WO2024085495A1 - Exosome-derived biomarker for diagnosing colon cancer and use thereof - Google Patents
Exosome-derived biomarker for diagnosing colon cancer and use thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Definitions
- the present invention relates to an exosome-derived biomarker composition for diagnosing or predicting prognosis of colon cancer, a composition for diagnosing or predicting prognosis, a kit, and a method for providing information.
- Cancer is a representative disease that threatens human health. Cancer is the single most common cause of death in industrialized countries. The cause of cancer is still not known. The cause of cancer may be a combination of internal factors, such as genetic factors, and external factors, such as carcinogenic chemicals that act as cancer triggers, continuous inflammation and damage, and cancer-causing viral infections.
- internal factors such as genetic factors
- external factors such as carcinogenic chemicals that act as cancer triggers, continuous inflammation and damage, and cancer-causing viral infections.
- Diagnosis of colon cancer is made through existing testing methods for diagnosing colon cancer, such as fecal testing, radiography, and colonoscopy.
- This has the problem of causing pain and danger to the patient.
- the conventional methods described above not only have low diagnostic accuracy, but also make it impossible to diagnose patients before colorectal cancer develops, and cause inconvenience to the subjects.
- CEA tumor protein is widely used as a non-invasive diagnostic method and recurrence prediction biomarker for colorectal cancer, but the CEA tumor protein has the disadvantage of low sensitivity and specificity. For this reason, there is a continuous need for the development of biomarkers for more accurate diagnosis of colon cancer.
- CEA tumor protein is widely used as a biomarker for the diagnosis of colon cancer.
- colorectal cancer diagnosis using CEA tumor protein has limitations such as low accuracy and sensitivity.
- tumor-derived 'exosomes' are very small membrane-structured substances produced by cancer cells and secreted into the serum, and are known to contain various proteins and genetic information inside.
- the present inventors noted that there are many advantages in diagnosing colon cancer using exosome-derived proteins, as the accuracy and sensitivity are high and colon cancer can be diagnosed using only blood, making the test easy.
- the present inventors used Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) on exosomes derived from preoperative blood, surgical tumor tissue, tumor surrounding tissue, and postoperative blood of colorectal cancer patients.
- LC-MS/MS Liquid Chromatography with tandem mass spectrometry
- the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins of the present invention were found to have significantly higher expression levels compared to normal controls from the early stage of colon cancer development, so accuracy and sensitivity in early colon cancer diagnosis can be expected. .
- the problem to be solved by the present invention is to provide a biomarker composition for diagnosing or predicting prognosis of colon cancer containing an exosome-derived protein.
- Another problem to be solved by the present invention is to provide a composition for diagnosing or predicting prognosis of colon cancer, including an agent for measuring the expression of exosome-derived proteins.
- Another problem to be solved by the present invention is to provide a kit for diagnosing or predicting prognosis of colon cancer, which includes an agent for measuring the expression level of exosome-derived proteins.
- Another problem to be solved by the present invention is to provide a kit for diagnosing or predicting prognosis of colon cancer, which includes an agent for measuring the expression level of exosome-derived proteins.
- a biomarker composition for diagnosing or predicting prognosis of colon cancer comprising an exosome-derived protein or a gene encoding the same is provided.
- it may include at least one protein selected from the group consisting of exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, or at least one gene encoding the same.
- the individual may be an individual suspected of having colon cancer or an individual having colon cancer.
- the biological sample may be exosomes isolated from at least one selected from the group consisting of tumor tissue, blood, serum, and plasma.
- exosome refers to a small type of endoplasmic reticulum secreted from cells, and is preferably an exosome present in blood such as whole blood, serum, and plasma.
- the term “diagnosis” means confirming the presence or characteristics of a pathological condition.
- it is to diagnose colon cancer. More specifically, in colon cancer, the expression of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins in exosomes increases, so the amount of protein in the exosomes is measured and if it is higher than the control sample, the individual has colon cancer. Diagnosis is possible. In addition, colon cancer can be diagnosed by measuring the mRNA expression level of the gene encoding the protein and if it is higher than that of the control sample.
- colon cancer that can be diagnosed using a biomarker according to an embodiment of the present invention may include early stage colon cancer.
- Colon cancer occurs in the mucosal layer and as it progresses, it gradually penetrates the lower layers and grows in that order: the submucosal layer, the muscle layer, and the serosal layer. Colon cancer that has penetrated all four layers of the large intestine invades organs and lymph nodes around the large intestine, and as it progresses further, it spreads to the liver or lungs far away from the large intestine through lymphatic vessels or blood vessels. The extent to which colon cancer has progressed is called the stage. The stage of colon cancer can be divided into stages 1 to 4 based on the TNM system according to the AJCC classification system.
- Stage 1 defines cases where the tumor is still located in the submucosa or has invaded the muscularis propria
- Stage 2 (StageII) defines cases where the tumor has expanded into the muscle layer or nearby structures but has not metastasized to lymph nodes
- Stage 3 (Stage III) represents the group positive for lymph node metastasis
- stage 4 (Stage IV) defines the group positive for distant metastasis.
- the stage In order to determine treatment methods for colon cancer and identify prognostic factors, the stage must be accurately determined.
- Exosome-derived proteins SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 according to an embodiment of the present invention were significantly expressed in all colon cancer stages classified from stage 1 to stage 4, especially referring to Figure 7. , It was confirmed that the stage 1 (Stage I) early colon cancer patient group showed significant differences compared to the normal control group (Healthy). On the other hand, CEA (carcinoembryonic antigen), a conventional diagnostic biomarker for colon cancer, was found to have minimal expression in the Stage I patient group.
- the exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein according to an embodiment of the present invention has a high sensitivity from the early stage of colon cancer development. Because it shows sensitivity, early colon cancer can be diagnosed with high accuracy.
- the early stage colon cancer may be stage 1 (Stage I), in which the tumor is still located in the submucosal layer or has invaded the muscularis propria, but is not limited thereto.
- Stage I stage 1
- Early stages 0 to 1 colon cancer is often asymptomatic, making it difficult to recognize, and as it progresses slowly, symptoms such as indigestion, difficulty in defecation, bleeding, abdominal pain, and weight loss appear.
- Colonoscopy is recommended for early detection of colon cancer, but it is accompanied by burdens such as side effects and costs associated with endoscopy.
- exosome-derived protein biomarker Using the exosome-derived protein biomarker according to the present invention, the development of colon cancer in the early stages when symptoms are not recognized can be diagnosed with a simple blood test.
- the diagnosis may include recurrence.
- the biomarker composition according to the present invention can be expected to be effective as a diagnostic marker for recurrence through a blood test alone through long-term follow-up of an individual who has been diagnosed with colon cancer and has undergone chemotherapy or surgery.
- prognosis refers to the outlook for future symptoms or progress determined by diagnosing a disease. Predicting prognosis is a very important clinical task because it provides clues to the direction of future anticancer treatment.
- the term "prediction" means to guess in advance about medical consequences, and for the purpose of the present invention, the course of the disease (disease progression, improvement, recurrence, tumor growth, drug resistance) of a patient diagnosed with colon cancer. means guessing in advance.
- the prognosis for colon cancer can be predicted to be good.
- the prognosis can be predicted to be good for colon cancer.
- the term "marker” refers to a molecule that is quantitatively or qualitatively associated with the presence of a biological phenomenon
- the biomarker for colon cancer diagnosis of the present invention refers to a protein whose expression increases when colon cancer develops or a gene encoding the same.
- a composition for diagnosing or predicting prognosis of colon cancer comprising a.
- protein expression level measurement refers to a process of confirming the expression or activity level of a protein of interest in a biological sample, for example, using an antibody that specifically binds to the protein of interest to determine the level of peptide. You can check the amount. Analysis methods for this include Western Blotting, Enzyme Linked Immunosorbent Assay (ELISA), Rodioimmunoassay (RIA), Radioimmunodiffusion, Ouchterolony Immunodiffusion, and Rocket. (Rocket) There are immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FASC), and protein chip. It is not limited.
- the agent for measuring protein expression is an oligopeptide, monoclonal peptide that specifically binds to at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins. It may be a raw antibody, polyclonal antibody, chimeric antibody, ligand, peptide nucleic acid (PNA), or aptamer, but is not limited thereto.
- the term “antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site.
- the antibody in the present invention refers to an antibody that specifically binds to the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein of the present invention, and the antibody can be prepared according to a conventional method in the art.
- the types of antibodies include polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant to be intact, with two full-length light chains and two full-length heavy chains. Additionally, the above antibodies also include special antibodies such as humanized antibodies.
- aptamer refers to a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity.
- DNA DNA, RNA, or modified nucleic acid
- ) refers to a type of polynucleotide composed of
- aptamers can specifically bind to antigenic substances in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easier to synthesize, so they can be used as replacements for antibodies. You can.
- the term "gene expression level measurement” refers to the process of confirming the expression or activity of a gene of interest in a biological sample, for example, using a primer or probe that specifically binds to the gene of interest. You can check the amount. Analysis methods for this include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), and Northern blotting. blotting) or DNA chips, but are not limited thereto.
- the agent for measuring gene expression is an antisense agent that specifically binds to a gene encoding at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins. It may be, but is not limited to, an oligonucleotide, primer (pair), probe, small molecule compound, or nucleic acid molecule.
- the term "primer” is a nucleic acid sequence with a short free 3' hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. It refers to a short nucleic acid sequence that performs a function.
- Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.
- vascular disease can be diagnosed by performing PCR amplification using sense and antisense primers of ZO-1 polynucleotide to determine whether the desired product is produced. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.
- probe refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundreds of bases, capable of forming a specific binding to mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed.
- Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
- Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, “capsation,” substitution of a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).
- uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidate, carbamate, etc.
- charged linkages e.g. phosphorothioate, phosphorodithioate, etc.
- the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.
- the present invention provides an agent for measuring the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, or at least one gene encoding the same.
- Diagnosis or prognosis of colon cancer including: A prediction kit is provided.
- the kit of the present invention confirms the expression level of one or more proteins selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 in exosomes, or one or more genes encoding them, which is higher compared to the control sample. In this case, it can be used to diagnose colon cancer. Additionally, if it is lower than that of the control sample, it can be used to determine that the prognosis for colon cancer is good.
