KR101654526B1 - Pharmaceutical composition for the treatment of colorectal cancers or inhibition of metastasis containing the expression or activity inhibitors of cadherin―11 - Google Patents

Abstract

In the present invention, when cadherin-11 is overexpressed in a colon cancer cell line and siRNA inhibits expression of carderin-11, cancer cell infiltration and cell mobility decrease, and epithelial-mesenchymal cells epithelial-mesenchymal transition (EMT), and inhibited cell signaling. In addition, carderine-11 was directly expressed by EMT-inducible transcription factor ZEB2 in colon cancer, liver cancer and stomach cancer cell line, suggesting that carderine-11 is a therapeutic composition for treating colorectal cancer, liver cancer, As shown in FIG.

Description

Pharmaceutical composition for the treatment of colorectal cancers or inhibition of metastasis containing the expression or activity inhibitors of cadherin-11}

The present invention relates to a pharmaceutical composition for treating or inhibiting metastasis of colon cancer, liver cancer or gastric cancer, comprising an inhibitor of expression or activity of a novel cancer treatment target, cadherin-11 protein.

Colorectal cancer is a refractory disease in which 1 million patients occur annually and 530,000 people die worldwide.According to aging and changes in dietary patterns, it is rapidly increasing, ranking 3rd with 12.7% of total cancer incidence in Korea. have. Currently, as effective anticancer agents for colon cancer, fluoropyrimidine drugs such as 5-FU, UFT, capecitabine (brand name: Geloda), drugs such as irinotecan (brand name: Gamputo) and oxaliplatin are widely used. In addition, the colorectal cancer treatment market is expected to grow rapidly from $900 million to $7.8 billion in 2017 in the seven major pharmaceutical markets in the world including the United States and Europe. The continuous use of these drugs always has a problem that resistance increases and side effects can occur, so it is time to develop new therapeutic agents and research on diagnosis.

Cancer-Fetal Antigen (CEA), which is currently used as a diagnostic marker for colorectal cancer, is a type of glycoprotein that is normally made during the fetal period. Because production of this substance is normally stopped before birth, it is higher in adults than newborns. If the antigen (CEA) level appears, this suggests that there is a possibility of colon cancer or other cancer, but this level may increase in patients with cirrhosis, liver disease, alcoholic pancreatitis, and even smokers, so it is being used as a supplement. .

Gastric cancer is a cancer with the highest cancer morbidity and cancer mortality in the world, and accounts for the most cause of cancer deaths in Korea, Japan, China, Russia, Central Europe, South and Central America, Hong Kong, and Scandinavia. It is reported that more than 16% of men in Korea or Japan are dying from stomach cancer. In particular, in the case of Asia such as Korea and Japan, it is reported that the incidence of gastric cancer is higher than that of races on other continents. It is reported to come from differences. In the case of Koreans, the daily intake of salt is about 20g, which is twice as much as that of Westerners. In particular, it is reported that the incidence of gastric cancer is high in Korea, Japan, Finland, Iceland, etc. In addition, genetic causes other than eating habits are involved as the cause of gastric cancer, and it is known that the incidence of gastric cancer is high in first-generation offspring of gastric cancer patients. In addition, the symptoms of gastric cancer show a variety of patterns from no symptoms at all to severe pain, and the symptoms of gastric cancer do not have any characteristics, but show general digestive symptoms, and in most cases there are no symptoms at the beginning of gastric cancer. And, even if there are symptoms, they are relatively mild, so they feel a little indigestion or discomfort in the upper abdomen, so it is easy for most people to overlook it, which increases the mortality rate of gastric cancer. Currently, as the main treatment for treating various cancers including gastric cancer, one or a combination of surgical surgery, irradiation, and chemotherapy is used, and surgery involves removing most of the diseased tissue. Surgical surgery is effective in removing tumor tissue or tissue around them, but cannot be used to treat tumors in difficult-to-operate areas such as the spine or to treat disperse tumors scattered throughout the body such as leukemia. Chemotherapy is to treat cancer by disrupting cell replication or cell metabolism, and can be used for the treatment of various tumors, but it also acts on normal cells, causing serious side effects. In particular, it may cause serious side effects that weaken the patient's immune system by acting on the hematopoietic organ where cell division and cell metabolism are actively occurring. These side effects have a great impact on the patient's life, and there is also an injectable dose limiting toxicity (DLT) that must be noted when administering the drug. As such, side effects caused by chemotherapeutic agents and radiation therapy become a major problem in clinical treatment of cancer patients, and therefore, the development of new anticancer drugs with excellent stability in the body that can reduce side effects of chemotherapy treatments is urgently needed.

Gastric cancer is a cancer with the highest cancer morbidity and cancer mortality in the world, and accounts for the most cause of cancer deaths in Korea, Japan, China, Russia, Central Europe, South and Central America, Hong Kong, and Scandinavia. However, anticancer drugs with excellent therapeutic effects for gastric cancer have not been developed.

Cadherin-11 is mainly expressed in breast cancer cells and prostate cancer cells, and has been reported to induce invasion of prostate cancer cells (Huang et al., (2010) Cancer Res. 70(11)4580-4589). On the other hand, the expression of cadherin-11 is suppressed in various cancer cell lines compared to normal epithelial cells, and the expression of cadherin-11 not only inhibits cancer expression and induces cancer cell apoptosis, but also inhibits EMT. It has been reported (Li et al., Oncogene 2011 Dec 5. doi: 10.1038/onc. 2011.541. Epub ahead of print). Therefore, it appears that the function of cadherin-11 in cancer cell metastasis is diverse. On the other hand, the function of cadherin-11 in colorectal cancer, gastric cancer and liver cancer has not been reported.

Accordingly, the present inventors have found that as a result of inhibiting the expression of cadherin-11 in colon cancer cell lines and inhibiting the expression of cadherin-11 in colon cancer, gastric cancer and liver cancer cell lines, cancer cell invasion and cell mobility decrease, epithelial cells-mesenchymal cells The present invention was completed by confirming that (epithelial-mesenchymal transition, EMT) was inhibited and cell signaling was inhibited.

An object of the present invention is a pharmaceutical composition for cancer treatment or metastasis inhibition selected from the group consisting of colorectal cancer, liver cancer and gastric cancer comprising an inhibitor of expression or activity of a novel cancer treatment target, cadherin-11 protein Is to provide.

In order to achieve the above object, the present invention contains an inhibitor of expression or activity of cadherin-11 protein as an active ingredient, treatment of a cancer selected from the group consisting of colorectal cancer, liver cancer and gastric cancer or inhibiting metastasis It provides a pharmaceutical composition for use.

In addition, the present invention

1) treating the test material on the cell line expressing the cadherin-11 protein;

2) measuring the level of expression of the cadherin-11 protein with respect to the cell line expressing the cadherin-11 protein; And

3) selecting a test substance whose expression level of the cadherin-11 protein is reduced compared to a control group not treated with the test substance;

It provides a method for screening a candidate material for inhibiting metastasis or treatment of cancer selected from the group consisting of colon cancer, liver cancer and gastric cancer, including.

In addition, the present invention

1) treating the test substance on the cadherin-11 protein;

2) measuring the degree of activity of the cadherin-11 protein; And

3) selecting a test substance whose activity level of the cadherin-11 protein is decreased compared to a control group not treated with the test substance;

A method for screening a candidate material for inhibiting metastasis or treatment of cancer selected from the group consisting of colorectal cancer, liver cancer and gastric cancer comprising a.

In addition, the present invention

1) measuring the expression or activity level of cadherin-11 protein from cells or tissues isolated from the subject; And

2) determining that the subject whose expression or activity level of the cadherin-11 protein is decreased compared to the normal control group is at high risk of developing cancer or cancer metastasis selected from the group consisting of colorectal cancer, liver cancer, and gastric cancer;

It provides a method for measuring the expression or activity level of a protein to provide information for monitoring or diagnosis of cancer development or metastasis selected from the group consisting of colorectal cancer, liver cancer and gastric cancer, including.

In the present invention, as a result of overexpressing cadherin-11 in colorectal cancer cell lines and inhibiting the expression of cadherin-11 with siRNA, cancer cell invasion and cell mobility decrease, and epithelial cells-mesenchymal cells (epithelial -mesenchymal transition, EMT) was inhibited and cell signaling was inhibited. In addition, by confirming that the expression of cadherin-11 is directly induced by the EMT-induced transcription factor ZEB2 in cell lines of colorectal cancer, liver cancer and gastric cancer, cadherin-11 will be usefully used as a pharmaceutical composition for cancer treatment or metastasis inhibition. I can.

