KR20180013213A - Composition and kit for diagnosing gastrointestinal cancer comprising Rbfox2 antibody as effective component and - Google Patents

Abstract

The present invention relates to a composition and a kit for diagnosing digestive organ cancer comprising an Rbfox 2 protein antibody as an active ingredient, and to a diagnosing method using the same. Specifically, the present invention relates to a composition for diagnosing digestive organ cancer and a method for diagnosing digestive organ cancer using the same, in which an Rbfox 2 protein expressed only in a nuclear cell in a normal cell is expressed not only in a nuclear cell but also in cytoplasm in a digestive organ cancer cell. According to the present invention, digestive organ cancer diagnosis having high sensitivity and specificity is possible with a method for checking a content of the Rbfox 2 protein in cytoplasm of suspected cancer tissues and surrounding tissues thereof, compared to a method of measuring a content of protein expression in blood. Therefore, the present invention is very effective for diagnosing digestive organ cancer, and can be applied as a candidate marker for different carcinoma discovered newly.

Description

The present invention relates to a composition and kit for diagnosing gastrointestinal cancer, which comprises an albifox 2 protein antibody as an active ingredient,

The present invention relates to a composition and a kit for diagnosing digestive organs cancer, which contains an albifox 2 protein antibody as an active ingredient.

Digestive system Cancer is cancer of digestive system. Digestive system starts from mouth and includes pharynx, esophagus, stomach, small intestine and large intestine and anus. According to National Cancer Information Center cancer statistics, the most common cancer in Korea in 2013 was thyroid cancer, followed by gastric cancer, colon cancer, lung cancer, breast cancer, liver cancer, and prostate cancer. In the case of men, gastric cancer, colorectal cancer, lung cancer, liver cancer and prostate cancer occurred in the order of thyroid cancer, breast cancer, colon cancer, gastric cancer and lung cancer in the order of women. Thus, gastrointestinal cancers arising in the gastrointestinal tract and large intestine are frequently found in Korea.

In addition, when the cell receives stress such as heat, it temporarily stops the translation process, thereby regulating the physiological activity. In the case of stress, there is a risk of error in the translation process, so that the translation process is not precisely controlled will be. This regulatory process is mainly performed early in the translation process, and it is known that the regulation of eIF2α activity is important. In other words, when the cell is stressed, phosphorylation of eIF2α occurs, and phosphorylation of eIF2α causes intracellular mRNA, translation initiators, and various ribonucleic acid binding proteins to aggregate in the cytoplasm called stress granules (SGs) To inhibit and control the translation process.

Generally, in the course of development of solid cancer, cancer cells rapidly grow due to the existing blood vessels, and the rapid metabolism thus exceeds the oxygen and nutrients to be supplied. Therefore, cancer cells have a difficulty in dealing with low oxygen, high osmotic pressure and nutrient depletion Environment. In addition, ER stress is caused by the rapid growth rate and the restriction of protein biosynthesis, which causes misfolding of the protein.

In addition, cancer cells adapt to uncontrolled environments such as low oxygen, high osmotic pressure and nutrient depletion through uncoupling glycolysis, resulting in a high level of reactive oxygen species (ROS) , Post-transcriptional mechanisms through stress granulation (SG) have been shown to play an important role in cancer cell survival in harsh environments (Stress granules, P-bodies and cancer, Biochim Biophys Acta. 2015 Jul; 1849 (7): 861-70.).

On the other hand, alternative splicing of precursor mRNA (pre mRNA) produces a variety of mRNAs from one gene, thereby creating very different isotope proteins from a single gene. Such alternative splicing is regulated by cis-regulatory elements and RNA-binding proteins (RBPs) depending on the type of cell, the stage of development of the cell, or the external environment.

