KR20070027659A - A breast cancer related protein, a gene encoding the same, and a method for diagnosing a breast cancer using the protein and gene - Google Patents

Abstract

A polynucleotide for diagnosing or treating breast cancer is provided to effectively diagnose the breast cancer by measuring the expression degree of BCRP(breast cancer related protein) gene. The polynucleotide or a complimentary polynucleotide thereof for diagnosing or treating breast cancer comprises at least 10 consecutive nucleotides derived from a polynucleotide having a nucleotide sequence described as SEQ ID : NO. 3 and is characterized in that the length is 10-100 nucleotides.

Description

Breast cancer related protein, a gene encoding the same, and a method for diagnosing a breast cancer using the protein and gene}

Figure 1 shows the results of northern blotting using a breast cancer related protein (BCRP) gene specific probe for a number of normal tissues.

2 shows the results of Northern blotting using BCRP gene specific probes for various cancer tissues.

3 to 5 show the expression of BCRP in the cells by observing FITC fluorescence for colon cancer cell lines Clone A (CA), primary cultured normal kidney cells, and HEK 293 cell lines transfected with BCRP-pFLAG vector DNA, respectively. It shows the result of looking at the position.

Figure 6 shows the expression level of BCRP in CA cell line transfected with BCRP gene, and shows the result of confirming the expression level of various genes related to apoptosis by RT-PCR when BCRP is overexpressed.

7 shows the results of evaluating the effect of BCRP overexpression on cell proliferation in HEK 293, CA, and CX-1 (A, B, and C) cell lines through cell proliferation assay (MTT assay).

8 to 10 shows apoptosis assay to see the effect of BCRP transfection overexpression on cell lines, HEK 293, CA and CX transfected with BCRP-pFLAG vector DNA, respectively. The results of FACS analysis on -1 are shown.

11 and 12 show RNAs isolated from breast cancer tissues and normal tissues of different breast cancer patients, and RT-PCR using oligonucleotides of SEQ ID NOs: 4 and 5 as primers shows the results of BCRP expression differences. .

FIG. 13 shows the results of Northern blotting analysis on RNA of breast tumor tissues of three different breast cancer patients. FIG.

14 is a diagram showing changes in cell morphology when MDA-MB-231 cell line is treated with Taxol, an anticancer agent, to induce cell death.

FIG. 15 is a diagram showing the result of confirming the effect of taxol treatment on the expression of BCRP gene using RT-PCR using RNA extracted from cells in FIG. 14.

The present invention relates to a microarray in which a BCRP protein having activity to induce cell death, a gene encoding the same, and a fragment of the BCRP gene are immobilized. The present invention also relates to a method for diagnosing breast cancer using an antibody that specifically recognizes a BCRP protein, and a method for diagnosing breast cancer by determining whether BCRP gene is expressed.

Breast cancer is the most commonly diagnosed female cancer in the world. Breast cancer is second only to lungs in terms of the number of cancer-related deaths. The incidence of breast cancer has increased steadily over the last 50 years, especially in Korea. A number of established factors can increase the risk of breast cancer in women, including factors such as aging, past breast cancer history, significant radiation exposure, strong breast cancer family history, socioeconomic upstream classes, babies, menopause, menopause, or ages older than 30 years. Early pregnancy.

Breast cancer is a heterogeneous disease, in which several breast tumors are known to be induced by female hormones, but there are many other recognized and unknown factors. Changes in oncogenes identified include amplification of the HER-2 and epidermal growth factor receptor genes and overexpression of cyclin D1. Overexpression of these oncozin is associated with a fairly weak prognosis. Similarly, genetic changes or loss of tumor suppressor genes, such as the p53 gene, are well documented in breast cancer and also associated with a weaker prognosis. The researchers looked for two genes called BRCA1 and BRCA2, predictors of premenopausal familial breast cancer. Currently, genetic risk assessments are available that can enhance the identification of candidates for chemoprevention testing. Early diagnosis of breast cancer is essential to ensure the best treatment results. Many countries with advanced healthcare systems have implemented screening programs for breast cancer. Information related to treatment choice and prognosis is also included, such as measuring the status of estrogen and progestrone receptors.

