KR101821455B1 - Biomarker composition for diagnosing cancer metastasis comprising AWP1 - Google Patents

Abstract

The present invention relates to a biomarker composition for the diagnosis of cancer metastasis comprising AWP1, and it can be used as a clinical indicator for cancer metastasis by confirming that the expression of AWP1 is reduced in a cancer which is more easily metastasized than a non-metastatic cancer. In addition, it was confirmed that the inhibition of AWP1 expression by siRNA increased cancer growth and metastasis, and it was confirmed that when overexpression of AWP1 inhibited cancer metastasis by reducing cancer growth and migration, AWP1 could be used as a cancer metastasis inhibitor have.

Description

[0001] The present invention relates to a biomarker composition for diagnosing cancer metastasis comprising AWP1,

The present invention relates to a biomarker composition for cancer metastasis diagnosis comprising AWP1.

Cells, the smallest units that make up the human body, divide by intracellular regulatory functions when normal, and maintain cell balance while growing, dying, and disappearing. When a cell is damaged for some reason, it is treated and regenerated to serve as a normal cell, but if it does not recover, it will die by itself. However, cancer is defined as a condition in which abnormal cells that do not control these proliferation and inhibition for many reasons are not only excessively proliferating but also invade surrounding tissues and organs, resulting in mass formation and normal tissue destruction. Cancer is a cell proliferation that can not be inhibited, and it destroys normal cell and organ structure and function, so its diagnosis and treatment are very important.

AWP1 is known to be a regulatory factor involved in NF-kB signaling by binding to tumor necrosis factor-associated factor 2 (TRAF2). It is reported that the NF-kB signal is an important signal transduction pathway in the mechanism of metastasis, and the regulatory factors of various NF-kB signals affect the metastasis of cancer cells.

Japanese Unexamined Patent Application Publication No. 2005-170796 (June 30, 2005)

The present invention relates to a biomarker composition for cancer metastasis diagnosis comprising an AWP1 gene or a protein encoded by the gene, a method for diagnosing cancer metastasis using the same, a pharmaceutical composition for preventing or treating cancer metastasis comprising an AWP1 protein expression promoter or activator as an active ingredient And a screening method for cancer metastasis through the measurement of AWP1 protein expression level.

In order to solve the above problems, the present invention provides a biomarker composition for cancer metastasis diagnosis comprising an AWP1 gene or a protein encoded by the gene.

The present invention also provides a cancer metastasis diagnostic kit comprising a primer or a probe specifically binding to an AWP1 gene, an antibody, a peptide, an aptamer or a compound specifically binding to a protein encoded by the gene.

In addition, the present invention provides a method for providing information necessary for cancer metastasis diagnosis by measuring the expression level of AWP1 protein.

The present invention also provides a pharmaceutical composition for preventing or treating cancer metastasis comprising an AWP1 protein, an AWP1 protein expression promoter or an activator as an active ingredient.

In addition, the present invention provides a screening method for cancer metastasis through measurement of the expression level of AWP1 protein.

The present invention relates to a biomarker composition for the diagnosis of cancer metastasis comprising AWP1, and it can be used as a clinical indicator for cancer metastasis by confirming that the expression of AWP1 is reduced in a cancer which is more easily metastasized than a non-metastatic cancer. In addition, it was confirmed that the inhibition of AWP1 expression by siRNA increased cancer growth and metastasis, and it was confirmed that when overexpression of AWP1 inhibited cancer metastasis by reducing cancer growth and migration, AWP1 could be used as a cancer metastasis inhibitor have.

