KR102427871B1 - Biomarker composition for diagnosing diffuse type gastric cancer and medical use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing diffuse gastric cancer comprising TINAGL1 protein or a gene encoding the protein as an active ingredient, and a medical use thereof, and more particularly, TINAGL1 in cancer-related fibroblasts isolated from diffuse gastric cancer tissue. It was confirmed that secretion was increased, and it was confirmed that TINAGL1 improved metastasis and tumor formation of diffuse gastric cancer by activating the FAK signal in cancer cells. Accordingly, the TINAGL1 may be provided as a biomarker for diagnosis and prognosis of diffuse gastric cancer, and an inhibitor of TINAGL1 expression or activity may be provided as an effective therapeutic agent for diffuse gastric cancer.

Description

Biomarker composition for diagnosing diffuse type gastric cancer and medical use thereof

The present invention relates to a biomarker composition for diagnosing diffuse gastric cancer comprising TINAGL1 protein or a gene encoding the protein as an active ingredient, and to a medical use thereof.

Stomach cancer is the most common cancer in Korea, but as gastroscopy became more common and gastric cancer was detected at an early stage, the 5-year survival rate of gastric cancer patients (the proportion of patients who were diagnosed with cancer and survived more than 5 years) improved to 75.4%. . As such, although the early detection rate of gastric cancer is increasing, diffuse type gastric cancer with a poor prognosis accounts for 40% of all gastric cancers.

Gastric cancer is divided into enteric gastric cancer and diffuse gastric cancer. In intestinal gastric cancer, the cancer grows in the form of a lump on the mucosal surface, and the growth rate is slower than that of the diffuse type. On the other hand, in diffuse gastric cancer, the changes in the surface of the mucosa are not clearly seen, and precancerous lesions such as adenomas are not easily identified because cancer cells that are small enough to be invisible penetrate the stomach wall and grow under the mucous membrane. However, the proliferation rate is fast, and when cancer is discovered, it is often already advanced. In addition, the possibility of distant metastasis is high, and it easily metastasizes to adjacent sites such as the esophagus or duodenum.

In particular, as for gastric cancer occurring at a young age, diffuse-type gastric cancer, which has a rapid progression and metastasis, accounts for 60-70% of gastric cancer.

The treatment of diffuse gastric cancer is not significantly different from intestinal gastric cancer, and is determined in consideration of the patient's condition and disease progression. By performing submucosal dissection, only cancer cells can be removed without resection of the stomach, so complications and sequelae can be minimized, and a cure rate similar to that of surgery can be expected.

According to the current gastric cancer screening recommendations in Korea, gastroscopy is recommended every two years for those over 40 years of age.

Republic of Korea Patent Publication No. 10-2016-0022273 (published on February 29, 2016)

As described above, diffuse gastric cancer can be expected to have a high cure rate through early detection, but it is difficult to diagnose with general endoscopic ultrasound. To provide TINAGL1, which is secreted and expressed in fibroblast, CAF), as a biomarker composition for diagnosing diffuse gastric cancer.

The present invention may provide a biomarker composition for diagnosing diffuse gastric cancer comprising TINAGL1 protein or a gene encoding the protein as an active ingredient.

The present invention may provide a kit for diagnosing diffuse gastric cancer containing, as an active ingredient, an agent capable of detecting the expression or activity level of TINAGL1 protein or the expression level of a gene encoding the protein.

The present invention provides a method for providing information for diagnosing diffuse gastric cancer, comprising comparing the expression or activity level of TINAGL1 protein, or the expression level of a gene encoding the protein, from a sample isolated from a sample and a normal control. can provide

The present invention can provide a biomarker composition for predicting the prognosis of diffuse gastric cancer comprising TINAGL1 protein or a gene encoding the protein as an active ingredient.

The present invention may provide a pharmaceutical composition for preventing or treating diffuse gastric cancer containing a TINAGL1 inhibitor as an active ingredient.

The present invention can provide a pharmaceutical composition for preventing or treating diffuse gastric cancer containing a TINAGL1 inhibitor and an anticancer agent as active ingredients.

