KR102110338B1 - Novel FOLR2 Fusion Gene As Colon Cancer Diagnosis Marker and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel fusion gene and a use thereof as a colon cancer marker, a composition for diagnosing colorectal cancer comprising an RNF121-FOLR2 fusion protein, the fusion gene, or an agent measuring the transcript of the fusion gene; Kit for diagnosing colorectal cancer; A method of providing information for the diagnosis of colorectal cancer; Provided is a method for screening a therapeutic agent for colon cancer and a pharmaceutical composition for preventing or treating colorectal cancer comprising RNF121-FOLR2 fusion inhibitor as an active ingredient. The present invention provides a novel RNF121-FOLR2 fusion gene as a diagnostic marker specific for colorectal cancer, enabling accurate colorectal cancer diagnosis, predicting clinical prognosis through stratification of colorectal cancer patients, and treatment for colorectal cancer It becomes possible to predict the effectiveness of, and to predict the effectiveness of the treatment of colorectal cancer with an RNF121-FOLR2 inhibitor. In addition, accordingly, it is possible to limit the administration of the drug to a patient with colorectal cancer, which is considered to be ineffective, so that it is possible to realize efficient and safe customized colorectal cancer treatment.

Description

Novel FOLR2 Fusion Gene As Colon Cancer Diagnosis Marker and Uses Thereof as a Colon Cancer Diagnosis Marker

The present invention relates to a novel FOLR2 fusion gene as a diagnostic marker for colorectal cancer and its use.

Colorectal cancer is the third most common cancer in the world, and it accounts for the third in total cancer occurrence in Korea with a 12.3% incidence rate (Central Cancer Registration Headquarters, National Cancer Registration Business Annual Report (2013 cancer) Registration Statistics), Ministry of Health and Welfare, 2015).

If a benign polyp that can cause colon cancer is limited to epithelial cells, it can be treated with an endoscope, but if its size gradually increases to develop cancer, it can be combined with chemotherapy or radiation therapy after surgical resection. To proceed.

In the anti-cancer drug treatment, recently, a method of selectively attacking only cancer cells using a molecular targeted anti-cancer agent has been spotlighted (MacConail LE and Vink-Borger ME).

However, due to the lack of research on the molecular cell biological properties of colorectal cancer, there are limitations in developing a molecular targeted therapy for colorectal cancer and applying it to clinical trials.

Cancer cells have different biological properties than normal cells. Normal cells repeat cell cycles of about 50 times or apoptosis occurs when DNA is damaged. On the other hand, cancer cells have a characteristic that the chromosome is modified, apoptosis does not occur, and continues to divide without contact inhibition. In addition, cancer cells induce angiogenesis, and cancer may metastasize through blood supply.

The difference between these cancer cells and normal cells appears at the gene level as well as the cells, and chromosomal aberration is found in most cancer cells. In particular, it has been reported that there are about 45,000 chromosomal abnormalities in human neoplasm. Most cancer gene mutations are caused by chromosomal translocation, which causes fusion genes to be expressed by fusion of different genes.

That is, the fusion gene is a hybrid gene formed by chromosome rearrangement (translocation, deletion, inversion), trans-splicing, and read-through events. Fusion genes are known to be involved in the development and activation mechanisms of various tumors, including blood cancer, sarcoma, and prostate cancer (Nowell PC, and et al.). In addition, even if it is the same carcinoma, each patient has tumor heterogeneity, and the fusion gene can help to develop a molecular target drug by providing criteria for distinguishing cancer diversity for personalized treatment. There is Imatinib, a therapeutic agent for chronic myelogenous leukemia that selectively inhibits tyrosin kinase activated by the BCR-ABL fusion gene as a molecular target therapy using the fusion gene (Goldman JM and Melo JV), and Crizotinib in non-small cell lung cancer patients expressing the ROS1 fusion gene. It has been reported that the reaction rate is 50% higher when using (Forde PM and Rudin CM).

Therefore, specific mutations in genes can be used as useful information for predicting and diagnosing cancer.

Accordingly, the present inventors have made great efforts to develop a carcinogenic fusion gene as a marker that can be a target of diagnosis, prognosis, and treatment of colorectal cancer through genomic studies of colorectal cancer. As a result, the present inventors performed RNA-sequencing based on NGS (next-generation sequencing) using tissues of a large number of colorectal cancer patients, and screened for carcinogenic fusion genes expressed only in colorectal cancer tissues, of which new novel fusion genes FOLR2 (hereinafter, RNF121-FOLR2) was selected. The RNF121-FOLR2 fusion gene and fusion transcripts and fusion proteins encoded therein are factors that determine clinical prognosis as well as diagnosis of colorectal cancer, and when it is overexpressed in cancer cells and normal cells, by confirming that the survival rate of the cells increases , The present invention has been completed.

Accordingly, it is an object of the present invention to provide a fusion protein of RNF121 and FOLR2 and a fusion gene polynucleotide encoding the same.

In addition, another object of the present invention is to provide a composition for diagnosing colorectal cancer comprising an agent for measuring the fusion protein, the fusion gene, or the transcript of the fusion gene.

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

Another object of the present invention is to provide a method for providing information necessary for the diagnosis of colorectal cancer, comprising the step of measuring the level of the RNF121-FOLR2 fusion gene, its transcript, or its protein.

Another object of the present invention is to provide a method for providing information necessary to predict the prognosis of colorectal cancer treatment, comprising measuring the level of the RNF121-FOLR2 fusion gene, its transcript, or its protein.

Another object of the present invention is to provide a method for screening for a treatment for colorectal cancer comprising the step of measuring the expression level of the RNF121-FOLR2 fusion gene.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising an RNF121-FOLR2 fusion protein inhibitor as an active ingredient.

Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, the present invention provides a fusion protein of RNF121 (Ring Finger Protein 121) and FOLR2 (Folate receptor beta) and a fusion gene polynucleotide encoding the same.

Specifically, the present invention provides a fusion protein composed of the following structural formula:

N- [fragment containing the N-terminal of RRN121 (Ring Finger Protein 121)]-[fragment containing the C-terminal of FOLR2 (Folate receptor beta)]-C.

In addition, the present invention provides a fusion gene polynucleotide consisting of the following structural formula:

5 '-[fragment containing the 5' end of the RRN121 (Ring Finger Protein 121) gene]-[fragment containing the 3 'end of the FOLR2 (Folate receptor beta) gene] -3'.

That is, the present invention is a fusion protein in which a fragment comprising the N-terminal of RNF121 and a fragment comprising the C-terminal of FOLR2 are linked; And a fragment containing the 5 'end of the RNF121 gene and a fragment containing the 3' end of the FOLR2 gene, wherein the RNF121 or FOLR2 may be human. The fusion protein of the present invention can be mixed with the RNF121-FOLR2 fusion protein.

The present inventors first discovered a fusion example of the RNF121 protein and the FOLR2 protein in human colorectal cancer patients, as shown in the Examples of the present invention. In addition, it was confirmed that the cell proliferation capacity is increased in the case of cells having a fusion of RNF121 gene and FOLR2 gene and a fusion transcription product and a fusion protein encoded therein.

In the present invention, the RNF121 gene encoding the RNF121 protein may be derived from human and is located on human chromosome 11 (q13.4), and the encoded RNF121 protein is a polypeptide having a total amino acid length of 327aa. The RNF121 protein or RNF121 protein fragment is an N-terminal fusion partner of the RNF121-FOLR2 fusion protein.

For example, RNF121 gene is GenBank accession no. It may be one having a nucleotide sequence provided by NM_018320, and the RNF121 protein may be a protein having an amino acid sequence encoded by NM_018320. In particular, the RNF121 protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, and the RNF121 gene may be composed of the nucleic acid sequence of SEQ ID NO: 2, but is not limited thereto.

In the present invention, the fragment comprising the N-terminal of RNF121 has an amino acid sequence encoded by the nucleotide sequence up to the first exon of NM_018320 (71639768 to 71640170 base position based on the (+) strand on chromosome 1). May be, in particular, the fragment containing the N-terminal of RNF121 may be composed of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

In the present invention, the FOLR2 gene encoding the FOLR2 protein may be derived from human and is located on human chromosome 11 (q13.4), and the encoded FOLR2 protein is a polypeptide having a total amino acid length of 255aa. The FOLR2 protein or FOLR2 protein fragment is a C-terminal fusion partner of the RNF121-FOLR2 fusion protein.

For example, the FOLR2 gene is GenBank accession no. It may be one having a nucleotide sequence provided in NM_000803, and the FOLR2 protein may be a protein having an amino acid sequence encoded by NM_000803. In particular, the FOLR2 protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, and the FOLR2 gene may be composed of the nucleic acid sequence of SEQ ID NO: 6, but is not limited thereto.

In the present invention, the fragment comprising the N-terminus of FOLR2 has an amino acid sequence encoded by the nucleotide sequence up to the third exon of NM_000803 (base positions 71931914 to 71932994 based on the (+) strand on chromosome 1). May be, in particular, the fragment comprising the N-terminal of FOLR2 may be composed of the amino acid sequence of SEQ ID NO: 8, but is not limited thereto.