- the kit of the present invention may include not only an antibody that selectively recognizes a marker for colon cancer diagnosis, but also one or more other component compositions, solutions, or devices suitable for the analysis method.
- the kit for measuring the expression level of a protein in the present invention may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of an antibody.
- the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass
- the coloring enzyme may be peroxidase or alkaline phosphatase. Fatase (alkaline phosphatase), etc.
- the fluorescent substance can be FITC, RITC, etc.
- the coloring substrate solution is ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphate).
- Ponic acid or OPD (O-phenylenediamine) or TMB (tetramethyl benzidine) can be used.
- the kit of the present invention includes an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with a substrate, a chromogenic substrate solution that will undergo color development with the label, a washing solution, and It may contain an enzyme reaction stopping solution, etc., and may be manufactured into a number of separate packaging or compartments containing the reagent components used.
- kit of the present invention includes the above-described biomarker composition and a composition for diagnosis or prognosis prediction, duplicate content is omitted to avoid excessive complexity of the specification.
- the present invention includes the steps of isolating exosomes from a biological sample isolated from an individual (S810), obtaining a protein from the isolated exosome (S820), and expressing the protein or at least one gene mRNA encoding the protein.
- a method for diagnosing colon cancer including measuring the level (S830) and comparing the measured expression level of the protein or at least one gene mRNA encoding the same with a normal control group (S840).
- the biological sample isolated from the subject may include, but is not limited to, samples such as tissue, cells, blood, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, cell culture, or urine, and preferably the biological sample is exo. It contains at least one selected from the group consisting of tumor tissue containing exosomes, blood, serum, and plasma, and more preferably, it is blood containing exosomes isolated from an individual suspected of colon cancer.
- the protein obtained in the step of obtaining a protein from the isolated exosome (S820) is at least one selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3. It can be included.
- the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 or at least one gene encoding the same is measured, and the expression level of at least one gene encoding the same is measured in a normal control sample. If the expression level is high compared to , it can be diagnosed as colon cancer.
- the present invention includes the steps of isolating exosomes from a biological sample isolated from an individual; Obtaining at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins from the isolated exosomes; Measuring the mRNA expression level of the selected at least one protein or at least one gene encoding the selected protein; and comparing the mRNA expression level of the selected at least one protein or at least one gene encoding the selected protein with a control sample.
- the method measures the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, or at least one gene encoding the same, and compares it with a control sample. Therefore, if it is low, the prognosis for colon cancer is judged to be good and information on this can be provided.
- the subject may be an individual who has received chemotherapy or surgery after being diagnosed with colon cancer, but is not limited thereto.
- the control sample may be a sample obtained from an individual suspected of having colon cancer or an individual having colon cancer, and preferably may be a sample obtained from an individual suspected of having colon cancer or an individual having colon cancer who has not undergone chemotherapy or surgery. It is not limited to this.
- the present invention provides a method for measuring the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins in samples collected from individuals with colon cancer. step; Processing the sample with a candidate material and measuring the expression level of the selected protein; And if the expression level of the selected protein decreases after treating the sample with the candidate material compared to before treatment with the candidate material, screening the candidate material as an agent for preventing or treating colorectal cancer, comprising the step of selecting the candidate material as an agent for preventing or treating colorectal cancer.
- SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins in samples collected from individuals with colon cancer.
- the method for screening for colon cancer prevention or treatment according to the present invention is not limited to the above steps.
- the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins can be measured in samples collected from individuals with colon cancer.
- the expression level of the same protein whose expression level was measured before processing the candidate material can be measured in a sample treated with the candidate material.
- the candidate material may be selected as a material for preventing or treating colon cancer.
- the present invention has the effect of providing an exosome-derived biomarker that can be used to diagnose or predict the prognosis of colorectal cancer with high accuracy and sensitivity.
- the present invention provides a biomarker for colorectal cancer diagnosis with high accuracy and sensitivity using at least one protein selected from the group consisting of exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3. You can. Accordingly, the present invention can be used for more accurate diagnosis from the early stage of colon cancer development than the conventional diagnostic method based on biomarkers for colon cancer diagnosis.
- the present invention can provide a biomarker for accurate prognosis prediction for colon cancer anticancer treatment using at least one protein selected from the group consisting of exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3. there is.
- Figure 1 shows the results of screening proteins that can be used as diagnostic biomarkers for colon cancer using LC/MS on tissue- and blood-derived exosomes from 50 colon cancer surgery patients.
- Figure 2 shows seven proteins that show significant expression level differences using ELISA in preoperative tissue and blood-derived exosomes of normal controls without colon cancer and colon cancer patients for proteins that can be used as screened biomarkers. It shows one result.
- Figure 3 shows the results of evaluation using ROC (Receiver Operating Characteristic Curve) to confirm the significance of seven proteins as biomarkers.
- Figure 4 shows the results of normalizing (z-score) the expression values of each protein for seven proteins and expressing them as a heat-map to confirm their significance as biomarkers.
- Figure 5 shows the results of measuring the change in expression level of each protein using ELISA in a group of colon cancer patients before and 6 weeks after removal of the colon cancer tumor tissue in order to confirm the significance of the seven proteins as biomarkers.
- Figure 6 shows the serum CEA value detected according to the stage in which colorectal cancer patients were classified by stage, and the diagnosis rate results using CEA.
- Figure 7 shows the expression level results of each of the seven exosome-derived proteins detected according to the stage of classification of normal controls and colon cancer patients.
- Figure 8 exemplarily shows the procedure of a method for providing information for diagnosing or predicting prognosis of colon cancer by measuring the expression level of exosome-derived protein according to an embodiment of the present invention.
- Exosomes isolated from blood samples of healthy control subjects provided by Yonsei University Severance Hospital and preoperative blood, surgical tumor tissue, tumor surrounding tissue, and postoperative blood samples of about 50 colorectal cancer patients were used using the LC-MS/MS method. Thus, proteomic data was generated. Exosomes were isolated using the following method. The obtained sample was centrifuged at 17000xg for 5 minutes to obtain plasma. Exoquick-Exosome Precipitation Solution (System bioscience) was added to the centrifuged plasma as much as 1/4 of the plasma volume, rested at 4 °C for 2 hours, and centrifuged again at 1500xg for 30 minutes. Afterwards, the supernatant was removed, and the exosome pellet was dissolved in PBS and used in the experiment.
- Exosome Precipitation Solution System bioscience
- LC/MS technology was used to screen the top proteins that were minimally detected in post-surgical blood, that is, blood-derived exosomes after tumor tissue removal, and the top proteins that were specifically detected in tumor tissue-derived exosomes. .
- 30 proteins have no or low expression levels in plasma samples from healthy controls, high expression levels in plasma samples before colorectal cancer surgery, and no or reduced expression levels in plasma samples after colorectal cancer surgery (top). image) and 30 proteins with high expression levels in tumor tissue and adjacent tissues (bottom image), proteins that can be clinically used as circulating tumor biomarkers were discovered.
- Candidate proteins selected through the screening in Example 1 were compared by ELISA method in preoperative blood-derived exosomes of 84 colorectal cancer patients and blood-derived exosomes of 20 healthy controls, and the top proteins with the most significant difference in expression level were identified. It was excavated. As a result, as shown in Figure 2, seven proteins, SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, were derived, and their significance as biomarkers for colon cancer diagnosis or prognosis was evaluated in subsequent experiments.
- SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 derived in Example 2 were evaluated for their significance as clinical diagnostic biomarkers for colon cancer.
- each value was standardized (z-score) and expressed as a heat-map. As a result, as shown in Figure 4, it was confirmed that seven proteins showed significant differences between the healthy control group and the colon cancer patient group.
- SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins can be effectively used not only as diagnostic biomarkers for colon cancer, but also as biomarkers for predicting prognosis for anticancer treatment in individuals with colon cancer. .
- Example 3 The seven proteins verified in Example 3 were compared with CEA (carcinoembryonic antigen), which is currently used in clinical trials to diagnose or confirm treatment efficacy for colon cancer.
- CEA carcinoembryonic antigen
- SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins are significantly expressed in all four stages of the disease. confirmed. In particular, it was confirmed that the Stage I early colon cancer patient group showed significant differences compared to the normal control group (Healthy).
- the exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein As a result, compared to the conventional CEA (carcinoembryonic antigen), which has low sensitivity in the early stages of colon cancer development, the exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein according to an embodiment of the present invention Since it shows high sensitivity from the beginning of colon cancer development, early colon cancer can be diagnosed with high accuracy.
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Abstract
The present specification provides a biomarker composition for diagnosing or predicting the prognosis of colon cancer and use thereof. A composition, a kit, and a method according to an embodiment enable diagnosis of colon cancer with high accuracy by using a sample containing exosomes, and accordingly, same can be useful in the field of colon cancer diagnosis.
Description
본 발명은 엑소좀 유래의 대장암 진단 또는 예후 예측용 바이오 마커 조성물, 진단 또는 예후 예측용 조성물, 키트 및 정보 제공 방법에 관한 것이다.The present invention relates to an exosome-derived biomarker composition for diagnosing or predicting prognosis of colon cancer, a composition for diagnosing or predicting prognosis, a kit, and a method for providing information.
암은 인류의 건강을 위협하는 대표적인 질병이다. 암은 산업화된 국가에서 단일 질병으로는 가장 대표적인 사망 원인이다. 암의 발생원인은 아직도 규명되지 않고 있다. 내적 요인인 유전적 요소와 외적 요인인 암 발생 유발요소로 작용되는 발암화학물질, 계속적인 염증과 손상, 암 유발 바이러스 감염의 복합적 요소가 암의 발생원인일 수 있다. Cancer is a representative disease that threatens human health. Cancer is the single most common cause of death in industrialized countries. The cause of cancer is still not known. The cause of cancer may be a combination of internal factors, such as genetic factors, and external factors, such as carcinogenic chemicals that act as cancer triggers, continuous inflammation and damage, and cancer-causing viral infections.