In addition, the present invention comprises the steps of culturing cancer-associated fibroblasts (CAF) isolated from diffuse gastric cancer tissue to determine the TINAGL1 expression level (step 1);

Checking the TINAGL1 expression level by treating the TINAGL1-expressed cancer-associated fibroblasts with a candidate material (second step); and

It is possible to provide a drug screening method for treating diffuse gastric cancer, comprising comparing the TINAGL1 expression level of the first step with the TINAGL1 expression level of the second step (third step).

According to the present invention, it was confirmed that TINAGL1 secretion was increased in cancer-associated fibroblasts isolated from diffuse gastric cancer tissue, and it was confirmed that TINAGL1 improved metastasis and tumor formation of diffuse gastric cancer by activating the FAK signal of cancer cells. , as it was confirmed that the increase in TINAGL1 expression is correlated with the prognosis of diffuse gastric cancer patients, the TINAGL1 can be provided as a biomarker for the diagnosis and prognosis of diffuse gastric cancer, and TINAGL1 expression or activity inhibitors are effective diffuse gastric cancer It may be provided as a therapeutic agent.

1 is a result of confirming the effect of cancer-associated fibroblasts (CAF) and normal fibroblasts (NAF) on cancer cells, the results of confirming the migration and metastatic ability of cancer cells in SNU668 cells treated with CAF or NAF.
2 is a result of confirming the effect of cancer-associated fibroblasts (CAF) and normal fibroblasts (NAF) on cancer cells, the results of confirming the tumorigenicity and migration ability of cancer cells in SNU601 cells treated with CAF or NAF.
3 is a transcriptome analysis and proteomics analysis results between cancer-associated fibroblasts (CAF) and normal fibroblasts (NAF).
4 is a quantitative RT-PCR, Western blot, real-time PCR and ELISA analysis results confirming the difference in TINAGL1 expression level in CAF and NAF isolated from diffuse gastric cancer patients.
5 is a Western blot analysis result confirming the effect of cancer-associated fibroblasts (CAF) and normal fibroblasts (NAF) on FAK signal activation in diffuse gastric cancer cells.
6 is a Western blot analysis result confirming the FAK signaling activity of a diffuse gastric cancer cell line treated with recombinant TINAGL1.
7 is a result confirming the tumorigenicity of the gastric cancer cell line SNU601 treated with recombinant TINAGL1.
FIG. 8 is a Western blot and RT-PCR analysis result confirming cancer-associated fibroblasts (CAFs) in which TINAGL1 expression was inhibited by transfection with TINAGL1 siRNA.
9 is an immunocytochemical staining result confirming cancer-associated fibroblasts (CAFs) in which TINAGL1 expression is suppressed by transfection with TINAGL1 siRNA.
10 is a result confirming the effect of TINAGL1 expression inhibition on the migratory ability of gastric cancer cells after co-culturing cancer-associated fibroblasts (CAFs) in which TINAGL1 expression is suppressed with gastric cancer cells.
11 is a western blot analysis result confirming the effect of TINAGL1 expression inhibition on gastric cancer cell signaling after co-culturing cancer-associated fibroblasts (CAFs) in which TINAGL1 expression is suppressed with gastric cancer cells.
12 is a Western blot analysis result confirming the expression level of TINAGL1 in cancer tissues and normal gastric tissues isolated from diffuse gastric cancer patients.
13 is a result of confirming the expression levels of TINAGL1 and COL1A1 in cancer tissues and normal gastric tissues isolated from patients with diffuse gastric cancer, COL1A1 (red, fibroblast marker) and It is the result of confirming the mRNA expression level of TINAGL1 (green).
14 is a result confirming the prognosis of patients with diffuse gastric cancer according to the TINAGL1 expression level.

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

Among various histological subtypes of gastric cancer, diffuse gastric cancer is known to have a poor prognosis, and as cancer-associated fibroblasts accumulated in diffuse gastric cancer are reported to be related to the prognosis of patients, the inventors of the present invention have It was confirmed that TINAGL1 secretion was increased in the isolated cancer-associated fibroblasts, and it was confirmed that the TINAGL1 activates the FAK signal in cancer cells to improve metastasis and tumor formation of diffuse gastric cancer, thereby completing the present invention.

The present invention may provide a biomarker composition for diagnosing diffuse gastric cancer comprising TINAGL1 protein or a gene encoding the protein as an active ingredient.