In the present specification, break points (or fusion sites) of fragments of a protein that is a fusion partner are described based on exons of genes encoding them, and the N-terminal of the fragment (in the case of a C-terminal fusion partner). Or, at any point in the intron region between the exon contained at the end of the C-terminal (for the N-terminal fusion partner) and the exon that is cleaved and removed, the amino acid sequence of the encoded protein fragment is not affected. The point to be cut may be any of the intron positions.

Meanwhile, in the present invention, the RNF121-FOLR2 fusion gene may have the nucleotide sequence of SEQ ID NO: 9, and the RNF121-FOLR2 fusion protein may have the sequence of SEQ ID NO: 10, but is not limited thereto.

Unless stated otherwise in the present invention, all nucleotide sequences determined by sequencing a gene, transcript or transcript derived cDNA or DNA molecule described herein can be determined using an automated next generation sequencing sequencer or a Sanger sequencing sequencer. And all amino acid sequences encoded by the determined nucleotide sequence can be determined using an automated peptide sequencer. The nucleotide sequence determined by this automated approach may contain some errors compared to the actual sequence. For example, the nucleotide sequences determined by automatic are typically about 90% or more, specifically about 95% or more, more specifically about 99% or more, more specifically about 99.9% or more with the actual nucleotide sequence of the sequenced cDNA or DNA molecule. It can have homology. A single insertion or deletion may be included in a nucleotide determined in comparison with an actual sequence, and such an insertion or deletion causes a frameshift in nucleotide sequence translation, so that the encoded amino acid sequence is completely identical to the actual amino acid sequence. Can be different.

According to another aspect of the present invention, the present invention provides a composition for diagnosing colorectal cancer comprising an agent measuring the fusion protein, the fusion gene, or the transcript of the fusion gene.

In the present invention, the fusion protein and fusion gene are as described above.

In the present invention, the term "diagnosis" means to identify the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether or not colorectal cancer has occurred.

In the present invention, the term, "colon cancer (colon cancer)" is a cancer that occurs in the mucosa, the innermost surface of the large intestine, and refers to a general term for rectal cancer, colon cancer, and anal cancer.

Meanwhile, in the present invention, the RNF121-FOLR2 fusion protein and fusion gene may be used as a diagnostic marker for diagnosing colorectal cancer. In the present invention, the term "diagnostic marker, diagnostic marker, or diagnostic marker" is called colon cancer. This is a substance that can be diagnosed by distinguishing cells from normal cells. Polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides) that show an increased pattern in cells with colorectal cancer compared to normal cells Organic biomolecules such as disaccharides, oligosaccharides, and the like). For the purposes of the present invention, the colon cancer diagnostic marker is an RNF121-FOLR2 fusion gene, a gene whose expression is increased in colon cancer tissues or cells.

The selection and application of significant diagnostic markers determines the reliability of the diagnostic results. Significant diagnostic marker means a marker with high reliability so that the result obtained by diagnosis is accurate and thus has high validity and consistent results even in repeated measurements. The colon cancer diagnostic marker of the present invention shows the same result in repeated experiments with genes whose expression is always increased as a direct or indirect factor with the onset of colorectal cancer, and the difference in expression level is very large compared to the control group, and the wrong result These are highly reliable markers with little probability of falling. Therefore, the results diagnosed based on the results obtained by measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable. At this time, except for genes that are expressed in almost the same amount in normal colorectal epithelial cells and colorectal cancer cells, for example, genes whose expression is more than doubled in colorectal cancer tissue compared to genes expressed in normal tissue used as a control. You can sort them out.

In a specific embodiment of the present invention, the fusion gene of the RNF121 gene and the FOLR2 gene is a causative gene in colorectal cancer, and it was confirmed that cell proliferation and mobility are activated by the fusion.

Therefore, the fusion gene or fusion transcript and fusion protein encoded therein were found to be specifically found or expressed in patients with solid cancer, specifically colorectal cancer, and thus the fusion gene or fusion transcript and fusion protein encoded therein Suggests that it can be usefully used as a diagnostic marker for prognostic prediction of solid cancer, specifically colorectal cancer, and can also increase the likelihood of treatment with the fusion protein inhibitor.

The level of expression of the fusion gene of the present invention in a biological sample can be confirmed by confirming the amount of the fusion protein of the fusion gene, particularly mRNA. Preferably, the agent for measuring the level of the fusion gene mRNA may be a composition for diagnosing colorectal cancer that includes a primer specifically binding to the gene.

In the present invention, "measurement of mRNA expression level" refers to a process of confirming the presence and expression level of mRNA of colorectal cancer marker genes in a biological sample in order to diagnose colorectal cancer. Analysis methods for this include reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction (Real-time RT-PCR), and RNase protection assay (RPA; RNase protection) assay), Northern blotting, DNA chip, etc., but are not limited thereto.

In the present invention, the primer specifically binding to the mRNA of the fusion gene is a primer that specifically binds to the mRNA of the fusion gene so that the region amplified by the primer includes the fusion point of two genes to be fused. It may be a primer designed to be interposed therebetween, and in the case of the RNF121 gene and the FOLR2 gene, two genes that are fusion targets constituting the fusion gene encode a primer specific to the base sequence encoding the N terminus of the RNF121 protein and the C terminus of the FOLR2 C terminus. It is a primer pair containing a primer specific to the base sequence, and may be, in particular, a primer pair composed of the base sequences of SEQ ID NO: 17 and SEQ ID NO: 18, but is not limited thereto.

On the other hand, the "primer" of the present invention refers to a short nucleic acid sequence that can form a complementary template and base pair with a nucleic acid sequence having a short free 3-terminal hydroxyl group and functions as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of a reagent for a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) at a suitable buffer and temperature and four different nucleoside triphosphates. The primers of the present invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific to each marker gene. Primers can incorporate additional features that do not change the basic properties of the primer, which serves as the starting point for DNA synthesis.

Primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well-known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, "capsulation", substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg methyl phosphonate, phosphotriester, phos. Poroamidate, carbamate, etc.) or modifications to charged linkages (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acids are one or more additional covalently attached residues, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (e.g., acridine, psoralen, etc.) ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequence of the present invention can also be modified using a label that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

In the present invention, "an agent that measures a fusion gene" is a process of confirming the presence of the fusion gene of the present invention, which is a colon cancer marker gene, in a biological sample to diagnose colorectal cancer, for example, specifically for the fusion gene. It may be to include a binding probe or primer. Any probe or primer can be used as long as it can specifically bind to the fusion gene.

In the present invention, "measurement of protein expression level" is a process of confirming the presence and expression level of a protein expressed from the fusion gene of the present invention, which is a colon cancer marker gene, in a biological sample to diagnose colorectal cancer, preferably, The amount of protein can be confirmed using an antibody that specifically binds to the protein of the fusion gene.

In particular, in the present invention, the antibody that specifically binds to the protein of the fusion gene may be bound to the C-terminal of FOLR2 or near the fusion site of RNF121 and FOLR2, but is not limited thereto. As an analysis method for this, Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (FACS), protein chip, etc., but are not limited to this. .

Preferably, the agent for measuring the level of the protein may be a composition for diagnosing colorectal cancer that includes an antibody specific for the protein.

In the present invention, "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.

As described above, in the present invention, a novel colorectal cancer marker fusion protein has been identified, and thus an antibody can be generated using the same can be easily produced using techniques well known in the art.

The polyclonal antibody can be produced by a method well known in the art to obtain the serum containing the antibody by injecting the above-mentioned colon cancer marker protein antigen into an animal and collecting blood from the animal. Such polyclonal antibodies can be produced from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, cow dog.

Monoclonal antibodies are hybridoma methods well known in the art (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624 -628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

In addition, the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.

 In another aspect of the present invention, a kit for diagnosing colorectal cancer comprising the composition for diagnosing colorectal cancer according to the present invention is provided.

Specifically, the kit for diagnosing colorectal cancer may be a reverse electron polymerase-PCR (RT-PCR) kit, a DNA chip kit, or a protein chip kit. In particular, the kit for diagnosing colorectal cancer may further include one or more other component compositions, solutions, or devices suitable for analytical methods.

As an embodiment, the diagnostic kit may be a diagnostic kit characterized by including essential elements necessary to perform a reverse transcriptase reaction. The reverse transcriptase reaction kit includes each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 bp to 50 bp long, more preferably about 10 bp to 30 bp long. In addition, primers specific to the nucleic acid sequence of the control gene may be included. Other reverse transcriptase reaction kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -It may include DEPC-water, sterile water, and the like.

In another embodiment, it may be a diagnostic kit, characterized in that it comprises the necessary elements necessary to perform the DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, and the like for preparing a fluorescent marker probe. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In addition, preferably, it may be a diagnostic kit, characterized in that it comprises the essential elements necessary to perform a protein chip or to perform an ELISA. Protein chip kits or ELISA kits contain antibodies specific for the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. In addition, the ELISA kit may include antibodies specific to the control protein. Other ELISA kits are capable of binding reagents capable of detecting bound antibodies, e.g. labeled secondary antibodies, chromophores, enzymes (e.g. conjugated with antibodies) and substrates or antibodies thereof Other materials and the like.