대장암의 진단은 기존 대장암 진단을 위한 검사방법인 분변검사, 방사선조영검사, 대장내시경검사 등을 통해 이루어진다. 그러나 이는 환자에게 고통과 위험을 초래할 수 있다는 문제점이 있다. 상기와 같은 종래의 방법들은 진단의 정확도가 낮을 뿐만 아니라, 대장암이 발병하기 이전의 환자들에 대한 조기 진단이 불가능하며, 피검체들에게 불편을 주는 문제점이 있다. 이러한 기존 진단방법의 단점을 보완하기 위한 방법으로, 대장암의 비침습적 진단방법 및 재발 예측 바이오마커로서 CEA 종양 단백질이 널리 사용되고 있으나 CEA 종양 단백질의 경우 민감도 및 특이도가 낮다는 단점을 가진다. 이러한 이유로 보다 정확한 대장암의 진단을 위한 바이오 마커에 대한 개발이 지속적으로 요구되고 있는 실정이다. Diagnosis of colon cancer is made through existing testing methods for diagnosing colon cancer, such as fecal testing, radiography, and colonoscopy. However, this has the problem of causing pain and danger to the patient. The conventional methods described above not only have low diagnostic accuracy, but also make it impossible to diagnose patients before colorectal cancer develops, and cause inconvenience to the subjects. As a way to compensate for the shortcomings of these existing diagnostic methods, CEA tumor protein is widely used as a non-invasive diagnostic method and recurrence prediction biomarker for colorectal cancer, but the CEA tumor protein has the disadvantage of low sensitivity and specificity. For this reason, there is a continuous need for the development of biomarkers for more accurate diagnosis of colon cancer.
발명의 배경이 되는 기술은 본 발명에 대한 이해를 보다 용이하게 하기 위해 작성되었다. 발명의 배경이 되는 기술에 기재된 사항들이 선행기술로 존재한다고 인정하는 것으로 이해되어서는 안 된다.The technology behind the invention has been written to facilitate easier understanding of the invention. It should not be understood as an admission that matters described in the technology underlying the invention exist as prior art.
대장암의 진단을 위한 바이오 마커로서 CEA 종양 단백질이 널리 이용되고 있다. 그러나 CEA 종양 단백질에 의한 대장암 진단은 정확도 및 민감도가 낮다는 한계를 가진다. CEA tumor protein is widely used as a biomarker for the diagnosis of colon cancer. However, colorectal cancer diagnosis using CEA tumor protein has limitations such as low accuracy and sensitivity.
한편, 종양 유래 '엑소좀(Exosome)'은 암세포에서 만들어져 혈청내로 분비되는 매우 작은 막구조 물질로서 내부에 여러 가지 단백질 및 유전정보를 포함하고 있는 것으로 알려져 있다. Meanwhile, tumor-derived 'exosomes' are very small membrane-structured substances produced by cancer cells and secreted into the serum, and are known to contain various proteins and genetic information inside.
본 발명자의 발명자들은, 엑소좀 유래의 단백질들을 이용하여 대장암을 진단하는 경우 정확도 및 민감도가 높으며 혈액만으로 대장암 진단이 가능하므로 검사가 용이하여 많은 장점이 있다는 것을 주목하였다. The present inventors noted that there are many advantages in diagnosing colon cancer using exosome-derived proteins, as the accuracy and sensitivity are high and colon cancer can be diagnosed using only blood, making the test easy.
이에, 본 발명자들은 강력한 예측력을 갖춘 후보를 선택하기 위하여 대장암 환자의 수술 전 혈액, 수술 종양조직, 종양주변조직, 수술 후 혈액 유래 엑소좀을 Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) 방법을 활용하여 단백체 데이터를 생산하고, 이를 이용하여 바이오마커로 임상활용 가능한 단백질을 스크리닝하여 본 발명의 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질을 바이오마커로 활용할 경우 대장암 진단의 정확도와 민감도가 우수함을 발견하였다.Therefore, in order to select candidates with strong predictive power, the present inventors used Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) on exosomes derived from preoperative blood, surgical tumor tissue, tumor surrounding tissue, and postoperative blood of colorectal cancer patients. By using the method to produce proteomic data and using this to screen proteins that can be used clinically as biomarkers, when the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins of the present invention are used as biomarkers, colorectal cancer diagnosis can be achieved. It was found that accuracy and sensitivity were excellent.
특히 본 발명의 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질은 대장암 발병의 초기 단계부터 정상 대조군 대비 현저히 높은 발현량을 나타내는 것으로 확인되어 초기 대장암 진단의 정확도와 민감도를 기대할 수 있다. In particular, the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins of the present invention were found to have significantly higher expression levels compared to normal controls from the early stage of colon cancer development, so accuracy and sensitivity in early colon cancer diagnosis can be expected. .
이에, 본 발명이 해결하고자 하는 과제는, 엑소좀 유래의 단백질을 포함하는 대장암 진단 또는 예후 예측용 바이오 마커 조성물을 제공하는 것이다. Accordingly, the problem to be solved by the present invention is to provide a biomarker composition for diagnosing or predicting prognosis of colon cancer containing an exosome-derived protein.
본 발명이 해결하고자 하는 다른 과제는, 엑소좀 유래의 단백질의 발현을 측정하는 제제를 포함하는 대장암 진단 또는 예후 예측용 조성물을 제공하는 것이다. Another problem to be solved by the present invention is to provide a composition for diagnosing or predicting prognosis of colon cancer, including an agent for measuring the expression of exosome-derived proteins.
또한 본 발명이 해결하고자 하는 다른 과제는, 엑소좀 유래의 단백질의 발현수준을 측정하는 제제를 포함하는 대장암 진단 또는 예후 예측용 키트를 제공하는 것이다. Another problem to be solved by the present invention is to provide a kit for diagnosing or predicting prognosis of colon cancer, which includes an agent for measuring the expression level of exosome-derived proteins.
또한 본 발명이 해결하고자 하는 다른 과제는, 엑소좀 유래의 단백질의 발현수준을 측정하는 제제를 포함하는 대장암 진단 또는 예후 예측용 키트를 제공하는 것이다. Another problem to be solved by the present invention is to provide a kit for diagnosing or predicting prognosis of colon cancer, which includes an agent for measuring the expression level of exosome-derived proteins.
본 발명의 과제들은 이상에서 언급한 과제들로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다. The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
본 발명의 일시예에 따르면, 엑소좀 유래의 단백질 또는 이를 암호화하는 유전자를 포함하는 대장암 진단 또는 예후 예측용 바이오 마커 조성물이 제공된다.According to one embodiment of the present invention, a biomarker composition for diagnosing or predicting prognosis of colon cancer comprising an exosome-derived protein or a gene encoding the same is provided.
구체적으로 엑소좀 유래의 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자를 포함할 수 있다.Specifically, it may include at least one protein selected from the group consisting of exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, or at least one gene encoding the same.
본 발명의 특징에 따르면, 개체는 대장암 의심 개체 또는 대장암 보유 개체일 수 있다.According to the features of the present invention, the individual may be an individual suspected of having colon cancer or an individual having colon cancer.
본 발명의 특징에 따르면, 생물학적 시료는 종양 조직, 혈액, 혈청 및 혈장으로 이루어진 그룹에서 선택된 적어도 하나에서 분리된 엑소좀일 수 있다.According to features of the present invention, the biological sample may be exosomes isolated from at least one selected from the group consisting of tumor tissue, blood, serum, and plasma.
본 명세서에서 사용되는 용어 "엑소좀"은 세포에서 분비되는 작은 형태의 소포체를 의미하는 것으로, 바람직하게는 전혈, 혈청 및 혈장 등 혈액 내에 존재하는 엑소좀이다.The term “exosome” used herein refers to a small type of endoplasmic reticulum secreted from cells, and is preferably an exosome present in blood such as whole blood, serum, and plasma.
본 발명에서 용어 "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 대장암을 진단하는 것이다. 보다 구체적으로는 대장암에서 엑소좀 내 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질의 발현이 증가하므로 상기 엑소좀 내 단백질의 양을 측정하여 대조군 시료 대비 높을 경우 해당 개체의 대장암에 대해 진단이 가능하다. 또한 상기 단백질을 암호화하는 유전자의 mRNA 발현량을 측정하여 대조군 시료 대비 높을 경우 대장암에 대해 진단이 가능하다.As used herein, the term “diagnosis” means confirming the presence or characteristics of a pathological condition. For the purpose of the present invention, it is to diagnose colon cancer. More specifically, in colon cancer, the expression of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins in exosomes increases, so the amount of protein in the exosomes is measured and if it is higher than the control sample, the individual has colon cancer. Diagnosis is possible. In addition, colon cancer can be diagnosed by measuring the mRNA expression level of the gene encoding the protein and if it is higher than that of the control sample.
본 발명의 특징에 따르면 본 발명의 일 실시예에 따른 바이오마커를 이용하여 진단이 가능한 대장암은 초기 대장암을 포함할 수 있다.According to a feature of the present invention, colon cancer that can be diagnosed using a biomarker according to an embodiment of the present invention may include early stage colon cancer.
대장암은 점막층에서 발생하고 진행되면 점점 아래층을 파고 들어가 점막하층, 근육층, 장막층 순서로 침투해 자라게 된다. 대장의 4개층을 다 뚫고 나간 대장암은 대장 주변의 장기와 림파절을 침범하게 되고 좀더 진행되면 림프관이나 혈관을 통해 대장에서 멀리 떨어진 간이나 폐로 퍼지게 된다. 이렇게 대장암이 진행해서 나간 정도를 병기(stage)라고 한다. 대장암의 병기는 AJCC 분류 체계에 따라 TNM 체계를 기반으로 1기 ~ 4기로 구분할 수 있다. 1기(Stage I)는 종양이 아직 점막하층에 위치하거나, 고유근층까지 침범한 경우를 정의하며, 2기(StageⅡ)는 근육층 또는 인근 구조물로 확장되었지만 림프절 전이는 아닌 경우를 정의하고, 3기(Stage Ⅲ)는 림프절 전이 양성인 그룹을 나타내며, 4기(Stage Ⅳ)는 원격 전이 양성인 그룹을 정의한다. 대장암의 치료 방법을 결정하고 예후 인자를 알아내기 위해서는 병기를 정확하게 판정해야 한다.Colon cancer occurs in the mucosal layer and as it progresses, it gradually penetrates the lower layers and grows in that order: the submucosal layer, the muscle layer, and the serosal layer. Colon cancer that has penetrated all four layers of the large intestine invades organs and lymph nodes around the large intestine, and as it progresses further, it spreads to the liver or lungs far away from the large intestine through lymphatic vessels or blood vessels. The extent to which colon cancer has progressed is called the stage. The stage of colon cancer can be divided into stages 1 to 4 based on the TNM system according to the AJCC classification system. Stage 1 (Stage I) defines cases where the tumor is still located in the submucosa or has invaded the muscularis propria, Stage 2 (StageⅡ) defines cases where the tumor has expanded into the muscle layer or nearby structures but has not metastasized to lymph nodes, and Stage 3 (Stage Ⅲ) represents the group positive for lymph node metastasis, and stage 4 (Stage Ⅳ) defines the group positive for distant metastasis. In order to determine treatment methods for colon cancer and identify prognostic factors, the stage must be accurately determined.
