KR20190020364A - Diagnosis of gastric cancer using gastrokine 1 protein within blood - Google Patents

Abstract

The present invention relates to a gastric cancer diagnostic composition comprising an agent for measuring an exosomal gastrokine-1 (GKN1) protein level, or a gastric cancer diagnostic kit comprising the composition. The present invention relates to a method for providing information for diagnosis or prognosis of gastric cancer, which comprises a step of measuring the GKN1 protein level from a biological sample isolated from a patient, and a step of comparing the GKN1 protein level with a reference value obtained from a control group sample.

Description

Diagnosis of gastric cancer using gastrokinetic 1 protein in the blood {

The present invention relates to a diagnostic composition for gastric cancer comprising an agent for measuring GKN1 protein (Gastrokine-1 protein) level.

Gastric cancer is a common cancer worldwide in Korea, China, and Japan. In the United States and Europe, the incidence rate is low. In Korea, cancer incidence is the first and stomach cancer is the second most common cancer. Gastric cancer is classified as an adenocarcinoma arising in the glandular cells of the mucous membrane of the stomach, and 95% of the gastric cancers are gastrointestinal stromal tumors arising from lymphoma and epilepsy.

Currently, three major treatment modalities for cancer are surgical treatment, pharmacotherapy, and radiotherapy. Pharmacotherapy is associated with less pain and is associated with recurrence of cancer after treatment compared to surgical or radiotherapy. This is relatively small, so expectations are gathering. Therefore, many anticancer drugs have been developed and used to cope with them. Most of these anticancer drugs exhibit anticancer effects by selectively killing cells that are actively dividing in consideration of abnormal growth of cancer. Such anticancer drugs have serious problems such as immune cells and hair follicle cells, which are actively dividing cells in the human body, and they are accompanied with serious side effects, so that they can not be used for a long time. Endoscopic mucosal resection has been used for the treatment of gastric cancer, including lymphadenectomy, endoscopic mucosal resection, and laparoscopic gastrectomy. Endoscopic mucosal resection has been used for the treatment of early gastric cancer in the stomach, This method is effective in avoiding the pain of gastrectomy due to the simple endoscopic procedure. However, if the possibility of lymph node metastasis is limited among mucosal-limited early gastric cancer, There is a limitation that it can be used.

In recent years, research on the function of genes related to the generation and treatment of cancer has been conducted worldwide, and researches have been conducted to find therapeutic targets and to use them for diagnosis and therapeutic drug development. With the activation of the genome research, studies on human gene DNA chip or proteome analysis have been actively conducted, and a large number of genes related to cancer have been discovered. Therefore, a list of many genes and related databases have been established, but most of these genes Biological function and cancer relatedness have not yet been studied or are uncertain. Therefore, there is considerable difficulty in finding genes that can effectively treat cancer, in addition to actual cancer-related or diagnosis and utilization as target genes. Therefore, in addition to the cancer-related genes identified so far, it is necessary to discover new genes.

Korean Patent No. 10-1215069

It is an object of the present invention to provide a diagnostic composition for gastric cancer comprising an agent for measuring GKN1 protein (Gastrokine-1 protein) level.

It is still another object of the present invention to provide a gastric cancer diagnostic kit comprising the diagnostic composition for gastric cancer.

Yet another object of the present invention is to provide a method for detecting GKN1 protein comprising: (a) measuring the level of GKN1 protein from a biological sample isolated from a patient; And (b) comparing the level of GKN1 protein with a reference value obtained from a control sample. The present invention also provides a method for providing information for the diagnosis or prediction of prognosis of gastric cancer.

In order to achieve the above object, the present invention provides a diagnostic composition for gastric cancer, comprising a preparation for measuring GKN1 protein (Gastrokine-1 protein) level.

In one embodiment of the present invention, the agent for measuring the GKK1 protein level may be an antibody that specifically binds to the protein.

In one embodiment of the present invention, the GKN1 protein may be present in exosomes in blood, plasma or serum.

In addition, the present invention provides a gastric cancer diagnostic kit comprising a diagnostic composition for gastric cancer.

