Novel Epithelial-Mesenchymal Transition Markers for Pancreatic Cancer

Abstract

To clarify the mechanism of pancreatic cancer malignancy of BACH1, RNA sequencing was used to reveal the gene expression changes caused by the expression level of BACH1 in pancreatic cancer cell lines. Furthermore, in vitro migration ability, invasion ability, and in vivo orthotopic transplantation experiments were conducted, and the results showed that BACH1 can be used as an excellent biomarker for epithelial-mesenchymal transition of pancreatic cancer alone or in combination with its downstream regulator FOXA1.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the epithelial mesenchymal transition (EMT) ability of pancreatic cancer, a method for evaluating the metastasis or invasion ability of pancreatic cancer, a method for predicting the prognosis of pancreatic cancer, a pancreatic cancer metastasis inhibitor, and a method for screening pancreatic cancer metastasis inhibitors. Background Art

[0002] Pancreatic cancer primarily develops in the pancreatic duct epithelium, and with a five-year survival rate of less than 10% after diagnosis, it has the worst prognosis among cancers. Surgical resection is the only treatment option that promises a cure, but currently, early detection is difficult, 80% of cases are inoperable at diagnosis, and chemotherapy is ineffective due to their high malignancy, leading to a lack of effective diagnostic and treatment methods.

[0003] Our understanding of the pathogenesis of pancreatic cancer continues to deepen. Beginning in the 1950s, a multi-stage carcinogenesis hypothesis was gradually deduced based on pathological observations of lesions. Recent advances in genomic mutation analysis have revealed the accumulation of mutant genes that correlate with the carcinogenic process of pancreatic cancer. This multi-stage carcinogenesis hypothesis, in which the accumulation of mutant genes leads to cancer, is now widely accepted. Among this carcinogenic process, mutations in the kirsten rat sarcoma viral oncogene (KRAS) are known to be observed early in precancerous lesions, with KRAS mutations found in over 90% of pancreatic cancers. Generally speaking, in response to external stimulation of receptor-type tyrosine kinase receptors, KRAS binds to guanosine triphosphate (GTP) and becomes activated, promoting the RAS (Rat Sarcoma Viral Oncogene Homolog) / RAF (Rapidly Accelerated Fibrosarcoma) / MEK (Mitogen-Activated Protein Kinase) / ERK (Extracellular signal regulated kinase) signal transduction pathway to trigger cellular responses such as cell proliferation, apoptosis, and differentiation. In addition, its activity is inactivated when GTP is hydrolyzed to guanosine diphosphate (GDP). However, KRAS gene mutations commonly found in cancer often block the hydrolysis of GTP, maintaining an activated state and promoting the proliferation and malignancy of cancer cells. Except for pancreatic cancer, approximately 30% of all human cancers contain this activating RAS mutation. Therefore, efforts have been made to date to study inhibitors targeting RAS, such as competitive inhibitors of RAS and GTP. However, due to the high concentrations of GTP and GDP in cells, sufficient effects have not been achieved, and none of these have yet been clinically applied.

[0004] Therefore, while further elucidation of the mechanisms of pancreatic cancer malignancy is crucial, specific gene mutations responsible for metastasis and recurrence have yet to be identified. Furthermore, the influence of cancer cell self-replication, epithelial-mesenchymal transition, and the surrounding cancer environment on gene expression fluctuations and signal transduction changes in primary tumors during the acquisition of metastatic capacity, and thus on factors promoting metastasis, remains largely unknown.

[0005] Under normal conditions, BACH1 (BTB and CNC homology 1, BTB-CNC homology 1) inhibits the expression of genes such as heme oxygenase-1 (HO-1), which reduces oxidative stress. Under oxidative stress, BACH1 is expelled from the nucleus, thereby inhibiting the increase of reactive oxygen species (ROS), etc., and helps maintain cell homeostasis (Non-Patent Document 1). In addition, BACH1 forms a complex with the cancer suppressor transcription factor p53, thereby inhibiting the expression of some of its target genes and inhibiting cell aging (Non-Patent Document 2). In an experiment in which mouse embryonic fibroblasts (MEFs) into which mutant Ras had been introduced were transplanted subcutaneously into mice, the formation and proliferation of tumors in Bach1-deficient MEFs were significantly inhibited compared to wild-type MEFs (Non-Patent Document 3). Regarding the relationship between BACH1 and cancer, there are reports that in colorectal cancer and malignant melanoma with BRAF mutations, BACH1 promotes CpG island methylation, inhibiting the expression of cancer suppressor genes (Non-Patent Document 4). Furthermore, there are reports that in breast cancer, BACH1 regulates the expression of multiple genes associated with metastasis, thereby promoting bone metastasis (Non-Patent Document 5).

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent literature 1: Nat Rev Immunol, 2017.17(7):p.437-450

[0009] Non-patent document 2: Nat Struct Mol Biol, 2008.15(12):p.1246-54

[0010] Non-patent literature 3: Oncogene, 2013.32(27):p.3231-45

[0011] Non-patent document 4: Proc Natl Acad Sci USA, 2016.113(5):p.1250-5

[0012] Non-patent literature 5: J Biol Chem, 2012. 287(40): p.33533-44 Summary of the Invention

[0013] Problems to be solved by the invention

[0014] The present invention aims to provide a novel pancreatic cancer epithelial-mesenchymal transition marker, a pancreatic cancer metastasis or infiltration marker, a pancreatic cancer prognosis marker, a pancreatic cancer metastasis inhibitor, and a method for screening a pancreatic cancer metastasis inhibitor.

[0015] Solutions for solving problems

[0016] The present inventors focused on BACH1 and, using the Cancer Genome Atlas (TCGA) database, studied the relationship between BACH1 expression and pancreatic cancer prognosis. Their findings indicate that pancreatic cancers with high BACH1 expression have a significantly poorer prognosis. These findings suggest that BACH1 contributes to the malignant progression of pancreatic cancer. Furthermore, immunostaining of BACH1 in human pancreatic cancer tissue samples also demonstrated a worse prognosis in patients with high BACH1 expression.

[0017] Therefore, in this study, to clarify the pancreatic cancer malignancy mechanism of BACH1, RNA sequencing was used to reveal the changes in gene expression caused by the expression of BACH1 in pancreatic cancer cell lines. Focusing on epithelial-mesenchymal transition, in vitro migration and invasion abilities as well as in vivo orthotopic transplantation experiments were conducted. In addition, it was shown that BACH1 can be used as an excellent biomarker for epithelial-mesenchymal transition of pancreatic cancer, an excellent biomarker for metastasis or invasion of pancreatic cancer, and an excellent biomarker for prognosis prediction of pancreatic cancer by combining with its downstream regulatory factors.

[0018] Furthermore, it was shown that pancreatic cancer metastasis can be suppressed by regulating the expression of metastasis-related genes induced by BACH1, and a method for screening drugs that can suppress pancreatic cancer metastasis was also shown.

[0019] The present invention has been completed based on the above-mentioned research results.

[0020] That is, the present invention has the following technical solutions.

[0021] [1] A method (data acquisition method) for evaluating the epithelial-mesenchymal transition ability of pancreatic cancer in vitro, wherein the method includes a step of measuring the expression level of BACH1.

[0022] [2] The method for evaluating the epithelial-mesenchymal transition ability of pancreatic cancer in vitro (data acquisition method) according to [1], wherein the method further comprises a step of measuring the expression level of FOXA1.

[0023] [3] A method (data acquisition method) for evaluating the metastatic or invasive ability of pancreatic cancer in vitro, wherein the method includes a step of measuring the expression level of BACH1.

[0024] [4] The method for evaluating the metastatic or invasive ability of pancreatic cancer in vitro (data acquisition method) according to [3], further comprising the step of measuring the expression level of FOXA1.

[0025] [5] A method for predicting the prognosis of pancreatic cancer in vitro (data acquisition method), wherein the method includes a step of measuring the expression level of BACH1.

[0026] [6] The method for predicting the prognosis of pancreatic cancer in vitro (data acquisition method) according to [5], wherein the method further comprises a step of measuring the expression level of FOXA1.

[0027] [7] A method for screening pancreatic cancer metastasis inhibitors, comprising: adding a drug candidate substance to cells expressing the BACH1 gene or a reporter gene linked to the promoter of the BACH1 gene; measuring the expression level of the BACH1 gene or the reporter gene; and selecting a substance that reduces the expression level.

[0028] [8] A pancreatic cancer metastasis inhibitor comprising as an active ingredient an agent that reduces the expression of the BACH1 gene.

[0029] [9] The pancreatic cancer metastasis inhibitor according to [8], wherein the pancreatic cancer metastasis inhibitor inhibits the epithelial-mesenchymal transition ability of pancreatic cancer cells.

[0030]

[10] The pancreatic cancer metastasis inhibitor according to [8] or [9], wherein the agent is a small interfering RNA (siRNA) targeting the BACH1 gene.

[0031] Effects of the Invention

[0032] According to the present invention, data for evaluating the epithelial-mesenchymal transition ability of pancreatic cancer, data for evaluating the metastatic or invasive ability of pancreatic cancer, and data for predicting the prognosis of pancreatic cancer can be obtained, enabling diagnosis of the prognosis of pancreatic cancer. Furthermore, pancreatic cancer metastasis inhibitors and methods for screening pancreatic cancer metastasis inhibitors can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Indicates that BACH1 is a prognostic predictor for pancreatic cancer. Figure 1In Figure A, RNA sequence data from 176 pancreatic ductal cancer cases with clinical information obtained from the TCGA database were divided into two groups based on the cutoff value obtained from the ROC curve (Receiver operating characteristic curve) based on the expression level of BACH1 (left figure), and the Kaplan Meier method (KM method) was used to analyze the overall survival period between the two divided groups (right figure). Figure 1 In Figure B, 166 pancreatic cancer clinical test samples were subjected to immunohistochemistry using anti-human BACH1 antibodies and divided into three groups based on the staining ratio (weak positive (weak): positive rate less than 10%; moderate positive (moderate): positive rate 10% to 50%; strong positive (strong): positive rate more than 50%). Representative staining images in this classification are shown (magnification, 200 times). It should be noted that in the subsequent analysis, weak positive and moderate positive will be classified as BACH1 low expression group, and strong positive will be classified as BACH1 high expression group. Figure 1 In Figure C, overall survival was analyzed using the product limit method between the BACH1 high and low expression groups as determined by immunohistochemistry. P values ​​were calculated using the log-rank test.

[0034] Figure 2 It indicates that BACH1 does not affect the proliferation of pancreatic cancer cells in vitro. Figure 2 Panel A shows the expression levels of BACH1 in pancreatic cancer cell lines (AsPC1 (human metastatic pancreatic adenocarcinoma cells) and SW1990 (human pancreatic cancer cells)) with BACH1 knockdown (siBACH1) and in control cells (siCont.), as detected by quantitative reverse transcription PCR (RT-qPCR). Each experiment was performed at least three times. In the bar graph, each experimental value is shown as a dot, the mean as a column, and the standard error as a bar. Statistically significant differences were tested using the Student's t test, and P values ​​are shown (**: P < 0.01). Figure 2In Figure B, cell counts were measured over time every 24 hours for BACH1 knockdown (siBACH1) cells, AsPC1 and SW1990, and their control group (siCont.) cells, up to 72 hours. Changes in cell proliferation are plotted in a line graph. The experiment was performed four independent times, and the mean is plotted as a line graph, with the standard error at each time point shown as a bar. Statistically significant differences were tested using a Student's t-test (ns: not significant).

[0035] Figure 3 Indicates that BACH1 promotes mesenchymal traits in pancreatic cancer cells. Figure 3 In A, the cell morphology of BACH1 knockdown (siBACH1) AsPC1 is shown in a microscope image. Figure 3 B shows a microscopic image of the cell morphology of a pancreatic cancer cell line (Panc1) overexpressing BACH1. The scale bar represents 100 μm. Figure 3 C shows the expression of epithelial genes (CDH1 (Cadherin 1, Cadherin 1), OCLN (Occludin, tight junction protein) and FOXA1 (Forkhead Box A1)) and mesenchymal genes (VIM (Vimentin, Vimentin) and SNAI2 (Snail Family Transcriptional Repressor 2, Snail Family Transcriptional Repressor 2)) in BACH1 knockdown (siBACH1) AsPC1 and SW1990, BACH1 overexpression (exBACH1) Panc1, and their control cells (siCont. or EV (EmptyVector, blank vector)). Each gene was detected by RT-qPCR. The mRNA expression level of each factor was corrected for the mRNA expression level of β-actin (β-actin, ACTB). The experiment was performed independently three times, and the mean value of each experiment is shown in the column and the standard error is shown in the bar. Statistically significant differences were tested by Student's t-test, and P values ​​are shown (*: P<0.05; **: P<0.01). Figure 3D shows the protein blot (photo) of E-cadherin, BACH1, and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) or ACTB as the internal control protein in BACH1 knockdown (siBACH1) AsPC1 and SW1990, BACH1 overexpression (exBACH1) Panc1, and their control cells (siCont. or EV) (photo). Figure 3 E shows immunofluorescence staining of E-cadherin upon BACH1 knockdown. Scale bar indicates 25 μm.