The family of RNA-binding Fox (Rbfox) is one of the ribonucleic acid binding proteins (RBPs) that regulate alternative splicing and is known to play an important role in central nervous system development. The family of RNA-binding Fox (Rbfox) consists of Rbfox1, Rbfox2 and Rbfox3. Unlike Rbfox 1 or Rbfox 3, which is expressed only in certain tissues, Rbfox2 exhibits features expressed from the stem cell stage in various tissues (Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development. Biochem Biophys Res Commun. 2014 Aug 8; 450 (4): 1662-7).

As an example of the technique related to albicopy protein, US Patent Publication No. 2015-0167082 discloses a method of diagnosing the tendency or occurrence of autism by SNP analysis of albufox 1 (Rbfox1) (Rbfox2) protein antibody as an active ingredient, and a diagnostic method using the kit.

The present invention provides a composition for diagnosing digestive organs cancer, which comprises an albifox 2 protein antibody as an active ingredient, a kit and a diagnostic method using the same, wherein the active ingredient albufox 2 (Rbfox2) is transferred from the nucleus to the cytoplasm in cancer tissues and the expression is increased in the cytoplasm, thus completing the present invention.

In order to achieve the above object, the present invention provides a composition for diagnosing digestive organs cancer, which comprises, as an active ingredient, an antibody specifically recognizing the protein of Rbfox2 contained in a specimen, Provide a diagnostic kit.

Further, the present invention provides a method for diagnosing cancer, comprising the steps of: (1) obtaining a tissue suspected of having cancer from a suspicious patient of a digestive organs cancer and surrounding tissues presumed to be normal tissue;

(2) reacting the obtained Rbfox2 protein contained in the tissue suspected of being cancer and the surrounding tissue with an antibody that specifically recognizes the Rbfox2 protein;

(3) measuring the respective content of albufox2 (Rbfox2) protein contained in the cytoplasm of the tissue suspected of being cancer and the surrounding tissue from the reaction of step (2); And

(4) In step (3), the amount of Rbfox2 protein contained in the cytoplasm of the suspected cancer tissue and the content of Rbfox2 protein contained in the cytoplasm of the surrounding tissues of the cancer suspected cancer Comparing the content of the Rbfox2 protein contained in the cytoplasm of the cancer suspected tissue to the content of the Rbfox2 protein contained in the cytoplasm of the surrounding tissue; A method for providing information necessary for diagnosis of digestive organs cancer.

The present invention relates to a composition for diagnosing digestive organs cancer, which comprises an antibody specifically recognizing an albufox 2 (Rbfox2) protein contained in a specimen as an active ingredient, and a method for diagnosing digestive organs cancer using the composition. The present invention relates to a composition for diagnosing digestive organs cancer, and a method for diagnosing digestive organs cancer using the composition, wherein the Rbfox2 protein expressed in the nucleus is expressed in not only nuclei but also cytoplasm in colon cancer cells. According to the present invention, a method of confirming the expression pattern of albufox 2 (Rbfox2) protein in the digestive organs cancer cell cytoplasm in comparison with a method of measuring the protein expression amount in the blood enables digestion with higher sensitivity and specificity Thereby enabling the diagnosis of mechanical cancer. Therefore, it is not only very effective in the diagnosis of digestive system cancer, but also can be applied as a candidate marker for newly discovered cancer species.

FIG. 1 (A) shows that when Hela cells were treated with arbi- zate and stressed, the Rbfox2 protein was expressed not only in the nucleus but also in the cytoplasm. The SFPQ protein (splicing factor, poly -glutamine rich) was a control, and it was confirmed that there was no change according to the stress or not, unlike albifox 2. In FIG. 1 (B), when the Hela cells were treated with the abiotic salt, FIG. 1 (C) shows the results of confirming the expression of TIA-1 protein and Rbfox2 protein at the same position in the cytoplasm. In FIG. 1 (C), when Hela cells were treated with arbutin, This is the result of confirming that the G3BP1 protein and the Rbfox2 protein are expressed at the same position in the cytoplasm.
FIG. 2 (A) shows the results of confirming expression of the Rbfox2 protein in the normal colon tissue, and FIG. 2 (B) shows the results of confirming that the Rbfox2 protein in the colon cancer tissue And the expression of the gene was confirmed in the cells.

