CD68 protein but has the same or similar function or activity as the wild-type CD68 protein, and the equivalent may include an amino acid sequence having a sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 2.

MATN1 (Matrilin 1, NCBI gene ID: 4146) protein is a cartilage matrix protein that is involved in the formation of filamentous networks in the extracellular matrix of various tissues, and mutations in this gene are known to be associated with various hereditary chondrodysplasias.

In the present invention, the MATN1 protein refers to not only the wild-type MATN1 protein consisting of the amino acid sequence of SEQ ID NO: 3, but also an equivalent that has a different sequence from the wild-type MATN1 protein but has the same or similar function or activity as the wild-type MATN1 protein, and the equivalent may include an amino acid sequence having a sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 3.

SERPING1 (Serpin family G member 1, NCBI gene ID: 710) protein is a protease inhibitor belonging to the serpin superfamily, also known as a C1 esterase inhibitor, and is known to regulate physiological pathways including complement activation, blood coagulation, fibrinolysis, and kinin production by inhibiting the activation of the C1 complex.

In the present invention, the SERPING1 protein refers to not only the wild-type SERPING1 protein consisting of the amino acid sequence of SEQ ID NO: 4, but also an equivalent that has a different sequence from the wild-type SERPING1 protein but has the same or similar function or activity as the wild-type SERPING1 protein, and the equivalent may include an amino acid sequence having a sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 4.

SNCG (Gamma-synuclein, NCBI gene ID: 6623) protein is a type of synuclein family protein that is related to the pathogenesis of degenerative brain diseases and is often observed to be overexpressed in breast cancer cells, and is known to have a high correlation with the development of breast cancer.

In the present invention, the SNCG protein refers to not only the wild-type SNCG protein consisting of the amino acid sequence of SEQ ID NO: 5, but also an equivalent that has a part of the sequence different therefrom but has the same or similar function or activity as the wild-type SNCG protein, and the equivalent may include an amino acid sequence having a sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 5.

VTN (Vitronectin, NCBI gene ID: 7448) protein is a glycoprotein of the hemopexin family synthesized in the liver. It is known to promote cell adhesion and migration by binding to integrin alpha-V beta-3 and connecting cells to the extracellular matrix, and to be involved in hemostasis as a component of platelets.

In the present invention, the VTN protein refers to not only a wild-type VTN protein consisting of an amino acid sequence of SEQ ID NO: 6, but also an equivalent that has a different sequence from the wild-type VTN protein but has the same or similar function or activity as the wild-type VTN protein, and the equivalent may include an amino acid sequence having a sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 6.

FBXO4 (F-box protein 4, NCBI gene ID: 26272) protein is a protein-coding factor characterized by an F-box motif of about 40 amino acids, and is known to directly contribute to cell transformation, tumorigenesis, and progression by generating Fbx proteins that contain other protein-protein interaction modules or lack recognizable motifs. In addition, the FBXO4 protein is a tumor suppressor, and its tumor suppression activity is reported to be associated with dysregulation of cyclin D1 protein degradation.

In the present invention, the FBXO4 protein refers to not only the wild-type FBXO4 protein consisting of the amino acid sequence of SEQ ID NO: 7, but also an equivalent that has some sequence differences but has the same or similar function or activity as the wild-type FBXO4 protein, and the equivalent may include an amino acid sequence having a sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 7.

SYTL4 (Synaptotagmin like 4, NCBI gene ID: 94121) protein has an N-terminal Rab27 binding domain and a C-terminal tandem C2 domain, which allows it to bind to Rab GTPases and participate in intracellular membrane trafficking, and is known to regulate the release of nucleolar granules into cells or the secretion of pancreatic and pituitary hormones.

In the present invention, the SYTL4 protein refers to not only the wild-type SYTL4 protein consisting of the amino acid sequence of SEQ ID NO: 8, but also an equivalent that has some sequence differences but has the same or similar function or activity as the wild-type SYTL4 protein, and the equivalent may include an amino acid sequence having a sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more to the amino acid sequence of SEQ ID NO: 8.

The composition for diagnosing radiation resistance of colon cancer according to the present invention may contain, as an active ingredient, an agent for measuring the expression level of mRNA of two genes, three genes, four genes, five genes, six genes, seven genes, or eight genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4, and SYTL4 genes, or proteins encoded by the above genes.

In addition, the composition for predicting the prognosis of radiation therapy for colon cancer according to the present invention may contain, as an active ingredient, an agent for measuring the expression level of mRNA of two genes, three genes, four genes, five genes, six genes, seven genes, or eight genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4, and SYTL4 genes, or proteins encoded by the above genes.

In a specific embodiment of the present invention, it was confirmed that mRNA of a gene selected from the group consisting of the CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by the genes have differential expression levels in radiation-resistant and -sensitive colon cancer cells and have a close correlation with a decrease in the survival rate of colon cancer patients.