[0036] Figure 4 It shows that BACH1 enhances the migration and invasion ability of pancreatic cancer cell lines in vitro. Figure 4 A shows representative microscopic images and a bar graph of the measured cell motility in a scratch assay of BACH1 knockdown (siBACH1) AsPC1 and its control cell (siCont.) Figure 4 B shows representative microscope images and a bar graph of the measured cell mobility in a scratch assay of Panc1-overexpressing (exBACH1) and control cells (EV). Figure 4 Figure C shows representative microscopic images and bar graphs of the number of migrated or infiltrated cells from the transwell assay and invasion assay for BACH1 knockdown of AsPC1 and SW1990. Each experiment was performed independently at least three times. In the bar graphs, each experimental value is represented by a dot, the mean is represented by a column, and the standard error is represented by a bar. Statistically significant differences were tested using the Student's t-test, and P values ​​are shown (*: P < 0.05; **: P < 0.01).

[0037] Figure 5 A diagram showing the establishment of a BACH1 knockout AsPC1 cell line. Figure 5 Figure A shows the DNA sequence results at the target sites of clones (sgBACH1-1 and sgBACH1-2) created based on two guide RNAs (gRNAs). The two guide RNAs target sequences within exon 2 of the BACH1 gene. sgBACH1-1 is designed to introduce a 13-base deletion, while sgBACH1-2 is designed to introduce a single-base insertion. The reference sequence used is the human reference genome hg19. Figure 5B shows Western blots of BACH1 knockout (sgBACH1) AsPC1 and its control cells (sgCont.) for BACH1, E-cadherin, and GAPDH as an internal control protein (photograph). Figure 5 Panel C shows the RT-qPCR results for BACH1 knockout of AsPC1. The mRNA expression levels of each factor were normalized to the mRNA expression level of ACTB. The experiment was performed independently three times. The figure shows each experimental value as a dot, the mean as a column, and the standard error as a bar. Statistically significant differences were tested using the Student's t-test, and P values ​​are shown (*: P < 0.05; **: P < 0.01, ns: not significant). Figure 5 D shows an immunofluorescence staining image of BACH1 in which AsPC1 was knocked out using guide RNAsgBACH1-2. Figure 5 E shows immunofluorescence staining images of E-cadherin. Scale bars indicate 7.5 μm and 75 μm, respectively.

[0038] Figure 6 This indicates that although BACH1 promotes metastasis in vitro, it does not affect tumor formation. Figure 6 Figure A shows representative examples of pancreatic tumor formation, liver metastasis, and mesenteric spread 5 weeks after transplantation of BACH1 knockout (sgBACH1-2) AsPC1 or control (sgCont.) cells into the pancreas of NOG mice (BACH1 wild-type AsPC1: n=17; BACH1 knockout AsPC1: n=18). The area encircled by white lines represents pancreatic tumor formation, and white arrows indicate metastatic foci. Figure 6 Figure B shows a box-and-whisker plot of the weight of the removed pancreas, the number of liver metastases, and the number of peritoneal metastases. The lower end of the box represents the 25th percentile of the data, and the upper end represents the 75th percentile. The central line represents the median, and the whiskers indicate the maximum and minimum values, respectively. Statistically significant differences were tested using the Student's t-test, and P values ​​were calculated.

[0039] Figure 7 This indicates that BACH1 becomes a more accurate prognostic predictive marker for pancreatic cancer by additional functional evaluation. Figure 7 In Figure A, the RNA sequence data of 176 test samples obtained from the TCGA database were divided into two groups according to the cutoff value obtained by the ROC curve based on the expression level of FOXA1. Figure 7In Figure B, RNA sequencing data from 176 samples obtained from the TCGA database were divided into four groups based on cutoff values ​​derived from ROC curves for BACH1 and FOXA1 expression. Overall survival was analyzed between the divided groups using the product limit method. Statistically significant differences were tested using the log-rank test, and P values ​​are shown. DETAILED DESCRIPTION

[0040] <Method for evaluating epithelial-mesenchymal transition ability in pancreatic cancer>

[0041] The present invention provides a method (data acquisition method) for evaluating the epithelial-mesenchymal transition ability of pancreatic cancer in vitro, wherein the method includes the step of measuring the expression level of BACH1.

[0042] The BACH1 gene may be any gene derived from a test subject, and specific examples thereof include genes containing the base sequences shown below.

[0043] When the test subject is a human, the base sequence of the BACH1 gene can be exemplified by the base sequence described in SEQ ID NO: 1. Furthermore, as long as the BACH1 gene encodes a protein that functions as a transcription factor, it can include DNA having a complementary sequence to the base sequence described in SEQ ID NO: 1 and DNA that hybridizes under stringent conditions. Stringent conditions include, for example, washing in 0.1× SDS (Sodium Dodecyl Sulphate), 0.1× SSC (Standard Sodium Citrate), and 68°C. Furthermore, when a different animal is used as the test subject, the homologous gene derived from that animal is used for measurement.

[0044] The BACH1 protein is not limited as long as it is derived from the subject, and specific examples include proteins containing the amino acid sequence shown below. BACH1 protein also includes protein fragments, analogs, and mutants that have the same pancreatic cancer association as the protein.

[0045] When the test subject is a human, the amino acid sequence of the BACH1 protein can be exemplified by the amino acid sequence set forth in SEQ ID NO: 2. Furthermore, as long as the protein functions as a transcription factor, a protein having an amino acid sequence in which one or several (e.g., 1 to 20) amino acids are substituted, deleted, inserted, or added to these amino acid sequences may also be used. Furthermore, when the test subject is a different animal, a homologous protein derived from that animal is used as the test subject.

[0046] The present invention may further include a step of measuring the expression level of an epithelial cell marker.

[0047] Examples of epithelial cell markers include FOXA1 (Forkhead Box Protein A1), OCLN (Occludin), PKP2 (Plakophilin 2), CLDN3 (Claudin 3), and CLDN4 (Claudin 4). The epithelial cell marker is preferably FOXA1.

[0048] This step may be a step of measuring the expression level of one epithelial cell marker selected from the group consisting of these epithelial cell markers, or a step of measuring the expression levels of two or more epithelial cell markers.

[0049] When the test subject is a human, the base sequence of FOXA1 can be exemplified by the base sequence described in SEQ ID NO: 3. Furthermore, the FOXA1 gene may include DNA having a complementary sequence to the base sequence described in SEQ ID NO: 3 and DNA that hybridizes under stringent conditions, as long as it encodes a protein that functions as a transcription factor. Stringent conditions include, for example, washing in 0.1×SDS, 0.1×SSC, and 68°C. Furthermore, when the test subject is a different animal, the homologous gene derived from that animal is used for measurement.

[0050] When the test subject is a human, the amino acid sequence of FOXA1 can be exemplified by the amino acid sequence set forth in SEQ ID NO: 4. Furthermore, as long as the protein functions as a transcription factor, a protein having an amino acid sequence in which one or several (e.g., 1 to 20) amino acids are substituted, deleted, inserted, or added to these amino acid sequences may also be used. Furthermore, when the test subject is a different animal, a homologous protein derived from that animal is used as the measurement target.

[0051] The measurement of the expression levels of BACH1 and FOXA1 is not particularly limited, and may include, for example, measurement of the expression levels of their mRNA or proteins.

[0052] The expression levels of the BACH1 and FOXA1 genes can be measured by, for example, quantitative PCR, RT-PCR, quantitative RT-PCR, microarray, high-throughput sequencing, Northern blotting, spectrophotometry, and fluorometry. The expression levels of the BACH1 and FOXA1 proteins can be measured by, for example, flow cytometric analysis, Western blotting, ELISA (Enzyme Linked Immunosorbent Assay), and other immunochemical methods.

[0053] Primers, probes, and the like for gene expression analysis can be designed or obtained using the base sequence of the coding region of the BACH1 gene or FOXA1 gene, i.e., the sequence described in SEQ ID NO: 1 or SEQ ID NO: 3. The base sequence may have an identity of 90% or greater, preferably 95% or greater, and more preferably 98% or greater, to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, respectively. Furthermore, the primers, probes, and the like may have an identity of 90% or greater, preferably 95% or greater, and more preferably 98% or greater, to the complementary sequence of the base sequence, as long as they are capable of specifically binding to the base sequence.

[0054] The quantitative PCR method can be performed using conventional methods without limitation, and can be performed using a method using an intercalator or a method using a fluorescently labeled probe. For example, SYBR (registered trademark) Green I can be used as an intercalator.

[0055] Antibodies used to measure the expression level of BACH1 protein or FOXA1 protein can be commercially available antibodies or antibodies prepared using a portion of the amino acid sequence of the BACH1 protein or FOXA1 protein described in SEQ ID NO: 2 or SEQ ID NO: 4 as an antigen. The amino acid sequence may be one that has 90% or greater, preferably 95% or greater, and more preferably 98% or greater homology to the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4, respectively. Furthermore, as long as the antibody specifically binds to the described amino acid sequence, it may be one that has 90% or greater, preferably 95% or greater, and more preferably 98% or greater homology to the complementary sequence of the amino acid sequence.

[0056] The antibody may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred for stable supply of antibodies of stable quality. It may also be a fragment of a monoclonal antibody such as Fab' or F(ab')2.

[0057] In addition, antibodies can be commonly used, but are not limited to, antibodies derived from mouse, rat, rabbit, goat, sheep, or bird. Any antibody can be used as long as it specifically binds to the BACH1 protein or FOXA1 protein. The antibody may be labeled, or a labeled secondary antibody may be used.

[0058] When using a labeled secondary antibody, any antibody can be used without particular limitation as long as it recognizes the primary antibody. For example, when the primary antibody is a rabbit antibody, a labeled anti-rabbit IgG antibody can be used as the secondary antibody, and when the primary antibody is a mouse antibody, a labeled anti-mouse IgG antibody can be used as the secondary antibody.

[0059] Examples of labeling substances include enzymes, radioactive isotopes, fluorescent substances, luminescent substances, and gold sol.

[0060] Among these, enzymes are preferred from the perspectives of sensitivity and ease of operation, with peroxidase (PO), horseradish peroxidase (HRP), alkaline phosphatase (AP), and glucose oxidase (GOD) being more preferred. When HRP is used as the labeling substance, a substrate such as TMB (3,3',5,5'-tetramethylbenzidine) can be used. When AP is used, a substrate such as AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane disodium salt) and 9-(4-chlorophenylthiophosphoryloxymethylene)-10-methylacridine disodium salt can be used. Alternatively, fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine can be used as labeling substances.

[0061] The detection and quantitative method of the assay object substance will be different according to different labeling methods, and can be carried out using customary methods well known to those skilled in the art, without particular limitation. For example, when PO, HRP, AP, GOD, etc. are used as labeling substances, the change of absorbance and luminous intensity can be measured by adding a chromogenic matrix or a luminescent matrix, so that the assay object substance is quantitatively measured. In addition, when a fluorescent substance is used as a labeling substance, the assay object substance can be quantitatively measured by measuring its fluorescence intensity. In addition, when a radioisotope is used as a labeling substance, the assay object substance can be quantitatively measured by measuring radioactivity. In addition, when a gold sol is used as a labeling substance, the assay object substance can be quantitatively measured by measuring absorbance.

[0062] When performing quantification, for example, a calibration curve (standard curve) can be prepared in advance using a sample of known concentration, and the expression level of BACH1 protein or FOXA1 protein in the sample can be calculated by comparing the measured value with the calibration curve.

[0063] The concentration of the antibody solution is not particularly limited as long as it does not inhibit the antigen-antibody reaction and is not too low relative to the expression level of the BACH1 protein or FOXA1 protein in the test sample.