본 발명의 일 실시예에 따른 엑소좀 유래 단백질 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3은 1기 ~ 4기로 분류한 대장암 병기 단계 모두에서 유의하게 발현되었으며, 특히 도 7을 참고하면, 1기(Stage Ⅰ)의 초기 대장암 환자군에서도 정상 대조군(Healthy)과 비교하여 유의미한 차이를 나타내는 것을 확인하였다. 반면에 종래의 대장암 진단 바이오마커인 CEA(carcinoembryonic antigen)는 Stage Ⅰ 환자군에서는 발현이 미미한 것으로 나타났다.Exosome-derived proteins SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 according to an embodiment of the present invention were significantly expressed in all colon cancer stages classified from stage 1 to stage 4, especially referring to Figure 7. , It was confirmed that the stage 1 (Stage I) early colon cancer patient group showed significant differences compared to the normal control group (Healthy). On the other hand, CEA (carcinoembryonic antigen), a conventional diagnostic biomarker for colon cancer, was found to have minimal expression in the Stage I patient group.
이러한 결과로, 대장암 발병 초기 단계에서는 민감도가 낮게 나타나는 CEA와 비교하여 본 발명의 일 실시예에 따른 엑소좀 유래 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질은 대장암 발병 초기부터 높은 민감도를 나타내므로 높은 정확도로 초기 대장암을 진단할 수 있다. As a result, compared to CEA, which shows low sensitivity in the early stages of colon cancer development, the exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein according to an embodiment of the present invention has a high sensitivity from the early stage of colon cancer development. Because it shows sensitivity, early colon cancer can be diagnosed with high accuracy.
상기 초기 대장암은 종양이 아직 점막하층에 위치하거나, 고유근층까지 침범한 1기(Stage Ⅰ) 병기일 수 있으나 이에 제한되는 것은 아니다. 0기~1기의 초기 대장암은 무증상인 경우가 많아 자각이 어렵고, 서서히 진행되면서 소화불량, 배변곤란, 출혈, 복부통증, 체중감소 등의 증상이 나타난다. 이러한 대장암의 조기 발견을 위해 대장내시경 검사를 권장하고 있으나, 내시경에 따른 부작용, 비용 등의 부담을 동반한다. The early stage colon cancer may be stage 1 (Stage I), in which the tumor is still located in the submucosal layer or has invaded the muscularis propria, but is not limited thereto. Early stages 0 to 1 colon cancer is often asymptomatic, making it difficult to recognize, and as it progresses slowly, symptoms such as indigestion, difficulty in defecation, bleeding, abdominal pain, and weight loss appear. Colonoscopy is recommended for early detection of colon cancer, but it is accompanied by burdens such as side effects and costs associated with endoscopy.
본 발명에 따른 엑소좀 유래 단백질 바이오마커를 이용하면 간단한 혈액 검사만으로 증상이 자각되지 않는 초기 단계의 대장암 발병을 진단할 수 있다.Using the exosome-derived protein biomarker according to the present invention, the development of colon cancer in the early stages when symptoms are not recognized can be diagnosed with a simple blood test.
본 발명의 특징에 따르면, 상기 진단은 재발을 포함할 수 있다.According to features of the invention, the diagnosis may include recurrence.
다양한 실시예에서, 본 발명에 따른 바이오마커 조성물은 대장암 진단을 받고 항암 화학 요법 또는 수술을 진행한 개체에 대해 장기적 추적을 통해 혈액 검사만으로도 재발 여부의 진단 마커로서의 효능을 기대할 수 있다.In various embodiments, the biomarker composition according to the present invention can be expected to be effective as a diagnostic marker for recurrence through a blood test alone through long-term follow-up of an individual who has been diagnosed with colon cancer and has undergone chemotherapy or surgery.
본 발명에서 용어 "예후(prognosis)"는 질병을 진단하여 판단된 장래의 증세 또는 경과에 대한 전망을 말한다. 예후의 예측은 향후 항암 치료의 방향에 대한 단서를 제시하므로 매우 중요한 임상적 과제이다. In the present invention, the term “prognosis” refers to the outlook for future symptoms or progress determined by diagnosing a disease. Predicting prognosis is a very important clinical task because it provides clues to the direction of future anticancer treatment.
본 발명에서 용어 "예측"은 의학적 귀추에 대하여 미리 헤아려 짐작하는 것을 의미하며, 본 발명의 목적상 대장암으로 진단받은 환자의 병의 경과(병의 진행, 개선, 재발, 종양 성장, 약 저항성)를 미리 짐작하는 것을 의미한다.In the present invention, the term "prediction" means to guess in advance about medical consequences, and for the purpose of the present invention, the course of the disease (disease progression, improvement, recurrence, tumor growth, drug resistance) of a patient diagnosed with colon cancer. means guessing in advance.
예를 들어, 개체의 엑소좀 내 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질의 양을 측정하여 대조군 시료 대비 높을 경우 해당 개체의 대장암에 대해 예후가 나쁠 것으로 예측하고, 대조군 시료 대비 낮을 경우 대장암에 대해 예후가 좋을 것으로 예측할 수 있다. 또한 상기 단백질을 암호화하는 유전자의 mRNA 발현량을 측정하여 대조군 시료 대비 높을 경우 해당 개체의 대장암에 대해 예후가 나쁠 것으로 예측하고, 대조군 시료 대비 낮을 경우 대장암에 대해 예후가 좋을 것으로 예측할 수 있다.For example, by measuring the amount of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein in the exosome of an individual, if it is high compared to the control sample, it is predicted that the individual will have a poor prognosis for colon cancer, and compared to the control sample, If it is low, the prognosis for colon cancer can be predicted to be good. In addition, by measuring the mRNA expression level of the gene encoding the protein, if it is high compared to the control sample, it can be predicted that the individual will have a poor prognosis for colon cancer, and if it is low compared to the control sample, the prognosis can be predicted to be good for colon cancer.
본 발명에서 용어 "마커"는 생물학적 현상의 존재와 정량적 또는 정성적으로 연관된 분자를 의미하며, 본 발명의 대장암 진단용 바이오마커는 대장암 발병시 발현이 증가하는 단백질 또는 이를 암호화하는 유전자를 지칭한다.In the present invention, the term "marker" refers to a molecule that is quantitatively or qualitatively associated with the presence of a biological phenomenon, and the biomarker for colon cancer diagnosis of the present invention refers to a protein whose expression increases when colon cancer develops or a gene encoding the same. .
또한 본 발명의 일시예에 따르면, 엑소좀 유래 단백질 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자 발현을 측정하는 제제;를 포함하는 대장암 진단 또는 예후 예측용 조성물을 제공한다. Also, according to one embodiment of the present invention, an agent for measuring the expression of at least one protein selected from the group consisting of exosome-derived proteins SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, or at least one gene encoding the same; Provided is a composition for diagnosing or predicting prognosis of colon cancer comprising a.
본 발명에 있어서 용어, "단백질 발현수준 측정"이란 생물학적 시료에서 목적하는 단백질의 발현 또는 활성 정도를 확인하는 과정으로, 예를 들어 상기 목적하는 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 펩타이드의 양을 확인할 수 있다. 이를 위한 분석 방법으로는 웨스턴 블랏팅(Western Blotting), ELISA(Enzyme Linked Immunosorbent Assay), 방사선면역분석(RIA: Rodioimmunoassay), 방사 면역 확산법(Radioimmunodiffusion), 오우크테로니(Ouchterolony) 면역 확산법, 로케 트(Rocket) 면역전기영동, 조직면역염색, 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법 (Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FASC), 단백질 칩(Protein Chip) 등이 있으나, 이에 제한되지는 않는다.In the present invention, the term "protein expression level measurement" refers to a process of confirming the expression or activity level of a protein of interest in a biological sample, for example, using an antibody that specifically binds to the protein of interest to determine the level of peptide. You can check the amount. Analysis methods for this include Western Blotting, Enzyme Linked Immunosorbent Assay (ELISA), Rodioimmunoassay (RIA), Radioimmunodiffusion, Ouchterolony Immunodiffusion, and Rocket. (Rocket) There are immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FASC), and protein chip. It is not limited.
본 발명의 특징에 따르면, 단백질의 발현을 측정하는 제제는 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질에 특이적으로 결합하는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체, 키메릭(chimeric) 항체, 리간드, PNA(Peptide nucleic acid) 또는 앱타머(aptamer)일 수 있으나, 이에 제한되지 않는다.According to a feature of the present invention, the agent for measuring protein expression is an oligopeptide, monoclonal peptide that specifically binds to at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins. It may be a raw antibody, polyclonal antibody, chimeric antibody, ligand, peptide nucleic acid (PNA), or aptamer, but is not limited thereto.
본 발명에서 용어 "항체"는 당해 기술분야에 공지된 용어로서 항원성 부위에 대하여 지시되는 특이적인 면역글로불린을 의미한다. 본 발명에서의 항체는 본 발명의 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 당해 기술분야의 통상적인 방법에 따라 항체를 제조할 수 있다. 상기 항체의 형태는 폴리클로날 항체 또는 모노클로날 항체를 포함하며, 모든 면역글로불린 항체가 포함된다. 상기 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 갖는 완전한 형태를 의미한다. 또한, 상기 항체는 인간화 항체 등의 특수 항체도 포함된다.In the present invention, the term “antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein of the present invention, and the antibody can be prepared according to a conventional method in the art. . The types of antibodies include polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant to be intact, with two full-length light chains and two full-length heavy chains. Additionally, the above antibodies also include special antibodies such as humanized antibodies.