1 is a result of confirming the change in cancer cell invasion ability and cell mobility by inhibiting the expression of cadherin-11 in the colorectal cancer cell line SW480, the invasion and cell mobility of the colon cancer cells decreased by inhibiting the expression of cadherin-11. This is a picture showing.
Figure 2 is a result of performing western blot analysis after introducing cadherin-11 specific siRNA in order to analyze changes in cell signaling and EMT due to inhibition of expression of cadherin-11 in colorectal cancer cell lines, cadherin-11 It is a figure showing that EMT of colon cancer cells is induced and cancer cell invasion and cell mobility are induced.
FIG. 3 is a reporter assay showing that ZEB2 is a representative E-cadherin transcription inhibitor and EMT-induced transcription and is associated with cancer cell invasion and metastasis, and thus the expression of cadherin-11 is induced by ZEB2, SW480, This is a picture confirming that the cadherin-11 promoter is activated by ZEB2 in AGS and HEK293E cells.
4 shows that the expression of cadherin-11 is induced by ZEB2 by real time qPCR analysis. As a result, after transforming ZEB2 into SW480 cells, cadherin-11 mRNA level was increased. , After transfection of ZEB2-specific siRNA in gastric cancer cells SNU-638 and liver cancer cells SNU-398 expressing endogenous ZEB2, the expression of cadherin-11 mRNA was confirmed.
5 shows that Sp1 is expected to be required for the induction of cadherin-11 by ZEB2, and as a result of real-time quantitative analysis of polymerase chain reaction and western blot analysis to verify this, the expression of cadherin-11 induced by ZEB2 is Sp1. This is a picture confirming that it is reduced by inhibition of activity.
Figure 6 is a result of confirming by real-time polymerase chain reaction to confirm the effect of Sp1-specific siRNA on the expression of cadherin-11 induced by ZEB2, the expression of cadherin-11 induced by ZEB2 is Sp1-specific siRNA This is a picture confirming that it is also reduced by.
7 is a result of performing ChIP to determine which site of ZEB2 and Sp1 specifically binds to the cadherin-11 promoter in SNU-638 cells, which are liver cancer cell lines, in the cadherin-11 promoter (-191/-62) region. This is a picture confirming that ZEB2 and Sp1 specifically bind.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing or treating colorectal cancer, liver cancer and gastric cancer, containing an inhibitor of expression or activity of cadherin-11 protein.

The cadherin-11 protein is preferably all of the amino acid sequence represented by SEQ ID NO: 1, a fragment thereof, or a mutant sequence in which several amino acids have been added, determined, or substituted, but is not limited thereto. Specifically, the cadherin-11 is the amino acid sequence of SEQ ID NO: 1, a fragment from which the amino acid sequences 1 to 22 are partially removed (SEQ ID NO: 24), and the amino acid sequence 23 to 53 is partially removed (SEQ ID NO: 25) , It is more preferable that the fragment (SEQ ID NO: 26) from which the amino acid sequence 1 to 53 is partially removed.

The expression of the cadherin-11 protein is preferably induced by ZEB2 (zinc finger E-box-binding homeobox 2), but is not limited thereto.

The expression inhibitor of the cadherin-11 protein is an antisense nucleotide complementarily binding to the mRNA of the cadherin-11 gene, short hairpin RNA (shRNA) or small interfering RNA (siRNA), ZEB2 gene Antisense nucleotides complementarily binding to mRNA of, shRNA, siRNA, or ZEB2 protein antibodies, or antisense nucleotides complementary to the mRNA of Sp1 (specificity protein 1) gene, shRNA, siRNA, or antibody of Sp1 protein It is preferably any one selected from, but is not limited thereto.

The inhibitor of activity of the cadherin-11 protein is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, or antibodies that complementarily bind to cadherin-11 protein, but limited thereto. It doesn't work.

The present inventors confirmed changes in cancer cell invasion capacity and cell mobility by inhibiting the expression of cadherin-11 in the colon cancer cell line SW480. As a result, the invasion and cellular mobility of colon cancer cells were reduced by inhibition of cadherin-11 expression (FIG. 1).

In order to analyze changes in cell signaling and EMT due to inhibition of expression of cadherin-11 in colorectal cancer cell lines, cadherin-11 specific siRNA was introduced into SW480, which is a colorectal cancer cell line, and Western blot analysis was performed. As a result, as a result of suppressing the expression of cadherin-11 with siRNA in SW480 cells, it was confirmed that the expression of mesenchymal cell markers integrin alpha5 and vimentin decreased, and the expression of epithelial cell marker E-cadherin was increased. I did. In addition, it was confirmed that the expression of the transcription factor Sp1 was decreased, and the phosphorylation of FAK, ERK and c-src was decreased. Through these results, EMT was inhibited and cell signaling was inactivated by inhibition of cadherin-11 expression. Therefore, it was confirmed that cadherin-11 functions to induce EMT of colon cancer cells and to induce cancer cell invasion and cell mobility (FIG. 2).

The present inventors confirmed that ZEB2 is a representative E-cadherin transcription inhibitor and EMT-induced transcription and is associated with cancer cell invasion and metastasis, and thus the expression of cadherin-11 is induced by ZEB2 by reporter assay. As a result, it was confirmed that the cadherin-11 promoter was activated by ZEB2 in SW480, AGS and HEK293E cells (FIG. 3).

ZEB2 is a representative E-cadherin transcription inhibitor and EMT-induced transcription, and is associated with cancer cell invasion and metastasis. Therefore, real time qPCR analysis showed that the expression of cadherin-11 is induced by ZEB2. Confirmed. As a result, the level of cadherin-11 mRNA was increased when transforming ZEB2 into SW480 cells, and after transforming ZEB2-specific siRNA into SNU-638, a gastric cancer cell expressing endogenous ZEB2, and SNU-398, a hepatocellular carcinoma cell It was confirmed by quantitative analysis of real-time polymerase chain reaction that the expression of cadherin-11 mRNA was decreased (FIG. 4).

It was confirmed that the transcription factor Sp1 is involved in the expression of mesenchymal cell markers integrin alpha5 and vimentin by ZEB2 (Nam et al., (2012) Carcinogenesis 33(3):563-571). Therefore, it was expected that Sp1 would be required for induction of cadherin-11 by ZEB2, and to verify this, quantitative analysis of real-time polymerase chain reaction and Western blot analysis were performed. As a result, it was confirmed that the expression of cadherin-11 induced by ZEB2 was reduced by inhibition of Sp1 activity (FIG. 5).

Real-time polymerase chain reaction was carried out to confirm the effect of Sp1-specific siRNA on the expression of cadherin-11 induced by ZEB2 through the results of confirming that the expression of cadherin-11 induced by ZEB2 is reduced by inhibition of Sp1 activity. I did. As a result, it was confirmed that the expression of cadherin-11 induced by ZEB2 was also reduced by Sp1-specific siRNA (FIG. 6).

ChIP was performed to determine which site of ZEB2 and Sp1 specifically binds to the cadherin-11 promoter in SNU-638 cells, which are liver cancer cell lines. As a result, it was confirmed that ZEB2 and Sp1 specifically bind to the cadherin-11 promoter (-191/-62) region (FIG. 7).

Therefore, as a result of inhibiting the expression of cadherin-11 in colorectal cancer cell lines with siRNA, cancer cell invasion and cell mobility decrease, epithelial-mesenchymal transition (EMT) is inhibited, and cell signaling is inhibited. It was confirmed that it was inhibited. In addition, cadherin-11 can be effectively used as a pharmaceutical composition for the treatment or metastasis inhibition of colon cancer, liver cancer, or stomach cancer through direct expression of the EMT-induced transcription factor ZEB2.

The pharmaceutical composition containing the expression or activity inhibitor of the cadherin-11 protein of the present invention as an active ingredient preferably contains 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition, but is not limited thereto.

The pharmaceutical composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components may be mixed and used. , Buffers, bacteriostatic agents, and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets, and can act specifically on target organs. Thus, a target organ-specific antibody or other ligand may be used in combination with the carrier. Further, it may be preferably formulated according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The nucleotides or nucleic acids used in the present invention may be prepared for oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal administration, or the like.

Preferably, nucleic acids or vectors are used in injectable form. Thus, it can be mixed with any pharmaceutically acceptable vehicle for injectable compositions, especially for direct injection into the area to be treated. The pharmaceutical composition of the present invention may comprise in particular a sterile isotonic solution or a freeze-dried composition which allows the composition of an injectable solution upon addition of dry in particular sterile water or appropriate physiological saline. Direct injection of the nucleic acid into the patient's tumor is advantageous as it allows treatment efficiency to be focused on the infected tissue. The dosage of the nucleic acid used can be adjusted by various parameters, in particular the gene, the vector, the mode of administration used, the disease in question or alternatively the duration of treatment required. In addition, the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage is about 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, and it is preferable to administer once to several times a day.