Therefore, the formulation for measuring the expression level of mRNA of one or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes of the present invention or a protein encoded by one or more genes can diagnose the radioresistance of colon cancer more effectively than mRNA of other genes and/or proteins encoded by the genes and combinations thereof, and can predict the prognosis of radiation therapy for colon cancer, thereby determining whether to perform radiation therapy and the optimal dose of radiation therapy, thereby improving the efficiency and treatment results of radiation therapy for colon cancer.

Specifically, in colon cancer cells, the expression of the genes CXCL5, CD68, MATN1, SERPING1, SNCG and VTN is upregulated by radiation exposure, and the expression of the genes FBXO4 and SYTL4 is downregulated.

More specifically, the CXCL5, CD68 and MATN1 genes are up-regulated in their expression in radioresistant colon cancer cells, and the SERPING1, SNCG and VTN genes are up-regulated in their expression in radiosensitive colon cancer cells.

Specifically, the CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4, and SYTL4 genes may be genes whose expression is upregulated, thereby decreasing the survival rate of colon cancer patients.

Therefore, if the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG and VTN genes or the expression level of proteins encoded by two or more genes is high compared to the normal control group, it can be determined that radioresistance is present, and if it is low, it can be determined that radioresistance is low or radiosensitivity is present. For example, if the mRNA of two to six genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG and VTN genes and/or the two to six proteins encoded by the genes are all highly expressed, radioresistance can be determined to be high, and if the mRNA of two to six genes and/or the two to six proteins encoded by the genes are all lowly expressed, radioresistance can be determined to be low.

In addition, if the expression levels of mRNA of the FBXO4 and SYTL4 genes or the proteins encoded by the genes are lower than those of the normal control group, it can be determined that radioresistance exists, and if they are higher, it can be determined that radioresistance is low or radiosensitivity exists.

Meanwhile, if the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG and VTN genes or the expression level of proteins encoded by two or more genes is high compared to the normal control group, the prognosis of radiation therapy for colon cancer can be determined to be negative, and if the expression level is low, the prognosis of radiation therapy for colon cancer can be determined to be positive. For example, if the mRNA of two to six genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG and VTN genes and/or the two to six proteins encoded by the genes are all highly expressed, the prognosis of radiation therapy for colon cancer can be determined to be negative, and if the mRNA of two to six genes and/or the two to six proteins encoded by the genes are all lowly expressed, the prognosis of radiation therapy for colon cancer can be determined to be positive.

In addition, when the expression levels of mRNA of the FBXO4 and SYTL4 genes or the proteins encoded by the genes are lower than those of the normal control group, the prognosis of radiation therapy for colon cancer can be judged to be negative, and when the expression levels are high, the prognosis of radiation therapy for colon cancer can be judged to be positive.

Meanwhile, the composition for diagnosing radiation resistance of colon cancer and the composition for predicting the prognosis of radiation therapy for colon cancer of the present invention may include an agent for measuring the expression level of mRNA of the SERPING1 or SNCG gene or a protein encoded by the gene.

In the present invention, "radio-resistance" means a state in which a substance is not easily affected by radiation exposure and there is little change in cell recovery or proliferation when exposed to radiation. As a term opposite to the above-mentioned radio-resistance, "radio-sensitive" means a state in which a substance is easily affected by radiation exposure and there is a change in cell recovery or proliferation when exposed to radiation.

In the present invention, "radiation therapy" means treating cancer through a mechanism of inducing apoptosis of cancer cells by inhibiting cell division through DNA damage of cancer cells by irradiating them with radiation. The radiation therapy can be performed alone or in combination with surgical treatment, or as a combination therapy with anticancer chemotherapy or a radiation sensitizer, for the curative, adjuvant, or palliative purposes of solid cancer.

In the present invention, colorectal cancer may include colon cancer, rectal cancer, etc.

In the present invention, “diagnosis” includes determining the susceptibility of an individual to a particular disease or condition, determining whether an individual currently has a particular disease or condition, determining the prognosis of an individual with a particular disease or condition, or therametrics (e.g., monitoring the condition of an individual to provide information about the efficacy of a treatment).

In the present invention, "prognosis" means an outlook or preliminary evaluation of the medical outcome of a disease, and includes a positive prognosis (positive prognosis) or a negative prognosis (negative prognosis). The positive prognosis includes improvement or stabilization of the disease, such as a state of no disease, tumor regression, long-term survival possibility, or disease-free survival rate, and the negative prognosis includes progression or mortality of the disease, such as decreased survival rate, recurrence, tumor growth, metastasis, and drug resistance.

In addition, in the present invention, "prediction" means to guess in advance about the medical outcome, such as the course of a disease (progression, improvement, recurrence, tumor growth, possibility of death or survival after drug resistance treatment, etc.) or responsiveness to treatment such as chemotherapy or radiation therapy.

Specifically, in the present invention, the diagnosis may be a diagnosis of radiation resistance of colon cancer, and the prognosis prediction may be a prognosis prediction of radiation treatment of colon cancer.