In more detail, the "TINAGL1" is "Tubulointerstitial nephritis antigen-like 1" (Gene ID: 64129; GenBank: AAH09048.1).

The TINAGL1 may have increased secretion and expression in cancer-associated fibroblasts (CAF) in gastric cancer tissue.

The present invention may provide a kit for diagnosing diffuse gastric cancer containing, as an active ingredient, an agent capable of detecting the expression or activity level of TINAGL1 protein or the expression level of a gene encoding the protein.

The agent may be selected from the group consisting of primers, probes, antibodies, peptides and aptamers.

The present invention provides a method for providing information for diagnosing diffuse gastric cancer, comprising comparing the expression or activity level of TINAGL1 protein, or the expression level of a gene encoding the protein, from a sample isolated from a sample and a normal control. can provide

The "biomarker" of the present invention is a substance capable of diagnosing by distinguishing the tissue or cell of a subject with gastric cancer from the tissue or cell of a normal control, and increases or decreases in the tissue or cell of the subject with the disease compared to the normal control. and organic biomolecules such as proteins or nucleic acids, lipids, glycolipids, glycoproteins, and the like.

The present invention can provide a biomarker composition for predicting the prognosis of diffuse gastric cancer comprising TINAGL1 protein or a gene encoding the protein as an active ingredient.

In the present invention, "diagnosis" in a broad sense means judging the actual condition of a patient's disease in all aspects. The content of the judgment is the disease name, etiology, disease type, severity, detailed mode of the disease, and the presence or absence of complications.

"Prognosis" of the present invention includes determining whether a subject, ie, a test subject, will have a disease in the future for a specific disease or disorder, or determining the responsiveness of a test subject to treatment.

The present invention may provide a pharmaceutical composition for preventing or treating diffuse gastric cancer containing a TINAGL1 inhibitor as an active ingredient.

The TINAGL1 inhibitor may inhibit the secretion and expression of TINAGL1 protein or a gene encoding the protein in cancer-associated fibroblasts (CAF) in diffuse gastric cancer tissue.

The TINAGL1 inhibitor may be any one selected from the group consisting of TINAGL1 protein or a peptide, aptamer, antibody, antisense nucleotide, siRNA, shRNA, and miRNA that specifically binds to a gene encoding the protein.

The present invention can provide a pharmaceutical composition for preventing or treating diffuse gastric cancer containing a TINAGL1 inhibitor and an anticancer agent as active ingredients.

The anticancer agent is doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, eto It may be selected from the group consisting of etoposide, teniposide, bisantrene, imatinib, cisplatin and 5-fluorouracil.

In one embodiment of the present invention, the pharmaceutical composition containing the TINAGL1 inhibitor as an active ingredient may be administered as an injection, granule, powder, tablet, pill, capsule, suppository, gel, suspension, emulsion, drop or drop according to a conventional method. Any one formulation selected from the group consisting of liquid formulations may be used.

In another embodiment of the present invention, the pharmaceutical composition containing the TINAGL1 inhibitor as an active ingredient includes a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, flavoring agent, and a suitable carrier, excipient, disintegrant, commonly used in the manufacture of pharmaceutical compositions; It may further include one or more additives selected from the group consisting of antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject.

The preferred dosage of the TINAGL1 inhibitor may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the administration route and duration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention comprises the steps of culturing cancer-associated fibroblasts (CAF) isolated from diffuse gastric cancer tissue to determine the TINAGL1 expression level (step 1); Checking the TINAGL1 expression level by treating the TINAGL1-expressed cancer-associated fibroblasts with a candidate material (second step); and comparing the TINAGL1 expression level of the first step with the TINAGL1 expression level of the second step (third step).

The TINAGL1 expression level is reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (radioimmunoassay), immunohistochemistry (immunohistochemistry), It may be measured by any one or more selected from the group consisting of microarray, western blotting, and flow cytometry (FACS).

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

<Example 1> Confirmation of gene expression difference between cancer-associated fibroblasts (CAF) and normal fibroblasts (NAF)

Gastric cancer has severe intertumoral heterogeneity according to histological subtypes.