In addition, the present invention provides a method for treating cancer or inhibiting cancer metastasis, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to an individual suffering from cancer.

The pharmaceutically effective amount is 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, but is not limited thereto. The dosage may vary depending on the weight, age, sex, health status, diet, administration period, administration method, elimination rate, disease severity, etc. of a specific patient.

The pharmaceutical composition can be administered orally or parenterally during clinical administration, and when administered parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or chest It can be administered by intramuscular injection, and can be used in the form of a general pharmaceutical formulation.

It is overexpressed in colon cancer cell lines, and the growth, migration, invasion and metastasis of colon cancer, liver cancer and gastric cancer cells are inhibited by inhibition of cadherin-11, and apoptosis is induced, so that cadherin-11 is a target of a novel anticancer agent. Since the possibility has been confirmed, a pharmaceutical composition containing an inhibitor of expression or activity of cadherin-11 protein as an active ingredient can be usefully used in the treatment or metastasis of colon cancer, liver cancer, or stomach cancer.

In addition, the present invention provides a method for screening a substance for treatment or metastasis inhibition of colon cancer, liver cancer or gastric cancer using cadherin-11 protein.

Specifically, the method

1) treating a cell line expressing cadherin-11 protein with a test substance;

2) measuring the expression level of the cadherin-11 protein in the cell line; And

3) It is preferable to include, but is not limited to, selecting a test substance whose expression level of the cadherin-11 protein is reduced compared to the control group not treated with the test substance.

In the above method, the cell line of step 1) is preferably any one selected from the group consisting of colon, liver and stomach cells or colon cancer, liver cancer or gastric cancer cell lines, and more preferably colon cancer, liver cancer and gastric cancer cell lines. .

In the above method, the expression level of the protein in step 2) is immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western Blotting, and It is preferable to measure by any one selected from the group consisting of flow cytometry (FACS), but any method for measuring the amount of a transcript or a protein encoded therefrom can be used.

In the above method, in steps 2) and 3)

a) measuring the expression level of ZEB2 or Sp1 protein; And

b) selecting a test substance whose expression level of ZEB2 or Sp1 protein is decreased compared to a control group not treated with the test substance;

It is preferable to include a candidate material for inhibiting metastasis or treatment of cancer selected from the group consisting of colorectal cancer, liver cancer or gastric cancer, characterized in that it additionally comprises, but is not limited thereto.

Another way is,

1) treating the test substance on the cadherin-11 protein;

2) measuring the degree of activity of the cadherin-11 protein; And

3) It is preferable to include, but is not limited to, selecting a test substance whose activity level of the cadherin-11 protein is reduced compared to the control group not treated with the test substance.

In the above method, the cell line of step 1) is preferably any one selected from the group consisting of colon, liver and stomach cells or colon cancer, liver cancer or gastric cancer cell lines, and more preferably colon cancer, liver cancer and gastric cancer cell lines. .

In the above method, the degree of activity of the protein in step 2) is preferably measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry, and protein chip, but is not limited thereto. .

In the above method, in steps 2) and 3)

a) measuring the degree of activity of the ZEB2 or Sp1 protein; And

b) selecting a test substance whose activity level of the ZEB2 or Sp1 protein is decreased compared to the control group not treated with the test substance;

It is preferable to include a candidate material for inhibiting metastasis or treatment of cancer selected from the group consisting of colorectal cancer, liver cancer or gastric cancer, characterized in that it additionally comprises, but is not limited thereto.

Another way is,

1) measuring the expression or activity level of cadherin-11 protein from cells or tissues isolated from the subject; And

2) determining that the subject whose expression or activity level of the cadherin-11 protein is decreased compared to the normal control group is at a high risk of developing cancer or cancer metastasis selected from the group consisting of colorectal cancer, liver cancer, and gastric cancer;

It is preferable to include a candidate material for inhibiting metastasis or treatment of cancer selected from the group consisting of colorectal cancer, liver cancer or gastric cancer, characterized in that it additionally comprises, but is not limited thereto.

In the above method, the subject is a vertebrate animal, preferably a mammal, more preferably an experimental animal such as a mouse, rabbit, guinea pig, hamster, dog, and cat, and most preferably, a chimpanzee or a gorilla. It is an ape animal.

In the above method, the cell line of step 1) is preferably any one selected from the group consisting of colon, liver and stomach cells or colon cancer, liver cancer or gastric cancer cell lines, and more preferably colon cancer, liver cancer and gastric cancer cell lines. .

In the above method, the level of protein expression in step 1) is immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western Blotting, and It is preferable to measure by any one selected from the group consisting of flow cytometry (FACS), but any method for measuring the amount of a transcript or a protein encoded therefrom can be used.

In the above method, the degree of activity of the protein in step 1) is preferably measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry, and protein chip, but is not limited thereto. .

In the above method, in steps 1) and 2)

a) measuring the expression or activity of ZEB2 (zinc finger E-box-binding homeobox 2) or Sp1 (specificity protein 1) protein; And

b) selecting a subject whose expression or activity of the ZEB2 or Sp1 protein is decreased compared to the normal control;

Characterized in that it additionally comprises, including the step of determining that the risk of developing a cancer selected from the group consisting of colorectal cancer, liver cancer and gastric cancer or cancer metastasis is high, but is not limited thereto.

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

However, the following examples specifically illustrate the present invention, and the contents of the present invention are not limited by the examples.

< Example 1> Cancer cell line culture

SW480 (colorectal cancer cell line), AGS (gastric cancer cell line), and HEK293E (Human Embryonic Kidney 293E) cell lines were provided by the American Seed Bank (USA). The cell lines SNU-638 (gastric cancer cell line) and SNU-398 (liver cancer cell line) were provided by the Korea Cell Line Bank (Seoul, Korea). SW480, AGS, SNU-638 and SNU-398 cell lines were cultured in RPMI1640 (GIBCO, USA) medium containing 10% FBS, penicillin-streptomycin, L-glutamine and sodium pyruvate. HEK293E was cultured in DMEM (Dulbeco's Modified Eagle Medium: GIBCO, USA) medium. All cell lines were cultured at 37°C and 5% CO ₂ conditions.

< Example 2> To cancer cell lines Cardherin -11 specific siRNA Transformation

In order to inhibit the expression of cadherin-11 in cancer cell lines and to confirm the morphological change of cells, an electroporation technique for cadherin-11 specific siRNA to SW480, a colorectal cancer cell line.

(Neon Transfection system, Invitrogen) was used to transform.

Specifically, after harvesting the cells, washing them with PBS, resuspending in R buffer at ^{a concentration of 3 × 10 5} cells/12 ul, adding 2 ul of 40 uM siRNA, and transfection using an electroporation device. Converted.

After 48 hours, cells were harvested to perform invasion and cell mobility experiments, cells were lysed to perform western blot experiments, or RNA was extracted to perform RT-PCR. A mixture of 4 types of Cadherin-11-specific siRNA (Dharmacon) used and 4 types of target sequences are as follows:

5'-GUG AGA ACA UCA UUA CUU A-3' (SEQ ID NO: 2)

5'-GGA CAU GGG UGG ACA UAU G-3' (SEQ ID NO: 3)

5'-GGA AAU AGC GCC AAG UUA G-3' (SEQ ID NO: 4), and

5'-CCU UAU GAC UCC AUU CAA A-3' (SEQ ID NO: 5).

In addition, the integrin alpha5-specific siRNA sequence is 5'-GGACCAAGGCAGAAGGCAGTT-3' (SEQ ID NO: 6), and the Sp1-specific siRNA sequence is 5'-GGUAGCUCUAAGUUUUGAUTT-3' (SEQ ID NO: 7), and ZEB2-specific siRNA (A mixture of 4 Dharmacon) and is described below.

5'-GAA CAG ACA GGC UUA CUU AUU-3' (SEQ ID NO: 8)

5'-GAA GCU ACG UAC UUU AAU AUU-3' (SEQ ID NO: 9)

5'-CAA CAU AUC CAC UCC AUU UUU-3' (SEQ ID NO: 10), and

5'-GGA GAC AGA UCA GUA AUA UUU-3' (SEQ ID NO: 11).

< Example 3> In cancer cell lines Cardherin -11 Gene expression confirmation

In order to confirm the expression of the cadherin-11 gene in cancer cell lines, total RNA was isolated from various cell lines cultured under the conditions of <Example 1> using Trizol (Invitrogen), and RT- PCR was performed.