In the present invention, measurement of the mRNA expression level means a process of confirming the presence and expression level of mRNA of the gene in a biological sample for the purpose of diagnosing the radiation resistance of colon cancer and/or predicting the prognosis of radiation treatment of colon cancer. As an agent for measuring the expression level of the mRNA, a primer, a probe, or an antisense nucleotide that specifically binds to the gene or mRNA can be used, and the measurement of the expression level of the mRNA can be performed by one or more methods selected from the group consisting of RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chips, but is not limited thereto.

In addition, in the present invention, the measurement of the protein expression level means a process of confirming the presence and expression level of the protein in a biological sample for the diagnosis of radiation resistance of colon cancer and/or prediction of the prognosis of radiation treatment of colon cancer, and a polypeptide, compound, antibody or aptamer capable of specifically binding to the protein may be used as an agent for measuring the expression level of the protein, and the measurement of the expression level of the protein may be performed by Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, dual luciferase reporter assay, immunoprecipitation assay, complement fixation assay, FACS (fluorescence activated cell sorter) and protein It can be performed by one or more methods selected from the group consisting of chips, but is not limited thereto.

In the present invention, the "sample" may mean a sample derived from an individual receiving radiation therapy (radiation irradiation) or for which a decision on whether to receive radiation therapy is required. For example, the sample may be a sample capable of specifying the expression level of mRNA of two or more genes selected from the group consisting of the CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or a protein encoded by two or more genes, and may include, but is not limited to, cells, tissues, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine of the individual.

In the present invention, the "subject" refers to a subject for which it is desired to confirm the radiation resistance or progression possibility of colon cancer or to predict the prognosis of radiation treatment. The subject is not limited to any type of animal that can develop colon cancer, but may specifically be a mammal, for example, a human (Homo sapiens).

In the present invention, the "expression level" may be used interchangeably with the expression signature and the expression profile, and may include the expression level of mRNA of two or more genes selected from the group consisting of the CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes, or the expression level of proteins encoded by two or more genes. The expression level may include an increase in expression or a decrease in expression, and the expression level may be measured from cancer cells isolated from a subject before and/or after radiation treatment. Meanwhile, the expression level can be confirmed by measuring the amount of mRNA of the gene and/or the protein in the sample.

In the present invention, the "primer" means a short nucleic acid sequence having a short free 3'-terminal hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer solution and temperature. The primer of the present invention is a primer that can complementarily bind to the biomarker gene or mRNA of the present invention, and can be a sense and antisense nucleic acid having a sequence of 7 to 50 nucleotides, and can incorporate additional features that do not change the basic properties of the primer that serves as the starting point for DNA synthesis. Meanwhile, the primer of the present invention can be chemically synthesized using a phosphoramidite solid support method or other well-known methods, and can be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more of the natural nucleotides with their analogues, and modifications between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acids may contain one or more additional covalently bonded moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. In addition, the primers of the present invention may be modified with labels that can directly or indirectly provide a detectable signal, such as radioisotopes, fluorescent molecules, biotin, etc.

In the present invention, the term "probe" refers to a nucleic acid capable of complementarily binding to mRNA of several to several hundred bases in length, which is produced through an enzymatic chemical separation and purification process or a synthetic process. The probe of the present invention is a probe capable of complementarily binding to the biomarker gene or mRNA of the present invention, and can confirm the presence or absence of mRNA by labeling it with a radioactive isotope or enzyme, and can be designed and modified by a known method and used.

In the present invention, the "antisense nucleotide" refers to DNA, RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, which binds to the complementary sequence in the mRNA and inhibits the translation of the mRNA into a protein. The sequence of the antisense nucleotide refers to a DNA or RNA sequence that is complementary to the mRNA of the biomarker gene of the present invention and can bind to the mRNA. This can inhibit essential activities for translation, translocation into the cytoplasm, maturation or all other overall biological functions of the mRNA. The antisense nucleotide can be synthesized in vitro by a conventional method and administered in vivo or the antisense nucleotide can be synthesized in vivo, and the antisense nucleotide that can be used in the present invention can be produced by a method known in the art with reference to the base sequence and/or amino acid sequence of the biomarker gene and/or protein of the present invention.

In the present invention, the "antibody" refers to a globulin-based protein that reacts when an antigen, which is a foreign substance, invades while circulating in the blood or lymph in the immune system of a living body, and means a protein that specifically binds to an antigen. For the purpose of the present invention, the antibody means an antibody that specifically binds to the biomarker protein of the present invention, and may include all of a polyclonal antibody, a monoclonal antibody, and a recombinant antibody, and includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule. A functional fragment of an antibody molecule means a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2, and Fv, etc. The antibody that specifically binds to the biomarker protein of the present invention may be produced by a known method known to those skilled in the art, and may be produced, for example, by injecting the biomarker protein, which is an immunogen, into an external host. The foreign host includes mammals such as mice, rats, sheep, and rabbits, and the immunogen can be injected intramuscularly, intraperitoneally, or subcutaneously, and is usually administered together with an adjuvant to increase antigenicity. Thereafter, blood is periodically collected from the foreign host, and serum showing specificity for the antigen can be collected to isolate antibodies.

