Using microRNAs as biomarkers for pancreatic cancer

Abstract

One purpose of the present invention is to provide a novel use of a microRNA as a pancreatic cancer biomarker.　A use of a microRNA in a biological sample derived from a subject, said microRNA containing a nucleotide sequence represented by any of SEQ ID NOS: 1 to 15, as a pancreatic cancer biomarker, wherein the pancreatic cancer biomarker is to be used for determining whether a subject suffers from pancreatic cancer or not, for determining the clinical conditions of a subject suffering or once suffered from pancreatic cancer, or identifying a test substance which can be used in treating pancreatic cancer.

Description

[Technical Field]

[0001] The present disclosure relates to the use of microRNA as a biomarker for pancreatic cancer.The present disclosure also relates to a method for selecting microRNA for determining whether a subject has pancreatic cancer, a method for determining whether a subject has pancreatic cancer, a method for determining the pathology of a subject who has or has previously had pancreatic cancer, and a method for identifying a test substance that can treat pancreatic cancer.The present disclosure also relates to a kit for use in the above-mentioned use or method, a biomarker for detecting pancreatic cancer or for monitoring the effect of pancreatic cancer treatment, and a composition for detecting pancreatic cancer. [Background technology]

[0002] Pancreatic cancer is one of the malignant tumors with an extremely poor prognosis. The importance of pancreatic juice cytology by endoscopic retrograde cholangiopancreatography (ERCP) for patients with focal pancreatic duct strictures has been reported. However, its diagnostic accuracy varies between institutions, ranging from 30% to 84.7%, and its usefulness is not consistent.

[0003] There have been reports on the use of exosomes as biomarkers for diagnosing pancreatic cancer. Non-Patent Document 1 presents a method for detecting Glypican-1-positive exosomes in the serum of pancreatic cancer patients using flow cytometry, with 100% sensitivity and 100% specificity.

[0004] Non-Patent Document 2 reports a method for detecting KRAS mutations from DNA derived from exosomes in the serum of pancreatic cancer patients, and reports that the mutations were found in 66.7% of resectable pancreatic cancer cases. Non-Patent Document 3 reports a study focusing on exosomes in the pancreatic juice of pancreatic cancer patients. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nature 2015;523:177-182 [Non-patent document 2] Ann Oncol 2017;28:741-747 [Non-patent document 3] Ann Surg Oncol 2019;26:2104-2111 Summary of the Invention [Problem to be solved by the invention]

[0006] The method described in Non-Patent Document 1 has problems such as the small number of cases of early-stage pancreatic cancer that truly contribute to extending prognosis, and follow-up studies using large-scale cohorts are considered necessary. In the invention described in Non-Patent Document 2, a positive rate of 7.4% was observed even in healthy individuals, and the sensitivity was 75.4%. The invention described in Non-Patent Document 2 has issues with its specificity and sensitivity. The invention described in Non-Patent Document 3 analyzes known microRNAs (miR-21 and miR-155).

[0007] One objective of the present disclosure is to provide a novel use of microRNA as a biomarker for pancreatic cancer. Another objective of the present disclosure is to provide a novel method for selecting microRNA to determine whether a subject has pancreatic cancer, a novel method for determining whether a subject has pancreatic cancer, a novel method for determining the pathology of a subject who has or has previously had pancreatic cancer, and a novel method for identifying a test substance that can treat pancreatic cancer. Another objective of the present invention is to provide a novel kit for use in the above uses or methods, a novel biomarker for detecting pancreatic cancer or for monitoring the effect of pancreatic cancer treatment, and a novel composition for detecting pancreatic cancer. [Means for solving the problem]

[0008] One aspect of the present disclosure provides use of at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15 in a biological sample derived from a subject as a biomarker for pancreatic cancer, wherein the biomarker for pancreatic cancer is used to determine whether a subject has pancreatic cancer, to determine the pathology of a subject who is or has had pancreatic cancer, or to identify a test substance that can treat pancreatic cancer.

[0009] One aspect of the present disclosure provides a method for selecting microRNAs for determining whether or not a subject has pancreatic cancer, the method comprising selecting, from group A of microRNAs in a biological sample derived from a subject with pancreatic cancer, group B of microRNAs in a culture medium in which pancreatic cancer cells have been cultured, and group C of microRNAs in a biological sample derived from a subject showing no symptoms related to pancreatic cancer, a microRNA that is common to group A of microRNAs and group B of microRNAs but not common to group C of microRNAs.

[0010] One aspect of the present disclosure provides a method for determining whether a subject is suffering from pancreatic cancer, the method comprising: comparing the amount of at least one microRNA comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 15 in a biological sample derived from the subject with at least one threshold value corresponding to the at least one microRNA; and determining that the subject is suffering from pancreatic cancer if the amount of the at least one microRNA is greater than the at least one threshold value.

[0011] One aspect of the present disclosure provides a method for determining the pathological condition of a subject suffering from or having suffered from pancreatic cancer, the method comprising: comparing a first amount of at least one type of microRNA comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 15 in a first biological sample derived from the subject with a second amount of the at least one type of microRNA in a second biological sample from the subject; and determining that the condition of the subject has improved if the first amount is smaller than the second amount; wherein the first biological sample is collected after the second biological sample is collected.

[0012] One aspect of the present disclosure provides a method for identifying a test substance capable of treating pancreatic cancer, the method comprising: administering the test substance to a subject suffering from pancreatic cancer; comparing a first amount of at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1-15 in a first biological sample derived from the subject after administration of the test substance with a second amount of the at least one microRNA in a second biological sample derived from the subject before administration of the test substance; and identifying the test substance as a test substance capable of treating pancreatic cancer if the first amount is smaller than the second amount.

[0013] One embodiment of the present disclosure provides a biomarker for detecting pancreatic cancer in a biological sample from a subject, the biomarker being at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15. One embodiment of the present disclosure provides a biomarker for monitoring the effectiveness of a pancreatic cancer treatment in a serum or plasma sample from a subject, the biomarker being at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 5, 9, and 15.

[0014] One aspect of the present disclosure provides a kit comprising a reagent for measuring at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15, for use as a biomarker for a microRNA according to the present disclosure or for use in a method according to the present disclosure.

[0015] One embodiment of the present disclosure provides a composition for detecting pancreatic cancer, comprising a reagent for measuring at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a bar graph showing the results of real-time PCR for microRNA miR-6858-5p. [Figure 2] FIG. 2 is a bar graph showing the results of real-time PCR for microRNA miR-4516 (SEQ ID NO: 1). [Figure 3] FIG. 3 is a bar graph showing the results of real-time PCR for microRNA miR-4674 (SEQ ID NO: 2). [Figure 4] FIG. 4 is a bar graph showing the results of real-time PCR for microRNA miR-6800-5p (SEQ ID NO: 3). [Figure 5] FIG. 5 is a bar graph showing the results of real-time PCR for microRNA miR-149-3p (SEQ ID NO: 4). [Figure 6] FIG. 6 is a bar graph showing the results of real-time PCR for microRNA miR-3621 (SEQ ID NO: 5). [Figure 7] FIG. 7 is a bar graph showing the results of real-time PCR for microRNA miR-4484 (SEQ ID NO: 6). [Figure 8] FIG. 8 is a bar graph showing the results of real-time PCR for microRNA miR-3940-5p (SEQ ID NO: 9). [Figure 9] FIG. 9 is a bar graph showing the results of real-time PCR for the microRNA miR-3656 (SEQ ID NO: 15). [Figure 10] FIG. 10 is a bar graph showing the results of real-time PCR for microRNA miR-4516 (SEQ ID NO: 1) in pancreatic juice samples. [Figure 11] FIG. 11 is a bar graph showing the results of real-time PCR for microRNA miR-4674 (SEQ ID NO: 2) in pancreatic juice samples. [Figure 12] FIG. 12 is a bar graph showing the results of real-time PCR for the microRNA miR-4516 (SEQ ID NO: 1) in multiple pancreatic juice samples. [Figure 13] FIG. 13 is a bar graph showing the results of real-time PCR for microRNA miR-4674 (SEQ ID NO: 2) in multiple pancreatic juice samples. [Figure 14] Figure 14A is a graph of the receiver operating characteristic (ROC) curve for miR-4516 (SEQ ID NO: 1) and Figure 14B is a graph of the receiver operating characteristic (ROC) curve for miR-4674 (SEQ ID NO: 2) in pancreatic juice samples. [Figure 15] FIG. 15 is a bar graph showing the results of real-time PCR for microRNA miR-4516 (SEQ ID NO: 1) in serum samples. [Figure 16] FIG. 16 is a bar graph showing the results of real-time PCR for microRNA miR-4674 (SEQ ID NO: 2) in serum samples. [Figure 17] FIG. 17 is a bar graph showing the expression levels of miR-4516 (SEQ ID NO: 1) in serum samples taken from PDAC-8 before and after surgery. [Figure 18] FIG. 18 is a bar graph showing the expression levels of miR-4674 (SEQ ID NO: 2) in serum samples taken from PDAC-4 before and after surgery. [Figure 19] FIG. 19 is a bar graph showing the expression levels of miR-4516 (SEQ ID NO: 1) in multiple serum samples. [Figure 20] FIG. 20 is a graph of the receiver operating characteristic (ROC) curve for miR-4516 (SEQ ID NO: 1) in multiple serum samples. DETAILED DESCRIPTION OF THE INVENTION