Specifically, cDNA was synthesized using reverse transcriptase (Bioneer, Korea). Real-time polymerase chain reaction quantification was performed using SYBR Green (PKT, Korea). As for the specific primer sequence of cadherin-11, forward primer: 5'-CCAACAGCCCGATAAGGTAT-3' (SEQ ID NO: 12) and reverse primer: 5'-TGGATTTCTGCTGCAAAGAC-3' (SEQ ID NO: 13) were used.

On the other hand, as an internal control of the PCR reaction, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used, and the primers used at this time were forward primer: 5'-CATGACCACAGTCCATGCCAT-3' (SEQ ID NO: 14) and reverse primer: 5'-AAGGCCATGCCAGTGAGCTTC-3 '(SEQ ID NO: 15), and the annealing temperature was performed at 58°C. The resulting PCR product was subjected to electrophoresis on 2% agarose gel or 5%, 8% polyacrylamide gel to confirm the presence or absence of expression.

< Example 4> Cardherin -11 promoter Construct making

For promoter reporter assay, The promoter construct of cadherin-11 was constructed.

Specifically, the human Cadherin-11 promoter using primers 5'-CCGCTCGAGTTTTTCGCGGGTCCAT-3' (SEQ ID NO: 16) and 5'-CCGCTCGAGCTGGGGCCCTTGAGG-3' (SEQ ID NO: 17) in genomic DNA of the colon cancer cell line SW480 PCR was performed to amplify the region. The amplification product obtained after performing the PCR was digested with restriction enzyme XhoI and inserted into a pGL3 basic vector (Promega, Southampton, UK) to prepare a pCad11-1350luc construct.

< Experimental Example 1> Confirmation of the effect of inhibiting the invasion and migration of colon cancer cells by inhibiting the expression of cadherin-11

In order to analyze changes in the invasion ability and cellular mobility of cancer cells by inhibiting the expression of cadherin-11 in the colorectal cancer cell line SW480, cadherin-11-specific siRNA (Dharmacon) and control siRNA (sense sequence 5'-CUU ACG CUG AGU ACU UCG ATT-3' (SEQ ID NO: 18), antisense sequence 5'-UCG AAG UAC UCA GCG UAA GTT-3' (SEQ ID NO: 19)) After 48 hours, invasion assay and migration assay were performed.

Specifically, the infiltration assay was performed by diluting a porous membrane of a 24-well transwell plate (8 µm pore size; Costar, USA) with a serum free medium at a concentration of 250 ug/ml 100 µl Matrigel (BD Biosciences, USA). USA), and allowed to stand at room temperature for 1 hour to solidify. The lower layer of the transwell plate was coated with 100 µl of collagen type I (Sigma) at a concentration of 5 ug/ml as a chemoattractant. ^{3 × 10 4} cells resuspended in a serum-free medium were dispensed into an upper chamber, and _{cultured at 37° C. and 5% CO 2} for 48 hours to move from the upper chamber to the lower chamber. Cells that did not migrate were removed from the surface of the upper chamber. Cells transferred to the lower chamber were fixed with 3.7% paraformaldehyde dissolved in PBS, and stained with 2% crystal violet solution. After washing the excess crystal violet solution with distilled water, a picture of the selected area (×100 magnification) was taken, and the number of moved cells was counted in five selected areas. The experiment showed a representative result of the number of repeated at least two or more times with two of the same conditions. The data values reflected the number of metastatic cell standard deviations per 200 magnification (HPF). In the cell mobility experiment, 1 × 10 ⁴ cells were dispensed into the upper chamber without the Matrigel coating, and the number of metastatic cell standard deviations per × 100 magnification was reflected.

As a result, after transforming the colorectal cancer cell line SW480 with cadherin-11-specific siRNA, invasion capability and cell mobility were performed using a matrigel-coated transwell to inhibit the expression of cadherin-11 It was confirmed that the mobility was reduced by 34% and 56%, respectively (FIG. 1).

< Experimental Example 2> Signaling and EMT of colon cancer cells by inhibition of expression of cadherin-11 Checking the inhibitory effect

In order to analyze changes in cell signaling and EMT due to inhibition of expression of cadherin-11 in colorectal cancer cell lines, cadherin-11 specific siRNA was introduced into SW480, which is a colorectal cancer cell line, and Western blot analysis was performed.

Cells were prepared in RIPA buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, complete protease inhibitor) Crushed in and cell lysates were quantified using a commercially available modified Bradford assay (Bio-Rad Laboratories, Hercules, CA). Thereafter, 30 ug of the obtained cell lysate was mixed with an SDS sample buffer and heated, followed by electrophoresis on an 8% SDS-PAGE gel. Protein separated on SDS-PAGE through electrophoresis was transferred to a nitrocellulose membrane and blocked with 5% skim milk. Afterwards, anti-phosphorylation-ERK1/2 and anti-ERK1/2 (cell signaling technology), anti-GAPDH (Santa Cruz), anti-integrin alpha 5 (BD Biosciences), anti-cadherin-11 (Invitrogen), anti -E-cadherin (R&D systems), anti-vimentin (Sigma) and anti-Sp1 (Santa Cruz) were reacted together, and horseradish peroxidase-conjugated secondary antibodies were reacted. After that, the ECL kit (ECL Plus, Amersham, USA) was treated according to the manufacturer's method to induce an evolutionary reaction.

As a result, as a result of suppressing the expression of cadherin-11 with siRNA in SW480 cells, it was confirmed that the expression of mesenchymal cell markers integrin alpha5 and vimentin decreased, and the expression of epithelial cell marker E-cadherin was increased. I did. In addition, the expression of the transcription factor Sp1 was decreased. In addition, it was confirmed that the phosphorylation of FAK, ERK, and c-src was decreased, and these results showed that EMT was inhibited and cell signaling was inactivated by inhibition of cadherin-11 expression. Therefore, cadherin-11 functioned to induce EMT of colon cancer cells and to induce cancer cell invasion and cell mobility (FIG. 2).

< Experimental Example 3> Induction of expression of cadherin- 11 by EMT- induced transcription factor ZEB2 in cancer cells

<3-1> On ZEB2 due to Cardherin Reporter analysis of induction of expression of -11

ZEB2 (Smad-interacting protein-1/Zinc finger E-box binding homeobox 2) is a representative E-cadherin transcription inhibitor and EMT-induced transcription and is related to cancer cell invasion and metastasis. This induction was confirmed by reporter assay.

Specifically, 2.5 × 10 ⁵ SW480, AGS, and HEK293E cells were dispensed into a 6-well plate, and after 24 hours, 2 ug of reporter plasmid DNA and 1.8 ug of ZEB expression vector prepared in Example 4 above. Transformed with pCS3 SIP1 (Dr. D, Huylebroeck, University of Leuven, Belgium; Nam et al., (2012) Carcinogenesis 33(3):563-571) and Lipofectamine 2000 reagent (invitrogen) Made it. 48 hours after transformation, luciferase activity was measured with a Dual-luciferase reporter assay system (Promega), and the transformation efficiency was 0.2 ug of Renilla luciferase ( Renila luciferase) vector pRL-TK (Promega) was confirmed by using the measured renilla luciferase activity measured by transforming together.

As a result, after cloning the cadherin-11 promoter region (-1350/+137) into pGL3basic, reporter analysis was performed. As a result, in SW480, AGS and HEK293E cells, the cadherin-11 promoter was 3.3 times and 3.6 by ZEB2, respectively. It was confirmed that it was activated twice and 1.5 times (Figure 3).

<3-2> Quantitative analysis of real-time polymerase chain reaction for induction of expression of cadherin- 11 by ZEB2

ZEB2 is a representative E-cadherin transcription inhibitor and EMT-induced transcription, and is related to cancer cell invasion and metastasis. -qPCR).

Specifically, after dispensing 2.5 × 10 ⁵ SW480 cells, 2 × 10 ⁵ SNU-638 cells, and 5 × 10 ⁵ SNU-398 cells into a 6-well plate, 24 hours later, 4 ug of ZEB expression vector, pCS3 SIP1 (Dr. D, Huylebroeck, University of Leuven, Belgium; Nam et al., (2012) Carcinogenesis 33(3):563-571) or 4.5 ul of siRNA at a concentration of 40 uM was added to Lipofectamine 2000 reagent (invitrogen ) Was transformed. 48 hours after transformation, total RNA was isolated from the cells using Trizol (Invitrogen), and RT-qPCR was performed using this. The RT-qPCR was performed in the same manner as in <Example 3>.

As a result, after transforming SW480 cells with ZEB2, the level of cadherin-11 mRNA increased 2.3 times, and ZEB2-specific siRNA was transfected into SNU-638, a gastric cancer cell expressing endogenous ZEB2, and SNU-398, a liver cancer cell. After conversion, it was confirmed by RT-qPCR that the expression of cadherin-11 mRNA decreased by 39% and 71%, respectively (FIG. 4).