[0064] The present invention is not particularly limited to the time for the antigen-antibody reaction, the temperature for carrying out the method of the present invention, the composition and pH of the sample and reagent dilution and the washing solution, and conditions applicable to conventional protein quantification methods can be employed.

[0065] The test subject is not particularly limited as long as it is a mammal that expresses BACH1, but is preferably a human.

[0066] The sample to be measured is, for example, a biological sample obtained from a subject or a healthy subject.

[0067] Biological samples can include, for example, pancreatic cells or tissues, cells or tissues surrounding the pancreas, and cells or tissues in any other locations to which pancreatic cancer may metastasize. Furthermore, biological samples can include body fluids such as blood and lymph that may contain pancreatic cancer cells, or cancer cells circulating in the blood. Circulating cancer cells in the blood are not particularly limited; for example, they can be collected by applying magnetic beads encoded with antibodies that recognize pancreatic cancer to the blood and utilizing positive magnetic selection.

[0068] Epithelial-mesenchymal transition (EMT) refers to the process by which epithelial cells lose their cell-cell adhesion ability and become mesenchymal cells with migration ability.

[0069] For epithelial-mesenchymal transition ability, for example, it can be evaluated by the following method. A receiver operating characteristic (ROC) curve is prepared using clinical data of pancreatic cancer clinical test samples obtained from the database of The Cancer Genome Atlas (TCGA), and a cutoff value of BACH1 expression is calculated by the ROC curve. When the expression level of BACH1 in the biological sample obtained from the subject is greater than the cutoff value, it can be evaluated that the epithelial-mesenchymal transition ability of pancreatic cancer is high, and when it is below the cutoff value, it can be evaluated that the epithelial-mesenchymal transition ability of pancreatic cancer is low.

[0070] Furthermore, the epithelial-mesenchymal transition ability of pancreatic cancer can be more accurately evaluated by combining the expression level of FOXA1 as an epithelial cell marker. Similarly to BACH1, the clinical data of pancreatic cancer clinical detection samples obtained from the TCGA database were used to make a receiver operating characteristic (ROC) curve, and the cutoff value of the expression level of FOXA1 was calculated by the ROC curve. Then, when the expression level of FOXA1 in the biological sample obtained by the subject is greater than the cutoff value, and the expression level of BACH1 is below the cutoff value, it can be evaluated that the epithelial-mesenchymal transition ability of pancreatic cancer is low. On the other hand, when the expression level of FOXA1 in the biological sample obtained by the subject is below the cutoff value, and the expression level of BACH1 is greater than the cutoff value, it can be evaluated that the epithelial-mesenchymal transition ability of pancreatic cancer is high.

[0071] <Methods for evaluating the metastatic or invasive ability of pancreatic cancer>

[0072] The present invention provides a method (data acquisition method) for evaluating the metastatic or invasive ability of pancreatic cancer in vitro, wherein the method includes the step of measuring the expression level of BACH1.

[0073] The metastatic or invasive ability of pancreatic cancer can be evaluated, for example, by using the following method: a receiver operating characteristic (ROC) curve is created using clinical data of pancreatic cancer clinical test samples obtained from the Cancer Genome Atlas (TCGA) database, and a cutoff value for the expression level of BACH1 is calculated using the ROC curve. If the expression level of BACH1 in a biological sample obtained from the subject is greater than the cutoff value, the metastatic or invasive ability of pancreatic cancer can be evaluated as high; if the expression level is below the cutoff value, the metastatic or invasive ability of pancreatic cancer can be evaluated as low.

[0074] Furthermore, the metastatic ability or infiltration ability of pancreatic cancer can be more accurately evaluated by combining the expression level of FOXA1 as an epithelial cell marker. Similarly to BACH1, a receiver operating characteristic (ROC) curve was prepared using the clinical data of pancreatic cancer clinical detection samples obtained from the TCGA database, and the cutoff value of the expression level of FOXA1 was calculated by the ROC curve. Then, when the expression level of FOXA1 in the biological sample obtained by the subject is greater than the cutoff value, and the expression level of BACH1 is below the cutoff value, it can be evaluated that the metastatic ability or infiltration ability of pancreatic cancer is low. On the other hand, when the expression level of FOXA1 in the biological sample obtained by the subject is below the cutoff value, and the expression level of BACH1 is greater than the cutoff value, it can be evaluated that the metastatic ability or infiltration ability of pancreatic cancer is high.

[0075] <Methods for predicting the prognosis of pancreatic cancer>

[0076] The present invention provides a method (data acquisition method) for predicting the prognosis of pancreatic cancer in vitro, wherein the method includes the step of measuring the expression level of BACH1.

[0077] In the present invention, prognosis generally refers to the future outlook for a disease. In prognostic prediction, for example, pancreatic cancer remission or improvement, long-term survival, or a long period without progression is considered a good prognosis. On the other hand, for example, pancreatic cancer recurrence, resurgence, or metastasis, short-term survival, or a short period without progression is considered a poor prognosis.

[0078] Specifically, for example, a receiver operating characteristic (ROC) curve is created using clinical data of pancreatic cancer clinical test samples obtained from the Cancer Genome Atlas (TCGA) database. The cutoff value of BACH1 expression is calculated using the ROC curve. If the BACH1 expression level in the biological sample obtained from the subject is greater than the cutoff value, a poor prognosis can be predicted. If the expression level is below the cutoff value, a good prognosis can be predicted.

[0079] Furthermore, the prognosis can be predicted more accurately by combining the expression level of FOXA1 as an epithelial cell marker. Similar to BACH1, a receiver operating characteristic (ROC) curve was prepared using the clinical data of pancreatic cancer clinical test samples obtained from the TCGA database, and the cutoff value of the expression level of FOXA1 was calculated by the ROC curve. Then, when the expression level of FOXA1 in the biological sample obtained from the subject is greater than the cutoff value and the expression level of BACH1 is below the cutoff value, it can be predicted that the prognosis is good. On the other hand, when the expression level of FOXA1 in the biological sample obtained from the subject is below the cutoff value and the expression level of BACH1 is greater than the cutoff value, it can be predicted that the prognosis is poor.

[0080] The treatment plan can be determined based on the above-mentioned prognostic prediction results. For example, it can be determined whether to adopt any of surgical treatment, radiotherapy, chemotherapy, and endocrine therapy (hormone therapy). In addition, it can be determined whether to use any other endocrine drug as endocrine therapy.

[0081] <Method for screening pancreatic cancer metastasis inhibitors>

[0082] The present invention provides a method for screening a pancreatic cancer metastasis inhibitor, comprising: adding a candidate pharmaceutical substance to cells expressing the BACH1 gene or a reporter gene linked to the promoter of the BACH1 gene; measuring the expression level of the BACH1 gene or the reporter gene; and selecting a substance that reduces the expression level.

[0083] For example, when the expression level of the BACH1 gene or a reporter gene linked to the promoter of the BACH1 gene is reduced in the presence of a candidate compound compared to a control to which no candidate compound is added, specifically, when the expression level is reduced by, for example, 20% or more, preferably 50% or more, and more preferably 90% or more, the candidate compound can be selected as a candidate for a therapeutic drug for pancreatic cancer.

[0084] Cells expressing the BACH1 gene may be any of biological samples containing cells expressing the BACH1 gene, cultured cell lines expressing the BACH1 gene, or cultured cell lines forced to express the BACH1 gene. Preferably, cultured cell lines forced to express the BACH1 gene are used.

[0085] The cultured cell line expressing the BACH1 gene is not particularly limited as long as it is a cell line expressing the BACH1 gene, and examples thereof include cell lines derived from pancreatic cancer cells.

[0086] Examples of cultured cell lines forcibly expressing the BACH1 gene include those in which the BACH1 gene is embedded in a plasmid or viral vector used for gene introduction into mammalian cells and transfected using conventional methods such as lipofection. Transfection can be transient or stable.

[0087] For cells expressing a reporter gene linked to the BACH1 gene promoter, for example, a cultured cell line in which the reporter gene is forced to be expressed can be used. Specifically, the reporter gene is linked to the BACH1 gene promoter, then incorporated into a plasmid or viral vector used for gene introduction into mammalian cells, and then transfected into the cultured cell line using conventional methods such as known liposome transfection. Transfection can be either transient or stable.

[0088] When using a reporter gene linked to the promoter of the BACH1 gene, the promoter of the BACH1 gene preferably includes a region about 1 kbp upstream of the transcription start site, and more preferably includes a region about 2 kbp upstream.

[0089] The reporter gene may be a luciferase gene, a green fluorescent protein (GFP) gene, a chloramphenicol acetyltransferase gene, or the like, preferably a luciferase gene or a GFP gene.

[0090] In the present invention, candidate compounds may be any of high molecular weight compounds and low molecular weight compounds, without particular limitation. Examples of high molecular weight compounds include proteins, antibodies, peptides, non-peptide compounds, nucleic acids, and RNA. These compounds may be novel compounds, known compounds, or fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like containing these compounds.

[0091] By selecting a compound that reduces the expression level of the BACH1 gene compared to the case where no candidate compound is added, a substance that can be used as a therapeutic drug for pancreatic cancer can be obtained from the candidate compounds.

[0092] The concentration of the candidate compound added is not particularly limited as long as it is a concentration at which the expression level of the BACH1 gene can be confirmed to be reduced. Furthermore, the addition method, reaction time, reaction temperature, etc. can be appropriately selected depending on the test sample used.

[0093] Pancreatic cancer metastasis inhibitor

[0094] The present invention provides a pancreatic cancer metastasis inhibitor comprising, as an active ingredient, a drug that reduces BACH1 gene expression.

[0095] The agent that reduces BACH1 gene expression may be any of a high molecular weight compound and a low molecular weight compound, and is not particularly limited. Examples of high molecular weight compounds include proteins, antibodies (anti-BACH1 antibodies), peptides, non-peptide compounds, nucleic acids, or RNA. RNA is preferred, and siRNA is more preferred.

[0096] The base sequence of the siRNA is not limited as long as it reduces the expression of the BACH1 gene, and examples thereof include the base sequence described in SEQ ID NO: 5 or SEQ ID NO: 6.

[0097] Example

[0098] Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is obviously not limited to the Examples.

[0099] <Example 1> In silico study of the relationship between BACH1 expression and prognosis

[0100] We investigated whether the expression of BACH1 in pancreatic cancer samples registered in the Cancer Genome Atlas (TCGA) database is associated with prognosis. RNA-sequence clinical data of 176 pancreatic cancer patients were obtained from the TCGA database. Using JMP pro v13.1.0 software, we calculated the cutoff value based on the receiver operating characteristic (ROC) curve based on the clinical data. The patients were divided into two groups based on the cutoff value: a low BACH1 expression group (n = 134) and a high BACH1 expression group (n = 42). Figure 1 The overall survival (OS) was analyzed using the product limit method.

[0101] The results showed that in the BACH1 high expression group, OS was significantly reduced (log-rank test; P < 0.0001, Figure 1 This result suggests that BACH1 may function as a malignant factor in pancreatic cancer. This study focused on analyzing the function of BACH1 in pancreatic cancer.

[0102] <Example 2> Study on BACH1 Expression in Pancreatic Cancer Clinical Specimens

[0103] To investigate the relationship between BACH1 protein expression and prognosis, immunohistochemistry was performed using an anti-human BACH1 monoclonal antibody (9D11, a self-produced antibody, diluted 1:100) on clinical pancreatic cancer specimens from patients undergoing surgery at the Department of Gastroenterology and Surgery at Tohoku University Hospital in Japan. The pancreatic cancer specimens were collected from 116 patients who underwent resection at the Department of Gastroenterology and Surgery at Tohoku University Hospital between 2007 and 2014. The specificity of this anti-human BACH1 monoclonal antibody was confirmed by experiments using knockdown and knockout cells (data not disclosed).