FIG. 1 shows the level of expression of AWP1 in mouse breast cancer cell line E0771 and the change of cancer cells after knockdown of AWP1.
FIG. 2 shows the expression levels of AWP1, proliferation assay and wound healing assay in human breast cancer cell lines.
FIG. 3 shows the change of cancer cells after knockdown of AWP1 in MCF7 cell line in which AWP1 expression is relatively high.
FIG. 4 shows the result of overexpression of AWP1 in the MDA-MB-231 cell line in which the expression of AWP1 is relatively small and the effect thereof is confirmed again.
Figure 5 shows miRNA screening results that regulate expression at the AWP1 protein level in breast cancer cell lines.
6 shows the results of expression of AWP1, cell proliferation and cell migration in the prostate cancer cell line.
7 shows the effect of AWP1 overexpression in epithelial mesenchymal transition (EMT) in PC3 cells.
8 shows the knockdown effect of AWP1 in epithelial mesemchymal transition (EMT) in PC3 cells.
FIG. 9 shows the knockdown effect of AWP1 in epithelial mesemchymal transition (EMT) in DU-145 cells.

The present invention provides a biomarker composition for cancer metastasis diagnosis comprising an AWP1 gene or a protein encoded by the gene. Preferably, the cancer may be breast cancer or prostate cancer, but is not limited thereto.

The "AWP1 gene or protein encoded by the gene" of the present invention can be obtained by NCBI accession no. NM019006. ≪ / RTI >

The "AWP1" of the present invention may also be named "ZFAND6 (Zinc Finger, AN1-type Domain 6) ".

The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).

The present invention also provides a cancer metastasis diagnostic kit comprising a primer or a probe specifically binding to an AWP1 gene, an antibody, a peptide, an aptamer or a compound specifically binding to a protein encoded by the gene. Preferably, the cancer may be breast cancer or prostate cancer, but is not limited thereto.

As used herein, the term "primer" refers to a short nucleic acid that can form base pairs with a complementary template with a nucleic acid sequence having a short free 3 'hydroxyl group and serves as a starting point for template strand replication It refers to the sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

As used herein, the term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to an mRNA. The presence or absence of a specific mRNA, You can check the amount. The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.

As used herein, the term "antibody" means a specific immunoglobulin as indicated in the art and directed against an antigenic site. An antibody in the present invention means an antibody that specifically binds to AWP1 of the present invention, and the antibody can be produced according to a conventional method in the art. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody.

In addition, the kit of the present invention comprises an antibody specifically binding to a marker component, a secondary antibody conjugate conjugated with a label that develops upon reaction with the substrate, a coloring substrate solution to be colored with the label, A reaction stop solution, and the like, and may be manufactured from a number of separate packaging or compartments including the reagent components used.

As used herein, the term "peptide" has a high binding capacity to the target material and does not cause denaturation during thermal / chemical treatment. Also, because of its small size, it can be used as a fusion protein by attaching it to other proteins. It can be used as a diagnostic kit and a drug delivery material because it can be specifically attached to a polymer protein chain.

The term "aptamer " as used herein refers to a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid having a stable tertiary structure by itself and having a characteristic capable of binding with high affinity and specificity to a target molecule ). ≪ / RTI > As described above, since the aptamer is composed of a polynucleotide which is capable of specifically binding to an antigenic substance like the antibody and is more stable than the protein, has a simple structure, and is easy to synthesize, .

(1) measuring the mRNA expression level of the AWP1 gene or the expression level of the protein encoded by the gene from the cancer patient sample; (2) comparing the mRNA expression level of the AWP1 gene or the expression level of the protein encoded by the gene with a control sample; And (3) determining that the mRNA expression level of the AWP1 gene or the expression level of the protein encoded by the gene is lower than that of the control sample, thereby determining that cancer metastasis is high. do. Preferably, the cancer may be breast cancer or prostate cancer, but is not limited thereto.

In detail, the method for measuring the mRNA expression level may be RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) ), Northern blotting and DNA chips, but are not limited thereto.

In detail, the method for measuring the protein expression level may be Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, But are not limited to, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, Complement Fixation Assays, FACS and protein chips.

As used herein, the term "patient sample" refers to a biological marker such as a tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine which is different from the control group in the expression level of the AWP1 gene or protein But are not limited to, samples.

