KR20210151502A - Radio-resistance biomarker and detecting method thereof - Google Patents

Abstract

The present invention relates to a method for providing information for predicting the prognosis of radiation therapy for cancer, a diagnostic composition, a diagnostic kit, and a cancer treatment method in combination with radiation therapy, which is based on prognostic prediction information according to the method for providing information for predicting prognosis of radiation therapy for cancer.

Description

Radiation resistance indicator protein and its detection method {RADIO-RESISTANCE BIOMARKER AND DETECTING METHOD THEREOF}

The present invention provides an information providing method, a diagnostic composition, a diagnostic kit, and a method for predicting the radiation treatment prognosis of cancer for predicting the radiotherapy prognosis of cancer by measuring the expression level of a gene panel capable of indicating the radiation resistance level of cancer cells It relates to a radiation treatment method for cancer based on prognostic prediction information according to the information provision method.

Cancer treatment methods include surgery, chemical drug therapy, radiation therapy, and the like, and the importance of radiation therapy is increasing in recent years. In cases where surgical operation is difficult, there have been reports of cases in which tumors are effectively cured with only radiation therapy, and tumor treatment methods using radiation are also developing over the years. has been established as an effective way to treat Statistically, about 30-50% of cancer patients receive radiation therapy at one point during the treatment period. Since 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, alone or in combination with chemotherapeutic agents, is commonly used to treat various forms of cancer. However, the effectiveness of radiation therapy varies depending on the nature of the cancer, the patient, and the radiation with which it is combined with other treatments. In general, cancers for which radiation therapy is widely used include uterine cancer, pharyngeal cancer, lung cancer, brain cancer, breast cancer, and the like. Although these carcinomas are the main targets of radiation therapy, the response to radiation therapy is often poor, so a strategy to increase the treatment efficiency is needed. In addition, although the initial response is good due to good radiation therapy, recurrence is frequent.

Moreover, the acquisition of radiation resistance of cancer cells and damage to normal tissues during high-dose radiation therapy are pointed out as problems that reduce the efficiency of radiation therapy, and thus research is needed to improve the efficiency of radiation therapy. Currently, radiation therapy is an effective cancer treatment along with surgery or chemical drug therapy, and is used for the purpose of killing cancer cells by treating a cancer tissue part of a patient with radiation.

Although there is a difference in the sensitivity of radiation therapy to cancer cells, if a technology that can predict individual radiation susceptibility is developed before radiation therapy, the most suitable radiation therapy is provided to the individual by adjusting the radiation dose for each individual to treat cancer. You can do it.

Under this background, the present inventors have discovered novel radiation resistance indicator proteins. Accordingly, the present invention was completed so that the expression level of the radiation-resistance gene panel of cancer can be measured during cancer treatment to predict whether there is resistance to radiation therapy or the prognosis of radiation therapy.

Korean Patent Publication No. 2012-0077567 Korean Patent Registration Publication No. 1228148

One object of the present invention is to provide a composition for diagnosing radiation resistance or sensitivity of cancer, comprising an agent for measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes.

In addition, another object of the present invention includes a composition for diagnosing radiation resistance or sensitivity of cancer, comprising an agent for measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes. To provide a kit for diagnosing radiation-resistant or sensitive cancer.

In addition, another object of the present invention is

(a) measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes;

(b) comparing the measured level of mRNA or protein thereof with the level of mRNA or protein of a normal control; to provide an information providing method for diagnosing radiation resistance or sensitivity of cancer patients, including.

In addition, another object of the present invention is

(a) classifying an object having radiation resistance or sensitivity with prediction information according to the information providing method for diagnosing radiation resistance or sensitivity of a cancer patient;

It is to provide a cancer treatment method according to the radiation treatment comprising; (b) treating the radiation-sensitive object to the radiation.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

All terms (including technical and scientific terms) used in this specification may be used with meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in a commonly used dictionary are not to be interpreted ideally or excessively unless clearly defined in particular.

Hereinafter, a composition and kit for diagnosing radiation resistance or sensitivity of cancer according to the present invention, a method for providing information using the same, and a method for treating cancer will be described in detail.

One aspect of the present invention for achieving the above object, comprising an agent for measuring the level of two or more mRNA or protein thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes, radiation resistance or sensitivity of cancer To provide a diagnostic composition.