It has been reported that cancer-associated fibroblasts accumulated in diffuse gastric cancer are associated with the prognosis of patients. In order to confirm the medical significance of cancer-associated fibroblast (CAF) accumulation in a specific subtype, signet ring cell carcinoma, from the previous study results, stromal collagen in 175 patients Alternatively, fibroblast marker staining was performed to classify them into two groups.

As a result, it was confirmed that the patient group with a large amount of fibrotic matrix showed a poorer prognosis than other patient groups.

From the above study results, the difference in the effect of cancer-associated fibroblast (CAF) and normal gastric tissue-associated fibroblast (NAF) on cancer cells was confirmed, and transcriptome and proteomic analysis of CAF and NAF was performed.

NAF or CAF was dispensed in a 24-well plate, and the diffuse gastric cancer cell line SNU668 was dispensed in a transwell chamber with a pore size of 8 μm. Thereafter, serum-free DMEM was added to the upper chamber and DMEM supplemented with 10% FBS was added to the lower chamber, and then co-cultured with NAF or CAF or cancer cells were cultured alone.

After 48 hours, cells were fixed with methanol and H&E staining was performed. After removing the cells present on the top of the membrane in the upper chamber, the membrane was separated from the chamber and mounted with the bottom side up on a slide glass. Cells passing through the membrane were counted by taking three random pictures per well at 100 magnification, and then the averages were compared.

A 1.0% agarose gel was made at the bottom of the 24-well plate using agarose (low gelling temperature), and when the gel at the bottom was hardened, SNU668 cancer cells and NAF or CAF were mixed with 0.5% agarose gel and dispensed on it to harden. .

When the gel at the top was hardened, 300 μl of RPMI with 10% FBS was dispensed. Thereafter, the medium was replaced with a new medium once every 3 days and observation was made for 20 days.

Four wells were used in each group, and the diameter of colonies was measured in all wells on the 20th day. The degree of tumorigenesis between the two groups was compared with the average diameter of the top 5 spheroids per well.

In addition, in the same manner as in the previous experiment, SUN601 cells were treated with NAF and CAF, respectively, and cultured together to confirm tumorigenicity and migration ability.

As a result, as shown in FIGS. 1 and 2 , it was confirmed that in the group co-cultured with CAF compared to NAF, the migrating ability of cancer cells increased more and the average size of the formed tumor spheroids was larger.

In addition, referring to the transcript analysis result of FIG. 3 , 13 genes were generally increased in CAF than in NAF, and 6 genes were identified as genes related to the extracellular region, especially TINAGL1 showing the highest ratio. Confirmed.

In addition, referring to the mass spectrometry results, 96 proteins were identified that were significantly increased in CAF-conditioned medium compared to NAF. As a result of IPA function analysis for 96 proteins, 27 proteins were classified into groups related to the extracellular region. and TINAGL1 was identified.

In order to confirm whether TINAGL1, whose expression level was changed, exhibits an expression difference in CAF and NAF of patients with diffuse gastric cancer, quantitative RT-PCR, Western blot, real-time PCR, and ELISA were performed.

As a result, it was confirmed that TINAGL1 expression was increased in CAF than in NAF of diffuse gastric cancer patients as shown in FIG. 4 .

An additional experiment was performed to confirm the relationship between TINAGL1 secreted from CAF confirmed from the above results and gastric cancer progression.

<Example 2> Confirmation of the effect of cancer-associated fibroblasts (CAF) and normal fibroblasts (NAF) on diffuse gastric cancer cell lines

To determine the effect of cancer-associated fibroblasts (CAF) and normal fibroblasts (NAF) on diffuse gastric cancer cell lines, Western blot analysis was performed.

First, NAF or CAF was cultured in a 10 cm culture dish with serum-free DMEM for 48 hours. The cultured medium was transferred to a new tube, centrifuged at 4°C at 2000 rpm for 10 minutes, and the supernatant was collected in a new tube.

Diffuse gastric cancer cell lines SNU601 (5.5×10 ⁵ ) and AGS (3.0×10 ⁵ ) were aliquoted into 6-well plates and incubated overnight with serum-free DMEM. After starvation, each cancer cell was cultured in DMEM, NAF or CAF conditioned medium supplemented with 5% FBS.