According to another aspect of the present invention, the present invention provides information necessary for diagnosing colorectal cancer, including measuring the level of the RNF121-FOLR2 fusion gene, a transcript thereof, or a protein thereof, in order to provide information necessary for diagnosing colorectal cancer. It provides a way to provide. Specifically, measuring the level of the RNF121-FOLR2 fusion gene, its transcript or its protein from an isolated sample of an individual suspected of having colon cancer; And comparing the measured fusion gene, its transcript, or its protein expression level with that of a normal control sample, the fusion gene, its transcript, or its protein expression level. to provide.

In the present invention, the RNF121-FOLR2 fusion gene is a polynucleotide constituting the 5 'end of the RNF121 gene; And a 3 ′ end of the FOLR2 gene.

In the present invention, "individual" is an animal that may develop colon cancer, and is not limited as long as colon cancer is suspected. In addition, as a disease in which information necessary for diagnosis can be obtained through the above method, the expression of the fusion gene between the RNF121 gene and the FOLR2 gene is not limited, and may be colon cancer in particular.

In the present invention, the term, a sample of an individual is a tissue having different levels of mRNA or protein of the RNF121-FOLR2 fusion gene, a colon cancer marker, for example, cells, tissues, organs, body fluids (blood, lymphatic fluid, etc.), digestive fluid, Includes sputum, alveolar-bronchial lavage fluid, urine, feces, as well as nucleic acid extracts (genetic extracts, transcript extracts, cDNA preparations or aRNA preparations prepared from transcript extracts) or protein extracts from their biological samples, etc. It includes, but is not limited to. do. Further, the sample may be subjected to formalin fixation treatment, alcohol fixation treatment, freezing treatment, or paraffin embedding treatment. In addition, genes, transcripts, cDNA, or proteins can be prepared by selecting a known method suitable therein, taking into account the type and condition of the sample.

Through the above detection methods, the expression level of the RNF121-FOLR2 fusion gene, its transcript, or its protein in the normal control group can be compared with the expression level in the suspected colorectal cancer to diagnose whether or not the colorectal cancer is suspected. have. That is, after measuring the expression level of the marker of the present invention for cells suspected of colorectal cancer, and measuring the expression level of the marker of the present invention for normal cells, and comparing the two, the expression level of the marker of the present invention is normal If it is expressed more from a cell that is supposed to be colon cancer than that of a cell, it is possible to determine a cell suspected to be colon cancer as colon cancer. That is, in the above method, when the level of expression of the fusion gene, its transcript, or its protein, measured from an isolated sample of an individual suspected of colon cancer is higher than that of the normal control sample, the level of expression of the fusion gene, its transcript, or its protein, It may be characterized by being diagnosed with cancer.

As an analytical method for measuring the level of mRNA of RNF121-FOLR2 fusion gene or protein thereof, Western blot, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunity proliferation method, rocket immunoelectrophoresis, tissue immunostaining, There are, but are not limited to, immunoprecipitation assays, complement fixation assays, FACS, protein chips, and the like. Through the above analysis methods, the amount of formation of the antigen-antibody complex in the normal control group and the amount of formation of the antigen-antibody complex in a suspected colorectal cancer patient can be compared, and a significant difference from the colorectal cancer marker gene to the RNF121-FOLR2 fusion protein. By determining whether the amount of expression is increased, it is possible to diagnose whether a person with suspected colorectal cancer actually develops colorectal cancer.

In the present invention, the term antigen-antibody complex means a combination of a colon cancer marker protein and an antibody specific to it, and the amount of formation of the antigen-antibody complex can be quantitatively measured through the magnitude of the signal of the detection label. .

The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but is not limited thereto. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholines Terase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphenolpyruvate deca Voxylase, β-lactamase, and the like. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoerytherin, phycocyanin, allopicocyanine, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. The emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. The microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) 3] 2+, [RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like, but are not limited thereto. Radioisotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re.

The protein expression level is preferably measured by ELISA. ELISA is a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, an indirect ELISA using a labeled antibody recognizing a capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen from the complex of the antibody and antigen, labeled with the antibody attached to the solid support, and reacted with another antibody that recognizes the antigen from the complex of the antigen It includes various ELISA methods, such as indirect sandwich ELISA using a secondary antibody. More preferably, for attaching an antibody to a solid support and reacting a sample, then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex to enzymatically develop an antibody or recognize an antigen of the antigen-antibody complex It is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and enzymatically developed. By confirming the degree of formation of a complex of the colon cancer marker protein and the antibody, it can be confirmed whether or not colon cancer has occurred.

In addition, preferably, Western blot using one or more antibodies against the colon cancer marker is used. After separating the whole protein from the sample, and electrophoresis to separate the protein according to size, it is transferred to a nitrocellulose membrane and reacted with an antibody. The amount of the protein generated by the expression of the gene can be confirmed by confirming the amount of the antigen-antibody complex produced by using the labeled antibody. The detection method consists of a method of examining the expression level of the marker gene in the control group and the expression level of the marker gene in the cell having colon cancer. Levels of peptide fragments can be expressed as absolute (eg, μg / ml) or relative (eg, relative intensity of signals) differences of the marker proteins described above.

In addition, preferably, one or more antibodies against the colon cancer marker are arranged at a predetermined position on the substrate to use a protein chip immobilized with high density. In the method of analyzing a sample using a protein chip, a protein is separated from a sample, and the separated protein is hybridized with a protein chip to form an antigen-antibody complex, and read and read to confirm the presence or expression level of the protein. It can be confirmed whether or not colon cancer has occurred.

Specifically, in order to detect the fusion protein, fusion gene or transcript in a clinical sample of a suspected colon cancer subject of the present invention, (a) the fusion protein in the clinical sample, corresponding to the fusion gene encoding the fusion gene and the fusion gene Treating (adding) and reacting a substance interacting with at least one selected from the group consisting of transcription products; And (b) detecting a reactant obtained through the above process. The step of preparing a clinical sample prior to step (a) may further include, and the step of preparing the clinical sample may include obtaining (separating) a clinical sample from a suspected colon cancer subject. In the step (a), the interacting substance is a compound, antibody, aptamer and the fusion gene or transcript (specifically or partially; for example, fusion) that specifically bind to the fusion protein (all or part; for example, a fusion site). Site) may be one or more selected from the group consisting of nucleic acid molecules (eg, primers, probes, aptamers, etc.), compounds, and the like. These interacting substances may be attached with one or more immunochemical labeling substances selected from the group consisting of free radicals, radioactive-isotopes, fluorescent dyes, chromogenic substrates, enzymes, bacteriophage, coenzymes, etc., or used together with the labeling substances. . In step (b), the reactant is a complex produced by interaction (binding) of one or more substances selected from the group consisting of the fusion protein, fusion gene, and transcript obtained in step (a) and a substance interacting therewith. (complex). When a reactant is detected in the step of detecting the reactant, it may be determined that the fusion protein, fusion gene or transcript is present.

The fusion gene encoding the RNF121-FOLR2 fusion protein or a substance that interacts with the corresponding transcript may be a nucleic acid molecule capable of hybridizing with the fusion gene or transcript. For example, the nucleic acid molecule is an antisense oligonucleotide (eg, siRNA, microRNA) capable of specifically hybridizing with the fusion gene, the fusion site of the fusion gene, or a transcript corresponding to the fusion gene or fusion site in a clinical sample. , Probe (eg, 5 to 100 bp, 5 to 50 bp, 5 to 30 bp, or 5 to 25 bp), aptamer, or the like. For example, a fusion gene encoding the fusion protein or a nucleic acid molecule capable of hybridizing with a transcript molecule corresponding to the fusion gene is 50 to 250 consecutively containing a fusion site in the fusion gene, specifically 100 to 200 bases 20 to 100 bp, 20 to 100 bp, or 25 to 50 bp in length capable of hybridizing with 20 to 100, specifically 25 to 50 nucleotide sequences or complementary sequences adjacent to both ends of the polynucleotide fragment to amplify the composed polynucleotide fragment It may be a primer pair. The 'hybridization is possible' means that the detection is performed by completely complementing the sequence to be detected, or having a sequence that is complementary to 80% or more (eg 80-100%), specifically 90% or more (eg 90-100%). It means that it can specifically bind to the target gene or transcript.

In another example, the substance that specifically interacts with the RNF121-FOLR2 fusion gene or a transcript thereof is one selected from the group consisting of free radicals, radioisotopes, fluorescent dyes, chromogenic substrates, enzymes, bacteriophage, and coenzymes. The above immunochemical labeling substances may be attached or used together with the labeling substances. For example, a primer pair for polymerase chain reaction can be used for each fusion gene.

In another example, detection of the RNF121-FOLR2 fusion protein is a substance that interacts with the RNF121-FOLR2 protein that is dependent on the fusion of the RNF121-FOLR2 gene in the colon protein or colon cancer, such as a substance that specifically binds (e.g. , Antibody, aptamer or compound, and the like, a conventional assay for detecting the interaction between the fusion protein and the substance (eg, antibody or aptamer), that is, complex formation, such as immunochromatography , Immunohistochemistry, enzyme linked immunosorbent assay, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay , Western blotting and flow cytometry cell sorting.

According to another aspect of the present invention, the present invention provides information required to predict the prognosis of colon cancer treatment, comprising measuring the level of an RNF121-FOLR2 fusion gene, a transcript thereof, or a protein thereof from an isolated sample of a colon cancer patient. It provides a way to provide.