CLK4 (CDC Like Kinase 4, NCBI gene ID: 57396) phosphorylates serine- and arginine-rich proteins and is known to be involved in regulating alternative splicing. It is also reported that it is involved in the abscission checkpoint to prevent damage to chromosomes during cell division. (PMID: 27126587)

ZNF354B (Zinc Finger protein 354B, NCBI gene ID: 117608) is a sequence-specific DNA-binding protein. Although its function in human cells is not well known so far, it is said that it binds to the promoter of a tumor suppressor gene and suppresses its expression in mice. is known (PMID: 24105743)

GPX3 (Glutathione peroxidase 3, NCBI gene ID: 2878) belongs to the glutathione peroxidase family and has detoxification and removal functions of active oxygen such as hydrogen peroxide.

ZNF80 (Zinc Finger protein 80, NCBI gene ID: 7634) is a DNA-binding protein that is thought to be involved in gene transcription, but its function is largely unknown to date.

The diagnostic composition according to the present invention includes an agent for measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes, an agent for measuring the level of three or more mRNAs or proteins thereof, CLK4, and agents for measuring the level of mRNA or protein thereof of ZNF354B, GPX3 and ZNF80 genes.

In the present invention, “radiation therapy” refers to treatment of cancer through a mechanism of inducing apoptosis by inhibiting cell division through DNA damage of cancer cells by irradiating radiation. Radiation therapy is a curative, adjuvant, or palliative purpose of solid cancer alone or in combination with surgical treatment, chemotherapy or radiation sensitizer for many cancer patients. is being implemented

In the present invention, "radio-resistance" refers to a state in which there is little change in cell repair or proliferation upon exposure to radiation and not easily affected by radiation.

In the present invention, "radio-sensitive" refers to a state that is easily affected by radiation and shows changes in cell repair or proliferation upon exposure to radiation.

The composition for diagnosing radiation resistance or sensitivity of the present invention is composed of a gene having a differential expression level in radiation-resistant cancer cells compared to normal cancer cells, so that it is possible to determine the radiation resistance of an individual or a cell. That is, an individual or cancer cell having a higher or lower expression level of the composition for diagnosis of radiation resistance of the present invention than a control group may be determined to have radiation resistance or sensitivity.

Specifically, the CLK4, ZNF354B, GPX3 and ZNF80 genes may be down-regulated genes in radiation-resistant carcinoma. Accordingly, when the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes is low compared to the normal control, it can be determined that radiation resistance exists, and when it appears high, radiation resistance This low or radiation sensitivity can be determined to exist. For example, when all of the selected 2, 3, or 4 genes are expressed low, radiation resistance may be determined to be high, and when all of the 2, 3 or 4 genes are highly expressed, radiation resistance It can be judged that the resistance is low.

By using the down-regulating panel as described above, if the radiation resistance or sensitivity of the cancer to be treated with radiation and the level thereof are determined, the radiation dose of radiation therapy can be determined by reflecting this, so that the efficiency and treatment results of radiation therapy can be improved. can

As used herein, the term "marker or diagnostic marker" refers to a substance that can distinguish and diagnose cancer cells or individuals with cancerous diseases from radiation-resistant cells or individuals, radiation-sensitive cells or individuals, and compared to normal cancer cells. Organic biomolecules such as polypeptides, proteins or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, or sugars (e.g., monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in radiation-resistant cancer cells or individuals include

As used herein, the term “cancer” is used interchangeably with tumor and neoplasm, and a cell or cell population superior to that of normal cells in which growth, proliferation or survival is utilized as a control, for example, cells proliferative or differentiation disorder. Typically, the growth is out of control. Specifically, the cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, perianal cancer, colon cancer, fallopian tube cancer Cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute Leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, etc. , more specifically lung cancer, but is not limited thereto. In addition, cancer may include malignant as well as pre-malignant cancer. According to one embodiment of the present invention, the cancer is lung cancer.

In the present invention, the term “sample” may refer to a sample derived from a subject receiving radiation treatment (irradiation) or needing to determine whether or not to receive radiation treatment. For example, as a biomarker, it refers to a sample capable of specifying the expression of CLK4, ZNF354B, GPX3 and/or ZNF80, and a sample such as a patient's tissue cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, etc. may include, but is not limited thereto.