In addition, cancer cells and NAF or CAF starvated overnight with serum-free DMEM were dispensed into a 6-well plate and a transwell chamber with a pore size of 0.4 μm, respectively, and co-cultured with DMEM supplemented with 5% FBS.

At each time point, cells were collected, proteins were extracted, electrophoresed by SDS-PAGE, and transferred to PVDF membrane. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour.

The primary antibody against the target protein was reacted at 4° C. overnight, and the HRP-conjugated secondary antibody against the primary antibody was reacted for 1 hour at room temperature, visualized using an ECL solution, and targeted with β-actin. Protein expression was normalized.

As a result, as shown in FIG. 5 , FAK signaling was activated in cancer cells cultured with NAF or CAF conditioned medium or cultured with NAF or CAF, and it was confirmed that the degree of FAK signaling activation was improved more by CAF than by NAF. The results were identical in the two cell lines SNU601 and AGS.

<Example 3> Confirmation of FAK signal activity by TINAGL1

As confirmed in the previous experiment, as it was confirmed that CAF activates the FAK signal in diffuse cancer cells, it was confirmed whether TINAGL1 secreted from CAF could also regulate the FAK signal in diffuse cancer cells.

The culture dish was coated with fibronectin solution (Fibronectin solution, 10 μg/ml in PBS) while incubating at 37° C. for 3 hours.

SNU601 (5.5×10 ⁵ ) and AGS (3.0×10 ⁵ ) cancer cells were aliquoted into 6-well plates coated with or without fibronectin, and the cancer cells were starvated overnight with serum-free DMEM. After starvation, culture was performed in a medium supplemented with 5% FBS, and fibronectin or recombinant TINAGL1 (100ng/ml or 200ng/ml) was directly added to the medium according to each condition.

Thereafter, cells were collected at each time point, proteins were extracted, and Western blot was performed in the same manner as in Example 2, FAK signal by fibronectin, one of the ligands of integrin known to be upstream of FAK signaling. The degree of activity was confirmed.

As a result, as shown in FIG. 6 , FAK was activated in the fibronectin-treated experimental group, and it was confirmed that the FAK signaling activity was further improved by the addition of TINAGL1. In addition, it was confirmed that FAK signaling was activated even when TINAGL1 was treated alone (last sample on the right).

<Example 4> Confirmation of tumorigenicity of TINAGL1

Agarose (low gelling temperature) was used to make a 1.0% agarose gel at the bottom of the 6-well plate, and when the gel at the bottom hardened, SNU601 cancer cells were mixed with 0.5% agarose gel and dispensed thereon to harden.

When the upper gel was hardened, 500 μl of RPMI (100 ng/ml) supplemented with 10% FBS with or without rhTINAGL1 was dispensed. After that, it was observed for 20 days by replacing it with a new medium suitable for each once every 3 days.

Three wells were used for each group, and the diameter of colonies was measured in all wells on the 20th day. The degree of tumorigenesis between the two groups was compared with the average diameter of the top 5 spheroids per well.

As a result, it was confirmed that the average size of the tumor spheroids formed in the group treated with TINAGL1 was larger than that of the control group as shown in FIG. 7 .

<Example 5> Confirmation of the effect of TINAGL1 expression-inhibited CAF on gastric cancer cells

1. Preparation of CAFs with TINAGL1 Expression Inhibited

Lipofectamine RNAiMAX was used to transfect CAF cells with 30 nM TINAGL1 or NC siRNA for 48 hours. After 48 hours, RNA and protein were prepared, respectively.

After synthesizing cDNA from the prepared RNA, RT-PCR was performed using a primer for the target gene, and the PCR product was confirmed by electrophoresis in 2% agarose gel, 100V for 20 minutes.

In addition, the protein was electrophoresed by SDS-PAGE, and then transferred to a PVDF membrane. Thereafter, Western blot analysis was performed in the same manner as in Example 2.

As a result, as shown in FIG. 8 , it was confirmed that the expression of TINAGL1 gene and protein was decreased in CAF transfected with siTINAGL1 (30 nM).

Meanwhile, NAF and CAF were each dispensed in a 24-well plate at 3×10 ³ cells/well, and 30 nM siRNA was transfected using Lipofectamine RNAiMAX for 48 hours.

