KR100525704B1 - Pharmaceutical composition for the treatment of gastric cancer comprising inhibitory agent against 9-27 gene - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating gastric cancer containing an inhibitor against the 9-27 gene as an active ingredient and a gastric cancer diagnostic kit comprising a gene complementary to the gene. Since the 9-27 gene is specifically overexpressed in gastric cancer and increases the cancer cell's invasion and migration ability, thereby increasing the metastatic capacity of the cancer, the pharmaceutical composition comprising an inhibitor for the transcription or protein function of the gene may be The diagnostic kit may be usefully used for the treatment, and the diagnostic kit including the gene sequence complementary to the gene may be useful for the diagnosis of gastric cancer.

Description

Pharmaceutical composition for the treatment of gastric cancer comprising inhibitory agent against 9-27 gene}

The present invention relates to a pharmaceutical composition for treating gastric cancer, comprising an inhibitor for the 9-27 gene as an active ingredient, and a kit for diagnosing gastric cancer comprising a gene complementary to the gene.

Tumors are a collection of cells that exhibit autonomous overgrowth, and are classified into malignant tumors (so-called cancers) that may lead to death and benign tumors that do not have many exceptions. In particular, cancer is a genetic disease caused by mutations in genes such as oncogenes and tumor suppressor genes, and is a disease caused at the cellular level. Cancer gene products (oncogene products) are networked with each other in the cells to play a role in regulating the signaling system of cell division or differentiation. In other words, if their regulation is abnormal, the cell differentiation process is interrupted and indefinite division continues. If the signal transduction pathway is reduced normally, anticancer drugs that can provide excellent treatment effects with fewer side effects are obtained. Can be developed.

Stomach cancer is the highest cancer morbidity and cancer mortality in the world and is the most common cause of death from cancer in Korea, Japan, China, Russia, Central Europe, South and Central America, Hong Kong and Scandinavia. However, anti-cancer drugs with excellent therapeutic effects on gastric cancer have not been developed.

Currently, the three main treatments for cancer are surgical treatment, drug therapy, and radiation therapy. In reality, these or a combination of these and laser therapies are widely used, but considering the pain and metastasis associated with the treatment, Naturally, expectations for drug therapy are high. Accordingly, many anticancer agents have been developed and used in response to this, and most of these anticancer agents have been developed based on the principle of anticancer effect by selectively killing actively dividing cells.

Intercellular interactions, on the other hand, play an important role in the proliferation, activity and differentiation of cells, which are regulated by cell membrane proteins and secretory proteins that transmit activation or suppression signals. Cancer cells also acquire various immune evasion abilities by regulating the expression of cell membrane proteins and secretory proteins. Immunosuppressants that act include water-soluble immunosuppressive substances such as TGF-β1, interleukin-10 and interferongamma (Liyanage). UK et al ., J Immunol , 2002 , 169 (5), 2756-61; Saito H et al ., Cancer , 1999 , 86 (8), 1455-62; Galizia G et al ., J Interferon Cytokine Res , 2002 , 22 (4), 473-82; Majima T et al ., J Surg Oncol , 2001 , 78 (2), 124-30). In addition, cancer cells such as melanoma induce immune cells against activated Fas by cell necrosis by overexpressing the Fas ligand, leading to inhibition of immune mechanisms (Redondo P et al ., Br J Dermatol , 2002 , 147 (1)). , 80-6; Greil R et al ., Leuk Lymphoma , 1998 , 31 (5-6), 477-90).

The 9-27 gene is a gene known to be induced by interferon-γ, which is derived from immune cells, epidermal cells and fibroblasts and is closely related to cell growth (Lewin AR et al ., Eur J Biochem , 1991 , 199). (2), 417-423; Wedrychowski A et al ., J. Biol. Chem ., 1992 , 267 (7), 4533-4540; Deblandre GA et al ., J. Biol. Chem. , 1995 , 270 (40 ), 23860-23866). In addition, although genes 9-27 have been shown to be reduced in expression by interferon-γ in cancer cells such as astrocytomas (Huang H et al , Cancer Res , 2000 , 60 (24), 6868-). 74), it is not known that the cancer cell migration and the body's immune function are avoided.