[0104] Immunohistochemistry was performed using an anti-human BACH1 monoclonal antibody. Paraffin-embedded sections fixed with 10% paraformaldehyde were cut into 3 μm slices and mounted on slides coated with an anti-stripping film. The sections were deparaffinized with xylene and then dehydrated with ethanol. Antigen activation was performed by boiling in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) for 30 minutes. Endogenous peroxidase activity was blocked by reacting in a 1% hydrogen peroxide / methanol environment for 10 minutes. Normal goat serum (DakoCytomation, Glostrup, Denmark) was used for blocking. The primary antibody reaction was performed at 4°C for 24 hours. The secondary antibody reaction was performed at room temperature for 2 hours using Histofine Simple Stain MAX-PO (M) (Nichirei Biosciences Inc., Tokyo, Japan). After that, 3,3'-diaminobenzidine and hydrogen peroxide dissolved in 0.1 M Tris buffer were used for color development for 5 minutes, and the nuclei were stained with hematoxylin. In microscopic observation, lymphocytes were used as internal standards, and the staining intensity was divided into three stages: weak positive (positive rate less than 10%), moderate positive (positive rate 10-50%), and strong positive (positive rate more than 50%). After evaluation, the strong positive was classified as the BACH1 high expression group (n = 28), and the weak positive and moderate positive were classified as the low expression group (n = 88) ( Figure 1 B).

[0105] <Example 3> Relationship between BACH1 expression and overall survival rate

[0106] To investigate the relationship between BACH1 protein expression based on immunohistochemical classification and prognosis, OS was analyzed using the product limit method.

[0107] The results showed that, consistent with the relationship between BACH1 expression and prognosis using clinical data registered in the TCGA database, OS was significantly reduced in the high-expression group of BACH1 protein (log-rank test; P = 0.0213), and BACH1 was a poor prognostic factor for pancreatic cancer ( Figure 1 C).

[0108] The results of univariate analysis showed that lymph node metastasis (P = 0.0094), distant metastasis (P = 0.0004), and BACH1 expression (P = 0.0231) showed significance as prognostic factors for OS. Therefore, tissue grade (P = 0.0638) was added to these prognostic factor candidates, and a multivariate analysis based on the Cox proportional hazards model was performed. The results showed that lymph node metastasis (P = 0.011) and BACH1 expression (P = 0.0296) showed significance as independent prognostic factors for OS (Table 1).

[0109] [Table 1]

[0110] Variable Analysis

[0111]

[0112] <Example 4> Relationship between BACH1 expression and cell proliferation

[0113] To investigate the effect of BACH1 expression on cancer cell proliferation, BACH1 knockdown was performed using siRNA-based RNA interference in pancreatic cancer cell lines AsPC1 and SW1990. siRNA-based knockdown was performed using the following method. BACH1 siRNA (Stealth RNAi (trademark) siRNA double-stranded oligoribonucleotides (Duplex Oligoribonucleotides), Invitrogen, CA, USA) was used. A Stealth RNAi (trademark) siRNA negative control (Negative Control), LowGC (Thermo Fisher Scientific Inc., MA, USA) was used as a control. siRNA was introduced using Lipofectamine RNAiMAX (Thermo Fisher Scientific Inc.) using liposome transfection according to the product instructions. The sequences of the siRNAs used are as follows.

[0114] siBACH1-1; 5′-GGUCAAAGGACUUUCACAACAUUAA-3′ (SEQ ID NO: 5).

[0115] siBACH1-2; 5′-GGGCACCAGGGAAGAUAGUAGUGUU-3′ (SEQ ID NO: 6).

[0116] As a result, no effect of BACH1 knockdown on cell proliferation was observed in either AsPC1 or SW1990 cell types ( Figure 2 ).

[0117] <Example 5> Cell morphological changes caused by BACH1 knockdown and overexpression

[0118] like Figure 3 As shown in A, the intercellular adhesion of the pancreatic cancer cell line AsPC1 after BACH1 knockdown was performed in the same manner as in Example 4 was enhanced, showing an epithelial cell morphology ( Figure 3 A).

[0119] Therefore, BACH1 overexpression was performed in Panc1, which has a lower expression level of BACH1 protein than AsPC1. Overexpression was performed using the commercially available transfection kit Fugene (registered trademark) HD, following the included product instructions, to introduce the plasmid into Panc1 cultured in a 6cm dish. As a control, 7.2 μg of the commercially available vector pcDNA3.1(-) (Addgene) was introduced. Meanwhile, for overexpression, 7.2 μg of the vector pCMV2-BACH1, containing the BACH1 gene, which contains the base sequence described in SEQ ID NO: 1, and 1 μg of pcDNA3.1(-) for drug selection, were introduced into the commercially available vector pCMV2. The cells were then cultured for two weeks in Panc1 culture medium supplemented with 2000 μg / ml of G418 (Calbiochem, Germany). AsPC1 was cultured in RPMI 1640 medium (serum-free cell freezing basal medium) (Sigma Aldrich, MO, USA) supplemented with 20% fetal bovine serum (FBS, Sigma Aldrich), 0.1 mg / ml penicillin / streptomycin (GIBCO, NY, USA), and 10 mM HEPES (hydroxyethylpiperazine ethanesulfonic acid, GIBCO). SW1990 and Panc1 were cultured in RPMI 1640 medium (Sigma Aldrich, MO, USA) supplemented with 10% FBS, 0.1 mg / ml penicillin / streptomycin, and 10 mM HEPES. After culture, colonies formed by surviving cells that had acquired drug resistance were isolated under a microscope and cloned.

[0120] As a result, in contrast to the case of BACH1 knockdown of AsPC1, the adhesion between cells in BACH1-overexpressing Panc1 was weakened, and the cells showed a pseudopodia-like deformed morphology ( Figure 3 B). This means that BACH1 can promote epithelial to mesenchymal transition (EMT).

[0121] Therefore, the expression of epithelial genes (CDH1, OCLN, FOXA1) and mesenchymal genes (VIM and SNAI2) was confirmed by quantitative RT-PCR according to the following steps. First, RNA was extracted using RNeasy (registered trademark) Plus Mini Kit (Qiagen, Germany) according to the attached product instructions. The extracted RNA was then reverse transcribed using Omniscript Reverse Transcription kit (Qiagen) according to the attached product instructions to synthesize cDNA. The reverse transcription reaction used 1 μg of RNA and 200 ng of random primers (Random primer) (Invitrogen) at 37°C for 60 minutes, and the reverse transcription reaction was stopped by heat treatment at 93°C for 5 minutes. The expression level of the target gene was determined by quantitative PCR using SYBR (registered trademark) GreenI as an intercalator. Using LightCycler (registered trademark) Fast Start DNA Master SYBR Green I (Roche, Switzerland), according to the accompanying product instructions, cDNA was used as a template and various gene-specific primers were used for quantitative PCR on a LightCycler (registered trademark) Nano (Roche). The expression level of the target gene mRNA was corrected by the expression level of the internal standard gene and evaluated by relative quantification compared between the test samples. The internal standard gene used was the β-actin gene (ACTB). The sequences of the primers are as follows.

[0122] ACTB

[0123] (Forward) 5'-ATTTGCGGTGGACGATGGAG-3' (SEQ ID NO: 7).

[0124] (Reverse) 5'-AGAGATGGCCACGGCTGCTT-3' (SEQ ID NO: 8).

[0125] BACH1

[0126] (Forward) 5'-AATCGTAGGCCAGGCTGATG-3' (SEQ ID NO: 9).

[0127] (Reverse) 5'-AGCAGTGTAGGCAAACTGAA-3' (SEQ ID NO: 10).

[0128] CDH1

[0129] (Forward) 5'-TCCTGGCCTCAGAAGACAGA-3' (SEQ ID NO: 11).

[0130] (Reverse) 5'-CCTTGGCCAGTGATGCTGTA-3' (SEQ ID NO: 12).

[0131] OCLN

[0132] (Forward) 5'-GAGTTGACAGTCCCATGGCA-3' (SEQ ID NO: 13).

[0133] (Reverse) 5'-CTGAAGTCATCCACAGGCGA-3' (SEQ ID NO: 14).

[0134] FOXA1

[0135] (Forward) 5′-GGTGGCTCCAGGATGTTAGG-3′ (SEQ ID NO: 15).

[0136] (Reverse) 5'-CCCAGGCCTGAGTTCATGTT-3' (SEQ ID NO: 16).

[0137] VIM

[0138] (Forward) 5'-GGACCAGCTAACCAACGACA-3' (SEQ ID NO: 17).

[0139] (Reverse) 5'-GGGTGTTTTCGGCTTCCTCT-3' (SEQ ID NO: 18).

[0140] SNAI2

[0141] (Forward) 5'-CAACGCCTCCAAAAAGCCAA-3' (SEQ ID NO: 19).

[0142] (Reverse) 5'-ACAGTGATGGGGCTGTATGC-3' (SEQ ID NO: 20).

[0143] As a result, in BACH1 knockdown cells (AsPC1, SW1990), the expression of epithelial genes increased compared to control cells, while the expression of mesenchymal genes decreased significantly. In addition, in BACH1 overexpression cells (Panc1), the expression of epithelial genes decreased, the expression of the mesenchymal gene VIM increased, but the expression of SNAI2 decreased ( Figure 3 C).

[0144] Furthermore, E-cadherin was detected in AsPC1 cells by Western blotting. The cells were suspended in SDS sample buffer (0.0625M Tris-HCl (pH 6.8), 2.2% SDS, 10% glycerol, 0.01% BPB (Bromophenol Blue), and 5% β-ME (β-mercaptoethanol). After solubilization, the proteins were denatured at 95°C for 5 minutes. The total protein extract from the heat-denatured cells was subjected to 7.5% to 12% polyacrylamide gel electrophoresis (PAGE) and analyzed by molecular weight. The gel was then wet-blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). The transferred PVDF membrane was blocked in T-TBS (TBS (25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 3 mM KCl) containing 0.05% Tween 20) with skim milk (Wako Jyun-Yaku Co., Osaka, Japan) added to a final concentration of 3% with shaking at room temperature for 1 hour. For the primary antibodies, anti-BACH1 antibody (1:1000, homemade), anti-GAPDH antibody (1:5000, ab8245, Abcam, England), anti-β-Actin antibody (1:1000, GTX109639, GeneTeX, CA, USA), and anti-E-cadherin antibody (1:1000, ab1416, Abcam) were diluted in the above blocking solution and reacted overnight at 4°C. HRP-conjugated anti-mouse IgG blotting reagents (1:5000, GE Healthcare, NJ, USA) diluted in the blocking buffer were prepared and added to the PVDF membrane after the primary antibody reaction. The membrane was allowed to react at room temperature for 60 minutes. After washing with wash buffer, the membrane was detected by chemiluminescence using SuperSignal West Pico (Thermo Fisher Scientific Inc.) or ECL Plus Western Blotting Substrate (Thermo Fisher Scientific Inc.), and then exposed to X-ray film (GE Healthcare).As a result, knockdown of BACH1 resulted in the downregulation of gene expression (CDH1, Figure 3 After C), protein expression also increased ( Figure 4 The same effect was also confirmed in AsPC1 and SW1990 ( Figure 3 Furthermore, it was found that when BACH1 overexpresses Panc1, the gene expression and protein expression of E-cadherin are reduced ( Figure 3 Here, considering that E-cadherin is a representative marker protein in epithelial cells and functions as an intercellular adhesion factor ( Figure 3 E), it can be suggested that it may be one of the factors that change cell morphology. These results suggest that BACH1 is important for the expression and maintenance of mesenchymal cell characteristics in pancreatic cancer.

[0145] <Example 6> Effects of BACH1 on migration and invasion abilities

[0146] In order to investigate whether BACH1 can promote the migration and invasion ability of pancreatic cancer cells, a scratch experiment was performed as follows to confirm its migration ability. First, a scratch (straight line) was formed on the cells after fusion (confluent) in a 6-well plate using a 1000μl pipette tip, and the cell movement area was measured by comparing the wound area when the scratch was just formed and 24 hours after formation. In addition, the movement area was measured by the number of pixels using Image J (NIH, https: / / imagej.nih.gov / ij / ) software, and the same experiment was performed independently more than 3 times. As a result, compared with the control cells, the migration ability was significantly reduced in BACH1 knockdown AsPC1 ( Figure 4 A), compared with the control cells, the migration ability was significantly increased in BACH1 overexpressing Panc1 ( Figure 4 B).