In one embodiment of the present invention, the kit may be an ELISA kit or a protein chip kit.

The present invention also provides a method for detecting a GKN1 protein, comprising: (a) measuring a GKN1 protein level from a biological sample isolated from a patient; And (b) comparing the GKN1 protein level with a reference value obtained from a control sample. The present invention also provides a method for providing information for diagnosis or prediction of prognosis of gastric cancer.

In one embodiment of the present invention, the GKN1 protein may further include a heat treatment at 60 to 80 ° C for 5 to 15 minutes, preferably a heat treatment at 75 to 85 ° C for 8 to 12 minutes Deg.] C, more preferably at 70 [deg.] C for 10 minutes.

In one embodiment of the present invention, it may further comprise a step of determining that the GKN1 protein level is lower than the reference value, and determining that the GKN1 protein level is a gastric cancer.

In one embodiment of the present invention, the method may further include a step of determining that the possibility of gastric cancer is high or the prognosis is poor if the GKN1 protein level is lower than the reference value.

In one embodiment of the present invention, the biological sample may be blood, plasma or serum, preferably serum.

In one embodiment of the present invention, the assay is performed using an enzyme immunoassay (ELISA), a radioimmunoassay (RIA), a sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, , Flow cytometry, fluorescence activated cell sorting (FACS), enzyme substrate staining, and antigen-antibody aggregation.

The GKN1 protein according to the present invention is a biomarker useful for predicting or diagnosing gastric cancer. If the total GKN1 protein level of the exosome including the GKN1 protein is lower than that of a normal human, The diagnosis of gastric cancer or prediction of prognosis may be possible.

FIG. 1A shows the result of identification of GKN1-binding protein, which shows a list of exosome proteins that bind to recombinant GKN1 protein in a protein microarray.
FIG. 1B shows the binding potency of GKN1 to COMT and YWHAZ protein by immunoprecipitation and Western blotting.
FIG. 2A shows the results of immunohistochemical staining for HFE-145 cells, exosomes isolated from AGS and MKN1 cells treated / untreated with recombinant GKN1 (rGKNl) protein, exosome-depleted and whole cell lysates (WCL) The results of Western blotting on GKN1, TSG-1 and CD81 are shown in FIG.
FIG. 2B shows the result of trypsin digestion assay by treatment / non-treatment of trypsin and Triton X-100 on exosome derived from HFE-145 cells.
FIG. 3A shows Western blotting results showing inhibition of Clathrin protein expression in AGS and MKN1 cells treated with siClathrin.
FIG. 3B shows the results of confirming isolated exosomes after treating recombinant GKN1 protein with AGS cells.
FIG. 3c shows localization results of exosomes containing GKN1 protein in AGS and MKN1 cells treated with siClathrin.
FIG. 3D shows Western blotting results of expression of GKN1 and CD81 in siClathrin-treated AGS and MKN1 cells.
FIG. 4A is a graph showing the results of measurement of the total medium, exosome, and exosome depleted media in the case where heat treatment is not performed (non-heating) or heat treatment is performed in HFE-145 cells GKN1 protein was analyzed.
FIG. 4B is a graph showing the relationship between total serum, exosomes (total serum), and total serum (total serum) of the 10 normal healthy subjects and 10 cancerous patients who were not heat- Exosome) and serum (W / O Esosome) from which exosome has been removed.
FIG. 4c shows the results of analysis of age and sex related to serum GKN1 protein concentration from 100 healthy subjects.
FIG. 4d shows the results of analysis of the concentration of GKN1 protein in healthy subjects, patients with atrophic gastritis (Atrophy), patients with atrophic gastritis accompanied by intestinal metaplasia (IM), and gastric cancer patients (P <0.0001).
Figure 4e shows the results of analysis of serum GKN1 levels in 100 healthy subjects, 150 human gastric cancer patients (GC serum), 45 liver cancer patients (LC serum) and 50 colon cancer patients (CC serum) .
Figure 4f shows the serum GKN1 levels in gastric cancer patients with a normal gastric mucosa, those with atrophic gastritis (Atrophy), and those with atrophic gastritis with intestinal metaplasia The results are shown in Fig.
FIG. 4g shows the results of ROC curve analysis as to whether serum GKN1 concentration can distinguish gastric cancer patients from liver cancer and colorectal cancer patients.
Figure 4h shows the expression of GKN1 protein in early gastric cancer and advanced gastric cancer among healthy subjects, patients with atrophic gastritis (Atrophy), patients with atrophic gastritis with intestinal metaplasia (IM) (P < 0.0001). &Lt; / RTI &gt;
Figure 4i shows the ROC curve for GKN1 levels in healthy subjects, patients with atrophic gastritis (Atrophy), patients with atrophic gastritis with intestinal metaplasia (IM), and patients with early gastric cancer among gastric cancer patients The results of the analysis are shown.
FIG. 5A shows the results of immunofluorescence assays to determine whether exosome derived from HFE-145 cells adheres to the cell membranes of Jurkat T cells, THP-1 protein aggregates, and U937 macrophages.
FIG. 5B shows the results of analysis of whether exosome GKN1 and CD81 proteins derived from HFE-145 cells are present in the cell membrane or cytoplasm or nucleus of Jurkat T cells, THP-1 protein, U937 macrophages.