The present invention relates to a composition for diagnosing digestive organs cancer, which comprises, as an active ingredient, an antibody specifically recognizing an albufox2 (Rbfox2) protein contained in a specimen.

The gastrointestinal cancer is preferably any one selected from gastric cancer, colon cancer, liver cancer, pancreatic cancer, esophageal cancer and oral cancer, but is not limited thereto.

The 'specimen' is a tissue that shows the expression of cancer diagnostic marker protein (in particular, Rbfox2 protein in the present invention) due to cancer development from nucleus to cytoplasm; But is not limited thereto. More preferably, it may be a tissue of a lesion (tumor) region separated from a patient suspected of having a cancer and a surrounding tissue thereof .

In addition, the 'tissue of the lesion (tumor) site separated from the patient suspected of having cancer and its surrounding tissue' can be collected by a biopsy method known in the art.

The above-mentioned albufox 2 (Rbfox2) is also known as FOX2, fox-2, HNRNPBP2, HRNBP2, RBM9, RTA, dj106I20, fxh and may be NCBI gene ID 23543, but is not limited thereto.

The above-mentioned albofox2 (Rbfox2) is expressed in the nucleus in normal cells, but is also expressed in cytosolic stress granule (SG) in colorectal cancer cells.

The substance for measuring the expression of the Rbfox2 protein is preferably an antibody specific to Rbfox2 protein or an aptamer, more preferably an antibody, but is not limited thereto. Any substance capable of specifically binding to Rbfox2 protein or a substance capable of measuring albumin 2 (Rbfox2) protein in a large intestine tissue or serum can be used freely.

Antibodies in the present invention are used in the broadest sense and specifically include monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies of any isotype such as IgG, IgM, IgA, IgD and IgE A chimeric antibody, and an antibody fragment. Antibodies that are reactive with a particular antigen can be produced by recombinant methods, e.g., by screening a library of recombinant antibodies in phage or like vectors, or by immunizing an animal with an antigen or antigen-encoding nucleic acid. A preferred antibody is a fragment of an antibody comprising a polyclonal or monoclonal antibody that specifically recognizes the Rbfox2 protein or a polypeptide having the binding domain of the Rbfox2 protein, I never do that.

 In addition to the primary antibody that specifically binds in the present invention, the antibody may further include a secondary antibody that binds to an antibody that specifically recognizes the Rbfox2 protein. The antibody of the present invention may be detected or detected Enzymes, fluorescent substances, radioactive substances, proteins, and the like. The modified antibody can be obtained by chemically modifying the antibody, and such a modification method is a method commonly used in the art.

The present invention also relates to a kit for the diagnosis of digestive organs cancer, which comprises the composition for diagnosing gastrointestinal cancer.

The diagnostic kit for digestive organs includes an antibody specifically binding to Rbfox2 protein, a secondary antibody conjugate conjugated with a marker that develops upon reaction with the substrate, a colorant capable of coloring with the label A substrate solution, a washing solution, and an enzyme reaction stop solution, etc., and may be manufactured from a number of separate packages or compartments including the reagent components used.

Further, the present invention provides a method for diagnosing cancer, comprising the steps of: (1) obtaining a tissue suspected of having cancer from a suspicious patient of a digestive organs cancer and surrounding tissues presumed to be normal tissue;

(2) reacting the obtained Rbfox2 protein contained in the tissues suspected of cancer and the surrounding tissues with an antibody specifically recognizing the Rbfox2 protein;

(3) measuring the respective content of albufox2 (Rbfox2) protein contained in the cytoplasm of the tissue suspected of being cancer and the surrounding tissue from the reaction of step (2); And

(4) In step (3), the amount of Rbfox2 protein contained in the cytoplasm of the suspected cancer tissue and the content of Rbfox2 protein contained in the cytoplasm of the surrounding tissues of the cancer suspected cancer Comparing the content of the Rbfox2 protein contained in the cytoplasm of the cancer suspected tissue to the content of the Rbfox2 protein contained in the cytoplasm of the surrounding tissue; The present invention relates to a method for providing information necessary for diagnosis of digestive system cancer.