<3-3> On ZEB2 due to Cardherin To the induction of expression of -11 Sp1 Impact Analysis

In a previous study by this research team, it was confirmed that the transcription factor Sp1 is involved in the expression of mesenchymal cell markers integrin alpha5 and vimentin by ZEB2 (Nam et al., (2012) Carcinogenesis 33(3):563). -571). Therefore, Sp1 was expected to be required for induction of cadherin-11 by ZEB2, and RT-qPCR and Western blot analysis were performed to verify this.

Specifically, SW480 and AGS cells were transformed with ZEB2, and after 24 hours, Sp1-DNA binding inhibitor (mithramycin A, 50 nM) was treated for 24 hours, and the real-time polymerase chain reaction quantitative test of <Example 3> And Western blot analysis. Cells were prepared in RIPA buffer (10 mM Tris, pH7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, complete protease inhibitor). Dissolved. Cytolysis was quantified by the Breadford analysis method (bio-Rad Laboratories, Hercules, CA). Cell lysis 30 ug was mixed with SDS sample buffer, heated, and run on an 8% SDS-PAGE gel. The separated protein was transferred to a nitrocellulose membrane and blocked with 5% skim milk. Then, it was reacted with anti-GAPDH (Santa Cruz) and anti-cadherin-11 (Invitrogen), and reacted with a secondary antibody to which horseradish peroxidase was bound. Thereafter, the ECL kit (ECL Plus, Amersham, USA) was treated according to the manufacturer's method to confirm the expression of cadherin-11.

As a result, it was confirmed that the expression of cadherin-11 induced by ZEB2 was reduced by inhibition of Sp1 activity (FIG. 5). Specifically, the level of cadherin-11 mRNA was increased 1.6-fold by ZEB2 in SW480 cells (FIG. 5A), and this increase was completely suppressed by inhibition of Sp1 activity (mithramycin A). In addition, ZEB2 in AGS cells increased the level of cadherin-11 mRNA by 6.3-fold (Fig. 5C), and this increase was completely suppressed by inhibition of Sp1 activity (misramycin A).

<3-4> Sp1 -specific siRNA in induction of expression of cadherin- 11 by ZEB2 Analyze the impact

RT-qPCR was performed to confirm the effect of Sp1-specific siRNA on the expression of cadherin-11 induced by ZEB2 through the results of confirming that the expression of cadherin-11 induced by ZEB2 was reduced by inhibition of Sp1 activity.

Specifically, after dispensing 2.5 × 10 ⁵ SW480 cells into a 6-well plate, 6 ul of siRNA at a concentration of 40 uM was introduced (transformed) after 24 hours using Lipofectamine 2000 reagent (invitrogen). 24 hours after transformation, 4 ug of ZEB expression vector pCS3 SIP1 (Dr. D, Huylebroeck, University of Leuven, Belgium; Nam et al., (2012) Carcinogenesis 33(3):563-571) was used as lipofectamine. It was transformed using 2000 reagent (invitrogen). After 24 hours, total RNA was isolated from the cells using Trizol (Invitrogen), and RT-qPCR was performed using this. The RT-qPCR was performed in the same manner as in <Example 3>.

As a result, it was confirmed that the expression of cadherin-11 induced by ZEB2 was also reduced by Sp1-specific siRNA (FIG. 6). Specifically, ZEB2 in SW480 cells increased cadherin-11 mRNA levels 3.0-fold, and this increase was reduced by 1.6-fold by Sp1-specific siRNA, so that ZEB2 induced cadherin-11 mRNA increased by Sp1-specific siRNA. Reduced by about 46%.

<3-5> In liver cancer cell lines ChIP (Chromatin immunoprecipitation ) Confirm

ChIP was performed to determine which site of ZEB2 and Sp1 specifically binds to the cadherin-11 promoter in SNU-638 cells, which are liver cancer cell lines.

Specifically, it was performed according to the instructions of the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY, USA). Rabbit anti-ZEB2 (H260, Santa Cruz Biotechnology) or rabbit anti-SP1 (PEP2, Santa Cruz Biotechnology) per 2×10 ^{6 SNU-638 cell equivalent was used for the ChIP reaction.} As a control antibody, normal rabbit IgG was used for the reaction. After destroying the cells and crushing the DNA, anti-ZEB2 or anti-Sp1 antibody was added and reacted to some extent at 4°C. Then, protein G-agarose beads were added and reacted at 4°C to obtain an antibody-DNA complex bound to the beads. Only DNA was separated and purified from this complex. And PCR was performed by designing a primer around the cadherin-11 promoter to be identified. The cadherin-11 promoter-specific primers are 5'-AGCCTCGGCTTCTCTCAG-3' (SEQ ID NO: 20) and 5'-GCTAGTGGCAGGAATGAGAA-3' (SEQ ID NO: 21) and -1122/-1007 that recognize the -191/-62 region. Region recognition 5'-AGTCCTTGGCGTGATTCCTA-3' (SEQ ID NO: 22) and 5'-CCTCACTGACGACTCGCTAC-3' (SEQ ID NO: 23) were used.