Major challenges in breast cancer treatment include improving early detection rates, finding new non-invasive markers that can be used to track disease progression and identify recurrences, and especially in more advanced diseases, where the 5-year survival rate is still very low. Finding a less toxic and improved treatment for There is a need to find more specific targets for cancer cells, ideally expressed on the surface of tumor cells, so that they can attack tumor cells by promising new methods such as immunotherapy and targeted toxins.

Accordingly, the present inventors came to complete the present invention by discovering a breast cancer specific expression protein while studying breast cancer specific SNP.

Accordingly, it is an object of the present invention to provide an isolated protein that is specifically expressed in breast cancer cells.

The present invention also provides a microarray in which a nucleic acid sequence encoding the protein and a nucleic acid sequence encoding the protein or a fragment thereof are immobilized.

The present invention also provides a method for diagnosing breast cancer using an antibody that specifically binds to the protein.

The present invention also provides a method for diagnosing breast cancer by determining whether the gene is expressed in breast tissue.

The present invention provides an isolated protein having an amino acid sequence of SEQ ID NO: 4, and having the activity of inducing apoptosis specifically expressed in breast cancer. The protein of the present invention (hereinafter also referred to as BCRP (breast cancer related protein) protein) is a membrane protein, and has a characteristic of being specifically expressed in heart tissue in normal tissue. In addition, the protein of the present invention has a characteristic of being specifically expressed in breast cancer tissues among various cancer tissues. Therefore, the presence or absence of the expression of the protein of the present invention can detect the presence of breast cancer.

In the present invention, the protein may increase the expression of one or more proteins selected from the group consisting of p53 and p21 in the cell, preferably when overexpressed.

The present invention also provides a method for detecting the presence of breast cancer, comprising reacting an anti-BCRP antibody having an amino acid sequence of SEQ ID NO: 4 with a polypeptide sample derived from human breast tissue. Provide a method. Preferably, when the amount of expression of the protein is greater than the amount of expression in normal tissue, preferably, at least 3%, 5%, 10% or 15% or more is determined to be breast cancer. Methods for producing antibodies to specific protein antigens are well known in the art, and the present invention can also produce anti-BCRP antibodies by such conventional antibody preparation methods.

The present invention also provides a polynucleotide having an amino acid sequence of SEQ ID NO: 4 and characterized by encoding an isolated protein having an activity of inducing apoptosis. Preferably, the polynucleotide is a polynucleotide having a nucleotide sequence of SEQ ID NO: 3. The polynucleotide of the present invention can be used not only to express the protein, but also for the purpose of determining the presence or absence of breast cancer by examining the expression of the protein.

Accordingly, the present invention also provides a polynucleotide for diagnosis or treatment of breast cancer or a complementary polynucleotide thereof comprising at least 10 consecutive nucleotides derived from a polynucleotide having the nucleotide sequence of SEQ ID NO: 3. Preferably, the polynucleotide is a polynucleotide, characterized in that 10 to 100 nucleotides in length, more preferably 10 to 50 nucleotides in length. Such polynucleotides can be used as polynucleotides that are primers or probes.

The present invention is also characterized by having a substrate on which a polynucleotide for diagnosis or treatment of breast cancer or a complementary polynucleotide thereof is immobilized, comprising 10 or more consecutive nucleotides derived from a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 It provides a microarray for diagnosing breast cancer. Preferably, the polynucleotide is characterized by having a length of 10 to 100 nucleotides, more preferably 10 to 50 nucleotides, but is not limited thereto.

The invention also provides a kit for diagnosing or treating breast cancer comprising at least 10 consecutive nucleotides derived from a polynucleotide having the nucleotide sequence of SEQ ID NO: 3.

Moreover, the present invention comprises the steps of obtaining a nucleic acid sample derived from breast tissue from a sample; And

Determining the degree of expression of the protein having the amino acid sequence of SEQ ID NO: 4 in the breast tissue sample, and determining the presence of breast cancer therefrom, provides a method for diagnosing breast cancer.

In the method of the present invention, the amount of expression of the protein can be measured using various methods known in the art. In the method of the present invention, the presence or absence of BCRP gene expression can be determined by Northern blotting using a BCRP gene specific probe at the mRNA level. In addition, the presence or absence of the expression of the BCRP gene may be determined by extracting the entire RNA, performing RT-PCR using a BCRP gene specific primer, and confirming the obtained product. However, the method of determining the presence or absence of expression is not limited to these examples. A nucleic acid sample derived from breast tissue does not necessarily mean a purely purified nucleic acid sample, and in some cases, a nucleic acid that can be used for analysis may exist depending on an analysis method. For example, when PCR is to be used to confirm the expression of the BCRP gene, a sample obtained by crushing cells without separating nucleic acids may be used as it is.