The present invention also provides a pharmaceutical composition for preventing or treating cancer metastasis comprising an AWP1 protein, an AWP1 protein expression promoter or an activator as an active ingredient. Preferably, the cancer may be breast cancer or prostate cancer, but is not limited thereto.

Specifically, the AWP1 protein expression promoter or activator may be a compound, a peptide, an aptamer, or an antibody that specifically binds to an AWP1 protein, but is not limited thereto.

Specifically, the AWP1 protein can be regulated by miRNA 181b, but is not limited thereto.

The pharmaceutical composition of the present invention may contain a chemical substance, a nucleotide, an antisense, an siRNA oligonucleotide and a natural product extract as an active ingredient. The pharmaceutical composition or combination preparation of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , A lubricant or a flavoring agent may be used. The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile solutions suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Pharmaceutical dosage forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, suspending agents or injectable solutions or suspensions . The pharmaceutical compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, ≪ / RTI > The effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, once to several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg when administered once to several times a day, In the case of protein or antibody, 0.1 ng / kg to 10 g / kg, antisense nucleotide, siRNA, shRNAi or miRNA can be administered at a dose of 0.01 ng / kg to 10 g / kg.

(1) contacting a test substance with a cancer cell; (2) measuring the expression or activity level of the AWP1 protein in cancer cells in contact with the test substance; And (3) selecting a test substance having increased expression or activity level of the AWP1 protein as compared with a control sample.

The term "test substance" used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening in order to examine whether it affects the expression amount of a gene or affects the expression or activity of a protein. do. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Total RNA extraction, cDNA synthesis, RT- PCR  And quantitative RT- PCR

Total RNA was extracted from cells using Qiazol reagent (Qiagen, Hilden, Germany) and reverse transcribed with 1-2 μg of RNA using RevertiAid First strand cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania). PCR was performed with Biorad thermal cycler. The sequences of the primers are as follows: AWP1 5'-CCA AGT GCC TAT GCT TTG TTC CA-3 '(sense) (SEQ ID NO: 1) and 5'- ACA GAG GTT GCA GGT GGG CTT AT-3' SEQ ID NO: 2); Vimentin 5'-CCG CAC ATT CGA GCA AAG AC-3 '(sense) (SEQ ID NO: 3) and 5'-CCT GCT GTC CCG CCG-3' (SEQ ID NO: 4); SMA 5'-CTT GTC CAG GAG TTC CGC TC-3 '(sense) (SEQ ID NO: 5) and 5'-ACG CTG GAG GAC TTG CTT TT-3' (SEQ ID NO: 6); (SEQ ID NO: 7) and 5'-CAT CGT CCA CCG GAC TCA AA-3 '(SEQ ID NO: 8); MMP9 5'-TCT ATG GTC CTC GCC CTG AA-3' 3 '(sense) (SEQ ID NO: 9) and 5'-GCC CAA TAC GAC CAA ATC C-3' (antisense) (SEQ ID NO: ).

Quantitative real-time RT-PCR (qRT-PCR) was performed using the Power SYBR Green 1-Step Kit and an ABI 7000 Real Time PCR System (Applied Biosystems, Carlsbad, CA, USA) Lt; / RTI &gt; The amplification protocol consisted of an initial reverse transcription step of 48 ° C for 30 minutes, denaturation step of 95 ° C for 15 seconds, 40 times and an extension and extension step of 60 ° C for 1 minute. Results were expressed as the ratio of target PCR products to GAPDH products. The values were normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) levels. Quantitation was performed using the ΔΔCT method (Applied Biosystems) with RQ manager 1.2 software. Data were expressed as means ± SD. All the samples were repeated three times for each experiment.