[0017] [Use as a biomarker for pancreatic cancer] One aspect of the present disclosure provides use of at least one microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15 in a biological sample derived from a subject as a biomarker for pancreatic cancer. In one embodiment, the biomarker for pancreatic cancer is used to determine whether a subject is suffering from pancreatic cancer. In one embodiment, the biomarker for pancreatic cancer is used to determine the pathological condition of a subject suffering from or having suffered from pancreatic cancer. In one embodiment, the biomarker for pancreatic cancer is used to identify a test substance capable of treating pancreatic cancer.

[0018] The term "pancreatic cancer" refers to cancer that originates in the pancreas. There are three main treatment methods for pancreatic cancer: surgery, radiation therapy, and chemotherapy. Surgery for pancreatic cancer involves, but is not limited to, removing the pancreas depending on the location of the cancer. Radiation therapy for pancreatic cancer includes, but is not limited to, external irradiation of the pancreatic cancer or intraoperative irradiation of the pancreatic cancer site. Chemotherapy for pancreatic cancer includes, but is not limited to, intravenous administration of anticancer drugs. Other treatment methods for pancreatic cancer include immunotherapy and gene therapy.

[0019] The term "biomarker for pancreatic cancer" refers to an entity that reflects information about pancreatic cancer. The information about pancreatic cancer may be, for example, information related to the presence or pathology of pancreatic cancer in a human or non-human mammal, or information for identifying a substance that can treat pancreatic cancer. The information about pancreatic cancer may be, for example, the concentration or content of a microRNA according to the present disclosure in a biological sample derived from a human or non-human mammal.

[0020] The term "subject" refers to a human or non-human mammal. The term "subject suffering from pancreatic cancer" refers to a human or non-human mammal who has been diagnosed with pancreatic cancer by a person with expertise, such as a physician or veterinarian, based on, for example, known diagnostic criteria. A subject suffering from pancreatic cancer may be a subject before or after undergoing treatment, such as surgery, radiation therapy, and chemotherapy. The term "subject suffering from pancreatic cancer" refers to a human or non-human mammal after undergoing treatment for pancreatic cancer. A subject suffering from pancreatic cancer may be a human or non-human mammal who has been diagnosed with pancreatic cancer by a person with expertise, such as a physician or veterinarian, based on, for example, known diagnostic criteria, and who has been cured of pancreatic cancer. The non-human mammal may be, for example, a mouse, rat, rabbit, dog, sheep, pig, or non-human primate. The subject is preferably a human. The subject may be, for example, a human or non-human mammal diagnosed with pancreatic cancer or chronic pancreatitis based on known diagnostic criteria. The subject may be, for example, a human or non-human mammal without symptoms associated with pancreatic cancer. The subject is preferably a human.

[0021] The term "biological sample" refers to a composition obtained from a subject that may contain at least one microRNA to be measured. Biological samples may be, for example, pancreatic juice samples, serum or plasma samples, stool samples, duodenal juice samples, or bile samples. Pancreatic juice samples may be pancreatic juice collected from a subject, or may be a sample that has been treated to separate and / or concentrate exosomes in the pancreatic juice that may contain at least one microRNA to be measured. The method for collecting pancreatic juice is not particularly limited, and it can be collected using known methods (e.g., a method of collecting the pancreatic juice by intubating the pancreatic duct). Serum or plasma samples may be serum or plasma obtained by removing blood cell components from whole blood collected from a subject using known methods such as centrifugation, or may be a sample that has been treated to separate and / or concentrate exosomes in the plasma or serum that may contain at least one microRNA to be measured. The stool sample, duodenal fluid sample, or bile sample may be stool, duodenal fluid, or bile collected from a subject, or may be a sample that has been treated to separate and / or concentrate exosomes that may contain at least one type of microRNA to be measured in stool, duodenal fluid, or bile.

[0022] The method for collecting pancreatic juice is not particularly limited and can be performed using known methods. The biological sample may contain additives to the extent that they do not interfere with the measurement of at least one type of microRNA to be measured. Such additives may be, for example, a buffer, a nuclease inhibitor (e.g., a DNase inhibitor and an RNase inhibitor), a pH adjuster, a surfactant, and a chelating agent, or a combination thereof.

[0023] In one embodiment, the use of the at least one microRNA as a biomarker for pancreatic cancer may include preparing a pancreatic juice sample, a serum or plasma sample, a stool sample, a duodenal juice sample, or a bile sample from pancreatic juice, serum or plasma, stool, duodenal juice, or bile fluid collected from a subject. In one embodiment, the biological sample is a pancreatic juice sample or a serum or plasma sample. In one embodiment, the biological sample is a pancreatic juice sample or a serum sample. In one embodiment, the biological sample is a pancreatic juice sample. In one embodiment, the biological sample is a serum or plasma sample, preferably a serum sample, for at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0024] The term "exosome" refers to a small vesicular granule of approximately 100 nm surrounded by a lipid bilayer membrane. Exosomes are not distinguished by their mechanism of origin or size. Exosomes in pancreatic juice, serum or plasma, feces, duodenal juice, or bile can be isolated and / or concentrated by known methods, such as centrifugation, or by commercially available extraction kits.

[0025] The term "microRNA" refers to RNA consisting of 16 to 25 bases. MicroRNAs are contained, for example, in exosomes and present in body fluids or blood. The microRNA to be measured comprises or consists of a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15. At least one microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15 can serve as a marker for pancreatic cancer. MicroRNAs can be isolated and / or concentrated using known methods or commercially available extraction columns. "MicroRNA consisting of" a predetermined nucleotide sequence means that the major component is composed solely of polynucleotides constituting the predetermined nucleotide sequence and does not contain other nucleotides. In one example, microRNA consisting of a predetermined nucleotide sequence contains only polynucleotides constituting the predetermined nucleotide sequence as nucleotide components and may further contain sugar chains, methyl groups, and other adducts.

[0026] The term "comprising" means that the recited elements and / or steps are present, and that other elements and / or steps may be added. The term "consisting essentially of" means that the recited elements and / or steps are present, and that other elements and / or steps may be added to the extent that they do not affect the novel technical features of the compositions, kits, and methods. For example, a microRNA "consisting essentially of" a given nucleotide sequence may further include 5 to 1 base, e.g., 3, 2, or 1 base, at the border of the given nucleotide sequence.

[0027] The at least one type of microRNA may be one type of microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15, or may be a combination of two or more types. The at least one type of microRNA may be a microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15, for example, a microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 5, 9, and 15, preferably a microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 3, 5, and 15, and more preferably a microRNA comprising or consisting of a nucleotide sequence set forth in SEQ ID NO: 1 or 2.

[0028] At least one microRNA in a biological sample may be, for example, a microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 15 in a pancreatic juice sample, serum, or plasma sample, or a combination of two or more types. In one embodiment, at least one microRNA in a biological sample may be, for example, a microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 5, 9, and 15 in a pancreatic juice sample, serum, or plasma sample, for example, a microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 3, 5, and 15, preferably a nucleotide sequence set forth in SEQ ID NOS: 1 or 2, or a combination of two or more types. In one embodiment, at least one microRNA in a biological sample may be, for example, a microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 3, 5, and 15 in a pancreatic juice sample, or a combination of two or more types. In one embodiment, at least one microRNA in a biological sample may be, for example, a microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3 to 5, 9, and 15 in a serum or plasma sample, or a combination of two or more types. In one embodiment, the at least one microRNA in the biological sample may be one of the microRNAs comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 in a serum or plasma sample.