The term " diagnosis " in the present invention means confirming the pathological state. For the purpose of the present invention, the diagnosis means confirming the progress of gastric cancer by confirming the expression level of GKN1 protein in exosome

As used herein, the term " antibody " as used herein refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to the GKN1 protein, which is a marker of the present invention. Also included are partial peptides that can be made from the protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 amino acids. The form of the antibody of the present invention is not particularly limited and any part thereof having a polyclonal antibody, a monoclonal antibody or an antigen-binding property is included in the antibody of the present invention and includes all the immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Such an antibody to the GKN1 protein of the present invention includes all antibodies that can be produced by a method known in the art.

The gastric cancer diagnostic kit of the present invention may include an antibody specifically binding to GKN1 protein of exosome and the kit for measuring protein level may be an ELISA kit or a protein chip kit used for " measurement of protein expression level & have.

Measurement of the protein expression using the antibody is performed by forming an antigen-antibody complex between the GKN1 protein and the antibody thereof, and quantitatively detected by measuring the formation amount of the complex by various methods.

The antigen-antibody complex can be assayed by enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, fluid But is not limited to, using one or more methods selected from the group consisting of flow cytometry, fluorescence activated cell sorting (FACS), enzyme substrate staining, and antigen-antibody aggregation.

The term " biological sample " in the present invention means a specimen capable of detecting the total GKN1 protein expression level, including the exo-somatic GKN1 protein, including blood, plasma or serum, Means serum.

The present invention can include a step of obtaining a biological sample separated from the patient, followed by heat treatment at 60 to 80 DEG C for 5 to 15 minutes. After the heat treatment, the expression level of GKN1 protein in the total sample or the exosome was measured and compared with the reference value obtained from the control sample, it was judged to be a gastric cancer when the level of GKN1 protein in the exosome was lower than the reference value, Do.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Experimental Method

1.1. Production of recombinant vector into which GKN1 was introduced

A recombinant vector was prepared as follows to prepare recombinant GKN1 protein. First, the human GKN1 cDNA was amplified by the PCR method and a recombinant vector pCMV6-AN-FC-hGKN1 was prepared using a restriction enzyme site in pCMV6-AN-FC (Origene ID PS100055) vector. More specifically, pCMV6-AN-FC vector and GKN1 cDNA were treated with restriction enzymes SgfI and MluI, and pCMV6-AN-FC and hGKN1 cDNA were ligated to introduce the GKN1 gene into the vector. Thereafter, E. coli was transformed to colonies formed on agar plates, followed by culturing and extracting DNA, followed by sequencing to confirm whether the GKNI gene was correctly introduced. Then, recombinant GKN1 protein was purified using protein A affinity chromatography, and the recombinant GKN1 protein was identified by Western blotting. Thereafter, the sequence of the GKNI gene is shown in SEQ ID NO: 1, and the amino acid sequence of the GKN1 protein encoded thereby is represented by 2.