In the present invention, the "aptamer" is a single-stranded oligonucleotide, and means a nucleic acid molecule having a size of about 20 to 60 nucleotides and having binding activity to a predetermined target molecule. The aptamer has various three-dimensional structures depending on the sequence, can have high affinity for a specific substance like an antigen-antibody reaction, and can inhibit the activity of a predetermined target molecule by binding to the predetermined target molecule. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and may be in a linear or cyclic form. The aptamer in the present invention can specifically bind to a protein encoded by the biomarker gene of the present invention, and can be prepared by a person skilled in the art from the base sequence of the biomarker gene of the present invention by a known method.

2. Kit for diagnosing radioresistance of colon cancer and kit for predicting prognosis of radiation therapy for colon cancer

Another aspect of the present invention provides a kit for diagnosing radiation resistance of colon cancer.

In addition, another aspect of the present invention provides a kit for predicting the prognosis of radiation therapy for colon cancer.

The kit for diagnosing radiation resistance of colon cancer of the present invention contains, as an active ingredient, a preparation for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

In addition, the kit for predicting the prognosis of radiation therapy for colon cancer of the present invention contains, as an active ingredient, a preparation for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

Since the overlapping description is the same as that described above in ' 1. Composition for diagnosing radiation resistance of colon cancer or predicting the prognosis of radiation therapy of colon cancer ', the description is omitted.

In the present invention, the "kit" means a tool which can diagnose the radioresistance of colon cancer or predict the prognosis of radiation therapy of colon cancer by confirming the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes. The kit of the present invention may include an agent for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes, specifically, a primer, a probe and an antisense nucleotide that specifically bind to the mRNA of the above genes, or a polypeptide, a compound, an antibody and an aptamer that specifically bind to the above proteins, and may include one or more other component compositions, solutions, or devices suitable for an analysis method.

In addition, the kit may additionally include a user manual describing optimal reaction performance conditions. The manual may include a guide booklet in the form of a pamphlet or leaflet, a label attached to the kit, and a description on the surface of a package containing the kit, and may include information disclosed or provided through electronic media such as the Internet.

In the present invention, the kit may be an RT-PCR kit, a microarray chip kit, a DNA kit, an ELISA kit, a protein chip kit, or a rapid kit.

3. Method for providing information for diagnosing radiation resistance of colon cancer and method for providing information for predicting prognosis of radiation therapy for colon cancer

Another aspect of the present invention provides a method for providing information for diagnosing radiation resistance of colon cancer.

In addition, another aspect of the present invention provides a method for providing information for predicting the prognosis of radiation therapy for colon cancer.

The method for providing information for diagnosing radiation resistance of colon cancer of the present invention comprises: (a) a step of measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes in a sample isolated from an individual; and (b) a step of comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control.

In addition, the method for providing information for predicting the prognosis of radiation therapy for colon cancer of the present invention comprises (a) a step of measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes in a sample isolated from an individual; and (b) a step of comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control.

Since the overlapping description is the same as that described above in ' 1. Composition for diagnosing radiation resistance of colon cancer or predicting the prognosis of radiation therapy of colon cancer ', the description is omitted.

In the information providing method for diagnosing radiation resistance of colon cancer of the present invention, step (a) may include a step of measuring the expression level of mRNA of the SERPING1 or SNCG gene or a protein encoded by the gene.

In addition, in the method for providing information for predicting the prognosis of radiation therapy for colon cancer of the present invention, step (a) may include a step of measuring the expression level of mRNA of the SERPING1 or SNCG gene or a protein encoded by the gene.

In the method for providing information for diagnosing radiation resistance of colon cancer of the present invention, in step (b), when the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG and VTN genes or the protein encoded by two or more genes is compared with the expression level of mRNA or protein of a normal control group, if the expression level is higher than the expression level of the normal control group, the colon cancer can be determined to have radiation resistance. The above high expression level may include that the expression level of the individual is increased by a similar level or by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% and 1000% or more compared to the expression level of a normal control.

In addition, in the information providing method for diagnosing radiation resistance of colon cancer of the present invention, if the expression level of the mRNA of the FBXO4 and SYTL4 genes or the protein encoded by the genes is compared with the expression level of the mRNA or protein of the normal control group in the step (b), and if it is lower than the expression level of the normal control group, the colon cancer can be determined to have radiation resistance. The low expression level may include that the expression level of the subject is reduced by a similar level or 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more compared to the expression level of the normal control group.

Meanwhile, in the method for providing information for predicting the prognosis of radiation therapy for colon cancer of the present invention, in step (b), if the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG and VTN genes or the protein encoded by two or more genes is compared with the expression level of mRNA or protein of a normal control group and is higher than the expression level of the normal control group, the prognosis of radiation therapy for colon cancer can be determined to be negative. The above high expression level may include that the expression level of the individual is increased by a similar level or by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% and 1000% or more compared to the expression level of a normal control.