[0029] Using microRNAs as biomarkers to determine whether someone has pancreatic cancer The use of microRNA as a biomarker for pancreatic cancer to determine whether a subject is suffering from pancreatic cancer includes comparing the amount of at least one type of microRNA containing or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15 in a biological sample derived from the subject with at least one threshold value corresponding to the at least one type of microRNA, and determining that the subject is suffering from pancreatic cancer if the amount of the at least one type of microRNA is greater than the at least one threshold value.

[0030] The term "microRNA amount" may refer to, for example, the content or concentration of the microRNA to be measured in a pancreatic juice sample, serum or plasma sample, stool sample, duodenal juice sample, or bile sample per unit amount. The amount of microRNA can be measured by a method that can quantitatively measure nucleotides having a specific sequence (e.g., quantitative PCR and immunoenzymatic assay). The amount of microRNA may be the signal intensity obtained by the measurement method, or the content or concentration calculated from the signal intensity obtained from a biological sample using the signal intensity obtained from a control sample with a known concentration.

[0031] In one embodiment, the use of the present invention as a pancreatic cancer biomarker for determining whether a subject has pancreatic cancer may include measuring the amount of at least one microRNA in a biological sample from the subject. The amount of microRNA can be measured, for example, by quantitative PCR or enzyme-linked immunosorbent assay (ELISA).

[0032] The term "threshold" refers to a value set for determining whether or not a subject has pancreatic cancer based on the amount of a microRNA to be measured. The threshold can be set appropriately depending on the type of microRNA to be measured, the type of sample, gender, age, and race. The threshold may be, for example, a value (diagnostic threshold) for distinguishing between pancreatic cancer and healthy subjects or chronic pancreatitis patients. The diagnostic threshold can be set, for example, by measuring the amount of the microRNA to be measured in a group of pancreatic cancer patients and the amount of the microRNA in a group of healthy subjects or chronic pancreatitis patients, and taking into account the false negative rate, false positive rate, cost, and prevalence. The threshold may be set using, for example, a receiver operator characteristic curve (ROC curve). The threshold may also be set empirically, for example.

[0033] The term "determination" can be performed automatically or mechanically without relying on the judgment of a person with specialized knowledge such as a doctor or a laboratory technician. Therefore, the determination is performed automatically or mechanically by comparing the amount (measured value) of the microRNA to be measured with a threshold value corresponding to that microRNA. For example, when the measured value is greater than the threshold value, the subject can be determined to have pancreatic cancer. In another example, when the measured value is less than the threshold value, the subject can be determined to have a low possibility of having pancreatic cancer.

[0034] When the threshold is normalized to zero, the comparison between the amount of the microRNA and the threshold may be whether or not the microRNA is present. In this example, the determination method may determine that the subject is suffering from pancreatic cancer if the microRNA is present.

[0035] The comparing may be a combination of, for example, comparing the amount of a microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 in two or more different biological samples (e.g., a pancreatic juice sample, or a serum or plasma sample) derived from a subject with a threshold value. In one embodiment, the comparing may include comparing a first amount of a first microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NO: 1) in a pancreatic juice sample derived from a subject with a first threshold value for the microRNA (SEQ ID NO: 1) for pancreatic juice; and comparing a second amount of a first microRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 in a serum sample derived from the subject with a second threshold value for the microRNA (SEQ ID NO: 1) for serum.

[0036] The comparing may be a combination of, for example, comparing the amount of a microRNA containing or consisting of two or more nucleotide sequences set forth in any of SEQ ID NOS: 1 to 15 in a specific biological sample (e.g., a pancreatic juice sample, or a serum or plasma sample) derived from a subject with a threshold value. In one embodiment, the comparing may include comparing the amount of a first microRNA consisting of a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 1) in a first pancreatic juice sample derived from the subject with a first threshold value for the first microRNA (SEQ ID NOS: 1) in the pancreatic juice; and comparing the amount of a second microRNA containing a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15 other than the SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 2) in a second pancreatic juice sample derived from the subject with a second threshold value for the second microRNA (SEQ ID NOS: 2) in the pancreatic juice.

[0037] The comparing may, for example, be a combination of comparing the amounts of two or more microRNAs containing or consisting of two or more nucleotide sequences set forth in any of SEQ ID NOS: 1 to 15 in two or more different biological samples (e.g., a pancreatic juice sample, or a serum or plasma sample) derived from a subject with a threshold value. In one embodiment, the comparing may include comparing the amount of a first microRNA consisting of a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 1) in a pancreatic juice sample derived from a subject with a first threshold value for the first microRNA (SEQ ID NOS: 1) for pancreatic juice; and comparing the amount of a second microRNA containing a nucleotide sequence set forth in any of SEQ ID NOS: 1 to 15 other than the SEQ ID NOS: 1 (e.g., SEQ ID NOS: 2) in a serum sample derived from the subject with a second threshold value for the second microRNA (SEQ ID NOS: 2) for serum.

[0038] In the above embodiment, the determining step may determine that the subject is suffering from pancreatic cancer if the first amount of the microRNA is greater than the first threshold and the second amount of the microRNA is greater than the second threshold. In the above embodiment, the determining step may determine that the subject requires re-examination or follow-up observation if the first amount of the microRNA is greater than the first threshold but the second amount of the microRNA is less than the second threshold. In the above embodiment, the determining step may determine that the subject is not suffering from pancreatic cancer if the first amount of the microRNA is less than the first threshold and the second amount of the microRNA is less than the second threshold.

[0039] Use of microRNAs as biomarkers for determining the disease state of subjects suffering from or having suffered from pancreatic cancer The use of microRNAs as biomarkers for determining the pathological condition of a subject suffering from or having suffered from pancreatic cancer includes comparing a first amount of at least one microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 in a first biological sample derived from the subject with a second amount of the at least one microRNA in a second biological sample derived from the subject, and determining that the subject's pathological condition has improved if the first amount is smaller than the second amount, wherein the first biological sample is measured after measuring the second biological sample. In one embodiment, the present invention provides the use of microRNAs as biomarkers for determining the pathological condition of a subject suffering from pancreatic cancer.

[0040] The first biological sample is preferably the same type of biological sample as the second biological sample. In one example, when the first biological sample is a pancreatic juice sample, the second biological sample is also a pancreatic juice sample. In another example, when the first biological sample is a serum sample, the second biological sample is also a serum sample. In another example, the first biological sample may be a different type of biological sample from the second biological sample. In one example, when the first biological sample is a pancreatic juice sample, the second biological sample may be a serum sample.

[0041] For example, the first biological sample may be collected after a subject suffering from or having suffered from pancreatic cancer has undergone treatment such as surgery, radiation therapy, and chemotherapy. In this example, the second biological sample may be collected before the treatment. The interval between the collection of the first biological sample and the collection of the second biological sample may be, for example, 2 weeks, 1 month, 2 months, or 3 months. In another example, the first biological sample and the second biological sample may each be collected after the treatment.

[0042] The "condition" of a subject refers to the state of pancreatic cancer in a human or non-human mammal that is or has been affected by pancreatic cancer. In one example, the condition of a subject can be a state of remission of pancreatic cancer in a human or non-human mammal that has been affected by pancreatic cancer.

[0043] For example, when the first amount is smaller than the second amount, the subject's condition can be determined to be improved. In another example, when the first amount is larger than the second amount, the subject's condition can be determined to be worsened. In another example, when the first amount is equal to the second amount, the subject's condition can be determined to be stable.

[0044] The comparing may, for example, be a combination of comparing a first amount of at least one microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 in two or more first biological samples (e.g., pancreatic juice samples, or serum or plasma samples) from a subject with a second amount of the at least one microRNA in a second biological sample from the subject. In one embodiment, the comparing may include comparing a first amount of a microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 1) in a first pancreatic juice sample from a subject with a second amount of the microRNA (SEQ ID NOS: 1) in a second pancreatic juice sample from the subject; and comparing a third amount of a microRNA consisting of the nucleotide sequence set forth in SEQ ID NOS: 1 in a first serum sample from a subject with a fourth amount of the microRNA (SEQ ID NOS: 1) in a second serum sample from the subject.