Thus, the present inventors conducted an experiment to find genes related to inhibition of immune function and metastasis among genes secreted from gastric cancer cell lines or expressed in cell membranes, and 9-27 genes were specifically expressed in gastric cancer cells. The present invention was completed by revealing that the inhibitor for the 9-27 gene can be used for the treatment of gastric cancer.

Disclosure of Invention It is an object of the present invention to provide a pharmaceutical composition for treating gastric cancer, comprising the inhibitor for the 9-27 gene specifically overexpressed in gastric cancer, and a kit for diagnosing gastric cancer comprising a gene sequence complementary to the gene.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment of gastric cancer containing an inhibitor for 9-27 gene specifically overexpressed in gastric cancer as an active ingredient.

In addition, the present invention provides a kit for diagnosing gastric cancer comprising a gene sequence complementary to the gene.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for treating gastric cancer, which contains an inhibitor for the 9-27 gene specifically overexpressed in gastric cancer as an active ingredient.

In the present invention, "an inhibitor for the 9-27 gene" means any substance that inhibits the expression of the 9-27 gene or inhibits the function of a protein produced by the expression of the 9-27 gene.

In the above, the inhibitor for the 9-27 gene includes all substances that inhibit the transcription of the 9-27 gene, substances that inhibit the translation of the transcribed 9-27 mRNA or substances that inhibit the function of the 9-27 protein. In this case, as a substance that inhibits the transcription of the 9-27 gene, a protein that binds to a promoter, an enhancer, and a transcription factor that binds to the promoter is known to regulate the transcription of the 9-27 gene. Or a compound, but is not necessarily limited thereto. In addition, a substance that inhibits the translation of mRNA includes an antisense molecule that specifically binds to the mRNA, but is not necessarily limited thereto. A substance that inhibits the function of 9-27 protein specifically binds to the protein. There are monoclonal or polyclonal antibodies, but is not limited thereto. Such antisense or monoclonal or polyclonal antibodies can be prepared using known conventional methods.

9-27 gene is a gene that is specifically overexpressed in gastric cancer, in a preferred embodiment of the present invention it was confirmed that the 9-27 gene is specifically overexpressed in gastric cancer tissue obtained from Korean gastric cancer cell line and surgical surgery patients ( Fig. 1 And FIG. 2 ). In addition, the 9-27 gene increases the immune evasion ability, migration ability and invasive ability of cancer cells, and when the interferon-γ is treated to cancer cells expressing the 9-27 gene, the immune evasion ability, migration ability and invasive ability of cancer cells is increased. It can be seen that the expression of the 9-27 gene is a gene that induces metastasis and progression of cancer (see FIGS . 3 and 4 ).

The pharmaceutical composition for treating gastric cancer containing an inhibitor against the 9-27 gene of the present invention as an active ingredient can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation.

That is, the pharmaceutical composition of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used Or with excipients. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, water Prepare a mixture of sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the pharmaceutical composition of the present invention is 1 to 5 mg / kg, preferably 1 to 1.5 mg / kg, may be administered 1 to 3 times a day

The present invention also provides a kit for gastric cancer diagnosis comprising a gene sequence complementary to the 9-27 gene.

As described above, since the 9-27 gene of the present invention is specifically expressed in gastric cancer and induces metastasis and progression of the cancer, the 9-27 gene in the sample of the subject using a gene sequence complementary to the 9-27 gene Cancer diagnostic kits for analyzing the expression level of can be prepared.

The diagnostic kit of the present invention can diagnose cancer, specifically gastric cancer, by quantitatively or qualitatively analyzing the 9-27 gene in a sample of a subject that binds to a gene complementary to the 9-27 gene included in the kit. For example, by preparing a gene complementary to the 9-27 gene, by binding the gene to a microchip to prepare a DNA chip, by binding a test sample thereto, the amount of the 9-27 gene present in the sample of the subject It is possible to prepare a diagnostic kit that can measure.