In the method of the present invention, when the amount of expression of the protein is higher than the amount expressed in normal breast tissue cells, it can be determined as breast cancer.

We searched for nucleic acids or proteins that are specifically expressed in breast cancer cells against several nucleic acid sequences selected from commercially available nucleic acid or protein databases. As a result, one nucleic acid or a protein, BCRP nucleic acid or protein which is found to be specifically expressed in breast cancer cells was selected. Next, the BCRP full-length gene was searched and sequenced from the cDNA library, and Northern analysis confirmed that this gene was specifically expressed in breast cancer cell lines and breast cancer cells of patients (FIGS. 12 and 13). Repeated measurements confirmed that the breast cancer cells are expressed about 2 times higher than normal cells (FIG. 13). In addition, the position expressed in the cells of the protein encoded by the BCRP gene was confirmed using the immunocytochemistry method (immunocytochemistry method). As one method, the gene of the protein was recombined into a pFLAG vector, the resulting recombinant vector was transfected into an animal cell line, and its expression was confirmed using the pFLAG fluorescence system (FIGS. 3, 4, and 5). . In addition, in order to confirm the function of the BCRP protein, it was overexpressed in the cell, and the effect of the BCRP protein on the cell was investigated. To this end, the gene encoding the BCRP protein was transfected into an animal cell line, and whole RNA was extracted therefrom and RT-PCR was performed using the template. By RT-PCR, the expression level of the BCRP gene and other genes related to cell death, such as p53 and p21, were monitored (FIG. 6). We also confirmed the structure of the promoter of the BCRP gene.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example 1 Search for BCRP Gene Based on SNP Data

1. Exploration of the BCRP Gene

For SNPs that have been found to be associated with breast cancer in a separate study, the database (NCBI) search and analysis can be used to find the nucleotide sequence of the site where the SNP exists and to make primers to amplify the gene. Was obtained.

2. Amplification of BCRP Gene Fragments

Using the obtained base sequence information, a primer for amplifying the DNA around the searched SNP was designed (SEQ ID NOs: 1 and 2).

Next, genomic DNA was used as a template, and PCR was performed using the primer set as a primer to amplify a BCRP gene fragment of 239 bp. The PCR conditions used were 10 pmol each of the forward and reverse primers, and 200-1 μg of the genomic DNA template. The reaction was carried out for 40 seconds at 95 ° C., 40 seconds at 57 ° C., and 1 minute at 72 ° C. Were repeated 35 times each. The result was confirmed by 1% agarose gel electrophoresis, it was confirmed that the expected DNA fragment of 239 bp was amplified. This was used as a probe for full-length BCRP gene search.

3. Screening of BCRP Full-length Genes through cDNA Library

The cDNA library used was the human fetal brain cDNA library (λtriplrEx library) (Clontech Corp.) and the search was in accordance with the manufacturer's experimental instructions (PT3003-1). The search process is briefly described as follows.

PCR was performed to obtain mRNA, and cDNA library search was performed using the PCR product obtained as a probe. As the cells used for the search, the commonly used E. coli XL-1 blue cells were used.

First, as a result of confirming the titer of the library, both sets were 2.0 × 10 ⁹ pfu / ml. Next, the cDNA library was plated on a plate of E. coli XL-1 blue. In general, the coating was carried out at 2 to 5 x 10 mPafu / 150 mm. Next, the lambda phage was transferred to a nylon membrane with a positive charge. Filter hybridization reaction was performed using a probe labeled by random primed DNA labeling with radioactive isotope labeled dCTP ([α- ³² P] dCTP, 3000 Ci / mmol) And detected. The result was a positive clone.

4. Sequencing and Protein Sequence Prediction of BCRP Full-length Gene

The base sequence of the BCRP full-length gene was confirmed by analyzing the nucleotide sequence of the BCRP gene sequence obtained from the obtained clone using a sequence automatic analyzer (ABI 3700). In addition, the putative protein sequence of the gene was confirmed using NCBI and the program (new GENSCANW web program). The nucleotide sequence of the BCRP gene is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO: 4. As a result, it can be seen that the SNP used for the search for BCRP is located in the promoter region, and the BCRP genomic gene spans from 16032 to 965546, and is composed of three introns and four exons.