[0045] The comparing may, for example, be a combination of comparing a first amount of microRNA containing or consisting of two or more nucleotide sequences set forth in any one of SEQ ID NOS: 1-15 in a first biological sample (e.g., a pancreatic juice sample, or a serum or plasma sample) from a subject with a second amount of the microRNA in a second biological sample (a pancreatic juice sample) of the same type from the subject. In one embodiment, the comparing may include comparing a first amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 1) in a first pancreatic juice sample from the subject with a second amount of the microRNA (SEQ ID NOS: 1) in a second pancreatic juice sample from the subject; and comparing a third amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2) in the first pancreatic juice sample from the subject with a fourth amount of the microRNA (SEQ ID NOS: 2) in the second pancreatic juice sample from the subject.

[0046] The comparing may, for example, be a combination of comparing a first amount of microRNA containing or consisting of two or more nucleotide sequences set forth in any one of SEQ ID NOS: 1-15 in two or more first biological samples (e.g., pancreatic juice samples, or serum or plasma samples) from a subject with a second amount of the microRNA in a second biological sample from the subject. In one embodiment, the comparing may include comparing a first amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 1) in a first pancreatic juice sample from the subject with a second amount of the microRNA (SEQ ID NOS: 1) in a second pancreatic juice sample from the subject; and comparing a third amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2) in a first serum sample from the subject with a fourth amount of the microRNA (SEQ ID NOS: 2) in a second serum sample from the subject.

[0047] In the above embodiment, the determining step may determine that the condition of the subject suffering from pancreatic cancer has worsened if the first amount of the microRNA is greater than the second amount of the microRNA and the third amount of the microRNA is greater than the fourth threshold. In the above embodiment, the determining step may determine that the condition of the subject suffering from pancreatic cancer has not changed or requires follow-up observation if the first amount of the microRNA is greater than the second amount of the microRNA but the third amount of the microRNA is less than the fourth amount of the microRNA. In the above embodiment, the determining step may determine that the condition of the subject suffering from pancreatic cancer has improved if the first amount of the microRNA is less than the second amount of the microRNA and the third amount of the microRNA is less than the fourth amount of the microRNA.

[0048] Use of microRNAs as biomarkers to identify test agents that can treat pancreatic cancer Use of microRNA as a biomarker for identifying a test substance capable of treating pancreatic cancer includes administering the test substance to a subject suffering from pancreatic cancer; comparing a first amount of at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15 after administration of the test substance with a second amount of the at least one microRNA in a second biological sample from the subject before administration of the test substance; and identifying the test substance as a test substance capable of treating pancreatic cancer if the first amount is smaller than the second amount.

[0049] The term "test substance capable of treating pancreatic cancer" refers to a substance that is expected to reduce or maintain the progression of pancreatic cancer, or to cause regression of pancreatic cancer. A test substance capable of treating pancreatic cancer may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, or RNA. A test substance capable of treating pancreatic cancer may be, for example, a drug for treating a disease or cancer other than pancreatic cancer. The test substance may be, for example, one type, or a mixture of two or more types.

[0050] The test substance can be administered by known methods, for example, orally, by infusion, or by injection.

[0051] The term "subject suffering from pancreatic cancer" in this embodiment refers to a non-human mammal, which may be, for example, a mouse, rat, rabbit, dog, sheep, pig, or non-human primate (e.g., monkey and orangutan).

[0052] The first biological sample is preferably the same type of biological sample as the second biological sample. In one example, when the first biological sample is a pancreatic juice sample, the second biological sample is also a pancreatic juice sample. In another example, when the first biological sample is a serum sample, the second biological sample is also a serum sample.

[0053] The interval between the collection of the first biological sample and the collection of the second biological sample may be, for example, 1 hour, 3 hours, 12 hours, 1 day, 3 days, 1 week, or 1 month.

[0054] For example, the test substance capable of treating pancreatic cancer can be identified as a test substance capable of treating pancreatic cancer when the first amount is smaller than the second amount. In another example, the test substance capable of treating pancreatic cancer can be identified as a test substance capable of exacerbating pancreatic cancer when the first amount is greater than the second amount.

[0055] The comparing may, for example, be a combination of comparing a first amount of at least one microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 in two or more first biological samples (e.g., a pancreatic juice sample, or a serum or plasma sample) from a subject after administration of a test substance with a second amount of the at least one microRNA in a second biological sample from the subject before administration of the test substance. In one embodiment, the comparing may include comparing a first amount of a microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 1) in a first pancreatic juice sample from the subject after administration of the test substance with a second amount of the microRNA (SEQ ID NOS: 1) in a second pancreatic juice sample from the subject before administration of the test substance; and comparing a third amount of a microRNA consisting of the nucleotide sequence set forth in SEQ ID NOS: 1 in a first serum sample from the subject after administration of the test substance with a fourth amount of the microRNA (SEQ ID NOS: 1) in a second serum sample from the subject before administration of the test substance.

[0056] The comparison may be combined with, for example, comparing a first amount of microRNA containing or consisting of two or more nucleotide sequences set forth in any of SEQ ID NOs: 1 to 15 in a first biological sample (e.g., a pancreatic juice sample, or a serum or plasma sample) derived from a subject after administration of a test substance with a second amount of the microRNA in a second biological sample (e.g., a pancreatic juice sample) of the same type derived from the subject before administration of the test substance. In one embodiment, the comparing may include comparing a first amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NO: 1) in a first pancreatic juice sample from a subject after administration of a test substance with a second amount of the microRNA (SEQ ID NO: 1) in a second pancreatic juice sample from the subject before administration of the test substance; and comparing a third amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NO: 2) in the first pancreatic juice sample from the subject after administration of the test substance with a fourth amount of the microRNA (SEQ ID NO: 2) in the second pancreatic juice sample from the subject before administration of the test substance.

[0057] The comparison may be, for example, a combination of comparing a first amount of microRNA containing or consisting of two or more nucleotide sequences set forth in any of SEQ ID NOs: 1 to 15 in two or more first biological samples (e.g., pancreatic juice samples, or serum or plasma samples) derived from a subject after administration of a test substance with a second amount of the microRNA in a second biological sample derived from the subject before administration of the test substance. In one embodiment, the comparing may include comparing a first amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NO: 1) in a first pancreatic juice sample derived from the subject after administration of a test substance with a second amount of the microRNA (SEQ ID NO: 1) in a second pancreatic juice sample derived from the subject before administration of the test substance; and comparing a third amount of microRNA consisting of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NO: 2) in a first serum sample derived from the subject after administration of the test substance with a fourth amount of the microRNA (SEQ ID NO: 2) in a second serum sample derived from the subject before administration of the test substance.

[0058] Identification of the test substance may be, for example, identification of the test substance as a test substance capable of treating pancreatic cancer when the first amount is smaller than the second amount. In another example, identification of the test substance may be identification of the test substance as a test substance incapable of treating pancreatic cancer when the first amount is greater than the second amount.

[0059] The terms and explanations of embodiments (e.g., combination of biological sample and microRNA) in "Use as a biomarker for pancreatic cancer" and its exemplary embodiments "Use for determining whether a subject has pancreatic cancer," "Use for determining the pathological condition of a subject who has or has had pancreatic cancer," or "Use for identifying a test substance that can treat pancreatic cancer" are applicable among them as appropriate unless otherwise specified.

[0060] [Method for selecting microRNAs] One aspect of the present disclosure provides a method for selecting microRNAs for determining whether or not a subject has pancreatic cancer, the method comprising selecting, from group A of microRNAs in a biological sample derived from a subject with pancreatic cancer, group B of microRNAs in a culture medium in which pancreatic cancer cells have been cultured, and group C of microRNAs in a biological sample derived from a subject showing no symptoms related to pancreatic cancer, a microRNA that is common to group A of microRNAs and group B of microRNAs but not common to group C of microRNAs.

[0061] The term "pancreatic cancer cells" may refer to, for example, known pancreatic cancer cell lines. Pancreatic cancer cells may be commercially available or may be prepared by culturing pancreatic cancer cells collected from a subject suffering from pancreatic cancer. Pancreatic cancer cells may be, for example, one type of cell line or a combination or mixture of two or more cell lines. Examples of pancreatic cancer cells include KLM-1, MIA Paca2, Panc-1, PK-1, PK-45H, PK-45P, PK-59, PK-8, T3M-4, 58-Pan, Hc48, HPC-YT, PAN-1-JCK, PAN-2-JCK, PAN-4-JCK, PAN-5-JCK, PAN-6-JCK, PAN-7-JCK, PAN-8-JCK, PCI-6, PCI-10, PCI-19, PCI-24, PCI-35, PCI-43, PCI-55, PCI-64, PCI-66, PCI-68, PK-9, AsPc-1, Capan-1, Capan-2, DAN-G, FAMPAC, FAMPAC-A, BxPC-3, and MIAPaCa-2. In one embodiment, the pancreatic cancer cells comprise at least one selected from the group consisting of human pancreatic cancer cell lines Panc-1, BxPC-3, and MIAPaCa-2, or a combination or mixture of two or more of these.