Measuring the amount of the 9-27 gene of the subject using the diagnostic kit of the present invention

1) preparing a DNA chip by binding a gene complementary to the 9-27 gene to a microchip;

2) extracting the mRNA from the test sample to prepare a cDNA to which the coloring material is bound;

3) reacting and attaching the amplified cDNA of step 2 to the DNA chip of step 1; And

4) washing the reacted DNA chip of step 3 and analyzing it with a reader.

Coupling the gene complementary to the 9-27 gene of the step 1 to the microchip, wherein the gene complementary to the 9-27 gene comprises any one of DNA, RNA and peptide nucleic acids (PNA), And covalently fix the position of 3 'to the microchip. Photolithography, inkjet, microspotting, microinjection, and the like are used to fix nucleic acids to microchips. In addition, the plate of the microchip is made of inorganic materials such as glass and silicon, or made of a polymer material such as acrylic, polyethylene terephtalate (PET), polycarbonate, polystyrene, and polypropylene.

The step of preparing cDNA from the test sample of step 2 is to extract the mRNA from the test sample, ie, blood or tissue, and then fluoresce or color by reverse transcription by attaching a fluorescent substance directly or by inserting a linked substance or base to attach a coloring substance. CDNA is synthesized to make it possible. Here, the fluorescent material may be Cy5 (red) and Cy3 (green).

Step 3 is a process of attaching the amplified fluorescence or color cDNA of the test sample prepared in step 2 to each other by attaching the DNA chip prepared in step 1 to each other while maintaining the appropriate temperature and humidity.

In step 4, the process of washing the surface of the DNA chip reacted in step 3 to wash away all the remaining products that are not attached correctly to accurately read the gene, and then the fluorescent material or color of the washed DNA chip. The location of the material is confirmed by a scanner and analyzed by a computer.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Identification of Genes Specificly Expressed in Gastric Cancer Cells

Cancer cell secretion proteins and cell surface proteins are known to be involved in immune evasion mechanisms (Greil R et al ., Leuk Lymphoma , 1998 , 31 (5-6), 477-90; Liyanage UK et al ., J Immunol , 2002 , 169 (5), 2756-61). Thus, the present inventors have searched for genes related to immune evasion mechanisms among proteins expressed in gastric cancer tissues.

<1-1> Signal Sequence Trap Library

The present inventors produced a signal sequence capture library after extracting RNA from the Korean gastric cancer cell line SNU-638. Specifically, Korean gastric cancer cell line SNU-638 (Korea Cell Line Bank) was cultured at 37 ° C. and 5% CO ₂ using RPMI 1640 medium (Life Technologies) to which 10% serum (HyClone) and antibiotics (Life Technologies) were added. It was. RNA was extracted from the cultured cells using a fast track kit (Invitrogen), and then a signal sequence capture library was prepared using a SuperScript Choice System (Life Technologies). The first strand cDNA and the second strand cDNA were synthesized using 250 ng of the library as a random starting fragment, followed by fractionation according to the fragment size by attaching a Bst XI adapter (Invitrogen). It was. Sized cDNAs were inserted into pMX-SST vectors (Kojima T et al ., Nat Biotechnol , 199 9, 17 (5), 487-90.) To DH5 E. coli competent cells (Life Technologies). ) Was transformed by an electric shock method. The transformed cells were grown in solid LB medium (DIFCO) for 18 hours to obtain 2 × 10 ⁵ transformed cells. After culturing the cells again in liquid LB medium for 3 hours, the plasmid was extracted using a Qiagen Maxi Kit (Quiagen), which was used again for the production of a retrovirus library.