In addition, in the information providing method for predicting the prognosis of radiation therapy for colon cancer of the present invention, if the expression level of the mRNA or the protein encoded by the FBXO4 and SYTL4 genes or the expression level of the protein encoded by the genes is compared with the expression level of the mRNA or the protein of the normal control group in the step (b), and if the expression level is lower than the expression level of the normal control group, the prognosis of the radiation therapy for colon cancer can be determined to be negative. The low expression level may include that the expression level of the subject is similar to the expression level of the normal control group or is reduced by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more.

Meanwhile, the information-providing method for diagnosing radiation resistance of colon cancer of the present invention may further include: (c-1) a step of determining that the colon cancer has radiation resistance when the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, and VTN genes or proteins encoded by two or more genes is high compared to a normal control; and/or (c-2) a step of determining that the colon cancer has radiation resistance when the expression level of mRNA of FBXO4 and SYTL4 genes or proteins encoded by the above genes is low compared to a normal control.

In addition, the method for providing information for predicting the prognosis of radiation therapy for colon cancer of the present invention may further include: (c-1) a step of determining that the prognosis of radiation therapy for colon cancer is negative when the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, and VTN genes or proteins encoded by the two or more genes is high compared to a normal control group; and/or (c-2) a step of determining that the prognosis of radiation therapy for colon cancer is negative when the expression level of mRNA of FBXO4 and SYTL4 genes or proteins encoded by the above genes is low compared to a normal control group.

In a specific embodiment of the present invention, the overall survival period (OS) was analyzed in TCGA data to identify genes or combinations having a P-value of 0.05 or less, and the HR (Hazard Ratio) value according to each gene or combination was identified. The HR represents risk, and a higher HR value in a group with high expression means a lower survival rate of the individual. When the HR is 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, or 2.0 or more, the prognosis of the individual's colon cancer after radiation therapy can be determined to be negative.

Another aspect of the present invention provides a method for diagnosing radioresistance of colon cancer, comprising the steps of: (a) measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes in a sample isolated from an individual, or proteins encoded by the two or more genes; and (b) comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control.

Another aspect of the present invention provides a method for predicting the prognosis of radiation therapy for colon cancer, comprising the steps of: (a) measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by the two or more genes in a sample isolated from an individual; and (b) comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control.

Another aspect of the present invention provides a use of a formulation for diagnosing radioresistance of colon cancer, the formulation comprising measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

Another aspect of the present invention provides a use of a formulation for predicting the prognosis of radiation therapy for colorectal cancer, the formulation comprising measuring the expression levels of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes.

In the present invention, mRNA of the CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or the proteins encoded by the genes have differential expression levels in radioresistant and sensitive colon cancer cells, and are closely correlated with the decreased survival rate of colon cancer patients. Therefore, the agent for measuring the expression level of mRNA of a gene selected from the group consisting of the CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes of the present invention or the protein encoded by the gene can effectively diagnose the radioresistance of colon cancer or predict the prognosis of colon cancer radiotherapy, and thereby determine whether to perform radiotherapy and the optimal dose of radiotherapy, thereby improving the efficiency and treatment results of radiotherapy for colon cancer.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

Figure 1 shows the results of radiosensitivity measurements for human colon cancer cell lines HT29 and HCT116.

Figure 2 shows the results of analyzing genes whose expression increased or decreased after irradiation with a dose of 8 Gy to human colon cancer cell lines HT29 and HCT116.

Figure 3 shows the results of analyzing the changes in protein expression of radiation-responsive genes in human colon cancer cell lines HT29 and HCT116.

Figure 4 shows the results of analyzing the expression levels and risk of CXCL5 and CD68 genes using the DESeq2 program for the RNAseq results (GSE107422) of 110 patients who relapsed after developing colorectal cancer.

Figure 5a shows Kaplan-Meier survival curves for the survival of patients with colon cancer according to VTN, SNCG, or SERPING1 expression.

Figure 5b shows Kaplan-Meier survival curves for the survival rate of patients with colon cancer according to the expression of VTN and MATN1, VTN and SERPING1, SNCG and SYTL4, SNCG and CD68, or SNCG and FBXO4.

Figure 6a shows the change in cell viability measured after knocking down the expression of radioresistance genes using siRNA in the human colon cancer cell line HCT116.

Figure 6b shows the change in cell viability measured after knocking down the expression of radioresistance genes using siRNA in the human colon cancer cell line HT29.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example 1. Measurement of radiation sensitivity of colon cancer cell lines

Human colon cancer cell lines HT29 and HCT116 were purchased from ATCC (American Type Culture Collection, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml) at 5% _CO2 and 37°C. To measure the radiation sensitivity of the colon cancer cells, each cell line was seeded in an appropriate number of cells in a 60 mm dish, and after 24 hours, radiation was irradiated at various doses and cultured for 10-14 days under conditions of 5% _CO2 and 37°C. After that, the formed cell colonies were washed with phosphate buffered saline (PBS) and stained with a 100% methanol mixture containing 1% methylene blue. The stained cell colonies (colonies with more than 50 cells) were dried at room temperature and then visually counted for each dish.