1.2. Cell culture

AGS and MKN1 gastric cancer cell lines that do not express the GKN1 (Gastrokine-1) protein and HFE-145 immortalized gastric epithelial cells that express GKN1 were cultured in 10% heat-inactivated FBS fetal bovine serum) in RPMI-1640 medium at 37 ° C and 5% CO ₂ . Jurkat T cells, THP-1 monocytes and U937 macrophages were also cultured under the same conditions.

1.3. Identification of GKN1 binding protein through HuProt (TM) microarray

Human Proteome Microarray (CDI Labs, USA) containing over 19,000 full-length recombinant human proteins was used to identify GKN1 binding proteins. Briefly, the protein microarray was treated with 1 μg of biotinylated GKN1 at 4 ° C for 8 h after 2 h of blocking buffer (5% BSA in PBS with 0.05% tween-20). The microarray was then treated with 1 μg of streptavidin-fluorescence (Alexa-Fluor 635 nm). Results were confirmed with a GenePix 4100A Microarray Laser Scanner (Molecular Devices, USA).

1.4. Exosome Isolation

To investigate the presence of GKN1 protein in exosomes, AGS and MKN1 cells were treated with recombinant GKN1 protein (rGKN1, ANRT, Daejeon, Korea). Exosomes were isolated from the supernatants of HFE-145, AGS and MKN1 cells. Briefly, cells from passage 3 to 8 were cultured in serum-free medium and cultured at 37 ° C, 5% CO 2 with 10% FBS and 1% penicillin / streptomycin for 48 hours before collecting the medium. The conditioned media was centrifuged at 2000 g for 10 min at 4 ° C to remove cell debris and then passed through a 0.22 μm filter. The filtered supernatant was transferred to a new glass tube and placed on ice. Then, 2 ml of supernatant was mixed with 0.75 ml of A / B / C solution (101Bio company, CA, USA) in a new glass tube, vortexed vigorously for 30 seconds and left at 4 ° C for 30 minutes. The mixture was separated into two layers and the upper layer was removed. The lower layer was transferred to a microcentrifuge tube and centrifuged at 5000 g for 3 minutes. The intermediate layer was transferred to a new microcentrifuge tube and centrifuged at 5000 g for 3 minutes. Lt; / RTI &gt; for 10 minutes. A total volume of 4X 1X PBS was added to the tube and strongly pipetted. Tubes were treated at high speed for 15 minutes in a horizontal shaker and centrifuged at 5000 g for 5 minutes. The supernatant was carefully transferred onto a PureExo Column (101Bio company, CA, USA) and centrifuged at 1000 g for 5 min. The passage liquid contains a separate exosomal fraction suspended in PBS.

1.5. Exosome Labeling

Exosomes were labeled with PKH26 (Sigma, St. Louis, USA) according to the manufacturer's protocol (slight modification). Briefly, the exosomal pellet was suspended in 1 ml of Diluent C, and 1 ml of Diluent C was mixed with 4 μl of PKH26 to make a Stain Solution. The exosomal suspension was mixed with the stain solution for 4 minutes, and the labeling reaction was stopped when the same amount of 1% BSA (bovine serum albumin) was added. Labeled exosomes were isolated using the Total Exosome Isolation Kit (Invitrogen) according to the manufacturer's protocol. Briefly, a 0.5 volume of Isolation Reagent was added to the labeled exosomes and mixed. Labeled exosomes were kept at 4 ° C overnight and subsequently centrifuged at 10,000 g for 1 hour at 4 ° C. Thereafter, the supernatant was discarded and each pellet was suspended in 100 μl of PBS.