Specifically, the present invention comprises the steps of measuring the level of the RNF121-FOLR2 fusion gene, its transcript or its protein from an isolated sample of colorectal cancer patients; And comparing the measured fusion gene, its transcript or protein expression level with the fusion gene, its transcript or protein expression level of the normal control sample.

The steps of measuring the RNF121-FOLR2 fusion gene, its transcript, or its protein level are almost the same as those described in the method for providing information necessary for the diagnosis of colorectal cancer.

However, the side of the sample to be measured is an isolated sample of a colon cancer patient rather than an isolated sample of an individual suspected of having colon cancer, and the normal control group in the present invention is a side group of the normal group and the colon cancer patient group in which the RNF121-FOLR2 fusion gene is not expressed. There is a difference.

In the present invention, a method for providing information necessary to predict the prognosis of treatment for colorectal cancer is a fusion gene of a RNF121-FOLR2 fusion gene, a transcript thereof or a protein expression level of a normal control sample from a separated sample of a colorectal cancer patient, a transcript thereof Or it may be characterized by predicting that the prognosis of colorectal cancer treatment is worse than when it is higher or lower than the protein expression level thereof. In addition, comparing the level of expression of the RNF121-FOLR2 fusion gene, its transcript, or its protein includes determining whether the RNF121-FOLR2 fusion gene, its transcript, or its protein is expressed. That is, the RNF121-FOLR2 fusion gene, its transcript, or its protein, includes those predicting that the colorectal cancer patient has a poorer prognosis for colorectal cancer than the unexpressed colorectal cancer patient.

In addition, in the present invention, a method of providing information necessary for predicting the prognosis of colorectal cancer treatment is a fusion gene, a transcript thereof, or a protein expression level thereof from an isolated sample of a colorectal cancer patient, a fusion gene, a transcript thereof, or a normal control sample When it is higher than its protein expression level, it may be characterized by predicting that the efficacy of RNF121-FOLR2 inhibitor (RNF121-FOLR2 fusion gene; its transcript or an inhibitor that inhibits protein expression or activity thereof) is high in treating colon cancer. .

On the other hand, in the present invention, a method of providing information necessary to predict the prognosis of colorectal cancer treatment is performed by treating an RNF121-FOLR2 inhibitor on isolated cells of a colorectal cancer patient, thereby increasing the level of the RNF121-FOLR2 fusion gene, its transcript, or its protein. It may be to include a step of measuring.

As an 'RNF121-FOLR2 inhibitor' which is a target for evaluating the effectiveness of treatment for colorectal cancer in the present invention, the RNF121-FOLR2 fusion gene; There is no particular limitation as long as it is a substance capable of directly or indirectly inhibiting its transcription product or its protein expression or activity.

According to another aspect of the present invention, the present invention provides a screening method of a treatment for colorectal cancer comprising the step of measuring the expression level of the NTRK1 fusion gene, specifically RNF121-FOLR2 fusion gene as a candidate for the treatment of colorectal cancer Treating the expressing cells; And measuring the expression level of the RNF121-FOLR2 fusion gene in the cell.

In the present invention, the RNF121-FOLR2 fusion gene is as described above.

In the present invention, measuring the expression level of the RNF121-FOLR2 fusion gene includes a method of measuring the level of the transcript of the fusion gene and a protein thereof, and a method or fusion of measuring the level of the transcript of the fusion gene. The method of measuring the protein level is as described above.

The screening method of the therapeutic agent for colorectal cancer of the present invention may be determined as a treatment for colorectal cancer, particularly when the expression level of the RNF121-FOLR2 fusion gene is lowered by treatment with a candidate for treatment of colorectal cancer.

Specifically, a method for comparing the increase or decrease in the expression of the RNF121-FOLR2 fusion gene in the presence and absence of a candidate for treatment for colorectal cancer can be usefully used to screen for the treatment of colorectal cancer. A substance that indirectly or directly reduces the concentration of the RNF121-FOLR2 fusion gene, or a protein expressed therefrom, can be selected as a therapeutic agent for colorectal cancer. That is, the expression level of the marker RNF121-FOLR2 fusion gene of the present invention in colorectal cancer cells in the absence of a candidate for treatment for colorectal cancer is measured, and the presence of the marker RNF121-FOLR2 fusion gene of the present invention in the presence of a candidate for treatment of colorectal cancer After measuring the expression level and comparing the two, a substance in which the expression level of the marker of the present invention when the candidate for treatment for colorectal cancer is present is lower than the marker expression level in the absence of the candidate for treatment for colorectal cancer is predicted as a therapeutic agent for colorectal cancer. It is possible.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising an RNF121-FOLR2 inhibitor as an active ingredient.

The RNF121-FOLR2 inhibitor is an RNF121-FOLR2 fusion gene; As an inhibitor that inhibits the expression or activity of its transcript or protein, it can act as a fusion protein or suppress the expression of a fusion gene. As described above, the RNF121-FOLR2 fusion protein inhibitor is not particularly limited as long as it is a substance capable of directly or indirectly inhibiting the function of the RNF121-FOLR2 gene or protein.

In addition, the RNF121-FOLR2 inhibitor is small interference RNA (siRNA), short hairpin RNA (shRNA), miRNA (microRNA), ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense oligonucleotides, antibodies, aptamers , It may be one or more selected from the group consisting of natural extracts and chemicals.

If the antibody has a polyclonal antibody, a monoclonal antibody or antigen-binding property, a part of it is included in the antibody of the present invention and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies, and antibodies known in the art may be included in addition to novel antibodies.

The composition of the present invention may be administered together with a pharmaceutically acceptable carrier, and when administered orally, a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, pigment, flavor, etc. may be used. In the case of injections, buffers, preservatives, painless agents, solubilizers, isotonic agents, stabilizers, etc. can be used in combination. For topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the composition of the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, it may be prepared in unit dosage ampoules or multiple dosage forms.

In another aspect, the present invention provides a method for preventing or treating colon cancer, comprising administering a pharmaceutical composition for preventing or treating colon cancer comprising the RNF121-FOLR2 inhibitor as an active ingredient to an individual.

RNF121-FOLR2 inhibitors, individuals and compositions of the invention are as described above.

Although the composition of the present invention can be used alone, radiation therapy or chemotherapy (cell growth arrest or cytotoxic substances, antibiotic substances, alkylating agents, anti-metabolites, hormones, immunizing agents, interferon types) to increase treatment efficiency Substances, cyclooxygenase inhibitors, metallomatrix protease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth receptor substances, anti-HER substances, anti-EGFR substances, anti-angiogenic substances, farnesyl transferase inhibitors , ras-raf signal conduction pathway inhibitor, cell cycle inhibitor, other cdk inhibitor, tubulin conjugate, topoisomerase I inhibitor, topoisomerase II inhibitor, and the like).

The term used in the present invention, administration means to introduce the composition of the present invention to the patient in any suitable way, the administration route of the composition of the present invention is selected according to the type of the inhibitor or the type of colorectal cancer, but the target tissue It can be administered via any general route as long as it can be reached. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraperitoneal administration, intraperitoneal administration, intrathecal administration may be performed, but are not limited thereto. Does not.

The effective dosage range of the composition of the present invention includes gender, body surface area, type and severity of disease, age, sensitivity to drugs, administration route and discharge rate, administration time, treatment period, target cells, expression level, and other medical fields. It can be varied according to various factors well-known to, and can be easily determined by experts in the field.

The present invention provides a novel RNF121-FOLR2 fusion gene as a diagnostic marker specific for colorectal cancer, enabling accurate colorectal cancer diagnosis, predicting clinical prognosis through stratification of colorectal cancer patients, and treatment for colorectal cancer It becomes possible to predict the effectiveness of, and to predict the effectiveness of the treatment of colorectal cancer with an RNF121-FOLR2 inhibitor. In addition, accordingly, it is possible to limit the administration of the drug to a patient with colorectal cancer, which is considered to be ineffective, so that it is possible to realize efficient and safe customized colorectal cancer treatment.

1 shows the fusion gene selection criteria.
Figure 2 shows the results of confirming the fusion gene connection site.
(M; DNA marker, 1; B4GALT1-BAG1, 2; GTF3A-CDK8, 3; NAGLU-IKZF3, 4; RNF121-FOLR2, 5; STRN-ALK, 6; TAF4-NDRG3)
Figure 3 shows the Sanger sequencing results of the fusion gene.
(The arrow means the cut point)
Figure 4 shows the schematic structure of the RNF121-FOLR2, TAF4-NDRG3 fusion gene.
5 shows the expression of the receptor gene and the fusion gene in the colorectal cancer cell line.
6 shows the Sanger sequencing results of the RNF121-FOLR2 fusion gene expressed in the HT29 cell line.
7 shows the results of cell proliferation when overexpressing the RNF121-FOLR2 fusion gene in colorectal cancer cell lines (DLD-1 and SW620). The MTT assay was repeated three times. EV; Empty vector, RF; RNF121-FORL2. (*; p <0.05 versus EV in a Student's t-test. #; p <0.05 versus each fusion gene expression vector in a Student's t-test)
Figure 8 shows the cell proliferation results when overexpressing the RNF121-FOLR2 fusion gene in the normal fibroblast line (NIH-3T3). The MTT assay was repeated three times. EV; Empty vector, RF; RNF121-FORL2. (*; p <0.05 versus EV in a Student's t-test. #; p <0.05 versus each fusion gene expression vector in a Student's t-test)

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Colon cancer As a marker New  Selection of candidates for fusion genes

In order to develop a marker for diagnosing colorectal cancer, the present inventors identified and selected a fusion gene that is specific for colorectal cancer through a large-scale colon cancer patient cohort analysis.