In the present invention, the “expression level” may be used interchangeably with an expression signature and an expression profile, and may include the expression level of two or more radioresistance gene panels. The expression level may include increased expression or decreased expression. In addition, the expression level may be measured from cancer cells isolated from a subject before and/or after radiation treatment.

The level of gene expression in a biological sample can be confirmed by determining the amount of mRNA or protein.

In the present invention, “mRNA expression level measurement” is a process of checking the presence and expression level of mRNA of the marker genes in a biological sample in order to diagnose radiation resistance and sensitivity, and the amount of mRNA is measured. Analysis 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.

"Measurement of protein expression level" in the present invention is 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 or sensitivity, preferably, the protein of the gene The amount of the protein can be confirmed by using an antibody that specifically binds to it. 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 level of the gene mRNA of the present invention can be prepared by those skilled in the art using various programs based on the sequence of the gene known in a known database for primers specifically binding to the gene.

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 the four different nucleoside triphosphates in appropriate buffers and temperatures. The primers of the present invention are sense and antisense nucleic acids having a sequence of 7 to 50 nucleotides as primers specific for each marker gene. Primers may incorporate additional features that do not change the basic properties of the primer to serve as the starting point of 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 phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked residues, e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, 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.

In addition, the agent for measuring the protein level of the gene may include an antibody specific for the protein.

In the present invention, 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. Since the radioresistance and sensitivity marker proteins have been identified as described above, antibody production using them can be easily prepared using techniques well known in the art. The polyclonal antibody can be produced by a method well known in the art for obtaining a serum containing the antibody by injecting the above-mentioned radioresistance or sensitivity marker protein antigen into an animal and collecting blood from the animal. 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.

In addition, the antibody of the present invention includes 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.

According to a specific embodiment of the present invention, the expression of the radioresistance gene panel CLK4, ZNF354B, GPX3 and/or ZNF80 was reduced in lung cancer compared to the normal control sample, and in the trsnscriptome analysis of two radiation-resistant lung cancer cell lines, Results of reduced expression of CLK4, ZNF354B, GPX3 and/or ZNF80 were confirmed.

Another aspect of the present invention for achieving the above object provides a kit for diagnosing radiation resistance or sensitivity of cancer comprising the composition for diagnosing radiation resistance or sensitivity. The kit is characterized in that it is 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 or sensitive diagnostic composition. In addition, the measurement of the expression change of the radiation resistance or sensitivity gene can be performed using the kit including the marker for diagnosis of radiation resistance or radiation sensitivity according to the present invention.

As used herein, the term "kit" refers to CLK4, ZNF354B, GPX3 and ZNF80 genes selected from the group consisting of two or more mRNA expression levels or protein expression levels to determine whether radiation resistance or sensitivity of cancer can be diagnosed. means tools. In the kit for diagnosing radiation resistance of cancer of the present invention, a primer for measuring the expression level of two or more genes selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes as a radioresistance diagnostic marker, a probe, or an antibody that selectively recognizes the marker as well as one or more other component compositions, solutions, or devices suitable for analytical methods may be included.

In addition, the kit may further include a user's manual describing optimal conditions for performing the reaction. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit.

In addition, the manual includes information published or provided through an electronic medium such as the Internet.

Another aspect of the present invention for achieving the above object is (a) measuring the level of two or more mRNA or protein thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes;

(b) comparing the measured level of mRNA or protein thereof with the level of mRNA or protein of a normal control; provides an information providing method for diagnosing radiation resistance or sensitivity of cancer patients, including.

Measuring the level of mRNA or protein thereof in step (a) can be applied to the contents of salpin in the composition and kit above.

The information providing method for diagnosing radiation resistance or sensitivity of cancer patients includes (b) comparing the measured level of mRNA or protein thereof with the level of mRNA or protein thereof of a normal control.

According to the comparison, when the measured level of mRNA or protein is low compared to the normal control group, it is determined that the radiation resistance is high.

Specifically, when the expression level of two or more, three or more or both selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes is lower than the normal level, the cancer patient may be determined to have high radiation resistance.