1.6. Expression of GKN1 protein in exosomes

Expression of GKN1 in cell lysate and exosomes of HFE-145, AGS and MKN1 cells was analyzed by Western blotting. Equivalent cell lysates and exosomes were separated by 12.5% SDS-PAGE and transferred to Hybond-PVDF (polyvinylidene difluoride) transfer membranes (Amersham). After blocking with 0.5% skim milk, the membranes were reacted with primary anti-GKN1 (Abcam, MA, USA), anti-TSG101 and anti-CD81 antibodies (Santa Cruz Biotechnology, TX, USA) peroxidase-conjugated secondary antibodies. Protein bands were detected on Westernsure ECL substrate (LI-COR Biosciences, NE, USA) and bands were visualized on LAS 4000 (Fuji Film, Japan).

1.7. Co-immunoprecipitation (co-immunoprecipitation)

AGS and MKN1 cell-derived exosomes treated with recombinant GKN1 (rGKNl) were washed with PBS and incubated with 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1.0 mM NaVO4 (J. Biol. Chem. 2010; 285: 20547-57) with PBS (pH 7.2) containing 1% protease inhibitor cocktail (Sigma, St. Louis, Mo., USA). As described previously, the same amount of protein aliquat (1.0 mg) was added to 2.0 μg of Protein A / G-protein coupled antibody to GKN1 (Abcam, Cambridge, UK), COMT and YWHAZ (Genetex, CA, USA) agarose (Santa Cruz Biotechnology) (EBioMedicine. 2016; 9: 97-109).

1.8. Trypsin digestion assay

Exosomes isolated from HFE-145 cells (35 μg protein in 7 μl of PBS) as previously described were incubated with 1% Triton X-100 for 30 min at 4 ° C to find the presence of GKN protein in the exosome Lipid membrane was degraded (Blood. 2009; 113: 1957-66). Exosomes were incubated under the same conditions without Triton X-100. The exosomal suspension was digested with trypsin at 37 ° C with two enzyme concentrations (15 and 0.01 μg / mL). Triton X-100 treatment had little effect on trypsin activity (data not shown). After the trypsin treatment, the samples were immediately mixed with an equal volume of Laemmli buffer and boiled for 3 minutes before gel loading.

1.9. Immunofluorescence studies

Mouse anti-GKN1 (abcam), anti-clathrin and anti-alexa-488 conjugated goat anti-mouse IgG (invitrogen) secondary antibodies were used to visualize the immune response. The specificity of the reaction was tested with Non-immune Mouse Serum (Invitrogen).

1.10. Serum samples

The study was conducted in a group of 150 gastric cancer patients who were scheduled to undergo gastrectomy and 100 healthy volunteers. In addition, 45 patients with hepatocellular carcinoma and 50 patients with colorectal cancer were used as controls. Whole blood was collected from 245 pre-operative cancer patients and 100 healthy volunteers. After sampling, the serum was separated and immediately stored at -80 ° C. The frozen serum was dissolved in ice, and the total protein concentration in the serum was diluted to 15 ug / ml with phosphate buffered saline (PBS). To investigate whether GKNl protein is present as culture medium of HFE-145 cells and as exoemia protein in human serum, the samples were not treated or treated at 70 &lt; 0 &gt; C. The concentration of GKN1 protein in the whole culture, whole serum, exosome-removed sample, and exosome fraction was calculated using an ELISA kit (USCN, Wuhan, China) according to the manufacturer's instructions. Notified consent was provided under the Helsinki Declaration, and written consent was obtained from all subjects. The present invention was approved by the Institutional Review Board of The Catholic University of Korea, College of Medicine (MC16SISI0132). There was no evidence of hereditary cancer in any of the cancer patients.

1.11. Statistical analysis

P values less than 0.05 were considered statistically significant. ROC (receiver operating characteristic) curve analysis was used to evaluate the diagnostic usefulness of markers based on EG classification. The ROC curve is a plot of true-positive fraction versus false-positive fraction, which is evaluated at all possible limits.

Example 2. Identification of GKN1 binding protein

The present inventors performed a protein microarray assay to identify the protein binding to GKN1. As a result, 27 exosome proteins with normal to strong binding ability to recombinant GKN1 (rGKN1) were found (Fig. 1A). To confirm the binding ability of GKN1 to exo-somatic protein, the present inventors extracted exo-somatic protein from AGS and MKN1 cells treated with HFE-145 cells and recombinant GKN1 (rGKN1), and co- The interaction between GKN1 and COMT in the protein and the interaction between GKN1 and YWHAZ was demonstrated (Fig. 1B).