[0062] The term "culturing pancreatic cancer cells" refers to maintaining or growing pancreatic cancer cells ex vivo. Pancreatic cancer cells can be cultured, for example, under known conditions using a known culture medium. The term "culture medium" refers to a liquid containing components necessary for cell culture. A culture medium can be prepared, for example, by mixing a predetermined amount of each component (solid) constituting the culture medium with an amount of purified water to achieve a predetermined concentration. The culture medium may be, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle's (BME), or DMEM / F12. The culture medium may further contain a predetermined additive (such as an antibiotic).

[0063] The term "group of microRNAs" includes information on the nucleotide sequences of at least two microRNAs. The group of microRNAs may further include information on the expression level of each microRNA. The group of microRNAs can be prepared, for example, by microarray analysis of microRNAs in a sample or culture medium. The group of microRNAs includes, for example, information on the nucleotide sequences of at least 100, 300, 500, 1000, 1500, or 2000 microRNAs.

[0064] The term "shared microRNA" means that at least two microRNAs are identical in terms of nucleotide sequence. In one example, shared microRNAs are at least two microRNAs that are 20 nucleotides long and that the types of nucleotides from the first to the twentieth are the same. The term "unshared microRNA" means that at least two microRNAs are different in terms of nucleotide sequence. In one example, unshared microRNAs are at least two microRNAs that are 18 nucleotides long and the other microRNA is 20 nucleotides long. In one example, unshared microRNAs are at least two microRNAs that are 20 nucleotides long and that the type of nucleotides from the first to the twentieth in one microRNA is different from the other microRNA at at least one position.

[0065] The term "subject without symptoms associated with pancreatic cancer" refers to a subject without symptoms suggestive of pancreatic cancer or whose symptoms do not reach a predetermined standard. Symptoms suggestive of pancreatic cancer can be examined, for example, by abdominal ultrasound, CT scan, MRI scan, endoscopic ultrasound (EUS), MR cholangiopancreatography (MRCP), or PET scan, or a combination thereof.

[0066] The term "select" or "selection" means selecting from a large number of microRNAs those that are suitable for a purpose, or removing from the large number of microRNAs those that are not suitable for a purpose. The term "selecting microRNAs" means selecting from a large number of microRNAs those microRNAs that are useful as indicators for determining whether a subject has pancreatic cancer, or removing from the large number of microRNAs those microRNAs that are not useful as indicators.

[0067] In one embodiment, selecting the microRNAs includes obtaining a first group of microRNAs that are common to the microRNA group A and the microRNA group B, and removing from the first group of microRNAs the microRNAs that are common to the microRNA group A, the microRNA group B, and the microRNA group C.

[0068] In one embodiment, selecting the microRNAs includes obtaining a second group of microRNAs from group A of microRNAs, excluding microRNAs that are common to group A of microRNAs and group C of microRNAs; obtaining a third group of microRNAs from group B of microRNAs, excluding microRNAs that are common to group B of microRNAs and group C of microRNAs; and selecting microRNAs that are common to the second group of microRNAs and the third group of microRNAs.

[0069] The method for selecting a microRNA may involve verifying the expression of the selected microRNA using either or both of a biological sample from a subject suffering from pancreatic cancer and a culture medium in which pancreatic cancer cells have been cultured. The method for selecting a microRNA may involve verifying the non-expression of the selected microRNA using a biological sample from a subject showing no symptoms related to pancreatic cancer. Verification of expression or non-expression can be carried out, for example, by quantitative PCR. The expression of the selected microRNA may be determined, for example, when the expression level of the selected microRNA is equal to or greater than a predetermined threshold. The non-expression of the selected microRNA may be determined, for example, when the expression level of the selected microRNA is less than a predetermined threshold.

[0070] [Method for determining whether a subject has pancreatic cancer] One aspect of the present disclosure provides a method for determining whether a subject is suffering from pancreatic cancer, the method comprising: comparing the amount of at least one type of microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15 in a biological sample derived from the subject with at least one threshold value corresponding to the microRNA; and determining that the subject is suffering from pancreatic cancer if the amount of the at least one type of microRNA is greater than the at least one threshold value.

[0071] A subject diagnosed with pancreatic cancer may be treated for pancreatic cancer. Accordingly, one embodiment of this aspect provides a method for treating pancreatic cancer in a subject. This embodiment provides a method for treating pancreatic cancer in a subject, the method comprising: comparing the amount of at least one microRNA containing a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 15 in a biological sample derived from the subject with at least one threshold value corresponding to the microRNA; determining that the subject has pancreatic cancer if the amount of the at least one microRNA is greater than the at least one threshold value; and treating the subject diagnosed as having pancreatic cancer for pancreatic cancer. The term "treatment for pancreatic cancer" may refer to, for example, chemotherapy, immunotherapy, gene therapy, radiation therapy, surgery, or a combination thereof for pancreatic cancer in a subject.

[0072] [Method for determining the pathological condition of a subject suffering from or having suffered from pancreatic cancer] One aspect of the present disclosure provides a method for determining the pathological condition of a subject suffering from or formerly suffering from pancreatic cancer, the method comprising: comparing a first amount of at least one microRNA comprising a nucleotide sequence set forth in any one of SEQ ID NOS: 1-15 in a first biological sample derived from the subject with a second amount of the at least one microRNA in a second biological sample from the subject; and determining that the subject's pathological condition has improved if the first amount is smaller than the second amount, wherein the first biological sample is collected after the second biological sample is collected. In one embodiment, the present invention provides a method for determining the pathological condition of a subject suffering from pancreatic cancer.

[0073] Methods for identifying test substances that can treat pancreatic cancer One aspect of the present disclosure provides a method for identifying a test substance capable of treating pancreatic cancer, the method comprising: administering the test substance to a subject suffering from pancreatic cancer; comparing a first amount of at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1-15 in a first biological sample derived from the subject after administration of the test substance with a second amount of the at least one microRNA in a second biological sample derived from the subject before administration of the test substance; and identifying the test substance as a test substance capable of treating pancreatic cancer if the first amount is smaller than the second amount.

[0074] [Biomarkers] One aspect of the present disclosure provides a biomarker for detecting pancreatic cancer or for monitoring the effectiveness of a pancreatic cancer treatment, the biomarker being at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOS: 1-15 in a biological sample from a subject. One aspect of the present disclosure provides a biomarker for monitoring the effectiveness of a pancreatic cancer treatment, the biomarker being at least one microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOS: 1-5, 9, and 15 in a serum or plasma sample from a subject. In one embodiment, the biomarker for monitoring the effectiveness of a pancreatic cancer treatment is a microRNA comprising a nucleotide sequence set forth in any of SEQ ID NOS: 1, 3-5, 9, and 15 in a serum or plasma sample from a subject. In one embodiment, the biomarker for monitoring the effectiveness of a pancreatic cancer treatment is a microRNA comprising a nucleotide sequence set forth in SEQ ID NOS: 1 in a serum or plasma sample from a subject.

[0075] The term "detecting pancreatic cancer" refers to obtaining information about pancreatic cancer. A "biomarker" for detecting pancreatic cancer or monitoring the effectiveness of a pancreatic cancer treatment refers to an entity that reflects information about pancreatic cancer and is used to obtain information about pancreatic cancer or the effectiveness of a pancreatic cancer treatment. Information about pancreatic cancer may be, for example, information related to the presence or condition of pancreatic cancer in a human or non-human mammal, information related to the effectiveness of a pancreatic cancer treatment, or information for identifying a substance that can treat pancreatic cancer. Information about pancreatic cancer may be, for example, the concentration or content of a microRNA according to the present disclosure in a biological sample derived from a human or non-human mammal. Biomarkers for detecting pancreatic cancer or monitoring the effectiveness of a pancreatic cancer treatment can be used, for example, in a method according to the present disclosure for determining whether a subject has pancreatic cancer, a method for determining the condition of a subject who has or has had pancreatic cancer, or a method for identifying a test substance that can treat pancreatic cancer. Biomarkers for monitoring pancreatic cancer treatment include, for example, the biomarkers disclosed herein in a composition obtained from a subject undergoing treatment for pancreatic cancer. The biomarkers can be prepared, for example, from a composition obtained from the subject (e.g., a pancreatic juice sample, a serum or plasma sample, a stool sample, a duodenal juice sample, or a bile sample) that may contain the microRNAs disclosed herein. The term "pancreatic cancer treatment" can be, but is not limited to, chemotherapy, immunotherapy, gene therapy, radiation therapy, or surgery, or a combination thereof.