Accordingly, the information providing method for diagnosing the radiation resistance or sensitivity of the cancer patient comprises the steps of (c) determining that the cancer patient exhibits resistance to radiation therapy when the measured mRNA or protein level is lower than that of the normal control group; may include more.

Based on this, prognostic information can also be provided, and "prognosis prediction" means predicting radiation-resistant or sensitive cancer cells through a gene that shows a difference in expression in a radiation-resistant cancer cell line compared to a normal control after irradiation. .

In addition, another aspect of the present invention for achieving the above object is the step of (a) classifying an object having radiation resistance or sensitivity as predictive information according to the information providing method for diagnosing radiation resistance or sensitivity of a cancer patient ;

It is to provide a cancer treatment method according to the radiation treatment comprising; (b) treating the radiation-sensitive object to the radiation.

Here, the subject with radiation sensitivity may mean a case in which the expression level of two or more, three or more, or both selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes is equal to or higher than the normal control level.

According to one embodiment of the present invention, each gene of the radiation-resistance gene panel with reduced expression is analyzed to have a close correlation with a decrease in survival rate as a high-risk group when expression is reduced in lung cancer patients, and can be utilized as a radiation-resistance biomarker in lung cancer patients. confirmed that there is. In addition, the decreased expression of the radioresistance gene panel genes CLK4, ZNF354B, GPX3 and ZNF80 was analyzed to have a close correlation with a decrease in the survival rate of lung cancer patients. It was confirmed that it can be used. In addition, the expression of these gene panels was analyzed to have a close correlation with the survival rate of cancer patients in the analysis targeting not only lung cancer but also cancer as a whole.

Accordingly, another aspect of the present invention for achieving the above object is CLK4, ZNF354B, GPX3 and ZNF80 gene comprising an agent for measuring the level of two or more mRNA or protein thereof selected from the group consisting of genes, cancer prognosis To provide a composition for prediction.

In addition, another aspect of the present invention for achieving the above object is CLK4, ZNF354B, GPX3 and ZNF80 gene comprising an agent for measuring the level of two or more mRNA or protein thereof selected from the group consisting of, predicting cancer prognosis to provide a kit for

Another aspect of the present invention for achieving the above object is (a) measuring the level of two or more mRNA or protein thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes;

(b) comparing the measured level of mRNA or protein thereof with the level of mRNA or protein of a normal control; provides a method of providing information necessary for predicting the prognosis of cancer, including.

In the present invention, the contents applied to the “composition for predicting cancer prognosis”, “kit for predicting cancer prognosis” and “method for providing information necessary for predicting the prognosis of cancer” refer to “for diagnosing radiation resistance or sensitivity of cancer” Composition”, “radiation resistance or sensitivity diagnostic kit”, and “information providing method for diagnosing radiation resistance or sensitivity in cancer patients” may be applied identically or modified for the purpose as long as they do not contradict each other.

For example, when at least two, three or more or all selected from the measured CLK4, ZNF354B, GPX3 and ZNF80 are expressed low compared to the normal control group, the prognosis of cancer may be judged to be poor, and the If the contrast is high, the prognosis of cancer may be good or it may be judged to be good.

For example, the composition for predicting cancer prognosis may include an agent for measuring the level of CLK4 gene mRNA or protein thereof. In addition, an agent for measuring the level of mRNA or protein thereof of the CLK4 gene; And it may be a composition for predicting cancer prognosis, including an agent for measuring the level of one or more mRNA or protein thereof selected from the group consisting of ZNF354B, ZNF80, or both genes.

Preferably, it may be a composition for predicting cancer prognosis, including an agent for measuring the level of mRNA or protein thereof of CLK4 and ZNF80 genes.

a kit for predicting cancer prognosis; In the method of providing information necessary for predicting the prognosis of cancer, the contents of the composition for predicting the prognosis of cancer may be appropriately modified and applied.

For example, the composition for predicting cancer prognosis may be a composition for predicting cancer prognosis for lung cancer. Specifically, the composition for predicting lung cancer prognosis may include an agent for measuring the level of GPX3 gene mRNA or protein thereof. In addition, an agent for measuring the level of mRNA or protein thereof of the GPX3 gene; And ZNF354B, ZNF80, or a composition for predicting lung cancer prognosis, including an agent for measuring the level of one or more mRNA or protein thereof selected from the group consisting of genes thereof. Preferably, it may be a composition for predicting lung cancer prognosis, including an agent for measuring the level of mRNA or protein thereof of GPX3, ZNF354B and ZNF80 genes.