Example 3 Detection of GKN1 Protein from Exocytosis from Stomach Cell Lines

In view of the fact that the recombinant GKN1 (rGKN1) protein binds to the exosomal protein, the present inventors investigated whether GKN1 is naturally secreted into the exosomal protein and internalized into cells, and cultured HFE-145, AGS and MKN1 cells Exosomes were separated from the supernatant to confirm the presence of GKN1. As a result, it was confirmed that GKN1 protein was contained in exosome derived from HFE-145 cell, but GKN1 protein was not expressed in exosome derived from AGS and MKN1 gastric cancer cell line (Fig. 2a). When AGS and MKN1 cells were treated with recombinant GKN1 protein, expression of GKN1 protein was mainly present in exosomes and there was little expression in the exosome-free medium and whole cell lysate (WCL) without exosome (Fig. 2a ). Among the two exosomal protein markers, TSG101 was identified in isolated exosomes and cell lysates, but CD81 was found to be present only in exosomes and CD81 was used as an exosomal marker in later experiments (FIG. 2a).

Next, trypsin digestion assays by treatment / non-treatment of trypsin and Triton X-100 were performed to confirm the presence of GKN1 protein in exosomes, and then Western blotting was performed (Blood. 2009; 113 : 1957-66). GKN1 did not undergo trypsin-dependent enzymatic degradation when Triton X-100 was untreated and only trypsin was treated with exosomes. However, when both Triton X-100 and trypsin were treated, GKN1 was completely degraded (Fig. 2B). From the above results, it was confirmed that GKN1 protein is present in the exosome.

Example 4. Clathrin-dependent internalization of exosomes containing GKN1 protein

The present inventors conducted an experiment to confirm whether Clathrin was involved in the internalization of GKN1 protein of exosomes. SiClathrin siRNA was used to knock down Clathrin, and clathrin expression was significantly reduced in siRNA and MKN1 cells infected with siClathrin (Fig. 3a). When the PKH26-positive exosome (FIG. 3b) containing GKN1 protein was treated on AGS and MKN1 cells, the exosomes were clearly internalized in the cytoplasm of AGS and MKN1 (FIG. 3c). However, the knockdown of clathrin in AGS and MKN1 cells significantly inhibited the presence of PKH26-positive exosome in the cell membrane and cytoplasm (Fig. 3c). In these cells, the expression of GKNl and CD81 protein in the cell membrane was markedly decreased, and not in the cytoplasm (Fig. 3d).

Thus, Clathrin-mediated endocytosis plays an important role in attaching and internalizing exocomes containing GKN1 protein to stomach cells.

Example 5 Diagnosis of Gastric Cancer by Serum GKN1 Protein Concentration

To determine whether GKN1 protein is present in the medium and human serum as exoemia protein, the concentration of GKN1 protein was measured by ELISA assay after treating HFE-145 cell culture medium and human serum at 70 ° C for 10 minutes. As a result, the non-heating GKN1 protein in the HFE-145 cells was not detected in the medium in which the whole medium, the exosome and the exosome were removed, while the heating was carried out at 70 ° C. for 10 minutes, , The concentrations of GKN1 protein were 1.13 ± 0.06, 1.08 ± 0.1, and 0.05 ± 0.04 ng / ml in the whole medium, exosome and exonome-free medium (FIG. 4A). Consistent with this, GKN1 protein was not detected in sera from which untreated whole serum, exosomes and exosomes were removed (Fig. 4B). Among 10 healthy subjects, GKN1 protein expression levels in the heat-treated whole serum and exosomes were 6.53 ± 0.58 ng / ml and 6.24 ± 0.55 ng / ml, respectively (FIG. 4b). In 10 gastric cancer patients, the expression levels of GKN1 protein in total serum and exosomes were 3.58 ± 0.54 ng / ml and 3.28 ± 0.82 ng / ml, respectively (FIG. 4b) It means that it exists. Based on the above results, we obtained total serum in 100 healthy subjects and 245 gastric cancer, colorectal cancer, and liver cancer patients, and then analyzed the GKN1 concentration after heat treatment at 70 ° C for 10 minutes.