<1-2> Retrovirus Library

The present inventors transduced the SNU-638 cDNA library prepared in Example <1-1> into Phoenix cells (ATCC) to prepare a retrovirus library. Specifically, 2 × 10 ⁶ Phoenix cells were cultured in 10% DMEM medium one day before transduction, and 20 mg of the SNU-638 cDNA library plasmid prepared in Example <1-1> was added to calcium phosphate kit (Clon Tech). ) Were transfected into the Phoenix cells. The transduced cells were incubated at 37 ° C., 5% CO ₂ for 18 hours, followed by replacement culture with fresh medium containing 10 mM sodium butyrate, and after 12 hours, the medium containing retroviruses. Was taken and mixed with 8 μl / ml of Plybrene (Sigma) and stored at −70 ° C. until use.

<1-3> Retroviral Library Insertion into Ba / F3 Cells

The present inventors inserted the retrovirus library prepared in Example <1-2> into Ba / F3 cells. Specifically, Ba / F3 cells (ATCC, USA) at 37 ° C., 5% CO ₂ using RPMI 1640 medium containing 10% bovine serum, antibiotics and 10 ng / ml of Interleukin-3 (IL-3, R & D) After incubation at, the cells were centrifuged and replaced with the same fresh medium. After incubating for 8 hours by mixing the medium containing the retrovirus library prepared in Example <1-2> to the medium, 5 ml of the medium containing the new retrovirus library was put again and incubated for 12 hours. Ba / F3 cells into which the retroviral library was inserted were washed with PBS to completely remove interleukin-3, and then evenly divided into 12 96-well plates and cultured to select clones that survive even without interleukin-3.

<1-4> cDNA fragment isolation from selected clones

We isolated cDNA fragments from Ba / F3 cells selected in Example <1-3>. Specifically, Ba / F3 cells selected in Example <1-3> were 0.2 ml lysis buffer (10 mM Tris-Cl pH 8.0, 0.1 M EDTA, 20 g / ml pancreatic RNase, 0.5% SDS and 100 g / ml). After dissolving at 37 ° C. for 30 minutes at 50 ° C. and 3 hours using proteinase K), genomic DNA was precipitated by adding twice the ethanol and 0.5 M NaCl to the supernatant extracted with the same amount of phenol / chloroform. The genomic DNA precipitated was dissolved in 100 μl of distilled water. The polymerase chain reaction was used to amplify the cDNA fragment inserted in the lysed genomic DNA. Primers used for the polymerase chain reaction were prepared based on the nucleotide sequences present in the pMX-SST vector used in Example <1-1>, and are described as SEQ ID NO: 1 and SEQ ID NO: 2 , respectively. The polymerase chain reaction was carried out for 5 minutes at 94 ° C. using genomic DNA obtained above, primers described in SEQ ID NOs: 1 and 2 , and Taq polymerase (TaKaRa), followed by 2 seconds at 98 ° C., 68 Repeated 30 times in two steps of 2 minutes at ℃. The cDNA fragment obtained by the polymerase chain reaction was analyzed for nucleotide sequence through the BLAST program.

As a result, 39 different clones were obtained and fragments of genes whose expression was induced by interferon-γ, which plays an important role in the dual cellular immune mechanisms.

<1-5> Cloning of 9-27 Gene cDNA

The present inventors analyzed the nucleotide sequence of the 9-27 gene fragment cDNA obtained in Example <1-4> and confirmed that it is a part of 9-27 gene. In order to find the entire nucleotide sequence of the 9-27 gene, a primer described in SEQ ID NO: 3 and a primer described in SEQ ID NO: 4 were synthesized using an EST data base, and then a pMX-SST vector (10 pmol) was used as a template. 10 pmol of the primers set forth in SEQ ID NO: 3 and SEQ ID NO: 4 , 0.5 unit of XD Taq DNA polymerase, 3 µl of 10 x polymerase chain reaction buffer solution, and 4 µl of 2.5 mM dNTP were added and reacted at 95 ° C for 10 minutes. Thereafter, the reaction was performed 30 times for 1 minute at 95 ° C., 1 minute at 55 ° C., and 1 minute at 72 ° C., and the polymerase chain reaction was carried out under the condition of reacting at 72 ° C. for 10 minutes.

As a result, the full length 9-27 gene described in SEQ ID NO: 5 was amplified, and the amplified 9-27 whole gene was inserted into the pLXSN plasmid (CLONTECH), which was named 'pLXSN-9-27'.