As a result, the survival rate of the HT29 cell line after radiation exposure was measured to be significantly higher than that of the HCT116 cell line, confirming that the HT29 cell line has higher radiation resistance than the HCT116 cell line (Fig. 1).

Example 2. Discovery of radiation-responsive genes in colon cancer

To discover radiation-responsive genes in colon cancer, genes were selected based on transcriptome results analyzed in HT29 and HCT116 cell lines, and the radiation responsiveness of each gene was measured by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The primer information used in this example is as shown in Table 1 below.

Target Sequence Official
Gene Name Product
length Dir. Sequence (5'->3') NM_002994.5 CXCL5 111 Forward CAG ACC ACG CAA GGA GTT CAT C
(sequence number 9) Reverse TTC CTT CCC GTT CTT CAG GGA G
(sequence number 10) NM_001040059.2 CD68 136 Forward CGA GCA TCA TTC TTT CAC CAG CT
(Sequence number 11) Reverse ATG AGA GGC AGC AAG ATG GAC C
(Sequence number 12) NM_002379.3 MATN1 92 Forward GGC TTC ACT CTG AAC AGC GAC G
(Sequence number 13) Reverse CCG TCA ATG AGG AAG ACC AGG T
(Sequence number 14) NM_012176.3 FBXO4 143 Forward GTC TTC GGG AAG ACC ATT GTT GG (SEQ ID NO: 15) Reverse GAG TTT CAG CCT CTG TAT CCT GG (SEQ ID NO: 16) NM_000062.3 SERPING1 157 Forward GCA TCA AAG TGA CGA CCA GCC A
(Sequence number 17) Reverse GTC TCT GTC AGT TCC AGC ACT G
(Sequence number 18) NM_001330120.2 SNCG 132 Forward TGT GGT GAG CAG CGT CAA CAC T
(Sequence number 19) Reverse TTG GAT GCC ACA CCC TCC TGT T
(sequence number 20) NM_000638.4 VTN 154 Forward TGG CTG TCC TTG TTC TCC AGT G
(Sequence number 21) 　 　 　 Reverse GTG TGC GAA GAT TGA CTC GGT AG (SEQ ID NO: 22) NM_001129896.3 SYTL4 94 Forward GGA GAC AGA AGC AAA TCC GTC C
(Sequence number 23) Reverse GGC GAT GTA ACT TCA CTA GGT GG (SEQ ID NO: 24) NM_022551.3 RPS18 107 Forward CAC GCC AGT ACA AGA TCC CA
(sequence number 25) Reverse TTC ACG GAG CTT GTT GTC CA
(Sequence number 26)

Thereafter, the HT29 and HCT116 cell lines were irradiated with 0 or 8 Gy of radiation, and then the expression of each gene was measured. As a result, the expression of CXCL5, CD68, MATN1, SERPING1, SNCG, and VTN was increased in both cell lines by radiation irradiation, while the expression of FBXO4 and SYTL4 was decreased in both cell lines by radiation irradiation (Fig. 2). In addition, CXCL5, CD68, and MATN1 were highly expressed in the relatively radioresistant HT29 cell line, SERPING1, SNCG, and VTN were highly expressed in the relatively radiosensitive HCT116 cell line, and FBXO4 and SYTL4 were similarly expressed in both cell lines.

Example 3. Confirmation of changes in protein expression of radiation response factors

In order to confirm the protein expression pattern according to radiation irradiation of the radiation response genes derived from the above Example 2, HT29 and HCT116 cell lines were irradiated with a dose of 8 Gy, and 24 hours later, proteins were extracted and Western blotting assay was performed. Information on antibodies used in this Example is as shown in Table 2 below.

Primary antibody manufacturing company Cat.No Host Size (kD) CXCL5 Invitrogen 700656 Rabbit 8 CD68 Santa Cruz sc-20060 Mouse 75-100 MATN1 Santa Cruz sc-14428 Goat 120 SERPING1 Invitrogen PA5-31541 Rabbit 100 SNCG Santa Cruz sc-65979 Mouse 80-90 VTN Santa Cruz sc-74484 Mouse 75 FBXO4 Santa Cruz sc-376372 Mouse 44 SYTL4 Invitrogen PA5-35992 Rabbit 80-90 ACTB Sigma A5316 Mouse 42

As a result, CXCL5 was highly expressed in the relatively radioresistant HT29 cell line, and SNCG was highly expressed in the relatively radiosensitive HCT116 cell line. In addition, VTN protein was significantly increased by radiation exposure, while FBXO4 protein was decreased (Fig. 3). Meanwhile, CD68, CXCL5, SNCG, and SERPING1 are all secreted proteins known to be closely related to the activation of immune cells around tumors, and thus, the decrease in the Western blot may mean that secretion into the medium has increased.