2. Gene silencing and transfection

Transfection of small interfering RNA (siRNA) targeting AWP1 was performed as previously reported. SMARTpool (ON-TARGET plus Human AWP1) of siRNA against AWP1, a combination of four siRNA oligonucleotide sequences selected, and negative control siRNA were purchased from Thermo Scientific Dharmacon (Lafayette, CO, USA). Cancer cells (eg, MDA-MB-231, MCF7, PC3, DU-145) were transfected with siRNA using transfection reagent RNAiMAX (Invitrogen, Carlsbad, Calif.). Silent efficiency was measured by RT-PCR. Cells were transfected with miRNAs using RNAiMAX and transfected with myc-tagged AWP1 over-expression vector by lipofectamine 2000 (Invitrogen, Carlsbad, Calif.).

3. Cell proliferation

Cells were inoculated at 96 plates for CCK-8 assay (Dojindo Molecular Technologies Inc, Japan) and then transfected with SiRNAs or myc-tagged AWP1 over-expressed constructs. The cultures were maintained at 37 [deg.] C for 24 or 48 hours. At the indicated times, cell growth was measured by CCK-8 colorimetric assay according to the manufacturer's instructions. All analyzes were repeated three or more times. 10 [mu] l cell counting kit-8 was added to each well and plates were incubated for an additional 1-4 hours at 37 [deg.] C. The absorbance of each fraction at 450 nm was measured using a microplate reader.

4. Cell migration scratch  analysis

Myc-tagged AWP1 transfected cancer cells were cultured in 48-well plates. Wounds were removed using a sterile 200 [mu] l pipette tip and the cells were incubated at 37 [deg.] C for an additional 24 hours. For AWP1 silencing experiments, cells were transfected with negative control or AWP1 siRNA and scraped off the cell layer. Cells were stained with CellTracker Green (CMFDA) (Invitrogen) according to the manufacturer's instructions and wound healing was measured using a fluorescence microscope (LSM700, Carl Zeiss). Percentage of filled wound area was measured using Adobe Photoshop 9.0.2 software and compared with baseline measurements. Wound healing was measured as a percentage of wound confluence compared to negative control. Each experiment was repeated three times.

5. Western Blat  analysis

Protein supernatants collected from the cells were boiled in 5 x SDS sample buffer and cell lysates were separated on 10-12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) . To exclude binding of unspecified proteins, the membrane was incubated for 1 hour with 5% I-block solution in Tris-buffered saline (20 mM Tris / HCl, pH 7.6, 150 mM NaCl, and 0.1% Triton X- After blocking, the reaction was carried out with a suitable primary antibody at 4 ° C overnight with gentle shaking. Immunoreactivity was detected using an enhanced chemiluminescence solution (Milli-pore, Billerica, MA) after HRP-conjugated secondary antibody was reacted at room temperature for 1 hour.

6. Animal studies

Six-week-old C57 / B6 was used for all xenotransplantation. For mammary-fat-pad tumor analysis, cells were harvested by try-oxidising, washed twice with PBS and counted. After that, the cells were resuspended in HBSS buffer (5 × 10 ⁵ cells / 50 μl). The mice were anesthetized, incised briefly to reveal the mammary gland, and 5 × 10 ⁵ cells were directly injected into the mammary-fat-pad. The incision was closed with the operating room (Silkam, BBRAUN, Rubi, Spain) and the primary tumor derivative was observed daily by measuring tumor length (L) and width (W). Tumor volume was calculated as length × width ² × 1/2.

& Lt; Example 1 > AWP1 through a controlled miR181b involved in breast cancer metastasis

The expression of AWP1 was observed in mouse breast cancer cell line E0771, and the change of cancer cells was observed by knockdown of AWP1 (FIG. 1A). It was confirmed that AWP1 knockdown did not affect the viability of E0771 cell line (Fig. 1B). In addition, E0771 knockdown of AWP1 was injected into a mammary fat pad to observe the progression and metastasis of cancer cells. Mice were observed for approximately 25 days after injection and the body weight and tumor size were measured. When comparing the AWP1 knockdown mouse group to the control group, there was no particular difference in body weight and tumor size (Fig. 1C and Fig. 1D). On the other hand, it was confirmed that mouse group in which knockdown of AWP1 in lung metastasis was significantly increased nodules formation as compared with control group (Fig. 1E). Therefore, it is thought that the expression of AWP1 is involved in cancer cell metastasis.