1-1. RNA-sequencing

The present inventors used patient tissues that were operated under the diagnosis of colorectal cancer at Pusan National University Hospital and Hwasun Chonnam National University Hospital from 2008 to 2012. Of the 150 patient tissues, 50 were also analyzed for normal tissues. Total RNA was extracted from patient tissue using RNAiso Plus (Takara Bio, Japan) and RNA-seq library was generated with TruSeq RNA sample Preparation Kit (Illumina, USA). The cDNA library was sequenced with HiSeq 2000 (Illumina, USA) and analyzed according to the human reference genome hg19 version.

In addition, the present inventors found in-frame shift fusion genes that do not appear in normal tissues using the GFP algorithm, and among them, spanning reads were 10 or more, and fusion genes having a distance between chromosomes of 85 kb or more were cross-verified by deFuse and FusionMap algorithms.

More specifically, RNA-sequencing was performed using 150 Korean colorectal cancer tissues, and among the 2460 fusion genes found by the GFP algorithm, 101 fusion genes were not expressed in normal tissue while maintaining the in-frame shift. Among them, 25 fusion genes with spanning reads of 10 or more and a distance between chromosomes of 85 kb or more are applied with deFuse and FusionMap algorithms for cross-validation, so 20 fusion genes appearing in common among the three algorithms, 21 GFP, 21 common deFuses, and GFP , FusionMap has 24 common. FPKM values and functions of receptor genes among 25 fusion genes from at least two algorithms were analyzed.

As a result, as shown in Figure 1 and Table 1, the final candidate oncogenic fusion gene 6 species (B4GALT1-BAG1, GTF3A-CDK8, NAGLU-IKZF3, RNF121-FOLR2, STRN-ALK) according to the expression value and function of the receptor gene , TAF4-NDRG3).

1-2. Fusion Gene Verification

The present inventors performed reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing using cDNA of a patient sample expressing a fusion gene to verify a fusion gene transcript.

At this time, RT-PCR conditions are shown in Table 2.

Primer sequence Product size (bp) Tm (℃) B4GALT1- BAG1 Forward: 5 '-CCACACCACCGCACTGTC-3' (SEQ ID NO: 11) 200 55 Reverse: 5 '-TTTCCTTCAGAGATTTTCCCTT-3' (SEQ ID NO: 12) GTF3A - CDK8 Forward: 5 '-ATCTGCTCCTTCCCTGACTG-3' (SEQ ID NO: 13) 208 55 Reverse: 5 '-TGAAACTTGATTATATGCCAGAGG-3' (SEQ ID NO: 14) NAGLU - IKZF3 Forward: 5 '-CTCTTCCTGCGAGAGCTGAT-3' (SEQ ID NO: 15) 248 55 Reverse: 5 '-GCGTTATTGATGGCTTGGTC-3' (SEQ ID NO: 16) RNF121 - FOLR2 Forward: 5 '-GAGGTGGAGGTTGGAGGTG-3' (SEQ ID NO: 17) 249 55 Reverse: 5 '-GCTCTGATTCACCTGCTGGA-3' (SEQ ID NO: 18) STRN - ALK Forward: 5 '-CCACAAGTTGAAATACGGGACAGAA-3' (SEQ ID NO: 19) 134 55 Reverse: 5 '-TCAGCTTGTACTCAGGGCTCT-3' (SEQ ID NO: 20) TAF4 - NDRG3 Forward: 5 '-AAAGGAGATGCAGCAACAGG-3' (SEQ ID NO: 21) 240 55 Reverse: 5 '-TCTCTGCGTCCATTGTAGGA-3' (SEQ ID NO: 22)

In more detail, each patient sample was used to amplify the junction portion of the fusion gene with RT-PCR to check whether it is a real fusion gene.

As a result of the gel loading, as shown in FIG. 2, two bands were identified, including the upper band at positions not predicted in RNF121-FOLR2 and TAF4-NDRG3, and the bands were identified at the expected fusion genes.

In addition, as a result of performing Sanger sequencing for all bands, as shown in FIG. 3, it was confirmed that the fusion gene connection site was consistent with the RNA-sequencing result.

In addition, as shown in FIG. 4, the upper band of the RNF121-FOLR2 band among the fusion genes contained the non-representation region of the FOLR2 2 exon region, and the upper band of the TAF4-NDRG3 band contained the TAF4 14 intron. .

Therefore, it was confirmed that all six fusion genes are fusion genes expressed in each patient. In the following example, the sequence of the lower band (corresponding to the accepting gene) including only the analysis site was used to confirm the biological function of the fusion gene.

Example  2. Expression of fusion gene in cell line

The present inventors performed RT-PCR using the primers disclosed in Table 3 to confirm expression of the fusion gene through expression of the receptor gene in various colon cancer cell lines (DLD-1, HT-29, SW480, SW620 and HCT15). Did.

Briefly, after denaturation for 4 minutes at 94 ° C, 94 ° C 40 seconds, 55 ° C 40 seconds, and 72 ° C 40 seconds were performed in 30 cycles. It was carried out using.

Primer sequence Product size (bp) Tm (℃) BAG1 Forward: 5 '-GGTTTTCTGCCCAAGGATTT-3' (SEQ ID NO: 23) 108 55 Reverse: 5 '-CAGTGTGTCAATCTCCTCCAAG-3' (SEQ ID NO: 24) CDK8 Forward: 5 '-AGCAGGGCAATAACCACACT-3' (SEQ ID NO: 25) 118 55 Reverse: 5 '-CCACCTGAGGTAGTGGTAGGA-3' (SEQ ID NO: 26) IKZF3 Forward: 5 '-ATCAACAAGGAAGGGGAGGT-3' (SEQ ID NO: 27) 195 55 Reverse: 5 '-GGCTCTGTGTTCTCCTCTGG-3' (SEQ ID NO: 28) FOLR2 Forward: 5 '-GTGAAGGCCTCTGGAGTCAC-3' (SEQ ID NO: 29) 168 55 Reverse: 5 '-CAGTCCCATGAAGCATCTCA-3' (SEQ ID NO: 30) ALK Forward: 5 '-GCAACATCAGCCTGAAGACA-3' (SEQ ID NO: 31) 192 55 Reverse: 5 '-GCCTGTTGAGAGACCAGGAG-3' (SEQ ID NO: 32) NDRG3 Forward: 5 '-CCACTCAACCTCGAGTAGCC-3' (SEQ ID NO: 33) 102 55 Reverse: 5 '-TCAGGGGACTCACAGGATTC-3' (SEQ ID NO: 34)

As a result, as shown in FIG. 5, BAG1, CDK8 and NDRG3 receptors were expressed in all colorectal cancer cell lines, whereas IKZF3, FOLR2 and ALK receptors were not expressed.

Also, interestingly, one of the carcinogenic fusion gene candidates, the RNF121-FOLR2 fusion gene, was expressed in the HT-29 cell line. This was analyzed by Sanger sequencing, which was consistent with the RNF121-FOLR2 reference sequence (Fig. 6). There was no colorectal cancer cell line expressing the fusion gene except RNF121-FOLR2 expressed in HT-29.

Thus, the carcinogenic fusion gene RNF121-FOLR2 was selected as a novel colorectal cancer diagnostic marker of the present invention, and RNF121-FOLR2 was used in the following examples.

Example  3. In cell lines RNF121 - FOLR2  Cells in Overexpression of Fusion Genes Proliferative capacity  Confirm

The present inventors confirmed the cell proliferation ability of the cell lines transformed with the selected carcinogenic fusion gene RNF121-FOLR2, in order to confirm whether the expression of the fusion protein encoded by the RNF121-FOLR2 fusion gene contributes to the carcinogenesis process. .

First, the inventors of the present invention, the recipient gene expressing the RNF121-FOLR2 fusion gene receptor (forward primer: 5'-GTGGCGGCCGctcgaATGGTCTGGAAATGGATGCCACTT-3 '(SEQ ID NO: 35), reverse primer: 5'-GCCCTCTAGActcgaGCCAAGGAGCCAGAGTTGCA-3' (SEQ ID NO: 36) And a fusion gene [forward primer: 5'-GTGGCGGCCGctcgaATGGCGGCAGTGGTGGAGGTGGA-3 '(SEQ ID NO: 37), reverse primer: 5'-GCCCTCTAGActcgaGCCAAGGAGCCAGAGTTGCA-3' (SEQ ID NO: 36)] was amplified. At this time, in order to make an overexpression vector, since the entire sequence of mRNA is required, it was prepared with primers that accurately hold the 5 'beginning and 3' ends of the mRNA sequence. In the primer sequence, the GTGGCGGCCGctcga and GCCCTCTAGActcga sequences consist of the vector sequence and the enzyme sequence of the part ligated to the sequence necessary for ligation of the mRNA into the vector by one step ligation method.