[0076] [kit] One aspect of the present disclosure provides a kit comprising a reagent for measuring at least one microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15, for use as a biomarker for pancreatic cancer as described above.

[0077] The terms "reagent for measurement" or "measurement reagent" include a probe capable of specifically binding to the microRNA to be measured. The probe may be labeled with, for example, a fluorescent substance. The probe may include, for example, at least one type of nucleotide (e.g., DNA or RNA) containing a sequence complementary to the nucleotide sequence of the microRNA. Such nucleotides are, for example, commercially available. The probe may be, for example, a primer set for use in a nucleic acid amplification method such as PCR. Such a primer set can be appropriately designed and prepared by a person skilled in the art based on the sequence of the microRNA to be amplified.

[0078] The kit may further include, for example, a liquid or solid buffer and a nuclease inhibitor (e.g., a DNase inhibitor and an RNase inhibitor). The characteristics of the measurement reagent can be appropriately determined depending on the method for measuring the microRNA to be measured. For example, when the measurement method is quantitative PCR, the measurement reagent includes, as a probe, a nucleotide having a sequence complementary to the microRNA to be measured, and the probe may be labeled with a fluorescent substance.

[0079] [Composition for detecting pancreatic cancer] One aspect of the present disclosure provides a composition for detecting pancreatic cancer, comprising a reagent for measuring at least one microRNA comprising or consisting of a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 15.

[0080] In one embodiment, the composition comprises a reagent for measuring at least one microRNA comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 5, or a combination thereof.

[0081] In one embodiment, the composition comprises both a reagent for measuring a microRNA comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a reagent for measuring a microRNA comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 2.

[0082] The terms and explanations of embodiments (e.g., combination of biological sample and microRNA) in the aspects provided by the present disclosure, [Use as a biomarker for pancreatic cancer], [Method for selecting microRNA], [Method for determining whether a subject has pancreatic cancer], [Method for determining the pathological condition of a subject who has or has had pancreatic cancer], [Method for identifying a test substance that can treat pancreatic cancer], [Biomarker], [Kit], and [Composition for detecting pancreatic cancer], are applicable among them as appropriate unless otherwise specified.

[0083] Specific examples will be described below, but they are intended to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims in any way.

[0084] Example [Test 1] Microarray analysis The human pancreatic cancer cell line Panc-1 was cultured, and exosomes secreted into the culture medium were extracted using the exoEasy Maxi Kit (QIAGEN, #76064). MicroRNAs were recovered from the extracted exosomes using NucleoSpin miRNA (Takara Bio). MicroRNAs were recovered from the culture medium of the human pancreatic cancer cell lines BxPC-3 and MIAPaCa-2 in the same manner as above. Pancreatic juice was collected from four pancreatic cancer patients using pancreatic duct intubation, and microRNAs were extracted from exosomes in the collected pancreatic juice samples in the same manner as above. Pancreatic juice was also collected from three patients with chronic pancreatitis in the same manner as above, and microRNAs were extracted from exosomes in the pancreatic juice samples.

[0085] Microarray analysis was performed on exosome-derived microRNAs secreted from the three human pancreatic cancer cell lines Panc-1, BxPC-3, and MIAPaCa-2, exosome-derived microRNAs in pancreatic juice samples from the four pancreatic cancer patients, and exosome-derived microRNAs in pancreatic juice samples from the three chronic pancreatitis patients. Microarray analysis was performed by Cell Innovator, Inc. More specifically, 1000 ng of total RNA for each microRNA was labeled using the FlashTag® Biotin HSR RNA Labeling Kit. The labeled RNA was hybridized to an Affymetrix GeneChip® miRNA 4.0 Array, and the hybridized array was scanned. The scanned data was analyzed using Affymetrix Transcriptome Analysis Console 4.0.

[0086] Microarray analysis of microRNA sequences derived from exosomes secreted from the three human pancreatic cancer cell lines was performed to compare them with microarray analysis of microRNA sequences derived from exosomes in pancreatic juice samples from the four pancreatic cancer patients. Multiple microRNA sequences that were highly expressed in both samples were identified. From the multiple microRNA sequences obtained, microRNA sequences that overlapped with the exosome-derived microRNA sequences in the pancreatic juice samples from the three chronic pancreatitis patients were removed. The resulting microRNAs were highly expressed in pancreatic cancer patients and / or pancreatic cancer cell lines and low in non-cancer patients (chronic pancreatitis patients), suggesting their potential use as biomarkers for pancreatic cancer. The 15 microRNAs identified in Study 1 are listed in the table below.

[0087] [Table 1]

[0088] The relationship between the sequence numbers referred to in this specification and the microRNAs listed in Table 1 is summarized in the table below. [Table 2]

[0089] [Test 2] Real-time PCR analysis The microarray analysis results in Study 1 showed that the microRNA miR-6858-5p did not meet the condition of being expressed at a higher level in pancreatic cancer patients and / or pancreatic cancer cell lines compared to non-cancer patients (chronic pancreatitis patients). Therefore, we quantified the microRNA in the pancreatic juice of four pancreatic cancer patients, two pancreatic juice of two chronic pancreatitis patients, and the culture medium of three pancreatic cancer cell lines using real-time PCR. The negative control for real-time PCR was a blank amplification reagent containing no pancreatic juice sample. These results are shown in Figure 1. Figure 1 shows that there was no significant difference in the amount of miR-6858-5p in the pancreatic juice of two patients with chronic pancreatitis, the pancreatic juice of four pancreatic cancer patients, or the culture medium of three pancreatic cancer cell lines, indicating that the condition of being expressed at a higher level in pancreatic cancer patients and / or pancreatic cancer cell lines compared to non-cancer patients (chronic pancreatitis patients) was not met. Figure 1 suggests that microRNA miR-6858-5p may not be useful as a biomarker for pancreatic cancer.

[0090] MicroRNA miR-4516 (SEQ ID NO: 1) listed in Table 2 was quantified by real-time PCR in the pancreatic juice of the above-mentioned patients and in the culture medium of cultured cells. The results are shown in Figure 2. Figure 2 shows that the amount of miR-4516 in the pancreatic juice (3 samples) and pancreatic cancer cell lines (2 samples) from pancreatic cancer patients was higher than the amount of miR-4516 in the pancreatic juice (2 samples) from non-cancer patients (chronic pancreatitis patients). These results satisfy the condition that the expression level of miR-4516 is higher in pancreatic cancer patients and / or pancreatic cancer cell lines than in non-cancer patients (chronic pancreatitis patients), suggesting that miR-4516 can be a biomarker for pancreatic cancer.

[0091] Similar to the above, real-time PCR was performed on several microRNAs (SEQ ID NOs: 2-6, 9, and 15) listed in Table 2. The results are shown in Figures 3-9. Figure 3 shows that the amount of miR-4674 (SEQ ID NO: 2) in the pancreatic juice (3 samples) of pancreatic cancer patients and the culture medium (3 samples) of pancreatic cancer cell lines was higher than the amount in the pancreatic juice (2 samples) of non-cancer patients (chronic pancreatitis patients). This result meets the condition that the expression level is higher in pancreatic cancer patients and / or pancreatic cancer cell lines compared to the expression level in non-cancer patients (chronic pancreatitis patients), suggesting that miR-4674 could be a biomarker for pancreatic cancer.

[0092] Figure 4 shows that the amount of miR-6800-5p (SEQ ID NO: 3) in the pancreatic juice (1 sample) of a pancreatic cancer patient and in the culture medium of a pancreatic cancer cell line (1 sample) is higher than the amount of miR-6800-5p in the pancreatic juice (2 samples) of a non-cancer patient (chronic pancreatitis patient). This result satisfies the condition that the expression level of miR-6800-5p is higher in a pancreatic cancer patient (1 sample) and / or a pancreatic cancer cell line (1 sample) than in a non-cancer patient (chronic pancreatitis patient), suggesting that miR-6800-5p can be a biomarker for pancreatic cancer.