As shown in FIG. 4c, serum GKN1 protein levels in normal healthy subjects tended to be age-related but not statistically significant (P = 0.338). There was also no difference in serum GKN1 levels between men and women (P = 0.074). Interestingly, those with atrophic gastritis and those with atrophic gastritis with intestinal metaplasia (IM) showed significantly lower serum GKN1 levels than those without gastritis, but significantly higher than those with gastric cancer (Figure 4d ).

GKN1 levels in total serum of 100 healthy subjects and 150 gastric cancer patients were 6.35 ± 0.82 ng / ml and 3.50 ± 0.57 ng / ml, respectively (FIG. 4e), indicating that serum GKN1 protein levels in healthy gastric cancer patients (P <0.0001) compared with the control group. Furthermore, serum GKN1 levels in liver cancer and colorectal cancer patients were 6.07 ± 0.92 and 6.26 ± 0.95 ng / ml, respectively, which were similar to those of healthy subjects and significantly higher than those of gastric cancer patients (FIG. 4e). In particular, the area values of the ROC curve were 1.0, 1.0, and 0.9964, respectively, indicating that patients with atypical gastritis accompanied by intestinal metaplasia (IM), healthy subjects, atrophic gastritis, (Fig. 4f). In order to confirm that the present inventors can distinguish gastric cancer patients from hepatocellular carcinoma and colorectal cancer patients by the level of serum GKN1, the ROC curve values were measured. The areas in the ROC curve were 0.9938 and 0.9987, respectively, It was confirmed that the risk of gastric cancer can be predicted and diagnosed by the GKN1 protein concentration (Fig. 4G).

Gastric cancer is divided into early gastric cancer and advanced gastric cancer according to the degree of gastric wall invasion. In the present study, the serum GKN1 concentration in early stage gastric cancer patients was 3.75 ± 0.47 ng / ml, which was higher than the serum GKN1 level in advanced gastric cancer patients (3.38 ± 0.58 ng / ml, P <0.001) intestinal metaplasia, IM) were lower than those of patients with atrophic gastritis (P <0.001) (Fig. 4h). The area of the ROC curve was 1.0, indicating that patients with atypical gastritis with intestinal metaplasia (IM) and early gastric cancer patients with healthy subjects, atrophic gastritis, and intestinal metaplasia (IM) (Fig. 4I).

From the above results, the present inventors confirmed that exosome GKN1 protein in human serum can be used as a biomarker useful for the diagnosis of gastric cancer.