본 발명에서 용어 "앱타머(aptamer)"란, 그 자체로 안정된 삼차 구조를 가지면서 표적 분자에 높은 친화성과 특이성으로 결합할 수 있는 특징을 가진 특별한 종류의 단일가닥 핵산(DNA, RNA 또는 변형핵산)으로 구성된 폴리뉴 클레오티드의 일종을 의미한다. 상술한 바와 같이, 앱타머는 항체와 동일하게 항원성 물질에 특이적으로 결합할 수 있으면서도, 단백질보다 안정성이 높고, 구조가 간단하며, 합성이 용이한 폴리뉴클레오티드로 구성되어 있으므로, 항체를 대체하여 사용될 수 있다.In the present invention, the term "aptamer" refers to a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. ) refers to a type of polynucleotide composed of As described above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easier to synthesize, so they can be used as replacements for antibodies. You can.
본 발명에 있어서 용어, "유전자 발현수준 측정"이란 생물학적 시료에서 목적하는 유전자의 발현 또는 활성을 확인하는 과정으로, 예를 들어 상기 목적하는 유전자에 대하여 특이적으로 결합하는 프라이머 또는 프로브를 이용하여 mRNA양을 확인할 수 있다. 이를 위한 분석 방법으로는 RT-PCR, 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting) 또는 DNA 칩 등이 있으나, 이에 제한되는 것은 아니다.In the present invention, the term "gene expression level measurement" refers to the process of confirming the expression or activity of a gene of interest in a biological sample, for example, using a primer or probe that specifically binds to the gene of interest. You can check the amount. Analysis methods for this include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), and Northern blotting. blotting) or DNA chips, but are not limited thereto.
본 발명의 특징에 따르면, 유전자의 발현을 측정하는 제제는 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질을 암호화하는 유전자에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머(쌍), 프로브, 소분자 화합물 또는 핵산 분자일 수 있으나, 이에 제한되지는 않는다.According to a feature of the present invention, the agent for measuring gene expression is an antisense agent that specifically binds to a gene encoding at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins. It may be, but is not limited to, an oligonucleotide, primer (pair), probe, small molecule compound, or nucleic acid molecule.
본 발명에서 용어 "프라이머"는 짧은 자유 3말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍(base pair)을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시할 수 있다. 구체적으로, ZO-1 폴리뉴클레오티드의 센스 및 안티센스 프라이머를 이용한 PCR 증폭을 실시하여 원하는 생성물의 생성 여부를 통해 혈관 질환을 진단할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 것을 기초로 변형할 수 있다.In the present invention, the term "primer" is a nucleic acid sequence with a short free 3' hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. It refers to a short nucleic acid sequence that performs a function. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. Specifically, vascular disease can be diagnosed by performing PCR amplification using sense and antisense primers of ZO-1 polynucleotide to determine whether the desired product is produced. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.
본 발명에서 용어 "프로브"란 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링 되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 프로브는 올리고뉴클로타이드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적당한 프로브의 선택 및 혼성화조건은 당업계에 공지된 것을 기초로 변형할 수 있다.In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundreds of bases, capable of forming a specific binding to mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed. there is. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.
본 발명의 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, "캡화", 천연 뉴클레오타이드 하나 이상의 동족체로의 치환, 및 뉴클레오타이드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, “capsation,” substitution of a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).
본 발명에서 용어 "검출" 또는 "측정"은 검출 또는 측정된 대상의 농도를 정량하는 것을 의미한다.In the present invention, the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.
또한 본 발명은 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자 발현수준을 측정하는 제제;를 포함하는 대장암 진단 또는 예후 예측용 키트를 제공한다.In addition, the present invention provides an agent for measuring the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, or at least one gene encoding the same. Diagnosis or prognosis of colon cancer, including: A prediction kit is provided.
구체적으로는 본 발명의 키트는 엑소좀에서 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 하나 이상의 단백질 또는 이를 암호화하는 하나 이상의 유전자 발현 수준을 확인함으로써, 대조군 시료 대비 높을 경우 대장암인 것으로 진단하는데 이용될 수 있다. 또한 대조군 시료 대비 낮을 경우 대장암에 대한 예후가 좋은 것으로 판단하는데 이용될 수 있다.Specifically, the kit of the present invention confirms the expression level of one or more proteins selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 in exosomes, or one or more genes encoding them, which is higher compared to the control sample. In this case, it can be used to diagnose colon cancer. Additionally, if it is lower than that of the control sample, it can be used to determine that the prognosis for colon cancer is good.
본 발명의 키트에는 대장암 진단을 위한 선택적으로 마커를 인지하는 항체뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액, 또는 장치가 포함될 수 있다.The kit of the present invention may include not only an antibody that selectively recognizes a marker for colon cancer diagnosis, but also one or more other component compositions, solutions, or devices suitable for the analysis method.
또한, 본 발명에서 단백질의 발현 수준을 측정하기 위한 키트는 항체의 면역학적 검출을 위하여 기질, 적당한 완충용액, 발색 효소 또는 형광물질로 표지된 2차 항체, 및 발색 기질 등을 포함할 수 있다. 상기에서 기질은 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96 웰 플레이트, 폴리스티렌 수지로 합성된 96 웰 플레이트 및 유리로 된 슬라이드 글라스 등이 이용될 수 있고, 발색효소는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase) 등이 사용될 수 있고, 형광물질은 FITC, RITC 등이 사용될 수 있고, 발색기질액은 ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-설폰산)) 또는 OPD(O-페닐렌디아민), TMB(테트라메틸 벤지딘)가 사용될 수 있다.In addition, the kit for measuring the expression level of a protein in the present invention may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of an antibody. In the above, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass, and the coloring enzyme may be peroxidase or alkaline phosphatase. Fatase (alkaline phosphatase), etc. can be used, the fluorescent substance can be FITC, RITC, etc., and the coloring substrate solution is ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphate). Ponic acid) or OPD (O-phenylenediamine) or TMB (tetramethyl benzidine) can be used.
또한, 본 발명의 키트는 마커 성분에 특이적으로 결합하는 항체, 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차 항체 접합체(conjugate), 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 효소반응 정지용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다.In addition, the kit of the present invention includes an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with a substrate, a chromogenic substrate solution that will undergo color development with the label, a washing solution, and It may contain an enzyme reaction stopping solution, etc., and may be manufactured into a number of separate packaging or compartments containing the reagent components used.
본 발명의 키트는 상술한 바이오마커 조성물 및 진단 또는 예후 예측용 조성물의 구성을 포함하므로, 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the kit of the present invention includes the above-described biomarker composition and a composition for diagnosis or prognosis prediction, duplicate content is omitted to avoid excessive complexity of the specification.
또한 본 발명은 개체로부터 분리된 생물학적 시료에 대하여 엑소좀을 분리하는 단계 (S810), 상기 분리한 엑소좀으로부터 단백질을 수득하는 단계 (S820), 상기 단백질 또는 이를 암호화하는 적어도 하나의 유전자 mRNA의 발현 수준을 측정하는 단계 (S830) 및 상기 측정된 단백질 또는 이를 암호화하는 적어도 하나의 유전자 mRNA의 발현 수준을 정상 대조군과 비교하는 단계 (S840)를 포함하는 대장암 진단 방법을 제공한다.In addition, the present invention includes the steps of isolating exosomes from a biological sample isolated from an individual (S810), obtaining a protein from the isolated exosome (S820), and expressing the protein or at least one gene mRNA encoding the protein. A method for diagnosing colon cancer is provided, including measuring the level (S830) and comparing the measured expression level of the protein or at least one gene mRNA encoding the same with a normal control group (S840).
상기 개체로부터 분리된 생물학적 시료는 조직, 세포, 혈액, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액, 세포 배양액 또는 뇨와 같은 시료 등을 포함할 수 있으며 이에 제한되지 않으며 바람직하게는 생물학적 시료는 엑소좀을 포함하는 종양 조직, 혈액, 혈청 및 혈장으로 이루어진 그룹에서 선택된 적어도 하나를 포함하며 더욱 바람직하게는 대장암 의심 개체로부터 분리된 엑소좀을 포함하는 혈액이다. The biological sample isolated from the subject may include, but is not limited to, samples such as tissue, cells, blood, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, cell culture, or urine, and preferably the biological sample is exo. It contains at least one selected from the group consisting of tumor tissue containing exosomes, blood, serum, and plasma, and more preferably, it is blood containing exosomes isolated from an individual suspected of colon cancer.
본 발명의 일 실시예에 따르면, 상기 분리한 엑소좀으로부터 단백질을 수득하는 단계 (S820)에서 수득하는 단백질은 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 적어도 하나를 포함할 수 있다.According to one embodiment of the present invention, the protein obtained in the step of obtaining a protein from the isolated exosome (S820) is at least one selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3. It can be included.
본 발명의 일 실시예에 따르면, 상기 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자 발현 수준을 측정하고, 정상 대조군 시료와 비교하여 발현 수준이 높은 경우 대장암으로 진단할 수 있다.According to one embodiment of the present invention, the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 or at least one gene encoding the same is measured, and the expression level of at least one gene encoding the same is measured in a normal control sample. If the expression level is high compared to , it can be diagnosed as colon cancer.
또한 본 발명의 특징에 따르면 본 발명은 개체로부터 분리된 생물학적 시료에 대하여 엑소좀을 분리하는 단계; 상기 분리된 엑소좀으로부터 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질을 수득하는 단계; 상기 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자의 mRNA 발현 수준을 측정하는 단계; 및 상기 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자의 mRNA 발현 수준을 대조군 시료와 비교하는 단계;를 포함하는, 대장암 예후 예측 방법을 제공한다.In addition, according to a feature of the present invention, the present invention includes the steps of isolating exosomes from a biological sample isolated from an individual; Obtaining at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins from the isolated exosomes; Measuring the mRNA expression level of the selected at least one protein or at least one gene encoding the selected protein; and comparing the mRNA expression level of the selected at least one protein or at least one gene encoding the selected protein with a control sample.