After 48 hours, cells were fixed with 4% paraformaldehyde and blocked with 2% BSA at room temperature for 1 hour. As a primary antibody, TINAGL1 (CUSABIO, 1:100) was bound at 4° C. overnight, and a secondary antibody Cy3-Rabbit (1:500) with fluorescence attached to the primary antibody was bound at room temperature for 2 hours. After processing the mounting solution together with DAPI, it was observed under a microscope.

As a result, as shown in FIG. 9 , more TINAGL1 (red) expression was confirmed in CAF than in NAF, and it was confirmed that TINAGL1 expression was decreased in CAF transformed with siTINAGL1.

2. Confirmation of gastric cancer cell migration ability by TINAGL1 expression-inhibited CAF

CAF was aliquoted in 24-well plates, and 30 nM TINAGL1 or NC siRNA was transformed using Lipofectamine RNAiMAX for 3 days.

SNU601 (2×10 ⁴ ) was dispensed into an 8 μm pored transwell chamber and starvated overnight with serum-free DMEM. Thereafter, serum-free DMEM was added to the upper chamber and DMEM supplemented with 10% FBS was added to the lower chamber, and co-cultured with the transformed NAF or CAF. After 48 hours, cells were fixed with methanol and H&E staining was performed. After removing the cells present on the top of the membrane in the upper chamber, the membrane was removed from the chamber and mounted with the bottom side up on a slide glass. Cells passing through the membrane were counted by taking pictures of three sites at random in each well at 200 magnification, and then the averages were compared.

As a result, as shown in FIG. 10 , in the control group culturing SNU601 alone, the migration of cells was hardly confirmed. In the experimental group co-cultured with NAF, cancer cell migration was confirmed, but it was confirmed that cancer cell migration was significantly increased in the experimental group co-cultured with CAF.

In addition, it was confirmed that cancer cell migration was reduced in the experimental group co-cultured with CAF, in which TINAGL1 expression was suppressed through siRNA transformation, compared to the negative control group.

3. Confirmation of Effect of TINAGL1 Expression-Inhibited CAF on Signaling Mechanism in Gastric Cancer Cells

CAF was dispensed into a transwell chamber with a pore size of 0.4 μm, and 30 nM TINAGL1 or NC siRNA was transformed using Lipofectamine RNAiMAX for 3 days.

SNU601 was aliquoted into 6-well plates and incubated overnight with serum-free DMEM. Thereafter, the cells were co-cultured with NAF or transformed CAF cells in DMEM supplemented with 5% FBS for 24 hours.

Thereafter, SNU601 cells were collected, proteins were extracted, and Western blot analysis was performed in the same manner as in Example 2.

As a result, FAK signaling increased by CAF co-culture was reduced by TINAGL1 knockdown of CAF as shown in FIG. 11A . In addition, as shown in FIG. 11B , when FAK signal activation was reduced by TINAGL1 knockdown, it was confirmed that the EMT marker Twist increased by CAF co-culture decreased together.

<Example 6> Confirmation of expression level of TINAGL1 in diffuse gastric cancer tissue

The expression level of TINAGL1 protein was confirmed by Western blot in normal gastric and gastric cancer tissues obtained in pairs from diffuse gastric cancer patients.

As a result, as shown in FIG. 12 , it was confirmed that TINAGL1 protein expression was higher than that of normal tissues in cancer tissues of most diffuse gastric cancer patients.

In addition, mRNA expression levels of COL1A1 (red, fibroblast marker) and TINAGL1 (green) were confirmed in formalin-fixed paraffin-embedded (FFPE) tissues of patients with diffuse gastric cancer by performing double-staining RNA-in situ hybridization.

As a result, as shown in FIG. 13 , TINAGL1 mRNA expression was increased in the tumor tissue, and it was confirmed that it was co-expressed with COL1A1.

From the above results, it was confirmed that the expression of TINAGL1 was increased in cancer tissues than in normal gastric tissues.

On the other hand, the association between the TINAGL1 expression level and the prognosis of gastric cancer patients was confirmed using the published transcriptome data of gastric cancer.