In the kit for predicting the prognosis of lung cancer as a kit for predicting the prognosis of cancer, the method of providing information necessary for predicting the prognosis of lung cancer as a method of providing the information necessary for predicting the prognosis of cancer, the content regarding the composition for predicting the prognosis of lung cancer is appropriately It can be modified and applied.

This "prognosis" refers to the course of and cure of a disease such as cancer, eg, recurrence, metastatic spread, and the likelihood of cancer-caused death or progression, including drug resistance.

"Good prognosis" means that cancer patients, particularly lung cancer patients, have a chance of being cured of the disease, and "poor prognosis" means that the cancer or tumor is relapse or recurrence, metastasis, or death. means there is Cancer patients classified as having good outcomes are free of cancer or tumors being battling.

As used herein, the term "prediction" means that a patient responds favorably or unfavorably to a treatment such as chemotherapy or radiotherapy, so that the patient is treated, for example, a specific therapeutic agent, and/or removal of the primary tumor by surgery; and/or survival and/or likelihood of survival after treatment with chemotherapy for a specified period of time without recurrence of the cancer. The prediction method of the present invention can be used clinically by selecting and applying the most appropriate treatment method for any specific patient. The predictive method of the present invention determines whether a patient responds favorably to a treatment regimen, such as, for example, a prescribed treatment regimen, including administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, or the like, or after a treatment regimen, the patient long-term survival or the possibility of systemic or local recurrence. In addition, through this, it is possible to minimize unnecessary adjuvant chemotherapy or plan to use more effective adjuvant chemotherapy for patients with predicted systemic or local recurrence.

As used herein, the term “composition for predicting prognosis” refers to a substance capable of predicting recurrence by distinguishing between patients with good prognosis and patients with poor prognosis after treatment for cancer, and includes polypeptides or nucleic acids (eg, mRNA), lipids , including organic biomolecules such as glycolipids and glycoproteins as markers for predicting prognosis.

The present invention can predict whether resistance to radiation therapy or the prognosis of radiation therapy with an excellent effect. In particular, it is possible to more effectively treat cancer when combined with radiation therapy while confirming the activity of the radiation resistance gene panel.

In addition, by providing clinical prognosis information for cancer, particularly lung cancer, cancer can be systematically managed with high accuracy and sensitivity.

1 is a result of confirming the cell shape and a method for preparing a radiation-resistant cell line from a lung cancer cell line.
2 is a result confirming the radiation sensitivity of the prepared lung cancer radiation-resistant cell line.
3 is a result of analyzing the radiation-resistance-related gene through a transcript analysis test of the prepared lung cancer radiation-resistant cell line.
FIG. 4 is the analysis of radiation-related genes whose expression was significantly changed in common in the two prepared lung cancer radiation-resistant cell lines.
5 is a result of selecting genes associated with the survival rate of lung cancer patients among the analyzed radiation resistance-related genes.
6 is a list of genes associated with the survival rate of lung cancer patients among the analyzed radiation resistance-related genes.
7 is a result of verifying the expression of the radiation-resistance-related gene associated with the survival rate of lung cancer patients in the radiation-resistant cells.
8 is a result confirming the correlation between the expression of each radiation resistance gene and the survival rate in a human lung cancer patient group.
9 is a result confirming the correlation between the expression of the radiation resistance gene combination and the survival rate in the entire human cancer patient group.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Example 1. Preparation of radiation-resistant lung cancer cell line and measurement of radiation sensitivity

To prepare a radiation-resistant lung cancer cell line, the human lung cancer cell lines H1975 and H2126 cells were irradiated with 2 Gy per time, and a total of 60 Gy was irradiated. Cells that have been irradiated are irradiated with radiation for each dose in order to measure radiation sensitivity, and then cultured for 10-14 days and evaluated through a clonogenic assay. The cell line in which radiation resistance was confirmed was established as a cell line after mycoplasma test.

A schematic diagram of the entire process is shown in FIG. 1A . In addition, the results of the cell shape change during the production process of the radiation-resistant lung cancer cell line is shown in FIG. 1B.