The pcDNA3.1-V5-His B (invitrogene, USA) vector was cut with XhoI (NEB, USA) enzyme to produce an overexpression vector through one step ligation method. As a control, pcDNA3.1-V5-His B was used.

After injecting the overexpression vector into the colon cancer cell lines DLD-1 and SW620 cell lines (Korea Cell Line Bank, Korea) and the normal cell line NIH-3T3, MTT assay was performed to compare cell proliferation rates.

As a result, as shown in FIG. 7, colon cancer cell lines transformed with the RNF121-FOLR2 fusion gene showed increased cell proliferation rate.

In addition, as shown in FIG. 8, the same result was observed in the normal cell line NIH-3T3 transformed with the RNF121-FOLR2 fusion gene.

This shows that the expression of the RNF121-FOLR2 fusion protein changed the characteristics of the cell line, and the cells transformed with the RNF121-FOLR2 fusion gene increased survival and proliferation rate by the RNF121-FOLR2 fusion protein.

Example  4. RNF121 - FOLR2  Fusion genes and encoded by them Transcription  And Fusion protein  analysis

The present inventors identified fusion genes caused by chromosomal inversion of colorectal cancer patients through the above examples, and the fusion genes were first described in the present invention as fusion examples of RNF121 protein and FOLR2 protein in colorectal cancer.

4-1. RNF121  And FOLR2  fusion

The novel fusion gene RNF121-FOLR2 specifically present in colon cancer tissues identified in the present invention was summarized in more detail.

Donor gene domain Receptor gene domain Distance (bp) RNF121 chr11: 71,640,170 FOLR2 chr11: 71,931,914 291,744

The RNF121-FOLR2 fusion gene of the present invention is cut and fused at a break point (or fusion site) of protein fragments summarized in Table 4 above. The fragment is encoded at any point in the intron region between the exon contained at the end of the N-terminal (for the C-terminal fusion partner) or the C-terminal (for the N-terminal fusion partner) and the exon that is cleaved and removed. Since the amino acid sequence of the protein fragment is not affected, the actual cleavage point may be any of the intron positions.

4-2. RNF121 - FOLR2  Fusion gene and RNF121 - FOLR2 Fusion protein

In the present invention, the RNF121 gene encoding the RNF121 protein was a human-derived RNF121 gene and is located on human chromosome 11 (q13.4), and the encoded RNF121 protein is a protein having a total amino acid length of 327aa. The RNF121 protein or RNF121 protein fragment is an N-terminal fusion partner of the RNF121-FOLR2 fusion protein.

In the present invention, the GenBank accession no. The RNF121 gene having the nucleotide sequence of NM_018320 was used.

The fragment of the RNF121 protein is GenBank accession no. The one having the amino acid sequence encoded by the nucleotide sequence up to the first exon of NM_018320 (71639768 to 71640170 base position based on the (+) strand on chromosome 1) was used.

The gene encoding the fragment of the RNF121 protein and the RNF121 protein encoded therefrom are summarized in Tables 5 and 6 below.

Gene encoding a fragment of RNF121 protein RNF121 gene
(Accession No.) RNF121 protein coding site
(CDS) RNF121 protein fragment coding site:
Exxon standard RNF121 protein fragment coding site:
cDNA standard On the chromosome
Cutting position 5 'end cutting position NM_018320 984 bp
(SEQ ID NO: 1) Exon 1 SEQ ID NO: 2 chr11: 71,640,170 1-63bp

Fragment of RNF121 protein Full length of RNF121 protein
(Accession No.) RNF121 protein fragment site N-end cutting position 327aa (Accession No. NP_060790)
(SEQ ID NO: 3) SEQ ID NO: 4 1-21aa

In the present invention, the FOLR2 gene encoding the FOLR2 protein uses a human-derived FOLR2 gene and is located on human chromosome 11 (q13.4), and the encoded FOLR2 protein is a protein having a total amino acid length of 255aa. The FOLR2 protein or FOLR2 protein fragment is a C-terminal fusion partner of the RNF121-FOLR2 fusion protein.

In the present invention, the GenBank accession no. The FOLR2 gene with the base sequence of NM_000803 was used.

The fragment of the FOLR2 protein is GenBank accession no. The one having the amino acid sequence encoded by the nucleotide sequence up to the third exon of NM_000803 (base positions 71931914 to 71932994 based on the (+) strand on chromosome 1) was used.

Genes encoding fragments of the FOLR2 protein and FOLR2 proteins encoded therefrom are summarized in Tables 7 and 8 below.

Gene encoding fragment of FOLR2 protein FOLR2 gene
Accession No. FOLR2 protein coding site
(CDS) FOLR2 protein fragment coding site:
Exxon standard FOLR2 protein fragment coding site:
cDNA standard On the chromosome
Cutting position 5 'end cutting position NM_000803 768bp
(SEQ ID NO: 5) Exon 3-Exon 5 SEQ ID NO: 6 chr11: 71,931,914 151-768bp

Fragment of FOLR2 protein Full length of FOLR2 protein
(Accession No.) FOLR2 protein fragment site C-end cutting position 255aa (Accession No. NP_000794)
(SEQ ID NO: 7) SEQ ID NO: 8 51-255aa

The fusion gene (RNF121-FOLR2 fusion gene or FOLR2 fusion gene) encoding the RNF121-FOLR2 fusion protein in which the RNF121 protein or fragment thereof and the FOLR2 protein or fragment thereof are fused is the RNF121 protein or fragment thereof as described above at the 5 'end. And a nucleotide molecule encoding a FOLR2 protein or fragment thereof as described above at the 3 'end.

Specifically, in the present invention, a nucleotide sequence of exon 1 of NM_018320 at the 5 'end side and a nucleotide sequence of exon 3 to exon 5 of NM_000803 at the 3' end side were linked to prepare an RNF121-FOLR2 fusion gene.

More specifically, in the present invention, nucleotide sequences 1 to 63 of NM_018320 (SEQ ID NO: 2) and 5 to 151 of 151 to 768 nucleotide sequences of NM_000803 (SEQ ID NO: 6) are linked to the 5 'end. The RNF121-FOLR2 fusion gene (SEQ ID NO: 9) was prepared.

In addition, in the present invention, the RNF121-FOLR2 fusion protein encoded from the prepared RNF121-FOLR2 fusion gene has amino acid sequences from 1 to 21 of the RNF121 protein (SEQ ID NO: 4) on the N-terminal side and FOLR2 protein on the C-terminal side. It was a fusion protein (SEQ ID NO: 10) to which the amino acid sequence of SEQ ID NO: 51 to 255 (SEQ ID NO: 8) was linked.

As a result of analyzing the RNF121-FOLR2 fusion transcript using reverse transcription chain reaction and raw sequencing, the RNF121-FOLR2 fusion gene was cut at positions chr11: 71,640,170 in the RNF121 gene, conserving SEQ ID NO: 2 in the 5 'end direction, FOLR2 It was confirmed that the gene was cut at the chr11: 71,931,914 position and fused in a form conserving SEQ ID NO: 8 in the 3 'end.

As described above, according to the present invention, the RNF121-FOLR2 fusion gene of the present invention or a fusion transcript encoded therein and a fusion protein are detected to diagnose colorectal cancer and prognosis of colorectal cancer patients, and furthermore, RNF121-FOLR2 It becomes possible to predict the effectiveness of the treatment of colorectal cancer with an inhibitor that targets and suppresses.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. 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 including all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.