<1-6> 9-27 gene expression analysis by Northern blot analysis

RNAzolB (Tel) from four normal, gastric and gastric cancer cell lines SNU-1, SNU-16, SNU-216, SNU-484, SNU-620 and SNU-638 cells obtained from Surgical Surgery patients at Chungnam National University with patient's consent. Test) was used to extract the total RNA. 20 μg of the RNA extracted above was electrophoresed on an agarose gel and then adsorbed onto a nylon membrane (Gene Screen Plus, NEN Life Science Products), and the 9-27 gene fragment was radiolabeled with α [ ³² P] -dCTP. It was. The membrane was reacted with a radiolabeled 9-27 gene fragment in 65 ° C. ExpressHyb solution (Clonetech) and washed sufficiently to specifically attach only 9-27 mRNA in total RNA attached to the membrane. The expression of 9-27 was confirmed by exposing the membrane to which the radiolabeled 9-27 gene was specifically attached to the X-ray film and then exposing it.

As a result, the 9-27 gene was specifically overexpressed in gastric cancer tissues compared to normal tissues ( A in FIG. 1 ). In addition, the SNU-1, SNU-216, SNU-620, and SNU-638 cells in gastric cancer cell lines showed significantly higher expression levels of 9-27 genes than other gastric cancer cell lines ( FIG . 2B ). From the above results, it can be seen that the 9-27 gene is a gene that is specifically overexpressed in gastric cancer.

<1-7> Expression analysis of 9-27 gene by In situ analysis

The present inventors analyzed the expression of 9-27 gene which was confirmed to specifically overexpress in gastric cancer patients in Example <1-6> through the In situ analysis method. Specifically, four normal tissues and gastric cancer tissues obtained from Surgical Surgery patients of Chungnam National University under the consent of the patient were fixed in 4% paraformaldehyde solution, replaced with 30% sucrose solution, and stored overnight. . The tissues were cut to a thickness of 30 μm, fixed on a slide, and then reacted at room temperature for 20 minutes by adding 0.4% Triton X-100 and 25 g / mL proteinase K. In addition, sense and antisense RNA probes of the 9-27 gene were obtained using T3 and T7 RNA transcriptases. The treated slides were mixed with a hybrid reaction solution (0.5 mg / ml tRNA, 20 mM Tris-HCl, pH 8.0), 2.5 mM EDTA, 1 x Denhardts solution, 0.3 M NaCl, 50% deionized formamide. (deionized formamide), 0.1% Tween 20 and 0.5 g / ml digoxigenin labeled 9-27 sense or antisense RNA probe) and reacted overnight at 55 ° C., followed by 2 × SSC / 50% formamide. 1 hour at 55 ° C., 1 × SSC / 50% formamide solution was added to the solution, followed by 1 hour at 55 ° C. and 0.5 × SSC / 50% formamide solution, and washed at 60 ° C. for 1 hour. The washed slides were placed in an anti-digoxigenin alkaline phosphate coupled serum (1: 500 dilution, Roche) solution and left overnight, NBT (nitroblue tetrazolium) / BCIP (5-bromo-4 -chloro-3-indolyl- The color reaction was performed in phosphate solution.

As a result, it was confirmed that 9-27 genes were less expressed in normal tissues, whereas 9-27 genes were overexpressed in gastric cancer tissues ( FIG . 1C ).

Example 2 Expression Control Analysis of 9-27 Gene

The present inventors confirmed the effect of the 9-27 gene specifically expressed in cancer cells on the cellular immune evasion mechanism through Example 1. Specifically, the SNU-216 cell line was treated with interferon-γ (100 U / mL) (R & D), TNF-α (100 U / mL) (R & D) or TGF-β1 (1 ng / mL) (R & D). Total RNA was isolated and Northern blot analysis was performed in the same manner as in Example <1-6> to analyze the expression of 9-27 genes.