The two human lung cancer cell lines and the two human lung cancer radiation-resistant cell lines were prepared in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-spreptomycin (10,000 U/ml) at 5%, respectively. Maintenance culture was carried out under the conditions of CO _{2 and 37 °C.}

Each cell line was aliquoted into an appropriate number of cells in a 60 mm dish, and 24 hours had elapsed, followed by irradiation with each dose and cultured for 10-14 days under conditions of _{5% CO 2 and 37°C.} Thereafter, the formed cell colonies (colonies) were washed with Phosphate Buffered Saline (PBS) and stained with a 100% methanol mixture containing 1% methylin blue. Stained cell colonies (cell colonies having more than 50 cells) were visually counted for each dish after drying at room temperature, and photographs were obtained, and the results are shown in FIG. 2 .

2A shows H1975 cells, B shows H2126 cells.

As can be seen in FIG. 2 , in the case of the resistant cell line (RR), apoptosis did not occur well despite irradiation, and the colony forming ability was high.

Example 2. Lung cancer radiation resistance-related gene discovery

To discover lung cancer radiation resistance-related genes, RNA was extracted from two radiation-resistant cell lines and their parental cell lines, and transcriptome analysis was repeated twice through RNA-sequencing. Before performing the transcript analysis, the quality of the extracted total RNA was checked using Agilent's 2100 bioanalyzer system. The gene library was constructed using Clontech's SMARTer Stranded RNA-Seq Kit and analyzed using Illumina HiSeq 2500. Gene expression data were corrected by quantile normalization. In order to discover significant genes with changed expression, genes whose P-value is 0.05 or less and Raw Count is 4 or more, which have changed 1.5 times, were selected from 25,737 gene data.

The results are shown in FIG. 3 .

3A shows the number of genes increased or decreased by 1.5-fold or more in the radiation-resistant human lung cancer cell line H1975 and the number of genes increased or decreased by 1.5-fold or more in the radiation-resistant human lung cancer cell line H2126. As can be seen here, in the H1975 cell line, a total of 2168 genes with increased expression and a total of 1545 genes with reduced expression were identified. In the case of the H2126 cell line, a total of 324 genes with increased expression and 392 genes with reduced expression were identified.

Genes with reduced expression in both cell lines were analyzed and shown in FIG. 3B, and a total of 42 genes (reduced by 1.5 fold or more) were identified.

As such, a list of genes with reduced expression levels in both cell lines is listed in FIG. 4 . Specifically, the list of genes with reduced expression was shown in descending order of expression based on H1975 cells.

Example 3. Excavation of radiation-resistance-related genes among genes affecting survival in lung cancer patients

In order to investigate the list of genes affecting the survival rate in lung cancer patients, TCGA (The Cancer Genome Atlas) data, an open genome database, were used and analyzed. TCGA data analysis was performed using LinkedOmics software. In order to secure a significant survival rate-related gene of lung cancer patients, survival rate was analyzed in TCGA LUAD (Lung Adenocarcinoma) data, and a gene having a P-value of 0.05 or less was identified and shown in FIG. 5A. 5A is a result of discovering a significant gene affecting the survival rate in a TCGA LUAD sample using LinkedOmics software. A total of 4637 genes were identified. Among them, 1843 genes showed a low survival rate as the expression was high, and 2,794 genes showed a low survival rate as the expression was low.

In addition, as shown in FIG. 5B, a gene that is expressed low in radiation-resistant cells and is expected to have a lower survival rate of a patient as the expression is lower was analyzed and shown in a Venn diagram, and a total of four genes were isolated. Here, FIG. 5b shows a total of four genes having a low survival rate of lung cancer patients while having radioresistance as the expression is lowered.

A list of these genes was selected as a panel of radiation resistance genes, and the list is shown in FIG. 6 . The LogHR value is a value obtained by taking the natural logarithm of HR (Hazard Ratio), which is the calculated patient survival rate. In a group with high gene expression, the higher this value, the worse the patient's survival rate. Therefore, a positive value indicates a poor survival rate when the gene expression is high, and a negative value indicates a poor survival rate when the gene expression is low. In addition, the expression of each gene in radiation-resistant cells was also expressed as a value obtained by taking the natural logarithm.