Example 2 Confirmation of Intracellular and Tissue Expression of BCRP Gene

1. Confirmation of Intracellular Expression of BCRP Gene by Northern Blotting

Using the PCR product obtained in Example 1 as a probe, Northern blotting analysis was performed on a plurality of human normal tissues and tumor tissues (Clontech corp.). The results are shown in FIGS. 1 and 2. As shown in Figures 1 and 2, it was confirmed that the BCRP gene is specifically expressed only in heart tissues in normal tissues, and specifically expressed in breast cancer tissues in tumor tissues. From these results, it can be seen that by examining the expression of the BCRP gene of the present invention, it can be used to detect the presence of breast cancer. In FIG. 1, the tissues used for northern blotting were brain heart, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and terminal blood leukocyte cells, FIG. 2 The cancer tissue used for Northern blotting was breast cancer, ovarian cancer, uterine cancer, lung cancer, kidney cancer, gastric cancer, colon cancer and rectal cancer tissue (using multiple tissue northern blot manufactured by clontech). Figures 1.00 kb and 1.2 kb in the upper part of Figs. 1 and 2 indicate the size markers, and the blotting result was 1.37 kb.

The specific process of the northern blotting was performed as follows.

 (1) Preparation of radiolabeled probe

Probes were constructed using random primed DNA labeling (Roche Corp. Random primed DNA labeling kit, # 1004760). About 25 ng of the BCRP PCR product was purified and isotope [α- ³² p] dCTP, 250 μCi (BMS Corp.) was used.

 (2) prehybridization using a hybridization bottle

7 ml of ExpressHyb solution (# 8015-1, BD Clontech Corp.) was preheated at 68 ° C. and the nylon membrane was prehybridized at 68 ° C. for 30 minutes.

 (3) denaturation of radiolabeled probes

The radiolabeled probe was boiled at 95 ° C.-100 ° C. for 8-10 minutes and then quickly placed on ice.

 (4) hybridization

The radioactive probe was mixed in 10 ml fresh ExpressHyb solution. Then, the prehybridization solution was discarded in a hybridization bottle containing nylon membrane and placed in a fresh ExpressHyb solution mixed with the radiation probe, followed by incubation with shaking at 68 ° C. for about 1 hour.

 (5) washing

Washing solution 1 was washed for 30-40 minutes at room temperature. Next, wash again with washing solution 2 at 50 ° C. for 40 minutes. Next, the nylon membrane was removed from the bottle, removed to dryness, and placed in a plastic wrap.

 (6) It was put in an X-ray film, exposed at -70 ° C, and taken out after 1-2 days to check the band.

2. Confirmation of Tissue Expression of BCRP Gene

The expression of the BCRP gene in the lesional and nonlesional parts of breast cancer patients was confirmed.

11 and 12 show RNAs isolated from cells derived from breast cancer tissues of different breast cancer patients and RT-PCR using oligonucleotides of SEQ ID NOs: 4 and 5 as primers. As shown in FIGS. 11 and 12, the expression of the BCRP gene was increased compared to normal tissue, and the expression of p53, which was previously found to be related to cancer, selected as a comparative gene, was markedly increased.

FIG. 13 is a diagram showing a result of Northern blotting analysis on RNA of breast normal tissue and tumor tissue. FIG. As shown in Figure 13, it can be seen that the expression of the BCRP gene (about 1.8kb) is increased. In Figure 13 lanes 1 to 3 are the results for other tumor donors, lane 4 is the result for normal breast tissue as a control. The average value of the expression levels in lanes 1 to 3 was calculated and compared with the expression levels in normal tissues. As a result, BCRP gene was expressed about 2.05 times higher in breast cancer cells than in normal cells.