As a result, it was confirmed that ZEB2 and Sp1 specifically bind to the cadherin-11 promoter (-191/-62) region (FIG. 7).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Pharmaceutical composition for the treatment of colorectal cancers or inhibition of metastasis containing the expression or activity inhibitors of cadherin-11 <130> 15p-10-31 <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 796 <212> PRT <213> cadherin-11 preproprotein (1..796) <400> 1 Met Lys Glu Asn Tyr Cys Leu Gln Ala Ala Leu Val Cys Leu Gly Met 1 5 10 15 Leu Cys His Ser His Ala Phe Ala Pro Glu Arg Arg Gly His Leu Arg 20 25 30 Pro Ser Phe His Gly His His Glu Lys Gly Lys Glu Gly Gln Val Leu 35 40 45 Gln Arg Ser Lys Arg Gly Trp Val Trp Asn Gln Phe Phe Val Ile Glu 50 55 60 Glu Tyr Thr Gly Pro Asp Pro Val Leu Val Gly Arg Leu His Ser Asp 65 70 75 80 Ile Asp Ser Gly Asp Gly Asn Ile Lys Tyr Ile Leu Ser Gly Glu Gly 85 90 95 Ala Gly Thr Ile Phe Val Ile Asp Asp Lys Ser Gly Asn Ile His Ala 100 105 110 Thr Lys Thr Leu Asp Arg Glu Glu Arg Ala Gln Tyr Thr Leu Met Ala 115 120 125 Gln Ala Val Asp Arg Asp Thr Asn Arg Pro Leu Glu Pro Pro Ser Glu 130 135 140 Phe Ile Val Lys Val Gln Asp Ile Asn Asp Asn Pro Pro Glu Phe Leu 145 150 155 160 His Glu Thr Tyr His Ala Asn Val Pro Glu Arg Ser Asn Val Gly Thr 165 170 175 Ser Val Ile Gln Val Thr Ala Ser Asp Ala Asp Asp Pro Thr Tyr Gly 180 185 190 Asn Ser Ala Lys Leu Val Tyr Ser Ile Leu Glu Gly Gln Pro Tyr Phe 195 200 205 Ser Val Glu Ala Gln Thr Gly Ile Ile Arg Thr Ala Leu Pro Asn Met 210 215 220 Asp Arg Glu Ala Lys Glu Glu Tyr His Val Val Ile Gln Ala Lys Asp 225 230 235 240 Met Gly Gly His Met Gly Gly Leu Ser Gly Thr Thr Lys Val Thr Ile 245 250 255 Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Lys Phe Pro Gln Ser Val 260 265 270 Tyr Gln Met Ser Val Ser Glu Ala Ala Val Pro Gly Glu Glu Val Gly 275 280 285 Arg Val Lys Ala Lys Asp Pro Asp Ile Gly Glu Asn Gly Leu Val Thr 290 295 300 Tyr Asn Ile Val Asp Gly Asp Gly Met Glu Ser Phe Glu Ile Thr Thr 305 310 315 320 Asp Tyr Glu Thr Gln Glu Gly Val Ile Lys Leu Lys Lys Pro Val Asp 325 330 335 Phe Glu Thr Lys Arg Ala Tyr Ser Leu Lys Val Glu Ala Ala Asn Val 340 345 350 His Ile Asp Pro Lys Phe Ile Ser Asn Gly Pro Phe Lys Asp Thr Val 355 360 365 Thr Val Lys Ile Ser Val Glu Asp Ala Asp Glu Pro Pro Met Phe Leu 370 375 380 Ala Pro Ser Tyr Ile His Glu Val Gln Glu Asn Ala Ala Ala Gly Thr 385 390 395 400 Val Val Gly Arg Val His Ala Lys Asp Pro Asp Ala Ala Asn Ser Pro 405 410 415 Ile Arg Tyr Ser Ile Asp Arg His Thr Asp Leu Asp Arg Phe Phe Thr 420 425 430 Ile Asn Pro Glu Asp Gly Phe Ile Lys Thr Thr Lys Pro Leu Asp Arg 435 440 445 Glu Glu Thr Ala Trp Leu Asn Ile Thr Val Phe Ala Ala Glu Ile His 450 455 460 Asn Arg His Gln Glu Ala Lys Val Pro Val Ala Ile Arg Val Leu Asp 465 470 475 480 Val Asn Asp Asn Ala Pro Lys Phe Ala Ala Pro Tyr Glu Gly Phe Ile 485 490 495 Cys Glu Ser Asp Gln Thr Lys Pro Leu Ser Asn Gln Pro Ile Val Thr 500 505 510 Ile Ser Ala Asp Asp Lys Asp Asp Thr Ala Asn Gly Pro Arg Phe Ile 515 520 525 Phe Ser Leu Pro Pro Glu Ile Ile His Asn Pro Asn Phe Thr Val Arg 530 535 540 Asp Asn Arg Asp Asn Thr Ala Gly Val Tyr Ala Arg Arg Gly Gly Phe 545 550 555 560 Ser Arg Gln Lys Gln Asp Leu Tyr Leu Leu Pro Ile Val Ile Ser Asp 565 570 575 Gly Gly Ile Pro Pro Met Ser Ser Thr Asn Thr Leu Thr Ile Lys Val 580 585 590 Cys Gly Cys Asp Val Asn Gly Ala Leu Leu Ser Cys Asn Ala Glu Ala 595 600 605 Tyr Ile Leu Asn Ala Gly Leu Ser Thr Gly Ala Leu Ile Ala Ile Leu 610 615 620 Ala Cys Ile Val Ile Leu Leu Val Ile Val Val Leu Phe Val Thr Leu 625 630 635 640 Arg Arg Gln Lys Lys Glu Pro Leu Ile Val Phe Glu Glu Glu Asp Val 645 650 655 Arg Glu Asn Ile Ile Thr Tyr Asp Asp Glu Gly Gly Gly Glu Glu Asp 660 665 670 Thr Glu Ala Phe Asp Ile Ala Thr Leu Gln Asn Pro Asp Gly Ile Asn 675 680 685 Gly Phe Ile Pro Arg Lys Asp Ile Lys Pro Glu Tyr Gln Tyr Met Pro 690 695 700 Arg Pro Gly Leu Arg Pro Ala Pro Asn Ser Val Asp Val Asp Asp Phe 705 710 715 720 Ile Asn Thr Arg Ile Gln Glu Ala Asp Asn Asp Pro Thr Ala Pro Pro 725 730 735 Tyr Asp Ser Ile Gln Ile Tyr Gly Tyr Glu Gly Arg Gly Ser Val Ala 740 745 750 Gly Ser Leu Ser Ser Leu Glu Ser Ala Thr Thr Asp Ser Asp Leu Asp 755 760 765 Tyr Asp Tyr Leu Gln Asn Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp 770 775 780 Leu Tyr Gly Ser Lys Asp Thr Phe Asp Asp Asp Ser 785 790 795 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> cadherin-11 siRNA <400> 2 gugagaacau cauuacuua 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> cadherin-11 siRNA <400> 3 ggacaugggu ggacauaug 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> cadherin-11 siRNA <400> 4 ggaaauagcg ccaaguuag 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> cadherin-11 siRNA <400> 5 ccuuaugacu ccauucaaa 19 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> integrin alpha5 siRNA <400> 6 ggaccaaggc agaaggcagt t 21 <210> 7 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> sp-1 siRNA <400> 7 gguagcucua aguuuugaut t 21 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> ZEB2 siRNA <400> 8 gaacagacag gcuuacuuau u 21 <210> 9 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> ZEB2 siRNA <400> 9 gaagcuacgu acuuuaauau u 21 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> ZEB2 siRNA <400> 10 caacauaucc acuccauuuu u 21 <210> 11 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> ZEB2 siRNA <400> 11 ggagacagau caguaauauu u 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cadherin-11 forward primer <400> 12 ccaacagccc gataaggtat 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cadherin-11 reverse primer <400> 13 tggatttctg ctgcaaagac 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 14 catgaccaca gtccatgcca t 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 15 aaggccatgc cagtgagctt c 21 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SW480 forward primer <400> 16 ccgctcgagt ttttcgcggg tccat 25 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SW480 reverse primer <400> 17 ccgctcgagc tggggccctt gagg 24 <210> 18 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> cadherin-11 control group siRNA <400> 18 cuuacgcuga guacuucgat t 21 <210> 19 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> cadherin-11 control group siRNA <400> 19 ucgaaguacu cagcguaagt t 21 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> cadherin-11 -191/-62 forward primer <400> 20 agcctcggct tctctcag 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cadherin-11 -191/-62 reverse primer <400> 21 gctagtggca ggaatgagaa 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cadherin-11 -1122/-1007 forward primer <400> 22 agtccttggc gtgattccta 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cadherin-11 -1122/-1007 reverse primer <400> 23 cctcactgac gactcgctac 20 <210> 24 <211> 774 <212> PRT <213> cadherin-11 proprotein (23..796) <400> 24 Phe Ala Pro Glu Arg Arg Gly His Leu Arg Pro Ser Phe His Gly His 1 5 10 15 His Glu Lys Gly Lys Glu Gly Gln Val Leu Gln Arg Ser Lys Arg Gly 20 25 30 Trp Val Trp Asn Gln Phe Phe Val Ile Glu Glu Glu Tyr Thr Gly Pro Asp 35 40 45 Pro Val Leu Val Gly Arg Leu His Ser Asp Ile Asp Ser Gly Asp Gly 50 55 60 Asn Ile Lys Tyr Ile Leu Ser Gly Glu Gly Ala Gly Thr Ile Phe Val 65 70 75 80 Ile Asp Asp Lys Ser Gly Asn Ile His Ala Thr Lys Thr Leu Asp Arg 85 90 95 Glu Glu Arg Ala Gln Tyr Thr Leu Met Ala Gln Ala Val Asp Arg Asp 100 105 110 Thr Asn Arg Pro Leu Glu Pro Pro Ser Glu Phe Ile Val Lys Val Gln 115 120 125 Asp Ile Asn Asp Asn Pro Pro Glu Phe Leu His Glu Thr Tyr His Ala 130 135 140 Asn Val Pro Glu Arg Ser Asn Val Gly Thr Ser Val Ile Gln Val Thr 145 150 155 160 Ala Ser Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser Ala Lys Leu Val 165 170 175 Tyr Ser Ile Leu Glu Gly Gln Pro Tyr Phe Ser Val Glu Ala Gln Thr 180 185 190 Gly Ile Ile Arg Thr Ala Leu Pro Asn Met Asp Arg Glu Ala Lys Glu 195 200 205 Glu Tyr His Val Val Ile Gln Ala Lys Asp Met Gly Gly His Met Gly 210 215 220 Gly Leu Ser Gly Thr Thr Lys Val Thr Ile Thr Leu Thr Asp Val Asn 225 230 235 240 Asp Asn Pro Pro Lys Phe Pro Gln Ser Val Tyr Gln Met Ser Val Ser 245 250 255 Glu Ala Ala Val Pro Gly Glu Glu Val Gly Arg Val Lys Ala Lys Asp 260 265 270 Pro Asp Ile Gly Glu Asn Gly Leu Val Thr Tyr Asn Ile Val Asp Gly 275 280 285 Asp Gly Met Glu Ser Phe Glu Ile Thr Thr Asp Tyr Glu Thr Gln Glu 290 295 300 Gly Val Ile Lys Leu Lys Lys Pro Val Asp Phe Glu Thr Lys Arg Ala 305 310 315 320 Tyr Ser Leu Lys Val Glu Ala Ala Asn Val His Ile Asp Pro Lys Phe 325 330 335 Ile Ser Asn Gly Pro Phe Lys Asp Thr Val Thr Val Lys Ile Ser Val 340 345 350 Glu Asp Ala Asp Glu Pro Pro Met Phe Leu Ala Pro Ser Tyr Ile His 355 360 365 Glu Val Gln Glu Asn Ala Ala Ala Gly Thr Val Val Gly Arg Val His 370 375 380 Ala Lys Asp Pro Asp Ala Ala Asn Ser Pro Ile Arg Tyr Ser Ile Asp 385 390 395 400 Arg His Thr Asp Leu Asp Arg Phe Phe Thr Ile Asn Pro Glu Asp Gly 405 410 415 Phe Ile Lys Thr Thr Lys Pro Leu Asp Arg Glu Glu Thr Ala Trp Leu 420 425 430 Asn Ile Thr Val Phe Ala Ala Glu Ile His Asn Arg His Gln Glu Ala 435 440 445 Lys Val Pro Val Ala