<110> Pusan National University Industry-University Cooperation Foundation          PUSAN NATIONAL UNIVERSITY HOSPITAL <120> Novel FOLR2 Fusion Gene As Colon Cancer Diagnosis Marker and Uses          Thereof <130> PNU1-328p <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 984 <212> DNA <213> Homo sapiens <400> 1 atggcggcag tggtggaggt ggaggttgga ggtggtgctg ctggggaacg ggagctggat 60 gaggttgata tgtcagatct ctctccagaa gagcaatgga gggtcgagca cgcacgcatg 120 catgccaagc accgtggcca tgaagctatg catgctgaaa tggtcctcat cctcatcgca 180 accttggtgg tggcccagct gctcctggtg cagtggaagc agaggcaccc acgctcctac 240 aatatggtga ccctctttca gatgtgggtt gttcccctct atttcacagt gaagctgcac 300 tggtggaggt tcctagtgat ctggatcttg ttctctgctg tcacagcctt tgttaccttc 360 cgagccaccc gaaaacctct agtacagaca accccaaggt tggtttataa gtggttcctg 420 ctaatctata aaatcagcta tgccactggc attgttggct acatggctgt catgtttacc 480 ctctttggtc ttaacttatt attcaagatc aaaccagaag atgccatgga ctttggcatc 540 tcccttctct tctatggcct ctactatgga gttctggaac gggactttgc agaaatgtgt 600 gcagactaca tggcatctac catagggttc tacagcgagt cgggcatgcc taccaaacat 660 ctttcagaca gtgtgtgtgc tgtgtgtggg cagcagatct ttgtggacgt cagtgaagag 720 gggatcattg agaacacgta taggctgtcc tgcaatcatg tcttccacga gttctgcatc 780 cgtggctggt gcatcgtggg aaagaagcaa acgtgtccct actgcaaaga gaaggtagac 840 ctcaagagga tgttcagcaa tccctgggag aggcctcacg tcatgtatgg gcaactgctg 900 gactggcttc gatacttggt agcctggcag cctgtcatca ttggtgtagt ccaaggcatc 960 aactacatcc tgggcctgga atag 984 <210> 2 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> RNF121 fragment cDNA <400> 2 atggcggcag tggtggaggt ggaggttgga ggtggtgctg ctggggaacg ggagctggat 60 gag 63 <210> 3 <211> 327 <212> PRT <213> Homo sapiens <400> 3 Met Ala Ala Val Val Glu Val Glu Val Gly Gly Gly Ala Ala Gly Glu   1 5 10 15 Arg Glu Leu Asp Glu Val Asp Met Ser Asp Leu Ser Pro Glu Glu Gln              20 25 30 Trp Arg Val Glu His Ala Arg Met His Ala Lys His Arg Gly His Glu          35 40 45 Ala Met His Ala Glu Met Val Leu Ile Leu Ile Ala Thr Leu Val Val      50 55 60 Ala Gln Leu Leu Leu Val Gln Trp Lys Gln Arg His Pro Arg Ser Tyr  65 70 75 80 Asn Met Val Thr Leu Phe Gln Met Trp Val Val Pro Leu Tyr Phe Thr                  85 90 95 Val Lys Leu His Trp Trp Arg Phe Leu Val Ile Trp Ile Leu Phe Ser             100 105 110 Ala Val Thr Ala Phe Val Thr Phe Arg Ala Thr Arg Lys Pro Leu Val         115 120 125 Gln Thr Thr Pro Arg Leu Val Tyr Lys Trp Phe Leu Leu Ile Tyr Lys     130 135 140 Ile Ser Tyr Ala Thr Gly Ile Val Gly Tyr Met Ala Val Met Phe Thr 145 150 155 160 Leu Phe Gly Leu Asn Leu Leu Phe Lys Ile Lys Pro Glu Asp Ala Met                 165 170 175 Asp Phe Gly Ile Ser Leu Leu Phe Tyr Gly Leu Tyr Tyr Gly Val Leu             180 185 190 Glu Arg Asp Phe Ala Glu Met Cys Ala Asp Tyr Met Ala Ser Thr Ile         195 200 205 Gly Phe Tyr Ser Glu Ser Gly Met Pro Thr Lys His Leu Ser Asp Ser     210 215 220 Val Cys Ala Val Cys Gly Gln Gln Ile Phe Val Asp Val Ser Glu Glu 225 230 235 240 Gly Ile Ile Glu Asn Thr Tyr Arg Leu Ser Cys Asn His Val Phe His                 245 250 255 Glu Phe Cys Ile Arg Gly Trp Cys Ile Val Gly Lys Lys Gln Thr Cys             260 265 270 Pro Tyr Cys Lys Glu Lys Val Asp Leu Lys Arg Met Phe Ser Asn Pro         275 280 285 Trp Glu Arg Pro His Val Met Tyr Gly Gln Leu Leu Asp Trp Leu Arg     290 295 300 Tyr Leu Val Ala Trp Gln Pro Val Ile Ile Gly Val Val Gln Gly Ile 305 310 315 320 Asn Tyr Ile Leu Gly Leu Glu                 325 <210> 4 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> RNF121 protein fragment <400> 4 Met Ala Ala Val Val Glu Val Glu Val Gly Gly Gly Ala Ala Gly Glu   1 5 10 15 Arg Glu Leu Asp Glu              20 <210> 5 <211> 768 <212> DNA <213> Homo sapiens <400> 5 atggtctgga aatggatgcc acttctgctg cttctggtct gtgtagccac catgtgcagt 60 gcccaggaca ggactgatct cctcaatgtc tgtatggatg ccaagcacca caagacaaag 120 ccaggtcctg aggacaagct gcatgaccaa tgcagtccct ggaagaagaa tgcctgctgc 180 acagccagca ccagccagga gctgcacaag gacacctccc gcctgtacaa ctttaactgg 240 gaccactgcg gcaagatgga gcccgcctgc aagcgccact tcatccagga cacctgtctc 300 tatgagtgct cacccaacct ggggccctgg atccagcagg tgaatcagag ctggcgcaaa 360 gaacgcttcc tggatgtgcc cttatgcaaa gaggactgtc agcgctggtg ggaggattgt 420 cacacctccc acacgtgcaa gagcaactgg cacagaggat gggactggac ctcaggagtt 480 aacaagtgcc cagctggggc tctctgccgc acctttgagt cctacttccc cactccagct 540 gccctttgtg aaggcctctg gagtcactca tacaaggtca gcaactacag ccgagggagc 600 ggccgctgca tccagatgtg gtttgattca gcccagggca accccaacga ggaagtggcg 660 aggttctatg ctgcagccat gcatgtgaat gctggtgaga tgcttcatgg gactgggggt 720 ctcctgctca gtctggccct gatgctgcaa ctctggctcc ttggctga 768 <210> 6 <211> 617 <212> DNA <213> Artificial Sequence <220> <223> FOLR2 fragment cDNA <400> 6 gcagtccctg gaagaagaat gcctgctgca cagccagcac cagccaggag ctgcacaagg 60 acacctcccg cctgtacaac tttaactggg accactgcgg caagatggag cccgcctgca 120 agcgccactt catccaggac acctgtctct atgagtgctc acccaacctg gggccctgga 180 tccagcaggt gaatcagagc tggcgcaaag aacgcttcct ggatgtgccc ttatgcaaag 240 aggactgtca gcgctggtgg gaggattgtc acacctccca cacgtgcaag agcaactggc 300 acagaggatg ggactggacc tcaggagtta acaagtgccc agctggggct ctctgccgca 360 cctttgagtc ctacttcccc actccagctg ccctttgtga aggcctctgg agtcactcat 420 acaaggtcag caactacagc cgagggagcg gccgctgcat ccagatgtgg tttgattcag 480 cccagggcaa ccccaacgag gaagtggcga ggttctatgc tgcagccatg catgtgaatg 540 ctggtgagat gcttcatggg actgggggtc tcctgctcag tctggccctg atgctgcaac 600 tctggctcct tggctga 617 <210> 7 <211> 255 <212> PRT <213> Homo sapiens <400> 7 Met Val Trp Lys Trp Met Pro Leu Leu Leu Leu Leu Val Cys Val Ala   1 5 10 15 Thr Met Cys Ser Ala Gln Asp Arg Thr Asp Leu Leu Asn Val Cys Met              20 25 30 Asp Ala Lys His His Lys Thr Lys Pro Gly Pro Glu Asp Lys Leu His          35 40 45 Asp Gln Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr      50 55 60 Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp  65 70 75 80 Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln                  85 90 95 Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln             100 105 110 Gln Val Asn Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu         115 120 125 Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His Thr Ser His     130 135 140 Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val 145 150 155 160 Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe                 165 170 175 Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys             180 185 190 Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe         195 200 205 Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala     210 215 220 Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly 225 230 235 240 Leu Leu Leu Ser Leu Ala Leu Met Leu Gln Leu Trp Leu Leu Gly                 245 250 255 <210> 8 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> FOLR2 protein fragment <400> 8 Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr Ala Ser Thr Ser Gln   1 5 10 15 Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn Phe Asn Trp Asp His              20 25 30 Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His Phe Ile Gln Asp Thr          35 40 45 Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp Ile Gln Gln Val      50 55 60 Asn Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp Val Pro Leu Cys Lys  65 70 75 80 Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His Thr Ser His Thr Cys                  85 90 95 Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr Ser Gly Val Asn Lys             100 105 110 Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu Ser Tyr Phe Pro Thr         115 120 125 Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His Ser Tyr Lys Val Ser     130 135 140 Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln Met Trp Phe Asp Ser 145 150 155 160 Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr Ala Ala Ala                 165 170 175 Met His Val Asn Ala Gly Glu Met Leu His Gly Thr Gly Gly Leu Leu             180 185 190 Leu Ser Leu Ala Leu Met Leu Gln Leu Trp Leu Leu Gly         195 200 205 <210> 9 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> RNF121-FOLR2 fusion <400> 9 atggcggcag tggtggaggt ggaggttgga ggtggtgctg ctggggaacg ggagctggat 60 gagtgcagtc cctggaagaa gaatgcctgc tgcacagcca gcaccagcca ggagctgcac 120 aaggacacct cccgcctgta caactttaac tgggaccact gcggcaagat ggagcccgcc 180 tgcaagcgcc acttcatcca ggacacctgt ctctatgagt gctcacccaa cctggggccc 240 tggatccagc aggtgaatca gagctggcgc aaagaacgct tcctggatgt gcccttatgc 300 aaagaggact gtcagcgctg gtgggaggat tgtcacacct cccacacgtg caagagcaac 360 tggcacagag gatgggactg gacctcagga gttaacaagt gcccagctgg ggctctctgc 420 cgcacctttg agtcctactt ccccactcca gctgcccttt gtgaaggcct ctggagtcac 480 tcatacaagg tcagcaacta cagccgaggg agcggccgct gcatccagat gtggtttgat 540 tcagcccagg gcaaccccaa cgaggaagtg gcgaggttct atgctgcagc catgcatgtg 600 aatgctggtg agatgcttca tgggactggg ggtctcctgc tcagtctggc cctgatgctg 660 caactctggc tccttggctg a 681 <210> 10 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> RNF121-FOLR2 fusion <400> 10 Met Ala Ala Val Val Glu Val Glu Val Gly Gly Gly Ala Ala Gly Glu   1 5 10 15 Arg Glu Leu Asp Glu Cys Ser Pro Trp Lys Lys Asn Ala Cys Cys Thr              20 25 30 Ala Ser Thr Ser Gln Glu Leu His Lys Asp Thr Ser Arg Leu Tyr Asn          35 40 45 Phe Asn Trp Asp His Cys Gly Lys Met Glu Pro Ala Cys Lys Arg His      50 55 60 Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro  65 70 75 80 Trp Ile Gln Gln Val Asn Gln Ser Trp Arg Lys Glu Arg Phe Leu Asp                  85 90 95 Val Pro Leu Cys Lys Glu Asp Cys Gln Arg Trp Trp Glu Asp Cys His             100 105 110 Thr Ser His Thr Cys Lys Ser Asn Trp His Arg Gly Trp Asp Trp Thr         115 120 125 Ser Gly Val Asn Lys Cys Pro Ala Gly Ala Leu Cys Arg Thr Phe Glu     130 135 140 Ser Tyr Phe Pro Thr Pro Ala Ala Leu Cys Glu Gly Leu Trp Ser His 145 150 155 160 Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile Gln                 165 170 175 Met Trp Phe Asp Ser Ala Gln Gly Asn Pro Asn Glu Glu Val Ala Arg             180 185 190 Phe Tyr Ala Ala Ala Met His Val Asn Ala Gly Glu Met Leu His Gly         195 200 205 Thr Gly Gly Leu Leu Leu Ser Leu Ala Leu Met Leu Gln Leu Trp Leu     210 215 220 Leu Gly 225 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B4GALT1-BAG1 primer F <400> 11 ccacaccacc gcactgtc 18 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> B4GALT1-BAG1 primer R <400> 12 tttccttcag agattttccc tt 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GTF3A-CDK8 primer F <400> 13 atctgctcct tccctgactg 20 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GTF3A-CDK8 primer R <400> 14 tgaaacttga ttatatgcca gagg 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NAGLU-IKZF3 primer F <400> 15 ctcttcctgc gagagctgat 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NAGLU-IKZF3 primer R <400> 16 gcgttattga tggcttggtc 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RNF121-FOLR2 primer F <400> 17 gaggtggagg ttggaggtg 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RNF121-FOLR2 primer R <400> 18 gctctgattc acctgctgga 20 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> STRN-ALK primer F <400> 19 ccacaagttg aaatacggga cagaa 25 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> STRN-ALK primer R <400> 20 tcagcttgta ctcagggctc t 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TAF4-NDRG3 primer F <400> 21 aaaggagatg cagcaacagg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TAF4-NDRG3 primer R <400> 22 tctctgcgtc cattgtagga 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BAG1 primer F <400> 23 ggttttctgc ccaaggattt 20 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BAG1 primer R <400> 24 cagtgtgtca atctcctcca ag 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDK8 primer F <400> 25 agcagggcaa taaccacact 20 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CDK8 primer R <400> 26 ccacctgagg tagtggtagg a 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IKZF3 primer F <400> 27 atcaacaagg aaggggaggt 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IKZF3 primer R <400> 28 ggctctgtgt tctcctctgg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOLR2 primer F <400> 29 gtgaaggcct ctggagtcac 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOLR2 primer R <400> 30 cagtcccatg aagcatctca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALK primer F <400> 31 gcaacatcag cctgaagaca 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALK primer R <400> 32 gcctgttgag agaccaggag 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 primer F <400> 33 ccactcaacc tcgagtagcc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 primer R <400> 34 tcaggggact cacaggattc 20 <210> 35 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer F <400> 35 gtggcggccg ctcgaatggt ctggaaatgg atgccactt 39 <210> 36 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer R <400> 36 gccctctaga ctcgagccaa ggagccagag ttgca 35 <210> 37 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer F <400> 37 gtggcggccg ctcgaatggc ggcagtggtg gaggtgga 38