Example 3 Identification of Intracellular Expression Location of BCRP Protein

In this example, the position of BCRP gene is expressed in the cell through the immunocytochemistry method. For this purpose, BCRP gene was first cloned into pFLAG vector (Sigma, Amherst, NY). The cloning process is as follows. BCRP gene of SEQ ID NO: 3 was digested with Not I enzyme and Sal I enzyme, and pFLAG vector was also digested with the same enzyme as BCRP gene and ligated. Cloned BCRP-pFLAG vector DNA was transfected into several cell lines using Lipofection 2000. Transfected cells were incubated at 5% CO ₂ , 37 ° C. for 48 hours to allow BCRP gene to be expressed, and then fixed on plates with 3.5% paraformaldehyde. Next, the cells were made permeable with 0.1% Triton X-100 to allow staining of the parts inside the cells. The margin was blocked using 1% BSA blocking solution. Flag specific antibodies were reacted to the plates in which the cells were immobilized to allow Flag specific antibodies (anti-FLAG M2) to specifically bind to BCRP protein-Flag. Finally, a FITC conjugated secondary antibody (anti-mouse IgG-FITC) was reacted with the BCRP protein-Flag-1 antibody complex. The result was confirmed the position where BCRP protein is expressed through fluorescence generated from FITC using a fluorescence microscope. For comparison, the cell lines to which nothing was transfected were experimented with the above method using Flag specific antibodies.

The results are shown in FIGS. 3 to 5. 3 to 5 show the results of observing FITC fluorescence for colon cancer cell line Clone A (CA), primary cultured normal kidney cells, and HEK 293 cell line transfected with BCRP-pFLAG vector DNA, respectively. As shown in Figures 3 to 5, BCRP in all three cell lines were observed in the cell membrane.

From these experimental results, it is believed that the BCRP protein of the present invention is specifically expressed in the cell membrane.

Example 4 Effect of BCRP Gene Expression on Gene Expression in Cell Lines

In this example, the effect of BCRP gene expression on the gene expression of the cell line was confirmed. For this, first, Northern blot analysis to determine the presence of mRNA of BCRP gene. Next, we examined the effect of BCRP gene expression on the expression of genes related to cancer and autologous death in cells. First, the BCRP-pFLAG vector DNA prepared above in a CA (Colon A colon cancer) cell line was transfected using Lipofection 2000, incubated for 48 hours at 5% CO ₂ , 37 ° C, and then RNA was extracted by RT. -PCR was performed. β-actin was used as a control. As primers, primer sets capable of amplifying the BCRP gene, β-actin, p53, p21, CytC, caspase 5, caspase 3 and Apaf 1 genes were used (Table 1), respectively. By monitoring the amount of PCR product obtained as a result of RT-PCR, the expression level of each gene was confirmed.

Table 1. Primer sequences used for amplification of each gene

gene Primer sequence BCRP F: SEQ ID NO: 5 R: sequence number 6  β-actin F: SEQ ID NO: 7 R: sequence number 8 p53 F: SEQ ID NO: 9 R: sequence number 10 p21 F: SEQ ID NO: 11 R: SEQ ID NO: 12 CytC F: SEQ ID NO: 13 R: SEQ ID NO: 14 Caspase 5 F: SEQ ID NO: 15 R: sequence number 16 Caspase 3 F: SEQ ID NO: 17 R: sequence number 18 Apaf 1 F: SEQ ID NO: 19 R: SEQ ID NO: 20

The RT-PCR process is as follows. Whole RNA extracted from the cell line overexpressed BCRP gene is reverse transcribed using Superecript II reverse transcriptase (invitrogen) to obtain cDNA. PCR was performed using Taq polymerase as a template of the obtained cDNA 1-200 μg with 10 pmol of forward and reverse primers (see Table 1), respectively. Genomic DNA used as a template was carried out PCR using a CA cell line transfected with BCRP-pFLAG vector DNA and cDNA synthesized from all RNAs extracted from control cells as a template. In this case, the PCR conditions used were repeated 40 times at 95 ° C, 40 seconds at different annealing temperatures depending on the amplification target gene, and 30 minutes at 72 ° C for 1 minute. The annealing temperature was annealed at 58 ° C. for beta-actin, caspase 5 and BCRP, 52 ° C. for p53, p21, Cyt C, caspase 3 and Apaf1. The results were confirmed by 1% agarose gel electrophoresis, the results are shown in FIG. As shown in FIG. 6, expression of p53 was increased and expression of p21, which is known to be a potential mediator of p53-dependent growth arrest, was increased. However, no difference in expression was found for the other genes.