In step (3), the content of Rbfox2 protein contained in the cytoplasm of the tissue suspected of being cancerous and its surrounding tissues is determined by measuring the interaction of a substance specifically binding to the Rbfox2 protein Can be detected by a general antigen-antibody reaction. Immunochromatography, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemical staining, and immunohistochemical staining are examples of analytical methods for this general antigen-antibody reaction. The enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, protein chip, the fluorescence activated cell sorter (FACS) method, and the immunofluorescent microscopy method.

Preferably, in step (3), the amount of the Rbfox2 protein contained in the cytoplasm of the cancer suspected of the cancer and the content of the Rbfox2 protein contained in the cytoplasm of the surrounding tissue of the cancer- An immunofluorescence microscope analysis of the Rbfox2 protein contained in the cytoplasm of the tissue suspected of cancer and its surrounding tissues was carried out to compare the content of cytoplasm of the Rbfox2 protein with naked eyes It is to contrast.

According to this method, it is possible to distinguish between cancer tissue and normal tissue. (Rbfox2) protein in the cytoplasm of cancerous lesions, while Rbfox2 protein in normal tissues is expressed only in the nucleus and provides information necessary for cancer diagnosis.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  1. Greek HeLa ) Culture of cell line

The HeLa cell line (purchased from ATCC) was cultured and maintained in DMEM medium containing 10% (v / v) fetal bovine serum at 37 ° C and 5% (v / v) carbon dioxide .

Example  2. Albie Fox  2 (Rbfox2) Protein Stress Granule  Confirmation of expression

The HeLa cell line of Example 1 was treated with 0.5 mM of Abbate for 30 minutes, washed with PBS (phosphate buffered saline, pH 7.4), washed with HeLa, (v / v) fetal bovine serum at 37 ° C and 5% (v / v) carbon dioxide for 24 hours.

The HeLa cell line of Example 1 and the HeLa cells treated with the abiotic salt were fixed with 4% paraformaldehyde for 10 minutes and then immersed in 0.5% (v / v) Triton X-100 ) For 15 minutes to increase the permeability of the cells. The non-reactive blocking staining was blocked by treatment with Tris-buffered saline (pH 6.0) containing 5% (w / v) bovine serum albumin for 1 hour.

The primary chlorine polyclonal antibody against SFPQ protein was diluted 1: 1,000 in PBS (phosphate buffered saline, pH 7.4) and incubated at 4 ° C for 24 hours. The primary chlorine polyclonal antibody against G3BP1 protein was diluted in PBS (phosphate buffered saline, pH 7.4) at a ratio of 1: 100 and incubated at 4 ° C for 24 hours. The primary chlorine The polyclonal antibody was diluted at a ratio of 1: 100 in PBS (phosphate buffered saline, pH 7.4), and incubated at 4 ° C for 24 hours. Primary chlorine polyclonal antibody against human albufox 2 (Rbfox2) (phosphate buffered saline, pH 7.4) diluted at a ratio of 1: 1,000 was cultured at 4 ° C for 24 hours.

Hela (HeLa) cells treated with primary chlorine polyclonal antibodies against each protein were rinsed with PBS and incubated with Alexa-488 or Alexa-594 conjugated secondary antibody at room temperature. I rinsed it. The DNA in the nucleus was stained with DAPI (4 ', 6' diamino-2-phenylindole), and then the expression of each protein was confirmed by immunofluorescent microscopy.

As a result, it was confirmed that stress granules were formed when the arbate was treated in HeLa cells, and that Rbfox2 was expressed at the position where the stress granules were formed (Fig. 1).