[0147] Furthermore, the migration and infiltration abilities were confirmed using the Boyden chamber method. First, a culture medium containing serum and suitable for the cells used (20% FBS for AsPC1 and 10% FBS for SW1990) was added to the lower 24-well plate, and then an insert was placed on it, and then cells suspended in serum-free culture medium (2×105 for AsPC1 and 1×105 for SW1990) were inoculated into the insert. After 24 hours, all cells on the upper surface of the insert membrane were removed with a cotton swab, and the cells that moved to the lower surface of the insert membrane were immersed in a crystal violet solution (4.2% formalin, 0.05% crystal violet, 10% ethanol) for 10 minutes for fixation and staining. The stained membrane was washed with distilled water, dried, and then the number of migrated and infiltrated cells was visually counted under a microscope. For one experiment, each test sample was repeated twice, and for AsPC1, the number of cells in the entire membrane was counted. For SW1990, the number of cells was counted in 5 independent visual fields and the average was taken. The same experiment was performed independently more than 3 times. The results showed that the migration and invasion abilities of AsPC1 and SW1990 cells were significantly reduced after BACH1 knockdown ( Figure 4 C).

[0148] These results indicate that BACH1 enhances the migration and invasion abilities of pancreatic cancer cells.

[0149] <Example 7> Establishment of BAHC1 knockout cell line

[0150] To verify the metastasis-promoting function of BACH1 in vivo through mouse transplantation experiments, a BACH1 knockout pancreatic cancer cell line was generated using the CRISPR / CAS9 system. The CRISPR / CAS9 system was constructed as follows: the guide RNA described below was ligated to the viral vector lentiCRISPR v2 (Addgene, MA, USA) treated with the restriction enzyme BsmB1 (recognition sequence: 5'CGTCTC3') to create a viral vector carrying the target guide RNA. 293T cells, derived from human fetal kidney epithelial cells, were transfected using Fugene (registered trademark) HD ​​(Promega, WI, USA) according to the product instructions. Two days later, the culture supernatant was collected as a viral fluid. The collected viral fluid was used to infect AsPC1 cells with the virus carrying the target guide RNA. 10 μg / ml of puromycin (Sigma Aldrich) was added to the culture medium for three weeks of drug selection. The resulting colonies were then isolated under a microscope to obtain single clones. Gene mutation and loss of protein expression were confirmed by genomic sequencing and Western blotting, respectively. Single guide RNA (sgRNA) targeted the following sequence of BACH1.

[0151] Guide RNA-1; 5'-CCGCGCUCACCGGUCCGUGCUGG-3' (SEQ ID NO: 21).

[0152] Guide RNA-2; 5'-CCACUCAAGAAUCGUAGGCCAGG-3' (SEQ ID NO: 22).

[0153] Sequence analysis confirmed that the BACH1 knockout cells produced Figure 5 The target sequence regions of the two guide RNAs in the reference sequence shown in A contain a 13-base deletion mutation (sgBACH1-1) or a 1-base insertion mutation (sgBACH1-2) as the target. Figure 5 In addition, Western blotting confirmed that the expression of BACH1 protein disappeared ( Figure 5 Furthermore, quantitative RT-PCR was used to confirm whether BACH1 knockout would lead to enhanced expression of epithelial genes and decreased expression of mesenchymal genes, as in the case of BACH1 knockdown. The results showed that although the expression of epithelial genes was significantly enhanced, no decrease in the expression of mesenchymal genes was observed ( Figure 5 In addition, the expression level of E-cadherin was confirmed by Western blotting. The results showed that although the expression level of CDH1 was increased in both sgBACH1-1 and sgBACH1-2 cells ( Figure 5C), but it was confirmed that the expression level of E-cadherin increased significantly only in sgBACH1-2 cells, similar to the results of siRNA interference ( Figure 5 B). Furthermore, immunofluorescence staining was performed to confirm changes in the expression levels of BACH1 protein and E-cadherin. Immunofluorescence staining was performed as follows: cells cultured on slides were fixed with 4% formaldehyde / PBS (Polybutylene succinate) for 10 minutes. The cells on the slides were immersed in an antibody solution containing the primary antibodies BACH1 (9D11, homemade) and E-cadherin (ab1416, abcam) diluted 200-fold with 1% bovine serum albumin (BSA) / PBS and reacted at 37°C for 1 hour. After washing with PBS, the cells were immersed in an antibody solution containing the secondary antibody (anti-mouse IgG antibody-FITC (F3008, SigmaAldrich)) diluted 1000-fold with 1% BSA / PBS and reacted at 37°C for 1 hour. After washing with PBS, the cells were stained with 20 μg / ml Hoechst 33342 / PBS for nuclear staining. The cells were then mounted with VECTASHIELD Mounting Medium (Funakoshi, Tokyo, Japan) and observed under a high-speed fluorescence imaging system AF6500 (Leica, Germany), which is an inverted fluorescence microscope. The results showed that the expression of BACH1 protein decreased and the expression of E-cadherin protein increased ( Figure 5 D, E).

[0154] <Example 8> Effects of BACH1 on Tumor Formation and Metastasis

[0155] In order to analyze the function of BACH1 in individual mice, in situ cancer cell transplantation of BACH1 knockout AsPC1 and its control cells was performed on the pancreatic tissue of hyperimmune deficient mice (NOG (NOD.Cg-PrkdcscidIl2rgtm1Sug / ShiJic) mice). The experiment used NOG mice aged 6 to 8 weeks. After administering three mixed anesthetics (medetomidine 0.3 mg / kg, midazolam 4 mg / kg, butorphanol 5 mg / kg) to the mouse peritoneal cavity, BACH1 wild-type AsPC1 and BACH1 knockout AsPC1 were orthotopically transplanted into the pancreas. For orthotopic transplantation, a 1.5 cm laparotomy was performed on the left upper abdomen, the pancreas was removed from the body, and then a 27G injection needle was used to inject 10 4Cells (suspended in 100 μl PBS) were transplanted under the capsule of the pancreatic tail. After transplantation, the pancreas was returned to the body and the abdomen was sutured with 3-0 absorbable sutures. Five weeks later, pancreatic tissue weight, the number of metastases to the liver, and the number of metastases to the mesentery, diaphragm, and peritoneum as peritoneal seeding were visually measured by dissection. Figure 6 A represents a representative image of the pancreas, liver, and mesentery during dissection. Consistent with the results of in vitro cell proliferation, no significant difference in tumor proliferation ability was observed in the pancreas (transplantation nest) ( Figure 6 However, in BACH1 knockout AsPC1, liver metastasis and peritoneal seeding were significantly reduced ( Figure 6 (Center and right panels of B). This result shows that BACH1 has no effect on primary tumor formation in pancreatic cancer cells, but enhances metastatic ability.