Example 4. Analysis of risk and survival curves according to expression of radiation response gene panel in colon cancer patients

In order to confirm the expression pattern of the radiation response genes derived from the above Example 2 in actual colon cancer patients, data (GSE107422) of 110 patients who relapsed after the onset of colon cancer were downloaded and analyzed using the DESeq2 program. As a result, as shown in Table 3 below, it was confirmed that the expression of CXCL5 and CD68 was significantly increased by 3.946 times and 1.46 times, respectively.

Symbol FC(fold change) P-value CXCL5 3.946 0.000006 CD68 1.464 0.00007

In addition, as a result of analyzing the overall survival period of cancer patients according to the expression of CXCL5 and CD68, it was found that as the expression of CXCL5 increased, the overall survival period significantly decreased, and the risk was 1.51, and as the expression of CD68 increased, the overall survival period significantly decreased, and the risk increased to 2.01 (Figure 4).

Thereafter, in order to analyze the effect of the expression of the radiation response genes derived from the above Example 2 on the survival rate of colon cancer patients, the publicly available genomic database data, The Cancer Genome Atlas (TCGA), was utilized to analyze the overall survival (OS) with GEPIA2 to identify genes or combinations with a P-value of 0.05 or less, and the results are shown in Table 4 below. In Table 4 below, HR (Hazard Ratio) represents the risk level, and a higher HR value in a group with high gene expression means a worse survival rate for the patient. In addition, the survival rate of colon cancer patients according to the expression of the genes or combinations was analyzed and visualized using a Kaplan-Meier survival curve (Figs. 5a and 5b).

Combination HR P-value VTN matn1 1.5 0.0084 VTN SNCG fbxo4 1.7 0.039 VTN SNCG fbxo4 CD68 1.7 0.038 VTN SERPING1 1.7 0.033 SERPING1 1.7 0.034 SNCG 1.7 0.042 SNCG SYTL4 1.7 0.029 SNCG CD68 1.7 0.028 SNCG CD68 Matn1 1.8 0.024 SNCG CD68 Matn1 VTN 1.8 0.02 SNCG CD68 fbxo4 1.8 0.018 SNCG CD68 VTN 1.8 0.017 SNCG fbxo4 2 0.0053

As a result of the analysis, the risk of survival rate of colon cancer patients due to increased SNCG expression and the risk due to increased SERPING1 expression were both 1.7, which means that increased expression of SNCG and SERPING1 can confirm increased risk and decreased survival rate alone, and can be classified as a radioresistance factor. In addition, in the case of SNCG, the risk was found to increase further when co-expressed with CD68, SYTL4, or FBXO4 increased. On the other hand, the risk of increased expression of VTN alone was 1.4, which was not statistically significant, and when co-expression of VTN/MATN1 or VTN/SERPING1 increased, the risk was 1.5 and 1.7, respectively, indicating a significant decrease in overall survival rate.

In addition, since FBXO4 and SYTL4 have a decreased expression due to radiation irradiation, and when their co-expression with other gene panels increases, the overall survival period of colorectal cancer patients decreases. Therefore, colorectal cancer patients with high expression of FBXO4 and SYTL4 can be considered as candidates for radiation therapy and have a positive prognosis of radiation therapy. On the other hand, CD68, CXCL5, MATN1, SERPING1, SNCG, and VTN have a increased expression due to radiation irradiation, and when their expression increases, they are directly linked to a decreased survival rate of colorectal cancer patients. Therefore, these genes have radiation resistance, and colorectal cancer patients with high expression of these genes can be considered as excluded candidates for radiation therapy and have a negative prognosis of radiation therapy.

Example 5. Analysis of cell viability by knockdown of radiation-responsive genes in human colon cancer cells

In order to confirm the change in the survival rate of colon cancer cells when the expression of the radiation resistance genes derived from Example 2 above was knocked down, the expression of the radiation resistance genes was knocked down with siRNA, and then the change in the survival rate of colon cancer cell lines HT29 and HCT116 was measured.

Specifically, each siRNA was transfected into colon cancer cell lines using Lipofectamine™ RNAiMAX transfection reagent (ThermoFisher scientific, USA) based on a 96-well plate, and then irradiated with 8 Gy of radiation. After 48 hours, the cell viability was measured by treating with CCK-8 (DOJINDO, Japan) reagent. The siRNA information used in this example is as shown in Table 5 below.

Official
Gene Name NCBI Gene ID Direction Sequence (5'->3') Scramble RNA Sense CCUCGUGCCGUUCCAUCAGGUAG (SEQ ID NO: 27) Anti-sense CUACCUGAUGGAACGGCACGAGG (SEQ ID NO: 28) SNCG 6623 Sense GACCAAGGAGAAUGUUGUACA (SEQ ID NO: 29) Anti-sense UGUACAACAUUCUCCUUGGUC(SEQ ID NO: 30) SYTL4 94121 Sense GAUGAGGGUGAGAUGAUAUUU(Sequence number 31) Anti-sense AAAUAUCAUCUCACCCUCAUC(SEQ ID NO: 32) CD68 968 Sense GCUUAUUGCUUUCUGCAUCAU(sequence number 33) Anti-sense AUGAUGCAGAAAGCAAUAAGC(SEQ ID NO: 34) VTN 7448 Sense GUAGUCAGUACUGGCGCUUUG (SEQ ID NO: 35) Anti-sense CAAAGCGCCAGUACUGACUAC (SEQ ID NO: 36) SERPING1 710 Sense GCCAAGUGGAAGACAACAUUU (SEQ ID NO: 37) 　 　 Anti-sense AAAUGUUGUCUUCCACUUGGC(SEQ ID NO: 38)