Example  3. Using colon cancer tissue Albie Fox  2 (Rbfox2) in the cytoplasm

Normal colon mucosa tissue near the cancer tissue (at least 1 cm away from the cancer tissue) was used as a normal control.

Colon cancer tissues and normal colon mucosal tissues were fixed with 4% paraformaldehyde for 10 minutes and then treated with 0.5% (v / v) Triton X-100 for 15 minutes to increase cell permeability , non-reactive blocking staining was blocked by treatment with Tris-buffered saline (pH 6.0) containing 5% bovine serum albumin for 1 hour.

After that, the primary chlorine polyclonal antibody against the Rbfox2 protein was diluted in PBS (phosphate buffered saline, pH 7.4) buffer at a ratio of 1: 1,000 and cultured at 4 ° C for 24 hours. After rinsing with PBS, secondary antibody conjugated with Alexa-488 or Alexa-594 was incubated at room temperature, washed again with PBS, and DNA was stained with DAPI (4 ', 6'diamino-2-phenylindole) . Subsequently, immunofluorescent microscopy confirmed the expression of the Rbfox2 protein.

As a result, it was confirmed that albufox 2 (Rbfox2) is expressed only in the nucleus in normal colon tissue but albicvox2 (Rbfox2) is mainly expressed in cytoplasm in colon cancer tissue (Fig. 2).

Claims (10)

A composition for diagnosing digestive organs cancer, which comprises, as an active ingredient, an antibody specifically recognizing an albufox 2 (Rbfox2) protein contained in a specimen. The composition for diagnosing gastrointestinal cancer according to claim 1, wherein the specimen is a tissue isolated from a patient suspected of having a digestive organs cancer. The composition for diagnosing gastrointestinal cancer according to claim 1, wherein the Rbfox2 protein migrates from the nucleus to the cytoplasm by the development of cancer cells. The composition for diagnosing gastrointestinal cancer according to claim 1, wherein the gastrointestinal cancer is any one selected from gastric cancer, colon cancer, liver cancer, pancreatic cancer, esophageal cancer and oral cancer. The method of claim 1, wherein the antibody is selected from the group consisting of polyclonal or monoclonal antibodies specific for Rbfox2 protein; Or a fragment of an antibody comprising a polypeptide having a binding domain of an albufox2 (Rbfox2) protein. The composition for diagnosing gastrointestinal cancer according to claim 1, further comprising a secondary antibody that binds to an antibody that specifically recognizes the Rbfox2 protein. A kit for the diagnosis of gastrointestinal cancer, comprising the composition for diagnosing gastrointestinal cancer according to any one of claims 1 to 6. (1) obtaining a tissue suspected of having cancer from a suspected patient of digestive organs cancer and surrounding tissues presumed to be normal tissue;
(2) reacting the obtained Rbfox2 protein contained in the tissue suspected of being cancer and the surrounding tissue with an antibody that specifically recognizes the Rbfox2 protein;
(3) measuring the respective content of albufox2 (Rbfox2) protein contained in the cytoplasm of the tissue suspected of being cancer and the surrounding tissue from the reaction of step (2); And
(4) In step (3), the amount of Rbfox2 protein contained in the cytoplasm of the suspected cancer tissue and the content of Rbfox2 protein contained in the cytoplasm of the surrounding tissues of the cancer suspected cancer And comparing the content of the Rbfox2 protein contained in the cytoplasm of the cancer suspected tissue to the content of the Rbfox2 protein contained in the cytoplasm of the surrounding tissue to discriminate the cancer; Wherein the information is provided for the diagnosis of digestive system cancer. 9. The method according to claim 8, wherein the content of the Rbfox2 protein contained in the cytoplasm of the tissue suspected of being cancer and the surrounding tissue in the step (3) is measured through an antigen-antibody reaction. A method of providing information necessary for cancer diagnosis. 10. The method according to claim 9, wherein the antigen-antibody reaction is measured using immunofluorescent microscopy.

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