Example 6. Delivery of exosomes to blood of GKN1 protein

Based on the evidence that the GKN1 protein produced in the phagemid is identified in the serum, the present inventors have found that lymphocytes, monocytes and macrophages in the gastric mucosa can function as transporters for exosomes containing GKN1 protein from phagocytes to blood . Interestingly, in the immunofluorescence assay, the exosomes derived from HFE-145 cells adhered to the membranes of THP-1 protein and U937 macrophages but not Jurkat T cells (Fig. 5a) (Fig. 5B). Fig. Therefore, the present inventors confirmed that transferring exosomes including GKN1 protein into blood is monocytes and macrophages.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Diagnosis of gastric cancer using gastrokinetic 1 protein within          blood <130> PN1706-210 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 600 <212> DNA <213> Artificial Sequence <220> <223> cDNA sequence of GKN1 <400> 1 atgcttgcct actcctctgt ccactgcttt cgtgaagaca agatgaagtt cacaattgtc 60 tttgctggac ttcttggagt ctttctagct cctgccctag ctaactataa tatcaacgtc 120 aatgatgaca acaacaatgc tggaagtggg cagcagtcag tgagtgtcaa caatgaacac 180 aatgtggcca atgttgacaa taacaacgga tgggactcct ggaattccat ctgggattat 240 ggaaatggct ttgctgcaac cagactcttt caaaagaaga catgcattgt gcacaaaatg 300 aacaaggaag tcatgccctc cattcaatcc cttgatgcac tggtcaagga aaagaagctt 360 cagggtaagg gaccaggagg accacctccc aagggcctga tgtactcagt caacccaaac 420 aaagtcgatg acctgagcaa gttcggaaaa aacattgcaa acatgtgtcg tgggattcca 480 acatacatgg ctgaggagat gcaagaggca agcctgtttt tttactcagg aacgtgctac 540 acgaccagtg tactatggat tgtggacatt tccttctgtg gagacacggt ggagaactaa 600                                                                          600 <210> 2 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of GKN1 <400> 2 Met Leu Ala Tyr Ser Ser Val His Cys Phe Arg Glu Asp Lys Met Lys   1 5 10 15 Phe Thr Ile Val Phe Ala Gly Leu Leu Gly Val Phe Leu Ala Pro Ala              20 25 30 Leu Ala Asn Tyr Asn Ile Asn Val Asn Asp Asp Asn Asn Asn Ala Gly          35 40 45 Ser Gly Gln Gln Ser Val Ser Val Asn Asn Glu His Asn Val Ala Asn      50 55 60 Val Asp Asn Asn Asn Gly Trp Asp Ser Trp Asn Ser Ile Trp Asp Tyr  65 70 75 80 Gly Asn Gly Phe Ala Ala Thr Arg Leu Phe Gln Lys Lys Thr Cys Ile                  85 90 95 Val His Lys Met Asn Lys Glu Val Met Pro Ser Ile Gln Ser Leu Asp             100 105 110 Ala Leu Val Lys Glu Lys Lys Leu Gln Gly Lys Gly Pro Gly Gly Pro         115 120 125 Pro Pro Lys Gly Leu Met Tyr Ser Val Asn Pro Asn Lys Val Asp Asp     130 135 140 Leu Ser Lys Phe Gly Lys Asn Ile Ala Asn Met Cys Arg Gly Ile Pro 145 150 155 160 Thr Tyr Met Ala Glu Glu Met Gln Glu Ala Ser Leu Phe Phe Tyr Ser                 165 170 175 Gly Thr Cys Tyr Thr Thr Ser Val Leu Trp Ile Val Asp Ile Ser Phe             180 185 190 Cys Gly Asp Thr Val Glu Asn         195

Claims (11)

A composition for the diagnosis of gastric cancer, comprising an agent for measuring GKN1 protein (Gastrokine-1 protein) level. The method according to claim 1,
Wherein the agent for measuring the GKN1 protein level is an antibody that specifically binds to the protein. The method according to claim 1,
Wherein said GKNl protein is present in exosomes of blood, plasma or serum. A kit for gastric cancer diagnosis comprising the composition according to claim 1. 5. The method of claim 4,
Wherein the kit is an ELISA kit or a protein chip kit. (a) measuring the level of GKN1 protein from a biological sample isolated from a patient; And
(b) comparing the GKN1 protein level with a reference value obtained from a control sample. The method according to claim 6,
Wherein the GKN1 protein further comprises heat-treating at 60 to 80 DEG C for 5 to 15 minutes. The method according to claim 6,
And determining that the gastric cancer is a gastric cancer when the GKN1 protein level is lower than a reference value. The method according to claim 6,
Further comprising the step of determining that there is a high possibility that the GKN1 protein level is high or the prognosis is poor when the GKN1 protein level is low compared to the reference value. The method according to claim 6,
Wherein the biological sample is blood, plasma or serum. The method according to claim 6,
The measurement may be performed by an enzyme immunoassay (ELISA), a radioimmunoassay (RIA), a sandwich assay, a Western blotting, an immunoprecipitation method, an immunohistochemical staining method, a flow cytometry method, , Fluorescence activated cell sorting (FACS), enzyme substrate staining, and antigen-antibody aggregation.

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