As a result, when each gene was knocked down compared to the scrambled RNA treatment group (scRNA) with a non-specific sequence as a control, a significant decrease in cell viability was observed by CD68, SERPING1, and VTN single gene knock-down in the HCT116 cell line, and among them, the effect of SERPING1 was the highest. In addition, it was shown that cell death increased when the co-gene knock-down of SNCG/SYTL4, SNCG/CD68, and SNCG/CD68/VTN was performed compared to the co-gene knock-down of SNCG/SYTL4, SNCG/CD68, and SNCG/CD68/VTN in the HCT116 cell line (Fig. 6a). On the other hand, in the HT29 cell line, no gene was found to significantly induce cell death when knocked down alone, but a significant cell death effect was observed when SNCG/SYTL4, SNCG/CD68, and SNCG/CD68/VTN were co-knocked down compared to the control. In addition, SNCG/CD68/VTN co-knockdown was shown to further increase cell death compared to SNCG knockdown alone, SNCG/CD68 co-knockdown compared to CD68 knockdown alone, and SNCG/CD68/VTN co-knockdown compared to SNCG/CD68 co-knockdown (Fig. 6b).

Through the above experiments, it was confirmed that by measuring the expression of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4, it is possible to effectively predict resistance to radiotherapy and the prognosis of radiotherapy. Therefore, it is expected that cancer can be treated more effectively when the activity of the above radioresistance gene panel is confirmed and combined with radiotherapy.

Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.

Claims (16)

A composition for diagnosing radiation resistance of colon cancer, comprising a preparation for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes. In claim 1, A composition for diagnosing radiation resistance of colon cancer, comprising a preparation for measuring the expression level of mRNA of SERPING1, CD68 or SNCG gene or a protein encoded by said gene. In claim 1, A composition for diagnosing radiation resistance of colon cancer, wherein the agent for measuring the expression level of mRNA of the above gene is at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to mRNA of the above gene. In claim 1, A composition for diagnosing radiation resistance of colon cancer, wherein the agent for measuring the expression level of the protein is at least one selected from the group consisting of a polypeptide, a compound, an antibody, and an aptamer that specifically bind to the protein. A kit for diagnosing radiation resistance of colon cancer, comprising a composition according to any one of claims 1 to 4. In claim 5, The above kit is a kit for diagnosing radioresistance of colon cancer, which is an RT-PCR kit, a microarray chip kit, a DNA kit, an ELISA kit, a protein chip kit or a rapid kit. (a) a step of measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes in a sample isolated from an individual; and (b) a step of comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control; A method for providing information for diagnosing radioresistance of colon cancer. In claim 7, A method for providing information for diagnosing radiation resistance of colon cancer, wherein the step (a) comprises a step of measuring the expression level of mRNA of SERPING1, CD68 or SNCG gene or a protein encoded by the gene. A composition for predicting the prognosis of radiation therapy for colon cancer, comprising an agent for measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes. In claim 9, A composition for predicting the prognosis of radiation therapy for colon cancer, comprising a preparation for measuring the expression level of mRNA of SERPING1, CD68 or SNCG gene or a protein encoded by said gene. In claim 9, A composition for predicting the prognosis of radiation therapy for colon cancer, wherein the agent for measuring the expression level of mRNA of the above gene is at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to mRNA of the above gene. In claim 9, A composition for predicting the prognosis of radiation therapy for colon cancer, wherein the agent for measuring the expression level of the above protein is at least one selected from the group consisting of antibodies and aptamers that specifically bind to the above protein. A kit for predicting the prognosis of radiation therapy for colon cancer, comprising a composition according to any one of claims 9 to 12. In claim 13, The above kit is a kit for predicting the prognosis of radiation therapy for colon cancer, which is an RT-PCR kit, a microarray chip kit, a DNA kit, an ELISA kit, a protein chip kit, or a rapid kit. (a) a step of measuring the expression level of mRNA of two or more genes selected from the group consisting of CXCL5, CD68, MATN1, SERPING1, SNCG, VTN, FBXO4 and SYTL4 genes or proteins encoded by two or more genes in a sample isolated from an individual; and (b) a method for providing information for predicting the prognosis of radiation therapy for colon cancer, comprising the step of comparing the expression level of the mRNA or protein with the expression level of the mRNA or protein of a normal control group. In claim 15, A method for providing information for predicting the prognosis of radiation therapy for colon cancer, wherein the step (a) comprises a step of measuring the expression level of mRNA of SERPING1, CD68 or SNCG gene or a protein encoded by the gene.

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