Example 5 Effect of BCRP Gene Overexpression on Cell Line Proliferation and Cell Death

1. MTT analysis

MTT analysis confirmed the effects of BCRP gene overexpression on cell line proliferation and cell death. The MTT assay is a method of measuring the absorbance of formazan produced by the reduction of MTT by living intracellular mitochondrial dehydrogenase, and the measured absorbance reflects the concentration of live and metabolically active cells. The cell lines used for analysis were Clone A and CX-1 (colon cancer cell line), and HEK 293, a normal kidney cell line. As in Example 4, pFLAG-BCRP vector DNA was transfected using Lipofection 2000, and as a control, a vector control transfected with pFLAG only, and none of the transfected but notching controls (stressed) that received the same stress ( nothing control, and a blank control without cells.

The experimental results were obtained from two replicates. There was less cell proliferation in the CA and CX-1 cell lines than in the control group. That is, cell growth was inhibited compared to the control. In addition, even in the HEK 293 cell line, relatively less cell proliferation occurred compared to two other controls, as in colon cancer cell lines (CA and CX-1). The results are shown in FIG. FIG. 7 shows the results of evaluating the effect of BCRP overexpression on cell line proliferation in HEK 293, CA, and CX-1 (A, B, and C, respectively) cell lines.

2. Apoptosis Analysis

In this assay, HEK 293, CA, and CX-1 cell lines were used to investigate the effect of BCRP gene expression on cell death using flowcytometry. As a control, a vector control in which only the pFLAG vector was transfected was used.

As a result of two repeated analyzes, both CA and CX-1 showed weak cell autologous cell death compared to control cells, but HEK 293 showed little or no cell death. The analysis results are shown in FIGS. 8 to 10. 8 to 10 show FACS analysis results for HEK 293, CA and CX-1, respectively.

Example 6 Effect of Apoptosis on Expression of BCRP Gene in Human Breast Cancer Cells

Conventionally, when the anticancer taxol is treated to human breast cancer cells, it is known that cell self-induction is induced. In the present embodiment, when cell self death occurs, in order to examine the expression level of the BCRP gene, the MDA-MB-231 cell line is treated with taxol to induce cell death, and the expression level of the BCRP gene is as described above. It was confirmed by performing RT-PCR.

Fig. 14 shows changes in cell morphology when taxol is treated in the MDA-MB-231 cell line. As shown in FIG. 14B, it can be seen that autologous cell death occurred by Taxol treatment.

15 is a diagram showing the results of confirming the effect of taxol treatment on the expression of BCRP gene using RT-PCR. As shown in Figure 15, it can be seen that the expression of the BCRP gene is increased by the treatment of taxol.

From the above results, it was found that the cell proliferation is inhibited by the BCRP gene of the present invention, the cell autologous is not strong, but weakly induced compared to the control. In addition, the results of BCRP expression in taxol-treated cell lines, which are indirect evidence, suggest that BCRP is involved in cell death. In addition, as confirmed in Example 4, it was found that the expression of p53 and p21 is significantly increased by the BCRP gene. Taken together, these results suggest that the expression level of p53 is increased by overexpression of BCRP gene, and the expression level of p21 is increased by activation of p53.

According to the isolated protein and the nucleic acid encoding the same according to the present invention, it can be usefully used for the diagnosis of breast cancer and the development of a medicament targeting the protein.

According to the method for diagnosing breast cancer using an antibody specifically binding to the BCRP protein of the present invention, breast cancer can be diagnosed effectively.

In addition, according to the method of the present invention for diagnosing breast cancer by measuring the expression level of BCRP gene in cells, breast cancer can be effectively diagnosed.

The microarray of the present invention can be usefully used in various analytical methods, such as the assay for detecting the presence of breast cancer.