Claims (27)

R-121 FOLR2 fusion protein consisting of the structural formula of N- [RF121 (Ring Finger Protein 121) N-terminal fragment fragment]-[FORLR2 (Folate receptor beta) C-terminal fragment fragment] -C;
The fragment comprising the N-terminal of RNF121, GenBank accession no. Is an RNF121 protein essentially comprising the contiguous amino acids of the 1st to 21st amino acids of the protein encoded by the polynucleotide of NM_018320;
The fragment comprising the C-terminal of FOLR2, GenBank accession no. A FOLR2 protein essentially comprising the sequential amino acids of the 51st to 255th amino acids of the protein encoded by the polynucleotide of NM_000803;
RNF121-FOLR2 fusion protein, characterized in that.
According to claim 1,
The RNF121-FOLR2 fusion protein is characterized in that consisting of the amino acid sequence of SEQ ID NO: 10, RNF121-FOLR2 fusion protein.
delete According to claim 1, The RNF121-FOLR2 fusion protein is characterized in that it is encoded from the RNF121-FOLR2 fusion gene consisting of the nucleotide sequence of SEQ ID NO: 9, RNF121-FOLR2 fusion protein.
According to claim 1, wherein the fragment comprising the N-terminal of RNF121 is characterized in that consisting of the amino acid sequence of SEQ ID NO: 4, RNF121-FOLR2 fusion protein.
delete delete According to claim 1, wherein the fragment comprising the C terminal of FOLR2 is characterized in that consisting of the amino acid sequence of SEQ ID NO: 8, RNF121-FOLR2 fusion protein.
A RNF121-FOLR2 fusion gene encoding the RNF121-FOLR2 fusion protein of claim 1.
10. The method of claim 9, The RNF121-FOLR2 fusion gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 9, RNF121-FOLR2 fusion gene.
delete A composition for diagnosing colorectal cancer comprising an RNF121-FOLR2 fusion protein according to claim 1, an RNF121-FOLR2 fusion gene according to claim 9, or an agent measuring the transcript of the fusion gene.
The composition for diagnosing colorectal cancer according to claim 12, wherein the transcript of the fusion gene is mRNA.
The composition for diagnosing colorectal cancer according to claim 13, wherein the agent for measuring the mRNA of the fusion gene comprises a primer that specifically binds to the mRNA of the fusion gene.
15. The method of claim 14, The primer specifically binding to the mRNA of the fusion gene is designed to sandwich the break point, so that the region amplified by the primer includes a break point of two genes to be fused. Characterized in that, the composition for the diagnosis of colorectal cancer.
The large intestine according to claim 15, wherein the primer is a primer pair comprising a primer specific for the nucleotide sequence encoding the N terminus of the RNF121 protein and a primer specific for the nucleotide sequence encoding the C terminus of FOLR2. Cancer diagnostic composition.
The composition for diagnosing colorectal cancer according to claim 16, wherein the primer pair is composed of the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18.
The composition for diagnosing colorectal cancer according to claim 12, wherein the agent for measuring the fusion gene comprises a probe or a primer that specifically binds to the fusion gene.
The composition for diagnosing colorectal cancer according to claim 12, wherein the agent for measuring the fusion protein comprises an antibody specific for the protein.
A kit for diagnosing colorectal cancer comprising the composition of claim 12.
The kit of claim 20, wherein the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit.
How to provide information for the diagnosis of colorectal cancer, including the following steps:
(a) measuring the level of the RNF121-FOLR2 fusion protein according to claim 1, the RNF121-FOLR2 fusion gene according to claim 9, or the transcript of the fusion gene from an isolated sample of an individual suspected of colon cancer; And
(b) the measured expression level of the RNF121-FOLR2 fusion protein, RNF121-FOLR2 fusion gene, or the transcript of the fusion gene of the normal control sample RNF121-FOLR2 fusion protein, RNF121-FOLR2 fusion gene, or of the fusion gene Comparing the expression level of the transcript.
The expression level of RNF121-FOLR2 fusion protein, RNF121-FOLR2 fusion gene, or transcript of the fusion gene measured from an isolated sample of an individual suspected of colorectal cancer is RNF121-FOLR2 of the normal control sample according to claim 22. When the fusion protein, RNF121-FOLR2 fusion gene, or the expression level of the transcript of the fusion gene is higher than the expression level, characterized in that diagnosed as colorectal cancer, method for providing information for colorectal cancer diagnosis.
A method of screening for a treatment for colorectal cancer comprising the following steps:
(A) treating a candidate for treating colorectal cancer to cells expressing the RNF121-FOLR2 fusion gene of claim 9; And
(b) measuring the expression level of the RNF121-FOLR2 fusion gene in the cell of step (a).
According to claim 24, When the expression level of the RNF121-FOLR2 fusion gene is lowered by treatment with the candidate for treatment of colorectal cancer, characterized in that it is determined as a treatment for colorectal cancer, a method for screening for treatment for colorectal cancer.
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