Then, we tried to observe whether it showed the same pattern in not only mouse but also human cancer cells. First, expression of AWP1 in human breast cancer cell line was confirmed by GEO profile. Expression of AWP1 was significantly inhibited in the MDA-MB-231 cell line as compared to MCF7 (Fig. 2A). As a result of Western blot analysis, the expression of AWP1 was less in the MDDA-MB-231 cell line than in the MCF7, as in the GEO profile (FIG. 2B). The proliferation assay revealed that MDA-MB-231 cells grow relatively faster than MCF7 (FIG. 2C), and wound healing assay also showed that MDA-MB-231 And the migration ability of the cells was also high (FIGS. 2D and 2E). Therefore, MDA-MB-231 cells with relatively low expression of AWP1 were found to proliferate or migrate faster.

AWP1 was knocked down on the MCF7 cell line with a relatively high expression of AWP1, and its effect was observed (Fig. 3A). The wound healing analysis showed that the AWP1 knockdown group was faster in wound healing than in the control group (FIG. 3B), and the statistical value was shown (FIG. 3C). It was observed that there was no effect on the viability of MCF7 cell line by AWP1 knockdown (FIG. 3D). Therefore, it was confirmed that cancer cell migration could be increased by knockdown of AWP1.

Overexpression of AWP1 in the MDA-MB-231 cell line, in which AWP1 expression was relatively low, was reaffirmed. After overexpression of AWP1, it was observed that AWP1 mRNA was increased by RT-PCR (Fig. 4A), and the protein level was also increased by Western blot (Fig. 4B). It was observed that AWP1 over-expression did not affect survival in the MDA-MB-231 cell line (Fig. 4C). On the other hand, over-expression of AWP1 reduced wound closure (Fig. 4D and Fig. 4E), confirming that cancer metastasis or EMT related markers were reduced (Fig. 4F). Thus, it was confirmed that overexpression of AWP1 could inhibit cancer cell migration and cancer metastasis or EMT-related markers.

Based on the above results, it was confirmed how the expression of AWP1 was inhibited. The expression level of AWP1 mRNA was significantly different from that of breast cancer cell line. Therefore, it was assumed that the expression level of AWP1 was regulated at the translational level. In order to prove this, AWP1 was targeted and miRNA which was highly expressed in MDA-MB-231 compared to MCF7 was selected (Fig. 5A). The luciferase activity was measured by cloning the portion of the AWP1 3'UTR that the miRNA was expected to target into a luciferase construct (Fig. 5B). Five miRNA mimics and inhibition tests, which are expected to target AWP1, have shown that miR181b most specifically targets AWP1.

Thus, the present invention has revealed that the protein level of AWP1 is important for cancer metastasis, and the protein level of AWP1 is regulated by miRNA 181b.

< Example  2> in prostate cancer cells On AWP1  Controlled by Transition of mesenchymal cells in epithelium (Epithelial-mesenchymal transition)

Similar to metastatic breast cancer, prostate cancer cells also showed that AWP1 has cancer-related functions.

Expression of AWP1 in human prostate cancer cell line was confirmed by GEO profile. It was confirmed that the expression of AWP1 was significantly inhibited in PC3 cell line compared to DU-145 (Fig. 6A). As a result of RT-PCR and Western blot analysis, AWP1 expression was less in the PC3 cell line than in the DU-145, as in the GEO profile (FIGS. 6B and 6C). Proliferation assay showed that PC3 cells grow relatively faster than DU-145 (Fig. 6D). Also, wound healing assay showed that PC3 cell migration ability compared to DU-145 (migration ability) was also high (Fig. 6E and Fig. 6F). Therefore, PC3 cells with relatively low expression of AWP1 were found to proliferate or migrate faster.