다양한 실시예에서, 방법은, 상기 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자 발현 수준을 측정하고, 대조군 시료와 비교하여 낮은 경우 대장암에 대한 예후가 좋은 것으로 판단하고 이에 대한 정보를 제공하도록 구성될 수 있다. 상기 개체는 대장암 진단을 받은 후 항암 화학 요법 또는 수술을 받은 개체일 수 있으나, 이에 제한되는 것은 아니다. 상기 대조군 시료는 대장암 의심 개체 또는 대장암 보유 개체로부터 수득한 시료일 수 있고 바람직하게는 대장암 의심 개체 또는 대장암 보유 개체로서 항암화학요법 또는 수술을 받지 않은 개체로부터 수득한 시료일 수 있으나, 이에 제한되는 것은 아니다.In various embodiments, the method measures the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, or at least one gene encoding the same, and compares it with a control sample. Therefore, if it is low, the prognosis for colon cancer is judged to be good and information on this can be provided. The subject may be an individual who has received chemotherapy or surgery after being diagnosed with colon cancer, but is not limited thereto. The control sample may be a sample obtained from an individual suspected of having colon cancer or an individual having colon cancer, and preferably may be a sample obtained from an individual suspected of having colon cancer or an individual having colon cancer who has not undergone chemotherapy or surgery. It is not limited to this.
또한 본 발명의 특징에 따르면 본 발명은 대장암이 발현된 개체로부터 채취한 시료에서 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질 발현 수준을 측정하는 단계; 상기 시료에 후보물질을 처리하고 상기 선택되는 단백질 발현 수준을 측정하는 단계; 및 상기 시료에 후보물질을 처리 후 후보물질 처리 전보다 상기 선택되는 단백질 발현 수준이 감소한 경우 상기 후보물질을 대장암 예방 또는 치료용 제제로 선별하는 단계를 포함하는, 대장암 예방 또는 치료용 제제의 스크리닝 방법을 제공한다.In addition, according to a feature of the present invention, the present invention provides a method for measuring the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins in samples collected from individuals with colon cancer. step; Processing the sample with a candidate material and measuring the expression level of the selected protein; And if the expression level of the selected protein decreases after treating the sample with the candidate material compared to before treatment with the candidate material, screening the candidate material as an agent for preventing or treating colorectal cancer, comprising the step of selecting the candidate material as an agent for preventing or treating colorectal cancer. Provides a method.
다양한 실시예에서, 본 발명에 따른 대장암 예방 또는 치료제의 스크리닝 방법은 상기 단계에 제한되지 않는다.In various embodiments, the method for screening for colon cancer prevention or treatment according to the present invention is not limited to the above steps.
다양한 실시예에서, 대장암이 발현된 개체로부터 채취한 시료에서 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질 발현 수준을 측정할 수 있다. In various embodiments, the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins can be measured in samples collected from individuals with colon cancer.
다양한 실시예에서 후보물질을 처리한 시료에서 후보물질을 처리하기 전에 발현 수준을 측정한 동일한 단백질의 발현 수준을 측정할 수 있다.In various embodiments, the expression level of the same protein whose expression level was measured before processing the candidate material can be measured in a sample treated with the candidate material.
다양한 실시예에서 측정한 단백질 발현 수준이 후보물질을 처리하기 전에 비해 감소한 경우 상기 후보물질을 대장암 예방 또는 치료용 물질로 선별할 수 있다.If the protein expression level measured in various examples is reduced compared to before processing the candidate material, the candidate material may be selected as a material for preventing or treating colon cancer.
본 발명은, 엑소좀 유래의 바이오마커를 제공하여 높은 정확도 및 민감도로 대장암을 진단 또는 예후 예측에 이용할 수 있는 효과가 있다.The present invention has the effect of providing an exosome-derived biomarker that can be used to diagnose or predict the prognosis of colorectal cancer with high accuracy and sensitivity.
보다 구체적으로, 본 발명은 엑소좀 유래의 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 적어도 하나의 단백질을 이용하여 정확도 및 민감도가 높은 대장암 진단용 바이오마커를 제공할 수 있다. 이에, 본 발명은 종래의 대장암 진단용 바이오마커들에 기초한 진단 방법보다, 대장암 초기 발병 단계에서부터 정확한 진단에 이용될 수 있다. More specifically, the present invention provides a biomarker for colorectal cancer diagnosis with high accuracy and sensitivity using at least one protein selected from the group consisting of exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3. You can. Accordingly, the present invention can be used for more accurate diagnosis from the early stage of colon cancer development than the conventional diagnostic method based on biomarkers for colon cancer diagnosis.
또한 본 발명은 엑소좀 유래의 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3으로 이루어진 군에서 선택되는 적어도 하나의 단백질을 이용하여 대장암 항암 치료에 대한 정확한 예후 예측용 바이오마커를 제공할 수 있다. In addition, the present invention can provide a biomarker for accurate prognosis prediction for colon cancer anticancer treatment using at least one protein selected from the group consisting of exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3. there is.
본 발명에 따른 효과는 이상에서 예시된 내용에 의해 제한되지 않으며, 더욱 다양한 효과들이 본 명세서 내에 포함되어 있다.The effects according to the present invention are not limited to the contents exemplified above, and further various effects are included in the present specification.
도 1은 50명의 대장암 수술 환자의 조직 및 혈액 유래 엑소좀을 대상으로 LC/MS를 사용하여 대장암 진단 바이오 마커로 활용 가능한 단백질을 스크리닝한 결과를 도시한 것이다. Figure 1 shows the results of screening proteins that can be used as diagnostic biomarkers for colon cancer using LC/MS on tissue- and blood-derived exosomes from 50 colon cancer surgery patients.
도 2는 스크리닝된 바이오마커로 활용 가능한 단백질을 대장암이 발생하지 않은 정상 대조군과 대장암 환자의 수술 전 조직 및 혈액 유래 엑소좀에서 ELISA를 이용하여 유의한 발현량 차이를 나타내는 7개 단백질을 스크리닝한 결과를 도시한 것이다. Figure 2 shows seven proteins that show significant expression level differences using ELISA in preoperative tissue and blood-derived exosomes of normal controls without colon cancer and colon cancer patients for proteins that can be used as screened biomarkers. It shows one result.
도 3은 7개 단백질에 대해 바이오마커로서의 유의성을 확인하기 위해 ROC(Receiver Operating Characteristic Curve)를 사용하여 평가한 결과를 도시한 것이다. Figure 3 shows the results of evaluation using ROC (Receiver Operating Characteristic Curve) to confirm the significance of seven proteins as biomarkers.
도 4는 7개 단백질에 대해 바이오마커로서의 유의성을 확인하기 위해 각 단백질 발현값을 표준화(z-score)하여 Heat-map으로 표현한 결과를 도시한 것이다.Figure 4 shows the results of normalizing (z-score) the expression values of each protein for seven proteins and expressing them as a heat-map to confirm their significance as biomarkers.
도 5는 7개 단백질에 대해 바이오마커로서의 유의성을 확인하기 위해 대장암 환자군에서 대장암 종양 조직 제거 전 및 제거 6주 후의 각 단백질 발현량 변화를 ELISA를 이용하여 측정한 결과를 도시한 것이다. Figure 5 shows the results of measuring the change in expression level of each protein using ELISA in a group of colon cancer patients before and 6 weeks after removal of the colon cancer tumor tissue in order to confirm the significance of the seven proteins as biomarkers.
도 6은 대장암 환자를 병기별로 분류한 단계에 따라 검출된 serum CEA값 및 CEA를 이용한 진단률 결과를 도시한 것이다.Figure 6 shows the serum CEA value detected according to the stage in which colorectal cancer patients were classified by stage, and the diagnosis rate results using CEA.
도 7은 정상 대조군 및 대장암 환자를 병기별로 분류한 단계에 따라 검출된 엑소좀 유래 7개 단백질의 각 발현량 결과를 도시한 것이다.Figure 7 shows the expression level results of each of the seven exosome-derived proteins detected according to the stage of classification of normal controls and colon cancer patients.
도 8은 본 발명의 일 실시예에 따른 엑소좀 유래의 단백질 발현수준을 측정하여 대장암 진단 또는 예후 예측을 위한 정보 제공 방법의 절차를 예시적으로 도시한 것이다.Figure 8 exemplarily shows the procedure of a method for providing information for diagnosing or predicting prognosis of colon cancer by measuring the expression level of exosome-derived protein according to an embodiment of the present invention.
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본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 첨부되는 도면과 함께 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나, 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다. The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments only serve to ensure that the disclosure of the present invention is complete and are within the scope of common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명한다. 다만, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것에 불과하므로 본 발명의 범위가 이들 실시예에 의해 한정되는 것으로 해석되어서는 아니된다.Hereinafter, the present invention will be described in more detail through examples. However, since these examples are only for illustrative purposes of the present invention, the scope of the present invention should not be construed as being limited by these examples.
실시예 1. 엑소좀 유래 대장암 특이 단백질 스크리닝Example 1. Screening of exosome-derived colorectal cancer-specific proteins
연세대학교 세브란스 병원에서 제공받은 건강 대조군의 혈액 샘플 및 50여명의 대장암 환자군의 수술 전 혈액, 수술 종양 조직, 종양 주변 조직, 수술 후 혈액 샘플로부터 분리한 엑소좀을 LC-MS/MS 방법을 활용하여 단백체 데이터를 생성하였다. 엑소좀의 분리는 다음의 방법을 이용하였다. 수득한 샘플을 17000xg에서 5분동안 원심분리하여 혈장을 얻었다. 상기 원심분리한 혈장에서 Exoquick-Exosome Precipitation Solution (System bioscience)을 혈장 부피의 1/4만큼 넣어준 후, 2시간 동안 4 °C에서 휴지시키고, 1500xg에서 30분간 다시 원심분리하였다. 이후 상층액을 제거한 뒤 엑소좀 펠렛을 PBS에 녹여 실험에 사용하였다. Exosomes isolated from blood samples of healthy control subjects provided by Yonsei University Severance Hospital and preoperative blood, surgical tumor tissue, tumor surrounding tissue, and postoperative blood samples of about 50 colorectal cancer patients were used using the LC-MS/MS method. Thus, proteomic data was generated. Exosomes were isolated using the following method. The obtained sample was centrifuged at 17000xg for 5 minutes to obtain plasma. Exoquick-Exosome Precipitation Solution (System bioscience) was added to the centrifuged plasma as much as 1/4 of the plasma volume, rested at 4 °C for 2 hours, and centrifuged again at 1500xg for 30 minutes. Afterwards, the supernatant was removed, and the exosome pellet was dissolved in PBS and used in the experiment.