[0093] Figure 5 shows that the amount of miR-149-3p (SEQ ID NO: 4) in the culture medium of pancreatic cancer cell lines (two samples) was higher than the amount of miR-149-3p in the pancreatic juice of a non-cancer patient (chronic pancreatitis patient) (one sample). This result meets the condition that the expression level of miR-149-3p is higher in pancreatic cancer patients and / or pancreatic cancer cell lines than in non-cancer patients (chronic pancreatitis patients), suggesting that miR-149-3p can be a biomarker for pancreatic cancer.

[0094] Figure 6 shows that the amount of miR-3621 (SEQ ID NO: 5) in the pancreatic juice (2 samples) from pancreatic cancer patients and the culture medium (2 samples) of pancreatic cancer cell lines is higher than the amount of miR-3621 in the pancreatic juice (2 samples) from non-cancer patients (chronic pancreatitis patients). This result satisfies the condition that the expression level of miR-3621 is higher in pancreatic cancer patients and / or pancreatic cancer cell lines than in non-cancer patients (chronic pancreatitis patients), suggesting that miR-3621 can be a biomarker for pancreatic cancer.

[0095] 7 shows that the amount of miR-4484 (SEQ ID NO: 6) in pancreatic juice (2 samples) from non-cancer patients (chronic pancreatitis patients) was higher than the amount of miR-4484 in pancreatic juice (4 samples) from pancreatic cancer patients and in the culture medium of pancreatic cancer cell lines (3 samples). This result does not satisfy the condition that the expression level of miR-4484 is higher in pancreatic cancer patients and / or pancreatic cancer cell lines compared to the expression level in non-cancer patients (chronic pancreatitis patients), and therefore indicates that miR-4484 in pancreatic juice cannot be used as a biomarker for pancreatic cancer.

[0096] Figure 8 shows that the amount of miR-3940-5p (SEQ ID NO: 9) in the culture medium of pancreatic cancer cell lines (two samples) was higher than the amount of miR-3940-5p in the pancreatic juice of non-cancer patients (chronic pancreatitis patients) (two samples). This result meets the condition that the expression level of miR-3940-5p is higher in pancreatic cancer patients and / or pancreatic cancer cell lines than in non-cancer patients (chronic pancreatitis patients), suggesting that miR-3940-5p can be a biomarker for pancreatic cancer.

[0097] 9 shows that the amount of miR-3656 (SEQ ID NO: 15) in the pancreatic juice (1 sample) of a pancreatic cancer patient and in the culture medium of a pancreatic cancer cell line (2 samples) is higher than the amount of miR-3656 in the pancreatic juice (1 sample) of a non-cancer patient (chronic pancreatitis patient). This result satisfies the condition that the expression level of miR-3656 is higher in pancreatic cancer patients and / or pancreatic cancer cell lines than in non-cancer patients (chronic pancreatitis patients), suggesting that miR-3656 can be a biomarker for pancreatic cancer.

[0098] Figures 2 to 6 and 8 to 9 show that the results of real-time PCR in Test 2 are consistent with the results of microarray analysis in Test 1 for microRNAs consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 5, 9, and 15. Figures 2 to 6 and 8 to 9 also show that the expression levels of microRNAs consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 3, 5, and 15 are higher in pancreatic cancer cell lines and pancreatic cancer patients than in non-cancer patients (chronic pancreatitis patients), suggesting that the microRNAs are suitable biomarkers for pancreatic cancer.

[0099] [Test 3] Verification with pancreatic juice samples I To further test the usefulness of the pancreatic cancer biomarkers miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2) suggested in Study 2, pancreatic juice from four additional pancreatic cancer patients, and pancreatic juice from four additional chronic pancreatitis patients, were used. The pancreatic juice samples used in Study 3 are shown in the table below. [Table 3]

[0100] The amounts of microRNA miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2) in pancreatic juice samples (6 samples) from patients a-f with chronic pancreatitis and 8 samples from patients A-H with pancreatic cancer were examined by quantitative real-time PCR. The results are shown in Figures 10 and 11, respectively. Figure 10 shows the relative miR-4516 levels in the pancreatic juice samples of each patient, normalized to the measured value of microRNA miR-4516 (SEQ ID NO: 1) in the pancreatic juice sample from patient a with chronic pancreatitis. Figure 10 shows that, when a relative value of 3 is used as the threshold, 5 out of 6 patients with chronic pancreatitis are judged to be negative (specificity: approximately 83.3%), 7 out of 8 patients with pancreatic cancer are judged to be positive (sensitivity: 87.5%), and 3 out of 3 types of pancreatic cancer cells are judged to be positive (positive rate: 100%). These results indicate that microRNA miR-4516 (SEQ ID NO: 1) in pancreatic juice samples is useful as a biomarker for pancreatic cancer.

[0101] Figure 11 shows the relative levels of miR-4674 (SEQ ID NO: 2) in pancreatic juice samples from each patient, normalized to the measured values ​​of the microRNA miR-4674 (SEQ ID NO: 2) in the pancreatic juice sample from chronic pancreatitis patient a. Figure 11 shows that, when a relative value of 2 is used as the threshold, 5 out of 6 patients with chronic pancreatitis are judged to be negative (specificity: 83.3%), 5 out of 8 patients with pancreatic cancer are judged to be positive (sensitivity: 62.5%), and 3 out of 3 types of pancreatic cancer cells are judged to be positive (positive rate: 100%). These results demonstrate that the microRNA miR-4674 (SEQ ID NO: 2) in pancreatic juice samples is useful as a biomarker for pancreatic cancer.

[0102] [Test 4] Verification with pancreatic juice samples II For miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2), whose usefulness as biomarkers for pancreatic cancer was demonstrated in Study 3, a similar study to Study 3 was conducted using pancreatic juice from 15 pancreatic cancer patients and 11 chronic pancreatitis patients as shown in the table below. [Table 4]

[0103] The results of a test similar to Test 3 are shown in Figures 12 and 13, respectively. Figure 12 shows the relative miR-4516 levels in the pancreatic juice samples of each patient, normalized to the measured values ​​of microRNA miR-4516 (SEQ ID NO: 1) in the pancreatic juice samples of chronic pancreatitis patient CP1. Figure 13 shows the relative miR-4674 levels in the pancreatic juice samples of each patient, normalized to the measured values ​​of microRNA miR-4674 (SEQ ID NO: 2) in the pancreatic juice samples of chronic pancreatitis patient CP1. Based on the results, receiver operating characteristic (ROC) curves were generated for miR-4516 and miR-4674 (Figure 14). Figure 14A shows that the area under the curve (AUC) for miR-4516 (SEQ ID NO: 1) was 0.82 (p = 0.026). Figure 14B shows that the AUC for miR-4674 (SEQ ID NO: 2) was 0.73 (p = 0.22).

[0104] The indices (sensitivity, specificity, positive predictive value, negative predictive value, and accuracy) of a test using miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2) as biomarkers for pancreatic cancer were calculated. [Table 5]

[0105] The sensitivity, specificity, and accuracy of the test using miR-4516 (SEQ ID NO: 1) alone were 80%, 81.8%, and 80.8%, respectively. The sensitivity, specificity, and accuracy of the test using miR-4674 (SEQ ID NO: 2) alone were 73.3%, 81.8%, and 76.9%, respectively. These results suggest that miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2) can be used for the diagnosis of pancreatic cancer, especially for early diagnosis, with high sensitivity, specificity, and accuracy, respectively.

[0106] The sensitivity, specificity, and accuracy of the test using the combination of miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2) (miR-4516 / 4674) were 93.3%, 72.7%, and 84.6%, respectively. These results suggest that the combination of miR-4516 and miR-4674 has higher sensitivity, specificity, and accuracy, and can be used for the diagnosis of pancreatic cancer, especially for early diagnosis.

[0107] The sensitivity, specificity, and accuracy of the test using a combination of miR-4516 (SEQ ID NO: 1) and pancreatic juice cytology (PJC) (miR-4516 / PJC) were 93.3%, 81.8%, and 88.5%, respectively. The sensitivity, specificity, and accuracy of the test using a combination of miR-4674 (SEQ ID NO: 2) and pancreatic juice cytology (PJC) (miR-4674 / PJC) were 100%, 81.8%, and 92.3%, respectively. The sensitivity, specificity, and accuracy of the test using a combination of miR-4516, miR-4674, and pancreatic juice cytology (PJC) (miR-4516 / 4674 / PJC) were 100%, 72.7%, and 88.5%, respectively. These results suggest that miR-4516 alone, miR-4674 alone, or the combination of miR-4516 and miR-4674, when further combined with PJC, can be used as an excellent biomarker for pancreatic cancer.