Overexpression of AWP1 in the PC3 cell line, in which AWP1 expression was relatively low, was reaffirmed. It was observed that AWP1 mRNA was increased by RT-PCR after overexpression of AWP1 (Fig. 7A). It was observed that AWP1 overexpression did not affect viability in PC3 cell lines (Fig. 7B). On the other hand, over-expression of AWP1 reduced the wound closure (Fig. 7C and Fig. 7D), and the epithelial mesenchymal transition (EMT), one of the important features of cancer metastasis Changes in the relevant markers (Vimentin, SMA, MMP9) were observed and they showed a decrease in expression due to AWP1 overexpression (Fig. 7E and Fig. 7F). Thus, it was confirmed that overexpression of AWP1 could inhibit cancer cell migration and EMT-related markers.

In the case of PC3, expression of endogenous AWP1 was small, but its function was also confirmed when it was almost inhibited. AWP1 was knocked down on the PC3 cell line to completely eliminate AWP1 expression and its effect was observed (Fig. 8A). AWP1 knockdown in the PC3 cell line had no effect on viability (Fig. 8B). Wound healing assay showed that wound healing in the AWP1 knockdown group was relatively fast (FIG. 8C) and statistical values were displayed (FIG. 8D). In order to confirm the epithelial mesemchymal transition (EMT) in epithelium, changes of related markers (Vimentin, SMA, MMP9) were observed and they showed an increase with AWP1 knockdown (FIGS. 8E and 8F) . Therefore, it was confirmed that cancer cell migration and EMT markers were increased by knockdown of AWP1.

Finally, AWP1 was knocked down to a DU-145 cell line with a relatively high expression of AWP1, and its effect was observed (FIG. 9A). It was observed that AWP1 knockdown had no effect on the viability of the DU-145 cell line (Fig. 9B). Wound healing assay showed faster wound closure of the AWP1 knockdown group (FIG. 9C) compared to the control group (FIG. 9C), and the statistical value was displayed (FIG. 9D). In addition, epithelial mesenchymal transition (EMT) -related markers (Vimentin, SMA, MMP9), one of the important features of cancer metastasis, have been shown to increase due to AWP1 knockdown (Fig. 9E). Therefore, it was confirmed that endogenous AWP1 expression pattern was involved in cancer cell migration and metastasis.

Having described the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> University of Ulsan Foundation for Industry Cooperation <120> Biomarker composition for diagnosing cancer comprising cancer metastasis          AWP1 <130> ADP-2015-0237 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ccaagtgcct atgctttgtt cca 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acagaggttg caggtgggct tat 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccgcacattc gagcaaagac 20 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cctgctgtcc cgccg 15 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cttgtccagg agttccgctc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acgctggagg acttgctttt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tctatggtcc tcgccctgaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 catcgtccac cggactcaaa 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agccacatcg ctcagaca 18 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcccaatacg accaaatcc 19

Claims (12)

A biomarker composition for predicting breast cancer metastasis or predicting metastasis risk comprising an AWP1 gene or a protein encoded by the gene. delete A primer or a probe specifically binding to an AWP1 gene, an antibody, a peptide, an aptamer or a compound specifically binding to a protein encoded by the gene, or a kit for predicting breast cancer metastasis diagnosis or metastasis risk. delete (1) measuring the mRNA expression level of the AWP1 gene or the expression level of the protein encoded by the gene from a breast cancer patient sample;
(2) comparing the mRNA expression level of the AWP1 gene or the expression level of the protein encoded by the gene with a control sample; And
(3) If the mRNA expression level of the AWP1 gene or the expression level of the protein encoded by the gene is lower than that of the control sample, it is determined that the metastatic potential of the breast cancer is high, thereby providing information necessary for diagnosis of breast cancer metastasis or prediction of metastasis risk. How to. delete delete delete delete delete (1) contacting a test substance to a breast cancer cell;
(2) measuring the level of expression or activity of an AWP1 protein in a breast cancer cell in contact with the test substance; And
(3) selecting a test substance having increased expression or activity level of the AWP1 protein as compared with a control sample. delete

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