엑소좀을 이용한 단백체 데이터로부터 LC/MS 기술을 사용하여 수술 후 혈액 즉, 종양조직 제거 후의 혈액 유래 엑소좀에서는 검출이 미미한 상위 단백질 및 종양조직 유래 엑소좀에서 특이적으로 검출되는 상위 단백질을 스크리닝하였다.From the proteomic data using exosomes, LC/MS technology was used to screen the top proteins that were minimally detected in post-surgical blood, that is, blood-derived exosomes after tumor tissue removal, and the top proteins that were specifically detected in tumor tissue-derived exosomes. .
도 1에 나타낸 바와 같이, 건강 대조군(healthy control)의 혈장 시료에서는 발현량이 없거나 적으며, 대장암 수술 전 혈장 시료에서는 발현량이 높고, 대장암 수술 후 혈장 시료에서는 발현량이 없거나 감소한 30개의 단백질(상단 이미지) 및 종양 조직 및 인접 조직에서 발현량이 높은 30개의 단백질(하단 이미지)로서 순환 종양 바이오마커로 임상활용 가능한 단백질을 발굴하였다.As shown in Figure 1, 30 proteins have no or low expression levels in plasma samples from healthy controls, high expression levels in plasma samples before colorectal cancer surgery, and no or reduced expression levels in plasma samples after colorectal cancer surgery (top). image) and 30 proteins with high expression levels in tumor tissue and adjacent tissues (bottom image), proteins that can be clinically used as circulating tumor biomarkers were discovered.
실시예 2. 대장암 진단 또는 예후 예측용 바이오마커 도출 Example 2. Derivation of biomarkers for colon cancer diagnosis or prognosis prediction
실시예 1의 스크리닝을 통해 선정된 후보 단백질을 84명의 대장암 환자의 수술 전 혈액 유래 엑소좀과 20명의 건강 대조군의 혈액 유래 엑소좀에서 ELISA 방법으로 비교하여 발현량이 가장 유의미하게 차이나는 상위 단백질을 발굴하였다. 그 결과 도 2에 나타낸 바와 같이, 7개 단백질 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3을 도출하고, 이 후 실험에서 대장암 진단 또는 예후 예측용 바이오마커로의 유의성을 평가하였다.Candidate proteins selected through the screening in Example 1 were compared by ELISA method in preoperative blood-derived exosomes of 84 colorectal cancer patients and blood-derived exosomes of 20 healthy controls, and the top proteins with the most significant difference in expression level were identified. It was excavated. As a result, as shown in Figure 2, seven proteins, SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3, were derived, and their significance as biomarkers for colon cancer diagnosis or prognosis was evaluated in subsequent experiments.
실시예 3. 대장암 진단 또는 예후 예측용 바이오마커 검증Example 3. Verification of biomarkers for colon cancer diagnosis or prognosis prediction
실시예 2에서 도출한 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3에 대해 대장암 임상 진단 바이오마커로서의 유의성을 평가하였다.SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 derived in Example 2 were evaluated for their significance as clinical diagnostic biomarkers for colon cancer.
먼저 ROC(Receiver Operating Characteristic Curve)를 사용하여 평가한 결과, 도 3에 나타낸 바와 같이 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3의 AUC(area under the ROC curve) 수치가 모두 0.80 이상의 값으로 확인되어 유의미한 결과를 나타냈으며, 특히 SRPK1, FCN1, THBS2, UBA1 또는 ATIC는 0.90 이상의 값을 가지는 것을 확인하였다. First, as a result of evaluation using ROC (Receiver Operating Characteristic Curve), as shown in Figure 3, the AUC (area under the ROC curve) values of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 were all above 0.80. It was confirmed that it showed significant results, and in particular, SRPK1, FCN1, THBS2, UBA1 or ATIC was confirmed to have a value of 0.90 or higher.
또한 건강 대조군과 수술 전 대장암 환자군에서 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3의 발현값 차이를 비교하기 위해 각 값을 표준화(z-score)하여 Heat-map으로 표현하였다. 그 결과 도 4에 나타낸 바와 같이 7개 단백질은 건강 대조군 및 대장암 환자군 간에 유의한 차이를 보이는 것을 확인하였다.Additionally, to compare the differences in expression values of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, or PSMB3 between the healthy control group and the preoperative colon cancer patient group, each value was standardized (z-score) and expressed as a heat-map. As a result, as shown in Figure 4, it was confirmed that seven proteins showed significant differences between the healthy control group and the colon cancer patient group.
또한 대장암 종양 조직의 제거 전 및 제거 후 6주가 경과하였을 때의 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질 발현량을 ELISA 방법으로 비교한 결과, 도 5에 나타낸 바와 같이 종양 조직 제거 전 실험군(Preop) 대비, 대장암 종양 조직을 제거 후 6주가 경과한 실험군(Postop)에서 7개 단백질 모두 발현이 감소한 것을 확인하였다. 특히 SRPK1, FCN1, THBS2, UBA1, CCT8 및 PSMB3 단백질의 발현이 유의하게 감소한 것을 확인하였다. In addition, as a result of comparing the protein expression levels of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 before and 6 weeks after removal of colon cancer tumor tissue by ELISA method, tumor tissue was removed as shown in Figure 5. Compared to the previous experimental group (Preop), it was confirmed that the expression of all seven proteins decreased in the experimental group (Postop) 6 weeks after removal of colon cancer tumor tissue. In particular, the expression of SRPK1, FCN1, THBS2, UBA1, CCT8, and PSMB3 proteins was confirmed to be significantly reduced.
이러한 결과로, SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질은 대장암에 대한 진단 바이오마커뿐만 아니라, 대장암을 갖는 개체의 항암 치료에 대한 예후 예측용 바이오마커로서 유효하게 사용될 수 있다.As a result, SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins can be effectively used not only as diagnostic biomarkers for colon cancer, but also as biomarkers for predicting prognosis for anticancer treatment in individuals with colon cancer. .
실시예 4. 종양 표지자 CEA와의 비교Example 4. Comparison with tumor marker CEA
현재 임상에서 대장암 진단 또는 치료 효능 확인을 위해 사용되고 있는 CEA(carcinoembryonic antigen)와 실시예 3에서 검증한 7개 단백질을 비교하였다.The seven proteins verified in Example 3 were compared with CEA (carcinoembryonic antigen), which is currently used in clinical trials to diagnose or confirm treatment efficacy for colon cancer.
84명의 대장암 환자를 병기별로 Stage Ⅰ, Stage Ⅱ, Stage Ⅲ 및 Stage Ⅳ로 분류하고 각 병기에 따라 CEA 검출을 확인한 결과를 도 6에 나타내고 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3의 검출을 확인한 결과를 도 7에 나타냈다.84 colon cancer patients were classified by stage into Stage Ⅰ, Stage Ⅱ, Stage Ⅲ, and Stage Ⅳ, and the results of CEA detection according to each stage are shown in Figure 6, and the detection results of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, or PSMB3 The results confirming detection are shown in Figure 7.
CEA의 경우, 도 6에 나타낸 바와 같이, serum CEA값(왼쪽)과 진단율(오른쪽)을 확인한 결과 대장암의 진행이 낮은 단계일수록 검출량이 적고 진단율이 낮은 것으로 확인되었다. 특히 Stage Ⅰ의 초기 대장암에서 serum CEA값과 진단율은 미미한 것으로 나타났다.In the case of CEA, as shown in Figure 6, as a result of checking the serum CEA value (left) and diagnosis rate (right), it was confirmed that the lower the stage of colon cancer progression, the lower the detection amount and the lower the diagnosis rate. In particular, the serum CEA value and diagnosis rate were found to be insignificant in Stage 1 early colon cancer.
반면에 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3의 경우, 도 7에 나타낸 바와 같이, 4단계의 병기 모두에서 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질이 유의하게 발현되는 것을 확인하였다. 특히, Stage Ⅰ의 초기 대장암 환자군에서도 정상 대조군(Healthy)과 비교하여 유의미한 차이를 나타내는 것을 확인하였다.On the other hand, in the case of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3, as shown in Figure 7, SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 proteins are significantly expressed in all four stages of the disease. confirmed. In particular, it was confirmed that the Stage I early colon cancer patient group showed significant differences compared to the normal control group (Healthy).
이러한 결과로, 대장암 발병 초기 단계에서는 민감도가 낮게 나타나는 종래의 CEA(carcinoembryonic antigen)와 비교하여 본 발명의 일 실시예에 따른 엑소좀 유래 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 또는 PSMB3 단백질은 대장암 발병 초기부터 높은 민감도를 나타내므로 높은 정확도로 초기 대장암을 진단할 수 있다. As a result, compared to the conventional CEA (carcinoembryonic antigen), which has low sensitivity in the early stages of colon cancer development, the exosome-derived SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 or PSMB3 protein according to an embodiment of the present invention Since it shows high sensitivity from the beginning of colon cancer development, early colon cancer can be diagnosed with high accuracy.
또한 대장암 수술을 한 개체에 대해 혈액 검사만으로 장기적 추적을 통해 재발 여부에 따른 진단 마커로서의 효능을 기대할 수 있다.In addition, through long-term follow-up of individuals who have undergone colon cancer surgery using only a blood test, efficacy as a diagnostic marker can be expected depending on whether or not there is recurrence.
이상 첨부된 도면을 참조하여 본 발명의 실시예들을 더욱 상세하게 설명하였으나, 본 발명은 반드시 이러한 실시예로 국한되는 것은 아니고, 본 발명의 기술사상을 벗어나지 않는 범위 내에서 다양하게 변형 실시될 수 있다. 따라서, 본 발명에 개시된 실시예들은 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예에 의하여 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 그러므로, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 보호 범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.Although embodiments of the present invention have been described in more detail with reference to the accompanying drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be made without departing from the technical spirit of the present invention. . Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but are for illustrative purposes, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of protection of the present invention should be interpreted in accordance with the claims below, and all technical ideas within the equivalent scope should be construed as being included in the scope of rights of the present invention.