6 shows the gene list of FIG. 5B, and the LogHR values are shown in ascending order.

Example 4. Confirmation of expression of radiation resistance gene panel

In order to confirm the expression level of the changed radiation-resistant gene panel in radiation-resistant cells, RNA was extracted from two radiation-resistant cell lines and their parental cell lines, and qPCR analysis was performed. After washing the cell line with PBS, RNA was isolated using TRIzol reagent. The separation method followed the reagent company's protocol. After quantifying RNA extracted with NanoDrop, 1 ug of RNA was reverse transcribed, and expression was confirmed using a LightCycler 96 device using primers (Table 1) of each gene. At this time, the expression ratio of each gene was shown in FIG. 7 using the GAPDH gene as an internal control for gene expression analysis.

[Table 1]

Figure 7A is H1975 cells and the radiation-resistant H1975 cell line, Figure 7B is the H2126 cells and the radiation-resistant H2126 cell line confirming the increase in gene expression, CLK4, ZNF354B, GPX3, ZNF80 in the two types of lung cancer parental cell lines compared to the radiation-resistant cell lines. all decreased.

Through the above results, it was confirmed that the expression of the radiation resistance gene panel appeared identical to the result of the transcript analysis.

Example 5. Survival curve analysis according to the expression of radiation resistance gene panel in lung cancer patients

In order to confirm the correlation with the survival rate of lung cancer patients according to the expression of the derived radiation resistance genes, the survival rate of lung cancer patients according to each gene expression was calculated using the TCGA LUAD data, which is a genome database data published with LinkedOmics software. It was analyzed and visualized through a Kaplan-Meier survival curve and is shown in Figure 8A. The risk of survival for lung cancer patients according to the increase in CLK4 was 0.764, indicating that a decrease in CLK4 was a significant factor in reducing the survival rate of lung cancer patients. In addition, using the TCGA LUAD data with GEPIA2 software, the survival rate of lung cancer patients according to the combination of radiation resistance genes was analyzed and visualized through the Kaplan-Meier survival curve. The dog gene combination and lung cancer patient survival rate, Figure 8D showed the four gene combinations and lung cancer patient survival rate. On the other hand, looking at the correlation analysis results with the lung cancer survival rate in the reduced gene panel group, the combination of ZNF80 + GPX3 + ZNF354B had the most significant risk of 0.68, and CLK4 + XNF80, GPX3 + ZNF354B, and GPX3 + ZNF80 were 0.70 each. , 0.71, 0.72. A combination of four reduced gene panels (CLK4 + XNF80 + GPX3 + ZNF354B) and the risk of survival for lung cancer patients were also confirmed to be 0.73.

Example 6. Analysis of survival curves according to the expression of radioresistance gene panel in total cancer patients

To analyze the effect of the radiation resistance gene panel on the survival rate of cancer patients as well as limited to lung cancer patients, the total TCGA data with GEPIA2 software was used to determine the survival rate of cancer patients according to the combination of radiation resistance genes. It was analyzed and visualized through the curve and shown in FIG. 9 . Looking at the results of correlation analysis with the overall cancer survival rate in the reduced gene panel group, the risk of the combination of CLK4 + XNF80 was most significant at 0.7, and the risk of the three combinations of ZNF354B + ZNF80 + CLK4 was 0.78, 4 With the reduced gene panel combination (CLK4 + XNF80 + GPX3 + ZNF354B), the cancer patient survival risk was found to be 0.85.

As can be seen above, in the present invention, in the case of CLK4, ZNF354B, GPX3, and/or ZNF80, it is possible to predict whether resistance to radiation therapy or the prognosis of radiation therapy. In particular, it is possible to more effectively treat cancer when combined with radiation therapy while confirming the activity of the radiation resistance gene panel.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> RADIO-RESISTANCE BIOMARKER AND DETECTING METHOD THEREOF <130> 112P <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ZNF80 Forward Primer <400> 1 gttttacagg agcaggtctc ca 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ZNF80 Reverse Primer <400> 2 gttaaacacg ctcccacatt cc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GPX3 Forward Primer <400> 3 tggtcattct gggctttccc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GPX3 Reverse Primer <400> 4 ccagaagagg cggtcagatg 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ZNF354B Forward Primer <400> 5 cgagacagtt cactgttgca ga 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ZNF354B Reverse Primer <400> 6 cacctcagca cccagacttt 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CLK4 Forward Primer <400> 7 gctatcgtgg aagtcacaag cg 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CLK4 Reverse Primer <400> 8 ctcgctcatt caaggacctt gc 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward Primer <400> 9 gtctcctctg acttcaacag cg 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse Primer <400> 10 accaccctgt tgctgtagcc aa 22