As a result, the treatment of TNF-α and TGF-β1 had no effect on the regulation of 9-27 gene expression, whereas the treatment of interferon-γ, which is known to be involved in cellular immune mechanisms, resulted in 9-27 gene. Was increased ( A in FIG. 2 ), and the expression of the 9-27 gene in the cell line was most increased after 24 hours after interferon-γ treatment ( B in FIG. 2 ).

In addition, since the 9-27 gene is known to inhibit the proliferation of lymphoid cells (Deblandre GA et al ., J Biol Chem , 1995 , 6; 270 (40), 23860-6.), Interferon-γ Cell proliferation experiments were performed to determine whether the expression of the 9-27 gene by the S-A gene influences the proliferation of SNU-216 cells.

First, 96-well culture by suspending SNU-216 cells, SNU-216 cells transduced with pLXSN or pLXSN-9-27 plasmid in 5 × 10 ³ cells / 200 μl in RPMI 1640 medium supplemented with 10% bovine serum 200 μl per well was added to the plate and incubated for 24 hours with or without interferon-γ (100 U / ml) for 12 hours. 0.5 μCi of ³ H-thymidine was added to each well containing the cultured cells, and further cultured for 24 hours, and then the cells were collected on glass fiber filter paper using a multiple cell harvester (Inotech). Intracellular content of ³ H-thymidine was measured with a liquid scintillation counter (Beckman).

As a result, the growth of the interferon-SNU-216 cells treated with -γ were untreated the SNU-216 cell proliferation and by no difference (Fig. 2 C), expression of the gene from the resulting lymphoid 9-27 It was found that SNU-216 cells are influenced by different signaling mechanisms than sex cells, and that they do not affect the proliferation of SNU-216 cells.

Example 3 Analysis of Effects of 9-27 Gene Overexpression on NK Cytotoxicity

<3-1> Overexpression of 9-27 Gene Through Transduction

Natural killer (NK) cells are associated with innate immune mechanisms against cancer cells, and recent studies have shown that NK cell activity against cancer cells is regulated by NK cell membrane receptors that receive amplification or cancellation signals (Menier C). et al ., Int J Cancer , 2002 , 100 (1), 63-70; Moretta L et al ., Curr Top Microbiol Immunol , 2002 , 266, 11-22). Therefore, in order to investigate what immunological and biological functions the 9-27 protein secreted from gastric cancer cells are delivered to the receptors of NK cells, the 9-27 gene was permanently transduced into SNU-216 cells, a Korean gastric cancer cell line. Cells were prepared. For this purpose, transfection is performed by first transfecting pLXSN or pLXSN-9-27 prepared in Example <1-5> to a Phoenix cell line to make a retrovirus, and then infecting the virus with SNU-216 cells. It was. The transduced cells were named 'SNU-216-vec' or 'SNU-216-9-27', respectively. Next, as a result of Northern blot by separating the entire RNA from the transduced cells in the same manner as in Example <1-6>, 9-27 gene expression was not confirmed in SNU-216-vec cells on the other hand, in the SNU-216-9-27 cells are identified, the expression of the gene was confirmed to be a 9-27 9-27 gene was introduced correctly transformed in SNU-216 cells (a in Fig. 3).

<3-2> Cytotoxicity of NK Cells by Overexpressed 9-27 Gene

The present inventors confirmed that the 9-27 gene was transduced and overexpressed in SNU-216 cells in Example <3-1>, and then the effect of 9-27 gene expression on cytotoxicity of NK cells was examined. The cytotoxicity test of SNU-216-9-27 cells was performed using NK92MI cells transfected with interleukin-2 in human NK cells. Specifically, SNU-216, SNU-216-vec or SNU-216-9-27 cells were treated with trypsin to detach the cells and suspended at a concentration of 1 × 10 ⁶ cells / ml, followed by 10 μCi of ⁵¹ Cr. The reaction was carried out at 37 ° C. and 5% CO ₂ for 2 hours. In the meantime, NK92MI cells (ATCC) were cultured at 37 ° C. and 5% CO ₂ using RPMI 1640 medium containing 10% bovine serum and antibiotics. Each ⁵¹ Cr-labeled SNU-216 cells were put in a 96-well culture plate at 1 × 10 ⁴ per well, and then cultured together for 4 hours by adding NK92MI cells: SNU-216 cell ratios of 10: 1 and 5: 1. . The radioactivity of ⁵¹ Cr released into the medium while SNU-216 cells were killed by NK92MI cells during the culture was measured by a γ-meter, and the killing ability of the SNU-216 cells was expressed as numerical values by substitution in Equation 1 .