<110> Samsung Electronics Co. LTD. <120> A breast cancer cell related protein, a gene encoding the same,          and a method for diagnosing a breast cancer using the protein and          gene <130> PN060423 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 acggacgagg gtgacaatag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 aggtaaaaga agggcatggg 20 <210> 3 <211> 672 <212> DNA <213> Homo sapiens <400> 3 atgaggctcc aaagaccccg acaggccccg gcgggtggga ggcgcgcgcc ccggggcggg 60 cggggctccc cctaccggcc agacccgggg agaggcgcgc ggaggctgcg aaggttccag 120 aagggcgggg agggggcgcc gcgcgctgac cctccctggg caccgctggg gacgatggcg 180 ctgctcgcct tgctgctggt cgtggcccta ccgcgggtgt ggacagacgc caacctgact 240 gcgagacaac gagatccaga ggactcccag cgaacggacg agggtgacaa tagagtgtgg 300 tgtcatgttt gtgagagaga aaacactttc gagtgccaga acccaaggag gtgcaaatgg 360 acagagccat actgcgttat agcggccgtg aaaatatttc cacgtttttt catggttgcg 420 aagcagtgct ccgctggttg tgcagcgatg gagagaccca agccagagga gaagcggttt 480 ctcctggaag agcccatgcc cttcttttac ctcaagtgtt gtaaaattcg ctactgcaat 540 ttagaggggc cacctatcaa ctcatcagtg ttcaaagaat atgctgggag catgggtgag 600 agctgtggtg ggctgtggct ggccatcctc ctgctgctgg cctccattgc agccggcctc 660 agcctgtctt ga 672 <210> 4 <211> 223 <212> PRT <213> Homo sapiens <400> 4 Met Arg Leu Gln Arg Pro Arg Gln Ala Pro Ala Gly Gly Arg Arg Ala   1 5 10 15 Pro Arg Gly Gly Arg Gly Ser Pro Tyr Arg Pro Asp Pro Gly Arg Gly              20 25 30 Ala Arg Arg Leu Arg Arg Phe Gln Lys Gly Gly Glu Gly Ala Pro Arg          35 40 45 Ala Asp Pro Pro Trp Ala Pro Leu Gly Thr Met Ala Leu Leu Ala Leu      50 55 60 Leu Leu Val Val Ala Leu Pro Arg Val Trp Thr Asp Ala Asn Leu Thr  65 70 75 80 Ala Arg Gln Arg Asp Pro Glu Asp Ser Gln Arg Thr Asp Glu Gly Asp                  85 90 95 Asn Arg Val Trp Cys His Val Cys Glu Arg Glu Asn Thr Phe Glu Cys             100 105 110 Gln Asn Pro Arg Arg Cys Lys Trp Thr Glu Pro Tyr Cys Val Ile Ala         115 120 125 Ala Val Lys Ile Phe Pro Arg Phe Phe Met Val Ala Lys Gln Cys Ser     130 135 140 Ala Gly Cys Ala Ala Met Glu Arg Pro Lys Pro Glu Glu Lys Arg Phe 145 150 155 160 Leu Leu Glu Glu Pro Met Pro Phe Phe Tyr Leu Lys Cys Cys Lys Ile                 165 170 175 Arg Tyr Cys Asn Leu Glu Gly Pro Pro Ile Asn Ser Ser Val Phe Lys             180 185 190 Glu Tyr Ala Gly Ser Met Gly Glu Ser Cys Gly Gly Leu Trp Leu Ala         195 200 205 Ile Leu Leu Leu Leu Ala Ser Ile Ala Ala Gly Leu Ser Leu Ser     210 215 220 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer of BCRP amplification <400> 5 cggacgaggg tgacaatag 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for BCRP amplification <400> 6 aggtaaaaga agggcatggg 20 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for beta-actin amplification <400> 7 aggactttga ttgcacattg ttgttt 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for beta actin amplification <400> 8 gagaccaaaa gccttcatac atctca 26 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p53 amplification <400> 9 atttgcgtgt ggagtatttg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p53 amplification <400> 10 ggaacaagaa gtggagaatg 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p21 amplification <400> 11 gtgagcgatg gaacttcgac tt 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p21 amplification <400> 12 ggcgtttgga gtggtagaaa tc 22 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for CytC amplification <400> 13 tttggatcca atgggtgatg ttgag 25 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for CytC amplification <400> 14 tttgaattcc tcattagtag cttttttgag 30 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for caspase 5 amplification <400> 15 ctgacattga aggaagagg 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for caspase 5 amplification <400> 16 gccaggtgat caaactttg 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for caspase 3 amplification <400> 17 tggaattgat gcgtgatgtt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for caspase 2 amplification <400> 18 ggcaggcctg aataatgaaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Apaf 1 amplification <400> 19 gggtttcagt tgggaaacaa 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Apaf 1 amplification <400> 20 cacccaagag tcccaaacat 20  

Claims (2)

A polynucleotide for diagnosis or treatment of breast cancer or a complementary polynucleotide thereof, comprising 10 or more consecutive nucleotides derived from a polynucleotide having a nucleotide sequence of SEQ ID NO: 3. The polynucleotide of claim 1, wherein the polynucleotide is 10 to 100 nucleotides in length.

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