[0156] <Example 9> Study on prognostic prediction by combining BACH1 and FOXA1

[0157] The relationship between FOXA1 expression and prognosis was analyzed using RNA-sequencing data from pancreatic cancer patients collected by TCGA. The results showed that the higher the expression of FOXA1, the better the prognosis ( Figure 7 A). Furthermore, by further analyzing the combination of BACH1 downstream factors and BACH1 expression, the function of BACH1 in prognosis was re-evaluated. The results showed that the prognosis was the worst in the combination of BACH1 high expression and FOXA1 low expression, in which BACH1 highly exerted its transcriptional repressor function, while the prognosis was good in the combination of BACH1 low expression and FOXA1 high expression, in which BACH1 lost its transcriptional repressor function ( Figure 7 B) These results demonstrate that BACH1 can be used as an excellent biomarker for predicting pancreatic cancer prognosis by combining its functional evaluation with the expression of its downstream gene, FOXA1. Furthermore, BACH1 is considered an indicator of the metastatic potential of pancreatic cancer. SEQUENCE LISTING <110> National University Corporation Tohoku University <120> A novel epithelial-mesenchymal transition marker in pancreatic cancer <130> OP-19065-PCT <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 5475 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (119)..(2329) <400> 1 tcgccccgc cgggcgct cgcttcagtc agtcggccg cgccgcgcct cagctctggt 60 tgatgataat tagagcatg ctttccactg aacttcccga siacattgt tatgcaga 118 atg tct ctg agt gag aac tcg gtt tt gcc tat gaa tct tct gtg cat 166 Met Ser Leu Ser Glu Asn Ser Val Phe Ala Tyr Glu Ser Val His 1 5 10 15 agc acc aat gtt tta ctc agc ctt aat gac cag cgg aag aaa gat gtg 214 Serve Thr Asn Val Leu Ser Leu Asn Asp Gln Arg Lys Asp Val 20 25 30 ctg tgc gat gtc acc atc ttt gtg gag gga cag cgg ttc cgc gct cac 262 Leu Cys Asp Val Thr Ile Phe Val Glu Gly Gln Arg Phe Arg Ala His 35 40 45 cgg tcc gtg ctg gcg gca tgc agc agt tac ttc cac tca aga atc ​​gta 310 Arg Ser Val Leu Ala Ala Cys Ser Tyr Phe His Ser Arg Ile Val 50 55 60 ggc cag gct gat gga gag ctg aac att act ctt cca gaa gag gtg aca 358 Gly Gln Ala Asp Gly Glu Leu Asn Ile Thr Leu Pro Glu Glu Val Thr 65 70 75 80 gtt aaa gga ttt gaa cct tta att cag ttt gcc tac act gct aaa ctg 406 Val Lys Gly Phe Glu Pro Leu Ile Gln Phe Ala Tyr Thr Ala Lys Leu 85 90 95 to tta agt aaa gag aat gtg gat gaa gtg tgc aaa tgt gtg gag ttt 454 Ile Leu Ser Lys Glu Asn Val Asp Glu Val Cys Lys Cys Val Glu Phe 100 105 110 tta agt gta cat aat att gag gaa tcc tgc ttt cag ttt ctg aaa ttt 502 Leu Ser Val His Asn Ile Glu Glu Ser Cys Phe Gln Phe Leu Lys Phe 115 120 125 aag ttt ttg gac tcc act gca gac cag caa gaa tgc cca aga aaa aaa 550 Lys Phe Leu Asp Ser Thr Ala Asp Gln Gln Glu Cys Pro Arg Lys Lys 130 135 140 tgc ttt tca tca cac tgt cag aaa aca gac ctt aaa ctt tca ctt ttg 598 Cys Phe Served His Cys Gln Lys Thr Asp Leu Lys Leu Served By Leu Leu 145 150 155 160 gac cag agg gat cta gaa act gat gaa gtg gag gaa ttt ctg gaa aat 646 Asp Gln Arg Asp Leu Glu Thr Asp Glu Val Glu Glu Phe Leu Glu Asn 165 170 175 aaa aat gtt cag act cct cag tgt aaa ctc cgc agg tat caa gga aat 694 Lys Asn Val Gln Thr Pro Gln Cys Leu Arg Arg Tyr Gln Gly Asn 180 185 190 gca aaa gcc tca cct cct cta caa gac agt gcc agt cag aca tat gag 742 Lys Ser Ser Pro Pro Leu Gln Asp Ser Ser Ala Gln Thr Tyr Glu 195 200 205 tcc atg tgc tta gag aag gat gct gct ctg gcc ttg cct tct tta tgc 790 Ser Met Cys Leu Glu Lys Asp Ala Ala Leu Ala Leu Pro Ser Leu Cys 210 215 220 ccc aaa tac aga aaa ttc caa aaa gca ttt gga act gac aga gtc cgt Pro Lys Tyr Arg Lys Phe Gln Lys Ala Phe Gly Thr Asp Arg Val Arg 225 230 235 240 act ggg gaa tct agt gtc aaa gac att cat gct tct gtt cag cca aat 886 Thr Gly Glu Ser Ser Val Lys Asp Ile His Ala Ser Val Gln Pro Asn 245 250 255 gaa agg tct gaa aat gaa tgc ctg gga gga gtc ccg gag tgt aga gat 934 Glu Arg Ser Glu Asn Glu Cys Leu Gly Gly Val Pro Glu Cys Arg Asp 260 265 270 ttg cag gtg atg tta aaa tgt gac gaa agt aaa tta gca atg gaa cct 982 Leu Gln Val Met Leu Lys Cys Asp Glu Ser Leu Lys Ala Met Glu Pro 275 280 285 gaa gaa acg aaag gat cct gct tct cag tgc cca act gaaaaa tca 1030 Glu Glu Thr Lys Lys Asp Pro Ala Ser Gln Cys Pro Thr Glu Lys Ser 290,295,300 gaa gtg act cct ttc ccc cac aat tct tcc ata gac cct cat gga ctt 1078 Glu Val Thr Pro Phe Pro His Asn Ser Ser Ile Asp Pro His Gly Leu 305 310 315 320 tat tct ttg tct ctt tta cac aca tat gac caa tat ggt gac ttg aat Tyr Ser Leu Ser Leu Leu His Thr Tyr Asp Gln Tyr Gly Asp Leu Asn 325 330 335 ttt gct ggt atg caa aac aca aca gtg tta aca gaa aag cct ttg tca 1174 Phe Ala Gly Met Gln Asn Thr Thr Val Leu Thr Glu Lys Pro Leu Ser 340 345 350 ggt aca gac gtc caa gaa aaa aca ttt ggt gaa agt cag gat tta cct 1222 Gly Thr Asp Val Gln Glu Lys Thr Phe Gly Glu Ser Gln Asp Leu Pro 355 360 365 ttg aaa tcc gac ttg ggc acc agg gaa gat agt agt gtt gca tct agt 1270 Leu Lys Ser Asp Leu Gly Thr Arg Glu Asp Ser Ser Val Ala Ser Ser 370 375 380 gat agg agt agt gtg gag cga gaa gtg gca gaa cac cta gca aaa ggc 1318 Asp Arg Ser Ser Val Glu Arg Glu Val Ala Glu His Leu Ala Lys Gly 385 390 395 400 ttc tgg agt gac att tgc agc acg gac act cct tgc caa atg cag tta 1366 Phe Trp Ser Asp Ile Cys Ser Thr Asp Thr Pro Cys Gln Met Gln Leu 405 410 415 tca cct gct gtg gcc aaa gat ggc tca gaa cag atc tca cag aaa cgg 1414 Ser Pro Ala Val Ala Lys Asp Gly Ser Glu Gln Ile Ser Gln Lys Arg 420 425 430 tct gag tgt ccg tgg tta ggt atc agg att agt gag agc cca gaa cca 1462 Ser Glu Cys Pro Trp Leu Gly Ile Arg Ile Ser Glu Ser Pro Glu Pro 435 440 445 ggt caa agg act ttc aca aca tta agt tct gtc aac tgc cct ttt ata 1510 Gly Gln Arg Thr Phe Thr Thr Leu Ser Ser Val Asn Cys Pro Phe Ile 450 455 460 agt act ctg agt act gaa ggc tgt tca agc aat ttg gaa att gga aac 1558 Ser Thr Leu Ser Thr Glu Gly Cys Ser Ser Asn Leu Glu Ile Gly Asn 465 470 475 480 gat gat tat gtt tca gaa ccc cag caa gaa cct tgc cca tat gct tgt 1606 Asp Asp Tyr Val Ser Glu Pro Gln Gln Glu Pro Cys Pro Tyr Ala Cys 485 490 495 gtc att agc ttg gga gac gac tct gag acg gac acc gaa gga gac agt 1654 Val Ile Ser Leu Gly Asp Asp Ser Glu Thr Asp Thr Glu Gly Asp Ser 500 505 510 gaa tcc tgt tca gcc aga gaa caa gaa tgt gag gta aaa ctg cca ttc 1702 Glu Ser Cys Ser Ala Arg Glu Gln Glu Cys Glu Val Lys Leu Pro Phe 515 520 525 aat gca caa cgg ata att tca ctg tct cga aat gat ttt cag tcc ttg 1750 Asn Ala Gln Arg Ile Ile Ser Leu Ser Arg Asn Asp Phe Gln Ser Leu 530 535 540 ttg aaa atg cac aag ctt act cca gaa cag ctg gat tgt atc cat gat 1798 Leu Lys Met His Lys Leu Thr Pro Glu Gln Leu Asp Cys Ile His Asp 545 550 555 560 att cga aga agat aag aaa aac aga att gct gca cag cgc tgt cgc aag 1846 Ile Arg Arg Arg Ser Lys Asn Arg Ile Ala Ala Gln Arg Cys Arg Lys 565 570 575 aga aaa ctt gac tgt ata cag aat ctt gaa tca gaa att gag aag ctg 1894 Arg Lys Leu Asp Cys Ile Gln Asn Leu Glu Ser Glu Ile Glu Lys Leu 580 585 590 caa agt gaa ag gag agc tg tg aag gaa aga gat cac att ttg tca 1942 Gln Ser Glu Lys Glu Ser Leu Leu Lys Glu Arg Asp His Ile Leu Ser 595,600,605 act ctg ggc gag aca aag cag aac cta act gga ctt tgc cag aaa gtt 1990 Thr Gly Glu Thr Lys Gln Asn Leu Thr Gly Leu Cys Gln Lys Val 610 615 620 tgt aaa gaa gca gct ctg agt caa gaa ca ata cag ata ctc gcc aag 2038 Cys Lys Glu Ala Leu Sere Gln Glu Gln Ile Gln Ile Leu Ala Lys 625 630 635 640 tac tca gct gca gat tgc cca ctt tca ttt tta att tct gaa aaa gat Tyr Ser Ala Ala Asp Cys Pro Leu Ser Phe Leu Ile Ser Glu Lys Asp 645,650,655 aaa agt act cct gat ggt gaa ctg gcg tta cca tca att ttc agt tta 2134 Lys Ser Thr Pro Asp Glu Glu Leu Ala Leu Pro Ser Ile Phe Ser Leu 660,665,670 tct gac cgg cct cca gca gtg ctg cct ccc tgt gcc aga gga aac agt 2182 Ser Asp Arg Pro Pro Ala Val Leu Pro Pro Cys Ala Arg Gly Asn Ser 675 680 685 gag cct ggc tac gcg cga ggg cag gag tcc cag cag atg tcc aca gcc 2230 Glu Pro Gly Tyr Ala Arg Gly Gln Glu Ser Gln Gln Met Ser Thr Ala 690 695 700 acc tct gag caa gct ggg cct gcg gaa cag tgt cgt cag agt ggt ggg 2278 Thr Ser Glu Gln Ala Gly Pro Ala Glu Gln Cys Arg Gln Ser Gly Gly 705 710 715 720 atc tca gat ttc tgt cag cag atg act gat aaa tgt act act gat gag 2326 Ile Ser Asp Phe Cys Gln Gln Met Thr Asp Lys Cys Thr Thr Asp Glu 725 730 735 taa acttgcattc acttccttca aaccatctaa ttttctcctg aagttttggc 2379 agcgtcttga aagcctaata tgaccatctg ttgctcaaca atactgtttt tttcctttag 2439 tagtttacca caagggaatt tcctttaagt caaccatgat ttctccttga tttctacaag 2499 agacaaagaa atgattttgc ctcctggata tcagaaaaat ccatgtgaaa atgtagtaaa 2559 cctttaaaac tcatgtttta aagaataata actctagtaa taactcttcc tgctattcag 2619 aataagtagg agaatgaaaa ctgcagcata tcagacagca atttaacagc ttgaaacatc 2679 tacagatagt tcctactaaa agaagtggcc tgcagaagtt taataatttg acttttttct 2739 aatatttag tttgaaagaa aatttcttcc caagcaatgc taatagagtt ctattcttag 2799 aagcagggtg tcagctactg ggaatatttt tgtagagctg cattgtgaaa aaaagatggt 2859 cttacctgaa tcttagggct ttgttcttcg gctcctaaaa tcaggcttta agctacattg 2919 ggaagattta gtaaataggc aagtggttgg cctaagacgg gggctgcttc tcctcttcag 2979 tatggactct agaaagtctg gctacatgaa tagattttaag tgtcactttc cctccctgcc 3039 ccccgcttca gtctctacca tatctggtcc catcatggac ttcctatttc ctggcatttt 3099 tgtccctttg gaagaagaaa taggactcag aatacagtgg catgagtgat tacactggca 3159 gcattatctc aggctcccta gaatctggag agcttaccaa catgtaaagc tgttcatttt 3219 tccaccgtgg gtcaccaatg ccagaaaacc agacatcacg gggaaagaat gttgcttact 3279 ttttaccagg agtgcagttc atttttca ccctgttttt gaagtcgtat tattcacttg 3339 taaaaatgat tgtaacagat aaaaaatgta tctgcagcaa ctctgcaggt ttgtgaaata 3399 ggatgaaact caatctttt ctattgtggg tttgcattg aaagcaggt tgaatccttg 3459 ctctctctc caaatttggt gtggtataaa vakacacaa tcattttaac ttggacattt 3519 aagatcagt cttagtgttt gttcagtcct gttacaaat agatactga gcacctatcg 3579 cataacattt tgcggtggct tttagccatg ctggggttag atgtgtttga gagtcaaatg 3639 aaagctatgg atctctcag cattaaaaa aaatgcatat attcacattc acagaaacat 3699 tggcagaacc cagttttaat ggtacagagg agtagtttat agtgttgatt tcaccaaaat 3759 cagagggctg aaagagacac ttctatagac tgcatcctga gcctagtgca gggcttgtct 3819 agctaatgtg ggcagccacc acccactgtg tatgacaag tctgaagcaa gttggccttg 3879 cccttgagag tatatgggga ccagtcttca tgtcttggag taatttgtca aatgttaccc 3939 ttttgatca gggtgtaggg ggaggatatt gctagtatat ttcagtggt ttgtatgttc 3999 tctctgtcac tgacttattt gtaagagaaa attagttgga cttgtttatt ttctagtagc 4059 ttttataagt acactcaaga atttgtcagg gagaataatt ctgatagtgc atcccatact 4119 gcaaaagaat ttgtgtgtgt gtgtgtgtgt gtgtgtgtgt atgtgtatgt atacatatat 4179 atctctccat ataggtattt ctttgatact tgtaatttta aatttcagct tcacgatata 4239 aaataatata agaacttctg gtttacaaaa tgtaaaatct taagccaatg gaacccttga 4299 tttcctacct cagtgtacac tcaactattg gttgtatcag tttgtgtatg tgcaaatgtc 4359 aaataatctt ttgctttaat tgctactgta cttgctttga aagattacct actattttat 4419 gataaaatgt agttgtctcc agagcttaaa tataatttgt aaagcacttg gtttaaattt 4479 ctctctacct ataaacagtt tagcattaag ggtttctatt aatgacacag aattattggc 4539 caagtgtaat ttcttaaaat ttagcattac tttaaatagc cagcatgtaa tacaagtaac 4599 tacactacct catatctaca tgattttcaa gttgtaatgc agatggacag ataaaaaaga 4659 ttttacgttt gtcttttggc cataagtggg aaagttttct gtatattgca tagcattaca 4719 catttatgcc tattttaaca ttaacttcta aagaagtttt ttctaagaaa atgtttcaag 4779 gcaatatttt tttgaggct gccgaagaca atgacagga ttatgagtat acagtgtatg 4839 ccttttcctt catgcagaat tttgaatgt ttcagttg tattgcat attcacatga 4899 tcattgctca ctattttag aactggcctt ctcaatgtttt gatgatttt taaaagctgt 4959 tatgttgaat tcagtaaaat aacattacct tattttttt cttattcaa ttctggaact 5019 atagcaaata attcgttaaa ttgtcatatt caaaacaat gtggatacag tcttggttct 5079 ccatctgtaa ttttttaa cagtttgcta tagcttactg cttaactaat ttataatag 5139 gaaataagta tgttagatgc agtagacgat acaggttgca tgtggacact cagtcacatt 5199 aacaacttgg gaaaaaaatg gcaatgttac ggtgaattct caggtgaact ttttcagtt 5259 aaaacatc tattttgaat ctgtaaatat tttaaatgtt ttattaggc atgtataaa 5319 ctattctttg aaacttgttg ggtagaatga aaattaaagc cataatggta aagatggca 5379 tactgatt aaagaagca gaaaaacatt gattttttta tactttcat atattaattt 5439 tctaacaatg caataaaacc actaaacttt tgtgtc 5475 <210> 2 <211> 736 <212> PRT <213> Homo sapiens <400> 2 Met Ser Leu Ser Glu Asn Ser Val Phe Ala Tyr Glu Ser Ser Val His 1 5 10 15 Ser Thr Asn Val Leu Leu Ser Leu Asn Asp Gln Arg Lys Lys Asp Val 20 25 30 Leu Cys Asp Val Thr Ile Phe Val Glu Gly Gln Arg Phe Arg Ala His 35 40 45 Arg Ser Val Leu Ala Ala Cys Ser Ser Tyr Phe His Ser Arg Ile Val 50 55 60 Gly Gln Ala Asp Gly Glu Leu Asn Ile Thr Leu Pro Glu Glu Val Thr 65 70 75 80 Val Lys Gly Phe Glu Pro Leu Ile Gln Phe Ala Tyr Thr Ala Lys Leu 85 90 95 Ile Leu Ser Lys Glu Asn Val Asp Glu Val Cys Lys Cys Val Glu Phe 100 105 110 Leu Ser Val His Asn Ile Glu Glu Ser Cys Phe Gln Phe Leu Lys Phe 115 120 125 Lys Phe Leu Asp Ser Thr Ala Asp Gln Gln Glu Cys Pro Arg Lys Lys 130 135 140 Cys Phe Ser Ser His Cys Gln Lys Thr Asp Leu Lys Leu Ser Leu Leu 145 150 155 160 Asp Gln Arg Asp Leu Glu Thr Asp Glu Val Glu Glu Phe Leu Glu Asn 165 170 175 Lys Asn Val Gln Thr Pro Gln Cys Lys Leu Arg Arg Tyr Gln Gly Asn 180 185 190 Ala Lys Ala Ser Pro Pro Leu Gln Asp Ser Ala Ser Gln Thr Tyr Glu 195 200 205 Ser Met Cys Leu Glu Lys Asp Ala Ala Leu Ala Leu Pro Ser Leu Cys 210 215 220 Pro Lys Tyr Arg Lys Phe Gln Lys Ala Phe Gly Thr Asp Arg Val Arg 225 230 235 240 Thr Gly Glu Ser Ser Val Lys Asp Ile His Ala Ser Val Gln Pro Asn 245 250 255 Glu Arg Ser Glu Asn Glu Cys Leu Gly Gly Val Pro Glu Cys Arg Asp 260 265 270 Leu Gln Val Met Leu Lys Cys Asp Glu Ser Lys Leu Ala Met Glu Pro 275 280 285 Glu Glu Thr Lys Lys Asp Pro Ala Ser Gln Cys Pro Thr Glu Lys Ser 290 295 300 Glu Val Thr Pro Phe Pro His Asn Ser Ser Ile Asp Pro