Ile Arg Val Leu Asp Val Asn Asp Asn Ala Pro 450 455 460 Lys Phe Ala Ala Pro Tyr Glu Gly Phe Ile Cys Glu Ser Asp Gln Thr 465 470 475 480 Lys Pro Leu Ser Asn Gln Pro Ile Val Thr Ile Ser Ala Asp Asp Lys 485 490 495 Asp Asp Thr Ala Asn Gly Pro Arg Phe Ile Phe Ser Leu Pro Pro Glu 500 505 510 Ile Ile His Asn Pro Asn Phe Thr Val Arg Asp Asn Arg Asp Asn Thr 515 520 525 Ala Gly Val Tyr Ala Arg Arg Gly Gly Phe Ser Arg Gln Lys Gln Asp 530 535 540 Leu Tyr Leu Leu Pro Ile Val Ile Ser Asp Gly Gly Ile Pro Pro Met 545 550 555 560 Ser Ser Thr Asn Thr Leu Thr Ile Lys Val Cys Gly Cys Asp Val Asn 565 570 575 Gly Ala Leu Leu Ser Cys Asn Ala Glu Ala Tyr Ile Leu Asn Ala Gly 580 585 590 Leu Ser Thr Gly Ala Leu Ile Ala Ile Leu Ala Cys Ile Val Ile Leu 595 600 605 Leu Val Ile Val Val Leu Phe Val Thr Leu Arg Arg Gln Lys Lys Glu 610 615 620 Pro Leu Ile Val Phe Glu Glu Glu Asp Val Arg Glu Asn Ile Ile Thr 625 630 635 640 Tyr Asp Asp Glu Gly Gly Gly Glu Glu Asp Thr Glu Ala Phe Asp Ile 645 650 655 Ala Thr Leu Gln Asn Pro Asp Gly Ile Asn Gly Phe Ile Pro Arg Lys 660 665 670 Asp Ile Lys Pro Glu Tyr Gln Tyr Met Pro Arg Pro Gly Leu Arg Pro 675 680 685 Ala Pro Asn Ser Val Asp Val Asp Asp Phe Ile Asn Thr Arg Ile Gln 690 695 700 Glu Ala Asp Asn Asp Pro Thr Ala Pro Pro Tyr Asp Ser Ile Gln Ile 705 710 715 720 Tyr Gly Tyr Glu Gly Arg Gly Ser Val Ala Gly Ser Leu Ser Ser Leu 725 730 735 Glu Ser Ala Thr Thr Asp Ser Asp Leu Asp Tyr Asp Tyr Leu Gln Asn 740 745 750 Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp Leu Tyr Gly Ser Lys Asp 755 760 765 Thr Phe Asp Asp Asp Ser 770 <210> 25 <211> 765 <212> PRT <213> cadherin-11 proprotein (23..54,54..796) <400> 25 Met Lys Glu Asn Tyr Cys Leu Gln Ala Ala Leu Val Cys Leu Gly Met 1 5 10 15 Leu Cys His Ser His Ala Gly Trp Val Trp Asn Gln Phe Phe Val Ile 20 25 30 Glu Glu Tyr Thr Gly Pro Asp Pro Val Leu Val Gly Arg Leu His Ser 35 40 45 Asp Ile Asp Ser Gly Asp Gly Asn Ile Lys Tyr Ile Leu Ser Gly Glu 50 55 60 Gly Ala Gly Thr Ile Phe Val Ile Asp Asp Lys Ser Gly Asn Ile His 65 70 75 80 Ala Thr Lys Thr Leu Asp Arg Glu Glu Arg Ala Gln Tyr Thr Leu Met 85 90 95 Ala Gln Ala Val Asp Arg Asp Thr Asn Arg Pro Leu Glu Pro Pro Ser 100 105 110 Glu Phe Ile Val Lys Val Gln Asp Ile Asn Asp Asn Pro Pro Glu Phe 115 120 125 Leu His Glu Thr Tyr His Ala Asn Val Pro Glu Arg Ser Asn Val Gly 130 135 140 Thr Ser Val Ile Gln Val Thr Ala Ser Asp Ala Asp Asp Pro Thr Tyr 145 150 155 160 Gly Asn Ser Ala Lys Leu Val Tyr Ser Ile Leu Glu Gly Gln Pro Tyr 165 170 175 Phe Ser Val Glu Ala Gln Thr Gly Ile Ile Arg Thr Ala Leu Pro Asn 180 185 190 Met Asp Arg Glu Ala Lys Glu Glu Tyr His Val Val Ile Gln Ala Lys 195 200 205 Asp Met Gly Gly His Met Gly Gly Leu Ser Gly Thr Thr Lys Val Thr 210 215 220 Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Lys Phe Pro Gln Ser 225 230 235 240 Val Tyr Gln Met Ser Val Ser Glu Ala Ala Val Pro Gly Glu Glu Val 245 250 255 Gly Arg Val Lys Ala Lys Asp Pro Asp Ile Gly Glu Asn Gly Leu Val 260 265 270 Thr Tyr Asn Ile Val Asp Gly Asp Gly Met Glu Ser Phe Glu Ile Thr 275 280 285 Thr Asp Tyr Glu Thr Gln Glu Gly Val Ile Lys Leu Lys Lys Pro Val 290 295 300 Asp Phe Glu Thr Lys Arg Ala Tyr Ser Leu Lys Val Glu Ala Ala Asn 305 310 315 320 Val His Ile Asp Pro Lys Phe Ile Ser Asn Gly Pro Phe Lys Asp Thr 325 330 335 Val Thr Val Lys Ile Ser Val Glu Asp Ala Asp Glu Pro Pro Met Phe 340 345 350 Leu Ala Pro Ser Tyr Ile His Glu Val Gln Glu Asn Ala Ala Ala Gly 355 360 365 Thr Val Val Gly Arg Val His Ala Lys Asp Pro Asp Ala Ala Asn Ser 370 375 380 Pro Ile Arg Tyr Ser Ile Asp Arg His Thr Asp Leu Asp Arg Phe Phe 385 390 395 400 Thr Ile Asn Pro Glu Asp Gly Phe Ile Lys Thr Thr Lys Pro Leu Asp 405 410 415 Arg Glu Glu Thr Ala Trp Leu Asn Ile Thr Val Phe Ala Ala Glu Ile 420 425 430 His Asn Arg His Gln Glu Ala Lys Val Pro Val Ala Ile Arg Val Leu 435 440 445 Asp Val Asn Asp Asn Ala Pro Lys Phe Ala Ala Pro Tyr Glu Gly Phe 450 455 460 Ile Cys Glu Ser Asp Gln Thr Lys Pro Leu Ser Asn Gln Pro Ile Val 465 470 475 480 Thr Ile Ser Ala Asp Asp Lys Asp Asp Thr Ala Asn Gly Pro Arg Phe 485 490 495 Ile Phe Ser Leu Pro Pro Glu Ile Ile His Asn Pro Asn Phe Thr Val 500 505 510 Arg Asp Asn Arg Asp Asn Thr Ala Gly Val Tyr Ala Arg Arg Gly Gly 515 520 525 Phe Ser Arg Gln Lys Gln Asp Leu Tyr Leu Leu Pro Ile Val Ile Ser 530 535 540 Asp Gly Gly Ile Pro Pro Met Ser Ser Thr Asn Thr Leu Thr Ile Lys 545 550 555 560 Val Cys Gly Cys Asp Val Asn Gly Ala Leu Leu Ser Cys Asn Ala Glu 565 570 575 Ala Tyr Ile Leu Asn Ala Gly Leu Ser Thr Gly Ala Leu Ile Ala Ile 580 585 590 Leu Ala Cys Ile Val Ile Leu Leu Val Ile Val Val Leu Phe Val Thr 595 600 605 Leu Arg Arg Gln Lys Lys Glu Pro Leu Ile Val Phe Glu Glu Glu Asp 610 615 620 Val Arg Glu Asn Ile Ile Thr Tyr Asp Asp Glu Gly Gly Gly Glu Glu 625 630 635 640 Asp Thr Glu Ala Phe Asp Ile Ala Thr Leu Gln Asn Pro Asp Gly Ile 645 650 655 Asn Gly Phe Ile Pro Arg Lys Asp Ile Lys Pro Glu Tyr Gln Tyr Met 660 665 670 Pro Arg Pro Gly Leu Arg Pro Ala Pro Asn Ser Val Asp Val Asp Asp 675 680 685 Phe Ile Asn Thr Arg Ile Gln Glu Ala Asp Asn Asp Pro Thr Ala Pro 690 695 700 Pro Tyr Asp Ser Ile Gln Ile Tyr Gly Tyr Glu Gly Arg Gly Ser Val 705 710 715 720 Ala Gly Ser Leu Ser Ser Leu Glu Ser Ala Thr Thr Asp Ser Asp Leu 725 730 735 Asp Tyr Asp Tyr Leu Gln Asn Trp Gly Pro Arg Phe Lys Lys Leu Ala 740 745 750 Asp Leu Tyr Gly Ser Lys Asp Thr Phe Asp Asp Asp Ser 755 760 765 <210> 26 <211> 743 <212> PRT <213> cadherin-11 muture protein (54..796) <400> 26 Gly Trp Val Trp Asn Gln Phe Phe Val Ile Glu Glu Tyr Thr Gly Pro 1 5 10 15 Asp Pro Val Leu Val Gly Arg Leu His Ser Asp Ile Asp Ser Gly Asp 20 25 30 Gly Asn Ile Lys Tyr Ile Leu Ser Gly Glu Gly Ala Gly Thr Ile Phe 35 40 45 Val Ile Asp Asp Lys Ser Gly Asn Ile His Ala Thr Lys Thr Leu Asp 50 55 60 Arg Glu Glu Arg Ala Gln Tyr Thr Leu Met Ala Gln Ala Val Asp Arg 65 70 75 80 Asp Thr Asn Arg Pro Leu Glu Pro Pro Ser Glu Phe Ile Val Lys Val 85 90 95 Gln Asp Ile Asn Asp Asn Pro Pro Glu Phe Leu His Glu Thr Tyr His 100 105 110 Ala Asn Val Pro Glu Arg Ser Asn Val Gly Thr Ser Val Ile Gln Val 115 120 125 Thr Ala Ser Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser Ala Lys Leu 130 135 140 Val Tyr Ser Ile Leu Glu Gly Gln Pro Tyr Phe Ser Val Glu Ala Gln 145 150 155 160 Thr Gly Ile Ile Arg Thr Ala Leu Pro Asn Met Asp Arg Glu Ala Lys 165 170 175 Glu Glu Tyr His Val Val Ile Gln Ala Lys Asp Met Gly Gly His Met 180 185 190 Gly Gly Leu Ser Gly Thr Thr Lys Val Thr Ile Thr Leu Thr Asp Val 195 200 205 Asn Asp Asn Pro Pro Lys Phe Pro Gln Ser Val Tyr Gln Met Ser Val 210 215 220 Ser Glu Ala Ala Val Pro Gly Glu Glu Val Gly Arg Val Lys Ala Lys 225 230 235 240 Asp Pro Asp Ile Gly Glu Asn Gly Leu Val Thr Tyr Asn Ile Val Asp 245 250 255 Gly Asp Gly Met Glu Ser Phe Glu Ile Thr Thr Asp Tyr Glu Thr Gln 260 265 270 Glu Gly Val Ile Lys Leu Lys Lys Pro Val Asp Phe Glu Thr Lys Arg 275 280 285 Ala Tyr Ser Leu Lys Val Glu Ala Ala Asn Val His Ile Asp Pro Lys 290 295 300 Phe Ile Ser Asn Gly Pro Phe Lys Asp Thr Val Thr Val Lys Ile Ser 305 310 315 320 Val Glu Asp Ala Asp Glu Pro Pro Met Phe Leu Ala Pro Ser Tyr Ile 325 330 335 His Glu Val Gln Glu Asn Ala Ala Ala Gly Thr Val Val Gly Arg Val 340 345 350 His Ala Lys Asp Pro Asp Ala Ala Asn Ser Pro Ile Arg Tyr Ser Ile 355 360 365 Asp Arg His Thr Asp Leu Asp Arg Phe Phe Thr Ile Asn Pro Glu Asp 370 375 380 Gly Phe Ile Lys Thr Thr Lys Pro Leu Asp Arg Glu Glu Thr Ala Trp 385 390 395 400 Leu Asn Ile Thr Val Phe Ala Ala Glu Ile His Asn Arg His Gln Glu 405 410 415 Ala Lys Val Pro Val Ala Ile Arg Val Leu Asp Val Asn Asp Asn Ala 420 425 430 Pro Lys Phe Ala Ala Pro Tyr Glu Gly Phe Ile Cys Glu Ser Asp Gln 435 440 445 Thr Lys Pro Leu Ser Asn Gln Pro Ile Val Thr Ile Ser Ala Asp Asp 450 455 460 Lys Asp Asp Thr Ala Asn Gly Pro Arg Phe Ile Phe Ser Leu Pro Pro 465 470 475 480 Glu Ile Ile His Asn Pro Asn Phe Thr Val Arg Asp Asn Arg Asp Asn 485 490 495 Thr Ala Gly Val Tyr Ala Arg Arg Gly Gly Phe Ser Arg Gln Lys Gln 500 505 510 Asp Leu Tyr Leu Leu Pro Ile Val Ile Ser Asp Gly Gly Ile Pro Pro 515 520 525 Met Ser Ser Thr Asn Thr Leu Thr Ile Lys Val Cys Gly Cys Asp Val 530 535 540 Asn Gly Ala Leu Leu Ser Cys Asn Ala Glu Ala Tyr Ile Leu Asn Ala 545 550 555 560 Gly Leu Ser Thr Gly Ala Leu Ile Ala Ile Leu Ala Cys Ile Val Ile 565 570 575 Leu Leu Val Ile Val Val Leu Phe Val Thr Leu Arg Arg Gln Lys Lys 580 585 590 Glu Pro Leu Ile Val Phe Glu Glu Glu Asp Val Arg Glu Asn Ile Ile 595 600 605 Thr Tyr Asp Asp Glu Gly Gly Gly Glu Glu Asp Thr Glu Ala Phe Asp 610 615 620 Ile Ala Thr Leu Gln Asn Pro Asp Gly Ile Asn Gly Phe Ile Pro Arg 625 630 635 640 Lys Asp Ile Lys Pro Glu Tyr Gln Tyr Met Pro Arg Pro Gly Leu Arg 645 650 655 Pro Ala Pro Asn Ser Val Asp Val Asp Asp Phe Ile Asn Thr Arg Ile 660 665 670 Gln Glu Ala Asp Asn Asp Pro Thr Ala Pro Pro Tyr Asp Ser Ile Gln 675 680 685 Ile Tyr Gly Tyr Glu Gly Arg Gly Ser Val Ala Gly Ser Leu Ser Ser 690 695 700 Leu Glu Ser Ala Thr Thr Asp Ser Asp Leu Asp Tyr Asp Tyr Leu Gln 705 710 715 720 Asn Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp Leu Tyr Gly Ser Lys 725 730 735 Asp Thr Phe Asp Asp Asp Ser 740