Specific release amount of ⁵¹ Cr = experimental release-(natural release / maximum release)-natural release

As a result, compared to the SNU-216-vec cells SNU-216-9-27 cells was increased the resistance to cytotoxicity NK92MI (B in Fig. 3). In addition, NK92MI cells were treated with 40: 1 and 10: 1 in ⁵¹ Cr-labeled SNU-216 cells treated with untreated or interferon-γ (100 U / mL), and cultured together for 4 hours, followed by NK92MI of SNU-216 cells. As a result of confirming resistance to cytotoxicity, resistance to NK cytotoxicity was further increased by interferon-γ inducing the expression of the 9-27 gene ( FIG . 3C ). From the above results, it can be seen that the expression of the 9-27 gene increases the resistance to NK cytotoxicity.

Example 4 Effect of Overexpression of 9-27 Gene on Migration and Infiltration of Gastric Cancer Cell Lines

Cell membrane proteins in cancer cells are important in immune metastasis as well as metastasis of cancer cells. In contrast, since NK cells inhibit the invasion and migration of cancer cells, increased resistance to NK cytotoxicity may eventually increase the metastatic capacity of cancer cells. Therefore, the present inventors performed cell invasion ability and mobility test to confirm whether overexpression of 9-27 gene encoding cell membrane protein affects the potential metastasis of gastric cancer cells.

<4-1> Effects of 9-27 Gene Overexpression on the Mobility of Gastric Cancer Cell Lines

We used a modified Boyden chamber and measured the ability of cells to migrate by passing the SNU-216-vec and SNU-216-9-27 cell lines through an uncoated filter. Specifically, 800 μl RPMI medium with 10% bovine serum was added under the Modified Bodene chamber transwell, and 3 × 10 ⁶ SNU-216-vec or SNU-216-9-27 was placed in the upper portion of the transwell. Cells were suspended in 100 μl RPMI with 0.5% BSA added. After culturing the cells at 37 ° C., 5% CO ₂ for 1 day, the cells that did not pass through the filter were completely removed with a cotton swab and only the cells that migrated to the bottom of the filter were fixed with methanol. The fixed cells were stained with hematoxylin and eosin and then counted 5 parts per filter on the microscope.

As a result, the SNU-216-9-27 cell line transfected with the 9-27 gene was more than twice as high as the control cell line into which only the vector was introduced ( A of FIG. 4 ).

<4-2> Effect of 9-27 Gene Overexpression on Invasiveness of Gastric Cancer Cell Line

We used a modified Boyden chamber and measured the ability of the SNU-216-vec and SNU-216-9-27 cell lines to infiltrate a filter coated with 20 μg of Matrigel (Becton Dickinson). . First, 6.5 μg fibronectin was treated on the bottom side of the filter, and then 800 μl of RPMI medium containing 10% bovine serum was placed under the transwell, and 3 × 10 ⁶ pieces were placed on the upper portion of the transwell. SNU-216-vec or SNU-216-9-27 cells were suspended in 100 μl of RPMI medium containing 10% bovine serum. After incubating the cells for 4 days at 37 ° C., 5% CO ₂ , the cells that did not pass through the filter were completely removed with a cotton swab and only the cells that migrated to the bottom of the filter were fixed with methanol. The fixed cells were stained with hematoxylin and eosin and then counted 5 parts per filter on the microscope.