His Gly Leu 305 310 315 320 Tyr Ser Leu Ser Leu Leu His Thr Tyr Asp Gln Tyr Gly Asp Leu Asn 325 330 335 Phe Ala Gly Met Gln Asn Thr Thr Val Leu Thr Glu Lys Pro Leu Ser 340 345 350 Gly Thr Asp Val Gln Glu Lys Thr Phe Gly Glu Ser Gln Asp Leu Pro 355 360 365 Leu Lys Ser Asp Leu Gly Thr Arg Glu Asp Ser Ser Val Ala Ser Ser 370 375 380 Asp Arg Ser Ser Val Glu Arg Glu Val Ala Glu His Leu Ala Lys Gly 385 390 395 400 Phe Trp Ser Asp Ile Cys Ser Thr Asp Thr Pro Cys Gln Met Gln Leu 405 410 415 Ser Pro Ala Val Ala Lys Asp Gly Ser Glu Gln Ile Ser Gln Lys Arg 420 425 430 Ser Glu Cys Pro Trp Leu Gly Ile Arg Ile Ser Glu Ser Pro Glu Pro 435 440 445 Gly Gln Arg Thr Phe Thr Thr Leu Ser Ser Val Asn Cys Pro Phe Ile 450 455 460 Ser Thr Leu Ser Thr Glu Gly Cys Ser Ser Asn Leu Glu Ile Gly Asn 465 470 475 480 Asp Asp Tyr Val Ser Glu Pro Gln Gln Glu Pro Cys Pro Tyr Ala Cys 485 490 495 Val Ile Ser Leu Gly Asp Asp Ser Glu Thr Asp Thr Glu Gly Asp Ser 500 505 510 Glu Ser Cys Ser Ala Arg Glu Gln Glu Cys Glu Val Lys Leu Pro Phe 515 520 525 Asn Ala Gln Arg Ile Ile Ser Leu Ser Arg Asn Asp Phe Gln Ser Leu 530 535 540 Leu Lys Met His Lys Leu Thr Pro Glu Gln Leu Asp Cys Ile His Asp 545 550 555 560 Ile Arg Arg Arg Ser Lys Asn Arg Ile Ala Ala Gln Arg Cys Arg Lys 565 570 575 Arg Lys Leu Asp Cys Ile Gln Asn Leu Glu Ser Glu Ile Glu Lys Leu 580 585 590 Gln Ser Glu Lys Glu Ser Leu Leu Lys Glu Arg Asp His Ile Leu Ser 595 600 605 Thr Leu Gly Glu Thr Lys Gln Asn Leu Thr Gly Leu Cys Gln Lys Val 610 615 620 Cys Lys Glu Ala Ala Leu Ser Gln Glu Gln Ile Gln Ile Leu Ala Lys 625 630 635 640 Tyr Ser Ala Ala Asp Cys Pro Leu Ser Phe Leu Ile Ser Glu Lys Asp 645 650 655 Lys Ser Thr Pro Asp Gly Glu Leu Ala Leu Pro Ser Ile Phe Ser Leu 660 665 670 Ser Asp Arg Pro Pro Ala Val Leu Pro Pro Cys Ala Arg Gly Asn Ser 675 680 685 Glu Pro Gly Tyr Ala Arg Gly Gln Glu Ser Gln Gln Met Ser Thr Ala 690 695 700 Thr Ser Glu Gln Ala Gly Pro Ala Glu Gln Cys Arg Gln Ser Gly Gly 705 710 715 720 Ile Ser Asp Phe Cys Gln Gln Met Thr Asp Lys Cys Thr Thr Asp Glu 725 730 735 <210> 3 <211> 3373 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (142)..(1560) <400> 3 ctcttcgccc gggtggcgtt gggcccgcgc gggcgctcgg gtgactgcag ctgctcagct 60 cccctccccc gccccgcgcc gcgcggccgc ccgtcgcttc gcacagggct ggatggttgt 120 attgggcagg gtggctccag g atg tta gga act gtg aag atg gaa ggg cat 171 Met Leu Gly Thr Val Lys Met Glu Gly His 1 5 10 gaa acc agc gac tgg aac agc tac tac gca gac acg cag gag gcc tac 219 Glu Thr Ser Asp Trp Asn Ser Tyr Tyr Ala Asp Thr Gln Glu Ala Tyr 15 20 25 tcc tcc gtc ccg gtc agc aac atg aac tca ggc ctg ggc tcc atg aac 267 Ser Ser Val Pro Val Ser Asn Met Asn Ser Gly Leu Gly Ser Met Asn 30 35 40 tcc atg aac acc tac atg acc atg aac acc atg act acg agc ggc aac 315 Ser Met Asn Thr Tyr Met Thr Met Asn Thr Met Thr Thr Ser Gly Asn 45 50 55 atg acc ccg gcg tcc ttc aac atg tcc tat gcc aac ccg ggc cta ggg 363 Met Thr Pro Ala Ser Phe Asn Met Ser Tyr Ala Asn Pro Gly Leu Gly 60 65 70 gcc ggc ctg agt ccc ggc gca gta gcc ggc atg ccg ggg ggc tcg gcg 411 Ala Gly Leu Ser Pro Gly Ala Val Ala Gly Met Pro Gly Gly Ser Ala 75 80 85 90 ggc gcc atg aac agc atg act gcg gcc ggc gtg acg gcc atg ggt acg 459 Gly Ala Met Asn Ser Met Thr Ala Ala Gly Val Thr Ala Met Gly Thr 95 100 105 gcg ctg agc ccg agc ggc atg ggc gcc atg ggt gcg cag cag gcg gcc 507 Ala Leu Ser Pro Ser Gly Met Gly Ala Met Gly Ala Gln Gln Ala Ala 110 115 120 tcc atg aat ggc ctg ggc ccc tac gcg gcc gcc atg aac ccg tgc atg 555 Ser Met Asn Gly Leu Gly Pro Tyr Ala Ala Ala Met Asn Pro Cys Met 125 130 135 agc ccc atg gcg tac gcg ccg tcc aac ctg ggc cgc agc cgc gcg ggc 603 Ser Pro Met Ala Tyr Ala Pro Ser Asn Leu Gly Arg Ser Arg Ala Gly 140 145 150 ggc ggc ggc gac gcc aag acg ttc aag cgc agc tac ccg cac gcc aag 651 Gly Gly Gly Asp Ala Lys Thr Phe Lys Arg Ser Tyr Pro His Ala Lys 155 160 165 170 ccg ccc tac tcg tac atc tcg ctc atc acc atg gcc atc cag cag gcg 699 Pro Pro Tyr Ser Tyr Ile Ser Leu Ile Thr Met Ala Ile Gln Gln Ala 175 180 185 ccc agc aag atg ctc acg ctg agc gag atc tac cag tgg atc atg gac 747 Pro Ser Lys Met Leu Thr Leu Ser Glu Ile Tyr Gln Trp Ile Met Asp 190 195 200 ctc ttc ccc tat tac cgg cag aac cag cag cgc tgg cag aac tcc atc 795 Leu Phe Pro Tyr Tyr Arg Gln Asn Gln Gln Arg Trp Gln Asn Ser Ile 205 210 215 cgc cac tcg ctg tcc ttc aat gac tgc ttc gtc aag gtg gca cgc tcc 843 Arg His Ser Leu Ser Phe Asn Asp Cys Phe Val Lys Val Ala Arg Ser 220 225 230 ccg gac aag ccg ggc aag ggc tcc tac tgg acg ctg cac ccg gac tcc 891 Pro Asp Lys Pro Gly Lys Gly Ser Tyr Trp Thr Leu His Pro Asp Ser 235 240 245 250 ggc aac atg ttc gag aac ggc tgc tac ttg cgc cgc cag aag cgc ttc 939 Gly Asn Met Phe Glu Asn Gly Cys Tyr Leu Arg Arg Gln Lys Arg Phe 255 260 265 aag tgc gag aag cag ccg ggg gcc ggc ggc ggg ggc ggg agc gga agc 987 Lys Cys Glu Lys Gln Pro Gly Ala Gly Gly Gly Gly Gly Ser Gly Ser 270 275 280 ggg ggc agc ggc gcc aag ggc ggc cct gag agc cgc aag gac ccc tct 1035 Gly Gly Ser Gly Ala Lys Gly Gly Pro Glu Ser Arg Lys Asp Pro Ser 285 290 295 ggc gcc tct aac ccc agc gcc gac tcg ccc ctc cat cgg ggt gtg cac 1083 Gly Ala Ser Asn Pro Ser Ala Asp Ser Pro Leu His Arg Gly Val His 300 305 310 ggg aag acc ggc cag cta gag ggc gcg ccg gcc ccc ggg ccc gcc gcc 1131 Gly Lys Thr Gly Gln Leu Glu Gly Ala Pro Ala Pro Gly Pro Ala Ala 315 320 325 330 agc ccc cag act ctg gac cac agt ggg gcg acg gcg aca ggg ggc gcc 1179 Ser Pro Gln Thr Leu Asp His Ser Gly Ala Thr Ala Thr Gly Gly Ala 335 340 345 tcg gag ttg aag act cca gcc tcc tca act gcg ccc ccc ata agc tcc 1227 Ser Glu Leu Lys Thr Pro Ala Ser Ser Thr Ala Pro Pro Ile Ser Ser 350 355 360 ggg ccc ggg gcg ctg gcc tct gtg ccc gcc tct cac ccg gca cac ggc 1275 Gly Pro Gly Ala Leu Ala Ser Val Pro Ala Ser His Pro Ala His Gly 365 370 375 ttg gca ccc cac gag tcc cag ctg cac ctg aaa ggg gac ccc cac tac 1323 Leu Ala Pro His Glu Ser Gln Leu His Leu Lys Gly Asp Pro His Tyr 380 385 390 tcc ttc aac cac ccg ttc tcc atc aac aac ctc atg tcc tcc tcg gag 1371 Ser Phe Asn His Pro Phe Ser Ile Asn Asn Leu Met Ser Ser Ser Glu 395 400 405 410 cag cag cat aag ctg gac ttc aag gca tac gaa cag gca ctg caa tac 1419 Gln Gln His Lys Leu Asp Phe Lys Ala Tyr Glu Gln Ala Leu Gln Tyr 415 420 425 tcg cct tac gc tct acg tg ccc gcc agc ctg cct cta gc agc gcc 1467 Ser Pro Tyr Gly Ser Thr Leu Pro Wing Ser Leu Pro Leu Gly Ser Wing 430 435 440 tcg gtg acc acc agg agc ccc atc gag ccc tca gcc ctg gag ccg gcg 1515 Ser Val Thr Thr Arg Ser Pro Ile Glu Pro Ser Wing Leu Glu Pro Wing 445 450 455 tac tac caa ggt gtg tat tcc aga ccc gtc cta aac act tcc tg 1560 Tyr Tyr Gln Gly Val Tyr Ser Arg Pro Val Leu Asn Thr Ser 460 465 470 ctcccgggac tggggttt gtctggcata gccatgctgg tagcaagaga gaaaaaatca 1620 aaaaaccaaaaaaccg aaaaaatcc storm 1680 ttttattca ttttcatgc acaactttc ccccagtgca aaagactgtt actttattat 1740 tgtattcaa attcattgtg tatattacta caagacaac cccaaccaa ttttttccct 1800 gcgaagttta atgatccaca agtgtatata tgaaattctc ctccttcctt gcccccctct 1860 ctttcttccc tctttcccct ccagacattc tagttgtgg agggttattt aaaaaaaaaa 1920 aaaaggaaga tggtcaagtt tgtaaaat ttgtttgtgc ttttccccc tccttacctg 1980 accccctacg agtttacagg tctgtggcaa tactcttaac cataagaatt gaaatggtga 2040 agaaacaagt atacactaga ggctcttaaa agtattgaaa gacaatactg ctgttatata 2100 gcaagacata aacagattat aaacatcaga gccatttgct tctcagttta catttctgat 2160 acatgcagat agcagatgtc tttaaatgaa atacatgtat attgtgtatg gacttaatta 2220 tgcacatgct cagatgtgta gacatcctcc gtatatttac ataacatata gaggtaatag 2280 ataggtgata tacatgatac attctcaaga gttgcttgac cgaaagttac aaggacccca 2340 acccctttgt cctctctacc cacagatggc cctgggaatc aattcctcag gaattgccct 2400 caagaactct gcttcttgct ttgcagagtg ccatggtcat gtcattctga ggtcacataa 2460 cacataaaat tagttcttat gagtgtatac catttaaaga attttttttt cagtaaaagg 2520 gatattaca atgttggagg agagataagt tagggagc tggatttca aacgtggtcc 2580 aagattcaaa aatcctattg atagtggcca ttttaatcat tgccatcgtg tgcttgtttc 2640 atccagtgtt atgcactttc cacagttgga catgtgtta gtatagccag acgggtttca 2700 attattc tctttgctt ctcaatgtta attattgca tggtttattc tttttctt 2760 cagctgaaat tgctttaaat gatggttaaa attacaaatt aaattgttaa ttttttacaa 2820 tgtgattgta atttaaaaata ttttgattta aaaaaaaaaataccag attttaagcc 2880 gtggaaaatg ttctgatca tttgcagtta aggacttta aataatcaa tgttaacaa 2940 agagcatttc tgttatttt tttcacttaa ctaaatccga agtgaatatt tctgaatacg 3000 atatttttca attctagaa ctgaataataa atgacaaaa tgaaaataa attgttttgt 3060 ctgttgttat atgaatgtg tagctagtaa aaggagtga aagaattca agtaagtgt 3120 ataagttgat ttaatattcc aagagttgag atttttaaga ttctttattc ccagtgatgt 3180 ttacttcatt tttttttt ttttgacac cggctagc cttctgtgtt tcctttgagc 3240 cttttcacta caaaatcaaa tattaattta actacctttc ctccttcccc aatgtatcac 3300 ttttctttat ctgagaattc ttccaatgaa aataaaatat cagctgtggc tgatagaatt 3360 aagttgtgtc caa 3373 <210> 4 <211> 472 <212> PRT <213> Homo sapiens <400> 4 Met Leu Gly Thr Val Lys Met Glu Gly His Glu Thr Ser Asp Trp Asn 1 5 10 15 Ser Tyr Tyr Ala Asp Thr Gln Glu Ala Tyr Ser Ser Val Pro Val Ser 20 25 30 Asn Met Asn Ser Gly Leu Gly Ser Met Asn Ser Met Asn Thr Tyr Met 35 40 45 Thr Met Asn Thr Met Thr Thr Ser Gly Asn Met Thr Pro Ala Ser Phe 50 55 60 Asn Met Ser Tyr Ala Asn Pro Gly Leu Gly Ala Gly Leu Ser Pro Gly 65 70 75 80 Ala Val Ala Gly Met Pro Gly Gly Ser Ala Gly Ala Met Asn Ser Met 85 90 95 Thr Ala Ala Gly Val Thr Ala Met Gly Thr Ala Leu Ser Pro Ser Gly 100 105 110 Met Gly Ala Met Gly Ala Gln Gln Ala Ala Ser Met Asn Gly Leu Gly 115 120 125 Pro Tyr Ala Ala Ala Met Asn Pro Cys Met Ser Pro Met Ala Tyr Ala 130 135 140 Pro Ser Asn Leu Gly Arg Ser Arg Ala Gly Gly Gly Gly Asp Ala Lys 145 150 155 160 Thr Phe Lys Arg Ser Tyr Pro His Ala Lys Pro Pro Tyr Ser Tyr Ile 165 170 175 Ser Leu Ile Thr Met Ala Ile Gln Gln Ala Pro Ser Lys Met Leu Thr 180 185 190 Leu Ser Glu Ile Tyr Gln Trp Ile Met Asp Leu Phe Pro Tyr Tyr Arg 195 200 205 Gln Asn Gln Gln Arg Trp Gln Asn Ser Ile Arg His Ser Leu Ser Phe 210 215 220 Asn Asp Cys Phe Val Lys Val Ala Arg Ser Pro Asp Lys Pro Gly Lys 225 230 235 240 Gly Ser Tyr Trp Thr Leu His Pro Asp Ser Gly Asn Met Phe Glu Asn 245 250 255 Gly Cys Tyr Leu Arg Arg Gln Lys Arg Phe Lys Cys Glu Lys Gln Pro 260 265 270 Gly Ala Gly Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Gly Ala Lys 275 280 285 Gly Gly Pro Glu Ser Arg Lys Asp Pro Ser Gly Ala Ser Asn Pro Ser 290 295 300 Ala Asp Ser Pro Leu His Arg Gly Val His Gly Lys Thr Gly Gln Leu 305 310 315 320 Glu Gly Ala Pro Ala Pro Gly Pro Ala Ala Ser Pro Gln Thr Leu Asp 325 330 335 His Ser Gly Ala Thr Ala Thr Gly Gly Ala Ser Glu Leu Lys Thr Pro 340 345 350 Ala Ser Ser Thr Ala Pro Pro Ile Ser Ser Gly Pro Gly Ala Leu Ala 355 360 365 Ser Val Pro Ala Ser His Pro Ala His Gly Leu Ala Pro His Glu Ser 370 375 380 Gln Leu His Leu Lys Gly Asp Pro His Tyr Ser Phe Asn His Pro Phe 385 390 395 400 Ser Ile Asn Asn Leu Met Ser Ser Ser Glu Gln Gln His Lys Leu Asp 405 410 415 Phe Lys Ala Tyr Glu Gln Ala Leu Gln Tyr Ser Pro Tyr Gly Ser Thr 420 425 430 Leu Pro Ala Ser Leu Pro Leu Gly Ser Ala Ser Val Thr Thr Arg Ser 435 440 445 Pro Ile Glu Pro Ser Ala Leu Glu Pro Ala Tyr Tyr Gln Gly Val Tyr 450 455 460 Ser Arg Pro Val Leu Asn Thr Ser 465 470 <210> 5 <211> 25 <212> RNA <213> Artificial sequence <220> <223> siRNA BACH1-1 <400> 5 ggucaaagga cuuucacaac auuaa 25 <210> 6 <211> 25 <212> RNA <213> Artificial sequence <220> <223> siRNA BACH1-2 <400> 6 gggcaccagg gaagauagua guguu 25 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ACTB primer forward <400> 7 atttgcggtg gacgatggag 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ACTB primer reverse <400> 8 agagatggcc acggctgctt 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> BACH1 primer forward <400> 9 aatcgtaggc caggctgatg 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> BACH1 primer reverse <400> 10 agcagtgtag gcaaactgaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CDH1 primer forward <400> 11 tcctggcctc agaagacaga 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CDH1 primer reverse <400> 12 ccttggccag tgatgctgta 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> OCLN primer forward <400> 13 gagttgacag tcccatggca 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> OCLN primer reverse <400> 14 ctgaagtcat ccacaggcga 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> FOXA1 primer forward <400> 15 ggtggctcca ggatgttagg 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> FOXA1 primer reverse <400> 16 cccaggcctg agttcatgtt 20 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <220> <223> VIM primer forward <400> 17 ggaccagcta accaacgaca 20 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <220> <223> VIM primer reverse <400> 18 gggtgttttc ggcttcctct 20 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SNAI2 primer forward <400> 19 caacgcctcc aaaaagccaa 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> SNAI2 primer reverse <400> 20 acagtgatgg ggctgtatgc 20 <210> twenty one <211> twenty three <212> RNA <213> Artificial sequence <220> <223> guide RNA BACH1-1 <400> twenty one ccgcgcucac cgguccgugc ugg 23 <210> twenty two <211> twenty three <212> RNA <213> Artificial sequence <220> <223> guide RNA BACH1-2 <400> twenty two ccacucaaga aucguaggcc agg 23 <210> twenty three <211> 65 <212> DNA <213> Artificial sequence <220> <223> hg19 <400> 23 ccgcgctcac cggtccgtgc tggcggcatg cagcagttac ttccactcaa gaatcgtagg 60 ccagg 65 <210> 24 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> sgBACH1-1 <400> 24 ccgcgctcac cggcatgcag cagttacttc cactcaagaa tcgtaggcca gg 52 <210> 25 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> sgBACH1-2 <400> 25 ccgcgctcac cggtccgtgc tggcggcatg cagcagttac ttccactcaa agaatcgtag 60 gccagg 66