Claims (19)

A composition for diagnosing radiation resistance or sensitivity of cancer, comprising an agent for measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes. The composition for diagnosing radiation resistance or sensitivity of cancer according to claim 1, wherein the agent for measuring the level of the mRNA or protein thereof comprises an agent for measuring the level of the mRNA or protein thereof of the GPX3 gene. The cancer of claim 1, wherein the cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, perianal cancer , colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary gland Any one or more selected from the group consisting of adenoma, a composition for diagnosing radiation resistance or sensitivity of cancer. The composition for diagnosing radiation resistance or sensitivity of cancer according to claim 3, wherein the cancer is lung cancer. The composition for diagnosing radiation resistance or sensitivity of cancer cells according to claim 1, wherein the agent for measuring the level of the gene mRNA comprises a primer that specifically binds to the gene. A kit for diagnosing radiation resistance or sensitivity of cancer, comprising the composition for diagnosing radiation resistance or sensitivity of cancer cells according to any one of claims 1 to 5. The kit for diagnosing radiation resistance or sensitivity of cancer according to claim 6, wherein the kit is any one kit selected from the group consisting of an RT-PCR kit, a DNA chip kit, a microarray, and a protein chip. (a) measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes;
(b) comparing the measured level of mRNA or protein thereof with the level of mRNA or protein of a normal control; providing information for diagnosing radiation resistance or sensitivity of a cancer patient comprising a. The method of claim 8, wherein (a) measuring the level of mRNA or protein thereof of the GPX3 gene; comprising, information providing method for diagnosing radiation resistance or sensitivity of cancer patients. 9. The method of claim 8, wherein the cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, perianal cancer , colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary gland Any one or more selected from the group consisting of adenomas, information providing method for diagnosing radiation resistance or sensitivity of cancer patients. A composition for predicting cancer prognosis, comprising an agent for measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes. The method according to claim 11, comprising: an agent for measuring the level of mRNA of the CLK4 gene or a protein thereof; And ZNF354B, ZNF80, or an agent for measuring the level of one or more mRNA or protein thereof selected from the group consisting of genes of both; Containing, cancer prognosis prediction composition. 12. The method of claim 11, wherein the cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, perianal cancer , colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary gland Any one or more selected from the group consisting of adenoma, a composition for predicting cancer prognosis. The composition for predicting cancer prognosis according to claim 13, wherein the cancer is lung cancer. 15. The method of claim 14, wherein the agent for measuring the level of the mRNA of the GPX3 gene or its protein; And ZNF354B, ZNF80, or a composition for predicting cancer prognosis, comprising an agent for measuring the level of one or more mRNA or protein thereof selected from the group consisting of genes of both. A kit for predicting cancer prognosis, comprising the composition for predicting cancer prognosis according to any one of claims 11 to 15. (a) measuring the level of two or more mRNAs or proteins thereof selected from the group consisting of CLK4, ZNF354B, GPX3 and ZNF80 genes;
(b) comparing the measured level of mRNA or protein thereof with the level of mRNA or protein of a normal control; a method of providing information necessary for predicting the prognosis of cancer comprising a. 18. The method of claim 17, wherein (a) the level of mRNA or protein thereof of the CLK4 gene; And ZNF354B, ZNF80, or the step of measuring the level of one or more mRNA or protein thereof selected from the group consisting of genes of both; comprising, a method of providing information necessary for predicting the prognosis of cancer. The method according to claim 17, wherein the method for providing information necessary for predicting the prognosis of lung cancer is to measure the level of mRNA or protein thereof of the GPX3 gene; And ZNF354B, ZNF80, or the step of measuring the level of one or more mRNA or protein thereof selected from the group consisting of genes of both; comprising, a method of providing information necessary for predicting the prognosis of cancer.

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