As a result, the invasion ability of the SNU-216-9-27 cell line into which the 9-27 gene was introduced was increased more than four times compared to the control cells ( B of FIG. 4 ). In addition, the invasion ability of the SNU-216 cell line was further increased by the treatment of interferon-γ, which induces the expression of the 9-27 gene ( FIG . 4C ). From the above results, it was confirmed that the expression of the 9-27 gene increases the invasive capacity and the migration capacity in addition to the cancer cell's resistance to NK cytotoxicity and eventually contributes to metastasis of the cancer cell.

As described above, the 9-27 gene is specifically overexpressed in gastric cancer and increases the metastasis capacity of cancer by increasing the invasion and migration capacity of cancer cells, and thus, includes an inhibitor for the transcription or function of the gene. The pharmaceutical composition may be usefully used for the treatment of gastric cancer, and a diagnostic kit including all or a part of the gene or a protein thereof may be usefully used for the diagnosis of gastric cancer.

1 is a photograph confirming the expression of 9-27 gene in normal tissue and gastric cancer tissue by Northern blot analysis (A), a photograph comparing the expression of 9-27 gene in Korean gastric cancer cell line by Northern blot analysis (B) and normal This is a picture (C) of 9-27 gene expression in tissue and gastric cancer tissue.

Figure 2 is a photograph (A) comparing the change in the expression of the 9-27 gene after treatment with interferon-γ, TNF-α or TGF-β1 to SNU-216 cells expressing the 9-27 gene (A), SNU-216, SNU-216-vec treated with interferon-γ and photograph (B) comparing the changes of 9-27 gene expression by time of Northern blot analysis after interferon-γ treatment to SNU-216 cells It is a graph (C) which analyzed the proliferation of SNU-216-9-27 cell by thyminin content.

Figure 3 is a Northern blot analysis (A), confirming the overexpression of 9-27 gene in SNU-216-9-27 cells transduced 9-27 gene, the NK cytotoxicity of SNU-216-9-27 cells A graph showing resistance (B) and a graph showing resistance to NK cytotoxicity of SNU-216 cells treated with interferon-γ (C).

Figure 4 is a graph comparing the mobility of SNU-216, SNU-216-vec and SNU-216-9-27 cells (A), SNU-216, SNU-216-vec and SNU-216-9-27 cells The graph comparing the invasion ability of (B) and the graph (C) comparing the invasion ability of cells after interferon-γ treatment to SNU-216 cells.

110> Korea research institute of bioscience and biotechnology <120> Pharmaceutical composition for the treatment of gastric cancer comprising inhibitory agent against 9-27 gene <130> 2p-08-18B <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for pMX-SST vector <400> 1 gggggtggac ccatcctcta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> backward primer for pMX-SST vector <400> 2 cgcgcagctg taaacggtag 20 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for 9-27 gene <400> 3 cggaattcat gcacaaggag gaacat 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> backward primer for 9-27 gene <400> 4 ccctcgagct agtaaccccg tttttc 26 <210> 5 <211> 378 <212> DNA <213> Homo sapiens <400> 5 atgcacaagg aggaacatga ggtggctgtg ctgggggcac cccccagcac catccttcca 60 aggtccaccg tgatcaacat ccacagcgag acctccgtgc ccgaccatgt cgtctggtcc 120 ctgttcaaca ccctcttctt gaactggtgc tgtctgggct tcatagcatt cgcctactcc 180 gtgaagtcta gggacaggaa gatggttggc gacgtgaccg gggcccaggc ctatgcctcc 240 accgccaagt gcctgaacat ctgggccctg attctgggca tcctcatgac cattggattc 300 atcctgttac tggtattcgg ctctgtgaca gtctaccata ttatgttaca gataatacag 360 gaaaaacggg gttactag 378

Claims (5)

delete delete 9-27 A pharmaceutical composition for the treatment of gastric cancer, comprising as an active ingredient an antisense molecule that specifically binds to mRNA. 9-27 A pharmaceutical composition for the treatment of gastric cancer, comprising a monoclonal or polyclonal antibody specific for a protein as an active ingredient. Gastric cancer diagnostic kit comprising gene sequence complementary to 9-27 gene.