Referring to FIG. 14 , high expression of TINAGL1 was not correlated with the prognosis of all patients and intestinal patients, but it was found that high expression of TINAGL1 was significantly associated with poor prognosis in diffuse gastric cancer with a high substrate ratio.

From the above results, it was confirmed that the expression of TINAGL1 gene in the stroma is very meaningful in the prognosis of diffuse gastric cancer.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

It comprises a TINAGL1 protein or a gene encoding the protein as an active ingredient, wherein the TINAGL1 protein or gene is a diffuse type characterized in that secretion and expression are increased in cancer-associated fibroblast (CAF) in gastric cancer tissue. A biomarker composition for diagnosing gastric cancer. delete An agent capable of detecting the expression or activity level of TINAGL1 protein or the expression level of a gene encoding the protein is contained as an active ingredient, and the agent is produced in cancer-associated fibroblasts (CAF) in gastric cancer tissue. A kit for diagnosing diffuse gastric cancer, characterized in that the TINAGL1 protein or gene with increased secretion and expression is detected. The kit for diagnosing diffuse gastric cancer according to claim 3, wherein the agent is selected from the group consisting of primers, probes, antibodies, peptides and aptamers. Comprising the step of comparing the expression or activity level of TINAGL1 protein, or the expression level of a gene encoding the protein from a sample isolated from a sample and a normal control,
The method for providing information for diagnosing diffuse gastric cancer, characterized in that the TINAGL1 protein or gene is increased in secretion and expression in cancer-associated fibroblasts (CAF) in gastric cancer tissue. TINAGL1 protein or a gene encoding the protein as an active ingredient, wherein the TINAGL1 protein or gene is characterized by increased secretion and expression in cancer-associated fibroblasts (CAF) in gastric cancer tissue. A biomarker composition for predictive prognosis of gastric cancer. It contains a TINAGL1 inhibitor as an active ingredient, wherein the TINAGL1 inhibitor inhibits the secretion and expression of TINAGL1 protein or a gene encoding the protein in cancer-associated fibroblast (CAF) in diffuse gastric cancer tissue. A pharmaceutical composition for preventing or treating diffuse gastric cancer. The method according to claim 7, wherein the TINAGL1 inhibitor is one selected from the group consisting of TINAGL1 protein or a peptide that specifically binds to a gene encoding the protein, an aptamer, an antibody, an antisense nucleotide, siRNA, shRNA, and miRNA. A pharmaceutical composition for preventing or treating diffuse gastric cancer. A pharmaceutical composition for the prevention or treatment of diffuse gastric cancer, comprising, as an active ingredient, cancer-associated fibroblasts (CAF) in which TINAGL1 expression is suppressed by transfection with siTINAGL1. A pharmaceutical composition for the prevention or treatment of diffuse gastric cancer, comprising, as active ingredients, cancer-associated fibroblasts (CAF) in which TINAGL1 expression is suppressed by transfection with siTINAGL1 and an anticancer agent. The method according to claim 10, wherein the anticancer agent is doxorubicin (doxorubicin), paclitaxel (paclitaxel), vincristine (vincristine), daunorubicin (daunorubicin), vinblastine (vinblastine), actinomycin-D (actinomycin-D), docetaxel (docetaxel), etoposide (etoposide), teniposide (teniposide), bisantrene (bisantrene), imatinib (imatinib), cisplatin (cisplatin) and 5-fluorouracil (5-fluorouracil) characterized in that it is selected from the group consisting of A pharmaceutical composition for the prevention or treatment of diffuse gastric cancer. Culturing cancer-associated fibroblasts (CAF) isolated from diffuse gastric cancer tissue to determine the TINAGL1 expression level (step 1);
Checking the TINAGL1 expression level by treating the TINAGL1-expressed cancer-associated fibroblasts with a candidate material (second step); and
A drug screening method for treating diffuse gastric cancer, comprising the step (third step) of comparing the TINAGL1 expression level of the first step with the TINAGL1 expression level of the second step. The method according to claim 12, wherein the TINAGL1 expression level is reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (radioimmunoassay), immune tissue Chemistry (immunohistochemistry), microarray (microarray), western blotting (western blotting) and flow cytometry (FACS), characterized in that the measurement by any one or more selected from the group consisting of a drug screening method for the treatment of gastric cancer.

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