Claims

1. Use of a primer or probe for measuring the expression level of the BACH1 gene or an antibody for measuring the expression level of the BACH1 protein in the manufacture of an article for in vitro evaluation of the epithelial-mesenchymal transition ability of pancreatic cancer.

2. Use of a combination of a primer or probe for measuring the expression level of the BACH1 gene or an antibody for measuring the expression level of the BACH1 protein and a primer or probe for measuring the expression level of the FOXA1 gene or an antibody for measuring the expression level of the FOXA1 protein in the manufacture of an article for in vitro evaluation of the epithelial-mesenchymal transition ability of pancreatic cancer.

3. Use of a primer or probe for measuring the expression level of the BACH1 gene or an antibody for measuring the expression level of the BACH1 protein in the manufacture of an article for in vitro evaluation of the metastatic ability or invasive ability of pancreatic cancer.

4. Use of a combination of a primer or probe for measuring the expression level of the BACH1 gene or an antibody for measuring the expression level of the BACH1 protein and a primer or probe for measuring the expression level of the FOXA1 gene or an antibody for measuring the expression level of the FOXA1 protein in the manufacture of an article for in vitro evaluation of the metastatic ability or invasive ability of pancreatic cancer.

5. Use of a primer or probe for measuring the expression level of the BACH1 gene or an antibody for measuring the expression level of the BACH1 protein in the manufacture of an article for in vitro prediction of the prognosis of pancreatic cancer.

6. Use of a combination of a primer or probe for measuring the expression level of the BACH1 gene or an antibody for measuring the expression level of the BACH1 protein and a primer or probe for measuring the expression level of the FOXA1 gene or an antibody for measuring the expression level of the FOXA1 protein in the manufacture of an article for in vitro prediction of the prognosis of pancreatic cancer.

7. A method for screening a pancreatic cancer metastasis inhibitor, wherein, comprises: a step of adding a pharmaceutical candidate substance to a cell expressing the BACH1 gene or a reporter gene linked to the promoter of the BACH1 gene; a step of measuring the expression level of the BACH1 gene or the reporter gene; and a step of selecting a substance that reduces the expression level.

8. Use of an agent for knocking out the BACH1 gene in the manufacture of a pancreatic cancer metastasis inhibitor, wherein the agent is siRNA for knocking out the BACH1 gene, and the base sequence of the siRNA is the base sequence described in SEQ ID NO: 5 or SEQ ID NO:

6.

9. The use in the manufacture of a pancreatic cancer metastasis inhibitor according to claim 8, wherein, the agent inhibits the epithelial-mesenchymal transition ability of pancreatic cancer cells.

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