[이 발명을 지원한 국가연구개발사업] [National research and development project that supported this invention]
[과제고유번호] 1465037152 [Assignment number] 1465037152
[과제번호] HI22C0353000022 [Assignment number] HI22C0353000022
[부처명] 보건복지부 [Ministry Name] Ministry of Health and Welfare
[과제관리(전문)기관명] 한국보건산업진흥원 [Project management (professional) organization name] Korea Health Industry Development Institute
[연구사업명] 신진의사과학자 양성지원 [Research project name] Support for nurturing young medical scientists
[연구과제명] 대장암 원발조직 및 혈액 유래 세포밖 소포체의 단백체 [Research project name] Proteome of colorectal cancer primary tissue and blood-derived extracellular vesicles
분석을 통한 진단적 순환 종양 바이오마커의 발굴 및임상적유효성 실험검증 Discovery of diagnostic circulating tumor biomarkers through analysis and experimental verification of clinical effectiveness
[기여율] 1/1 [Contribution rate] 1/1
[과제수행기관명] 연세대학교 산학협력단 [Name of project carrying out organization] Yonsei University Industry-Academic Cooperation Foundation
[연구기간] 2022.07.01 ~ 2023.12.31 [Research period] 2022.07.01 ~ 2023.12.31
Claims (21)
- SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질; 또는 이를 암호화하는 적어도 하나의 유전자;를 포함하는,At least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins; Or at least one gene encoding the same;대장암 진단 또는 예후 예측용 바이오마커 조성물.Biomarker composition for diagnosing or predicting prognosis of colon cancer.
- 제1항에 있어서, According to paragraph 1,상기 단백질은 엑소좀 유래인 것인, The protein is derived from exosomes,대장암 진단 또는 예후 예측용 바이오마커 조성물.Biomarker composition for diagnosing or predicting prognosis of colon cancer.
- 제1항에 있어서, According to paragraph 1,상기 대장암은 초기 대장암을 포함하는,The colon cancer includes early colon cancer,대장암 진단 또는 예후 예측용 바이오마커 조성물.Biomarker composition for diagnosing or predicting prognosis of colon cancer.
- 제1항에 있어서,According to paragraph 1,상기 진단은 재발을 포함하는,The diagnosis includes recurrence,대장암 진단 또는 예후 예측용 바이오마커 조성물.Biomarker composition for diagnosing or predicting prognosis of colon cancer.
- SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질; 또는 이를 암호화하는 적어도 하나의 유전자;의 발현을 측정하는 제제를 포함하는, At least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins; Or at least one gene encoding the same; comprising an agent for measuring the expression of,대장암 진단 또는 예후 예측용 조성물.Composition for diagnosing or predicting prognosis of colon cancer.
- 제5항에 있어서, According to clause 5,상기 단백질은 엑소좀 유래인 것인, The protein is derived from exosomes,대장암 진단 또는 예후 예측용 조성물.Composition for diagnosing or predicting prognosis of colon cancer.
- 제5항에 있어서, According to clause 5,상기 단백질의 발현을 측정하는 제제는 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질에 특이적으로 결합하는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체, 키메릭(chimeric) 항체, 리간드, PNA(Peptide nucleic acid) 또는 앱타머(aptamer)인, Agents for measuring the expression of the protein include oligopeptides, monoclonal antibodies, and polyclonal antibodies that specifically bind to at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins. An antibody, chimeric antibody, ligand, peptide nucleic acid (PNA), or aptamer,대장암 진단 또는 예후 예측용 조성물.Composition for diagnosing or predicting prognosis of colon cancer.
- 제5항에 있어서,According to clause 5,상기 유전자의 발현을 측정하는 제제는 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질을 암호화하는 유전자에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머(쌍), 프로브, 소분자 화합물 또는 핵산 분자인 것인, The agent for measuring the expression of the gene is an antisense oligonucleotide, a primer (pair) that specifically binds to a gene encoding at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins. ), which is a probe, small molecule compound or nucleic acid molecule,대장암 진단 또는 예후 예측용 조성물.Composition for diagnosing or predicting prognosis of colon cancer.
- 제5항에 있어서, According to clause 5,상기 대장암은 초기 대장암을 포함하는,The colon cancer includes early colon cancer,대장암 진단 또는 예후 예측용 조성물.Composition for diagnosing or predicting prognosis of colon cancer.
- 제5항에 있어서, According to clause 5,상기 진단은 재발을 포함하는,The diagnosis includes recurrence,대장암 진단 또는 예후 예측용 조성물.Composition for diagnosing or predicting prognosis of colon cancer.
- 제5항 내지 제10항 중 어느 한 항의 조성물을 포함하는,Comprising the composition of any one of claims 5 to 10,대장암 진단 또는 예후 예측용 키트.Kit for diagnosing or predicting prognosis of colon cancer.
- 제11항에 있어서,According to clause 11,상기 키트는 RT-PCR 키트, 마이크로어레이 칩 키트, DNA 키트, ELISA 키트, 단백질 칩 키트 또는 래피드(rapid) 키트인, The kit is an RT-PCR kit, microarray chip kit, DNA kit, ELISA kit, protein chip kit, or rapid kit,대장암 진단 또는 예후 예측용 키트.Kit for diagnosing or predicting prognosis of colon cancer.
- 개체로부터 분리된 생물학적 시료에 대하여 엑소좀을 분리하는 단계;Isolating exosomes from a biological sample isolated from an individual;상기 분리된 엑소좀으로부터 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질을 수득하는 단계;Obtaining at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins from the isolated exosomes;상기 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자의 mRNA 발현 수준을 측정하는 단계; 및Measuring the mRNA expression level of at least one protein selected from the group consisting of the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins or at least one gene encoding the same; and상기 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자의 mRNA 발현 수준을 대조군 시료와 비교하는 단계;를 포함하는, Comprising the step of comparing the mRNA expression level of at least one protein selected from the group consisting of the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 and PSMB3 proteins or at least one gene encoding the same with a control sample,대장암 진단 또는 예후 예측 방법.Methods for diagnosing or predicting prognosis for colon cancer.
- 제13항에 있어서, According to clause 13,상기 시료는 종양 조직, 혈액, 혈청 및 혈장으로 이루어진 군에서 선택되는 적어도 하나에서 유래한 엑소좀인 것인,The sample is an exosome derived from at least one selected from the group consisting of tumor tissue, blood, serum and plasma,대장암 진단 또는 예후 예측 방법.Methods for diagnosing or predicting prognosis for colon cancer.
- 제13항에 있어서, According to clause 13,상기 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자의 mRNA 발현 수준이 대조군 시료 대비 높은 경우, 대장암인 것으로 판단하는, When the mRNA expression level of at least one protein selected from the group consisting of the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins or at least one gene encoding the same is higher than the control sample, it is judged to be colon cancer. ,대장암 진단 또는 예후 예측 방법.Method for diagnosing or predicting prognosis for colon cancer.
- 제15항에 있어서,According to clause 15,상기 개체는 대장암 의심 개체이고, 상기 대조군은 대장암이 발병되지 않은 정상 대조군인 것인, 대장암 진단 또는 예후 예측 방법.A method for diagnosing or predicting prognosis of colon cancer, wherein the object is a suspected colon cancer patient, and the control group is a normal control group that does not develop colon cancer.
- 제13항에 있어서,According to clause 13,상기 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질 또는 이를 암호화하는 적어도 하나의 유전자의 mRNA 발현 수준이 대조군 시료 대비 낮은 경우, 대장암 예후가 좋은 것으로 판단하는,When the mRNA expression level of at least one protein selected from the group consisting of the SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins or at least one gene encoding the same is lower than the control sample, the prognosis for colon cancer is good. judging,대장암 진단 또는 예후 예측 방법.Methods for diagnosing or predicting prognosis for colon cancer.
- 제17항에 있어서,According to clause 17,상기 개체는 대장암 진단 후 항암화학요법 또는 수술을 받은 개체이고, 상기 대조군 시료는 대장암 의심 개체 또는 대장암 보유 개체로부터 수득한 시료인 것인, The individual is an individual who has undergone chemotherapy or surgery after being diagnosed with colon cancer, and the control sample is a sample obtained from an individual suspected of colon cancer or an individual with colon cancer,대장암 진단 또는 예후 예측 방법.Methods for diagnosing or predicting prognosis for colon cancer.
- 제13항 또는 제15항에 있어서, According to claim 13 or 15,상기 대장암은 초기 대장암을 포함하는,The colon cancer includes early colon cancer,대장암 진단 또는 예후 예측 방법.Methods for diagnosing or predicting prognosis for colon cancer.
- 제13항 또는 제15항에 있어서,According to claim 13 or 15,상기 진단은 재발을 포함하는,The diagnosis includes recurrence,대장암 진단 또는 예후 예측 방법.Methods for diagnosing or predicting prognosis for colon cancer.
- 대장암이 발병된 개체로부터 채취한 시료에서 SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8 및 PSMB3 단백질로 이루어진 군에서 선택되는 적어도 하나의 단백질 발현 수준을 측정하는 단계; Measuring the expression level of at least one protein selected from the group consisting of SRPK1, FCN1, THBS2, UBA1, ATIC, CCT8, and PSMB3 proteins in a sample collected from an individual with colon cancer;상기 시료에 후보물질을 처리하고 상기 선택되는 단백질 발현 수준을 측정하는 단계; 및Processing the sample with a candidate material and measuring the expression level of the selected protein; and상기 시료에 후보물질을 처리 후 후보물질 처리 전보다 상기 선택되는 단백질 발현 수준이 감소한 경우 상기 후보물질을 대장암 예방 또는 치료용 제제로 선별하는 단계;를 포함하는, 대장암 예방 또는 치료용 제제의 스크리닝 방법.Screening of an agent for the prevention or treatment of colorectal cancer, including: selecting the candidate material as an agent for preventing or treating colon cancer when the expression level of the selected protein is reduced after treating the sample with the candidate material compared to before treatment with the candidate material. method.
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