[0108] [Test 5] Verification with serum samples To further test the usefulness of miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2), whose usefulness as biomarkers for pancreatic cancer was demonstrated in Studies 2 and 3, sera from seven pancreatic cancer patients (PDAC-2, 4, 5, 8, 9, 10, and 11), five chronic pancreatitis patients (CP-2 to 6), and two healthy controls (HD-1 and 2) were used. The amounts of microRNA miR-4516 (SEQ ID NO: 1) and miR-4674 (SEQ ID NO: 2) in the serum of the chronic pancreatitis patients (5 samples), the healthy controls (2 samples), and the pancreatic cancer patients (7 samples) were examined by quantitative real-time PCR. The results are shown in Figures 15 and 16, respectively.

[0109] Figure 15 shows the relative levels of microRNA miR-4516 (SEQ ID NO: 1) in the serum of each patient. Figure 15 shows that, when a relative value of 1.5 is used as the threshold, two out of seven pancreatic cancer patients are judged as positive (sensitivity: approximately 28.6%), and five out of five chronic pancreatitis patients or two out of two healthy individuals are judged as negative (specificity: 100%). These results demonstrate that serum microRNA miR-4516 (SEQ ID NO: 1) is useful as a biomarker for pancreatic cancer.

[0110] Figure 16 shows the relative levels of microRNA miR-4674 (SEQ ID NO: 2) in the serum of each patient. Figure 16 shows that, when a relative value of 1.5 is used as the threshold, 0 out of 7 pancreatic cancer patients are judged as positive (sensitivity: 0%), and 5 out of 5 chronic pancreatitis patients and 2 out of 2 healthy individuals are judged as negative (specificity: 100%). These results indicate that serum microRNA miR-4674 (SEQ ID NO: 2) cannot be used as a biomarker for pancreatic cancer.

[0111] In Study 5, of four pancreatic cancer patients (PDAC-2, 4, 5, and 8) from whom both pancreatic juice and serum were collected, miR-4516 expression was high in two cases. In addition, of five non-pancreatic cancer patients (CP-2 to 6) from whom both pancreatic juice and serum were collected, miR-4516 expression was low in all cases.

[0112] [Study 6] Treatment effect monitoring Pancreatic cancer patient PDAC-8 underwent tumor removal surgery after chemotherapy. The expression level of miR-4516 (SEQ ID NO: 1) was high in both pancreatic juice and serum collected from PDAC-8 before surgery (Figures 12 and 15). Serum was collected from PDAC-8 after surgery, and the expression level of miR-4516 (SEQ ID NO: 1) in the serum was examined (Figure 17). Figure 17 shows that the expression level of miR-4516 (SEQ ID NO: 1) decreased after treatment for pancreatic cancer.

[0113] Pancreatic cancer patient PDAC-4 underwent tumor removal surgery. The expression level of miR-4516 (SEQ ID NO: 1) was low in pancreatic juice collected from PDAC-4 before surgery (Figure 12), but high in serum collected from PDAC-4 (Figure 15). Serum was collected from PDAC-4 after surgery, and the expression level of miR-4516 (SEQ ID NO: 1) in the serum was examined (Figure 18). Figure 18 shows that the expression level of miR-4516 (SEQ ID NO: 1) decreased after treatment for pancreatic cancer.

[0114] The results of Study 6 suggest that serum miR-4516 (SEQ ID NO: 1) is useful as a biomarker for managing the efficacy of pancreatic cancer treatment.

[0115] [Study 7] Potential use as a serum marker specific to pancreatic cancer We investigated whether miR-4516 (SEQ ID NO: 1) could be used as a serum biomarker to distinguish pancreatic cancer patients from other subjects. Serum samples were collected from 39 pancreatic cancer patients (PDAC) and 32 other subjects. The 32 subjects included two healthy controls (HD), five patients with chronic pancreatitis (CP), one patient with intraductal papillary mucinous neoplasm (IPMN), three patients with intraductal papillary mucinous adenocarcinoma (IPMC), eight patients with hepatocellular carcinoma (HCC), six patients with colorectal cancer (CR), and seven patients with gastric cancer (GC). The expression levels of miR-4516 (SEQ ID NO: 1) in the collected serum were examined (Figure 19). Based on the results, a receiver operating characteristic curve (ROC) was constructed (Figure 20). Figure 20 shows that the area under the curve for miR-4516 (SEQ ID NO: 1) was 0.53. Furthermore, the sensitivity and accuracy of the test using miR-4516 (SEQ ID NO: 1) were 15.4% and 53.5%, respectively, while the specificity was a favorable result of 100%.

Claims

1. Use of at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in a biological sample from a subject as a biomarker for pancreatic cancer, comprising: The pancreatic cancer biomarker is A device that presents information for determining whether a subject has pancreatic cancer; Presents information for determining the disease status of a subject who has or has had pancreatic cancer; or 1. A use for identifying a test substance capable of treating pancreatic cancer, comprising:

10. The use of a test substance as a biomarker for pancreatic cancer to identify a test substance capable of treating pancreatic cancer, comprising administering the test substance to a non-human subject suffering from pancreatic cancer.

2. the biomarker for pancreatic cancer provides information for determining whether a subject is suffering from pancreatic cancer; comparing the amount of at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in the biological sample from the subject with at least one threshold value corresponding to the at least one microRNA; and The use of claim 1, comprising presenting information for determining that the subject is suffering from pancreatic cancer if the amount of the at least one microRNA is greater than the at least one threshold.

3. The biomarker for pancreatic cancer provides information for determining the pathological condition of a subject who is or has been affected by pancreatic cancer; comparing a first amount of at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in a first biological sample from the subject with a second amount of the at least one microRNA in a second biological sample from the subject; and presenting information for determining that the pathological condition of the subject has improved when the first amount is smaller than the second amount; The use of claim 1 , wherein the first biological sample is collected after the second biological sample is collected.

4. the biomarker for pancreatic cancer is for identifying a test substance capable of treating pancreatic cancer; administering the test agent to a non-human subject suffering from pancreatic cancer; comparing a first amount of at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in a first biological sample from the non-human subject after administering the test substance with a second amount of the at least one microRNA in a second biological sample from the non-human subject before administering the test substance; and The use of claim 1, comprising identifying the test substance as a test substance capable of treating pancreatic cancer if the first amount is less than the second amount.

5. The use according to any one of claims 1 to 4, wherein the biological sample is a pancreatic juice sample, a serum or plasma sample, a stool sample, a duodenal juice sample or a bile sample.

6. The use according to any one of claims 1 to 4, wherein the biological sample is a pancreatic juice sample or a serum or plasma sample.

7. A method for presenting information for determining whether a subject is suffering from pancreatic cancer, comprising: comparing the amount of at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in the biological sample from the subject with at least one threshold value corresponding to the at least one microRNA; and presenting information for determining that the subject is suffering from pancreatic cancer when the amount of the at least one type of microRNA is greater than the at least one threshold value.

8. A method for presenting information for determining the pathological condition of a subject who has or has had pancreatic cancer, comprising: comparing a first amount of at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in a first biological sample from the subject with a second amount of the at least one microRNA in a second biological sample from the subject; and presenting information for determining that the pathological condition of the subject has improved when the first amount is smaller than the second amount; The method, wherein the first biological sample is collected after collecting the second biological sample.

9. 1. A method for identifying a test substance capable of treating pancreatic cancer, comprising: administering the test agent to a non-human subject suffering from pancreatic cancer; comparing a first amount of at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in a first biological sample from the non-human subject after administering the test substance with a second amount of the at least one microRNA in a second biological sample from the non-human subject before administering the test substance; and identifying the test substance as a test substance capable of treating pancreatic cancer if the first amount is less than the second amount.

10. A kit comprising a reagent for measuring at least one microRNA comprising the nucleotide sequence shown in SEQ ID NO: 1, for use in the use according to any one of claims 1 to 6 or the method according to any one of claims 7 to 9.

11. A composition for detecting pancreatic cancer, comprising a reagent for measuring at least one microRNA comprising the nucleotide sequence set forth in SEQ ID NO:1.

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