Claims (8)

delete delete 1) treating a test substance with an expression cell line of cardherin-11 protein which is composed of the amino acid sequence of SEQ ID NO: 1 and whose expression is induced by ZEB2 (zinc finger E-box-binding homeobox 2);
2) measuring the expression level of cardherin-11 protein and measuring the expression level of Sp1 (specificity protein 1) protein in the cell line expressing cardherin-11 protein; And
3) The test substance whose expression level of the cardherin-11 protein is decreased compared to the control group in which the test substance was not treated was selected, and then the test substance whose expression level of Sp1 protein was decreased compared to the control group not treated with the test substance, ;
Wherein the candidate substance for inhibiting metastasis of colon cancer is selected from the group consisting of:
delete 1) treating the test substance with a cardherine-11 protein composed of the amino acid sequence of SEQ ID NO: 1 and induced to be expressed by ZEB2;
2) measuring the activity of the cardiolin-11 protein and measuring the activity of the Sp1 protein; And
3) The test substance reduced in activity of the cardiolin-11 protein as compared to the control group not treated with the test substance was selected, and then the test substance decreased in activity level of the Sp1 protein compared to the control group in which the test substance was not treated, ;
Wherein the candidate substance for inhibiting metastasis of colon cancer is selected from the group consisting of:
delete 1) measuring the expression or activity level of cardioline-11 protein from cells or tissues separated from the subject and measuring the expression or activity level of Sp1 protein; And
2) determining that the test subject whose expression or activity level of cardiolin-11 and Sp1 protein is higher than that of the normal control group is considered to have a high risk of colon cancer metastasis;
Wherein the method comprises the steps of: (a) determining the level of expression or activity of a protein; delete

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