WO2014003053A1 - 膵臓がんの検出方法及び検出用キット - Google Patents
膵臓がんの検出方法及び検出用キット Download PDFInfo
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- WO2014003053A1 WO2014003053A1 PCT/JP2013/067508 JP2013067508W WO2014003053A1 WO 2014003053 A1 WO2014003053 A1 WO 2014003053A1 JP 2013067508 W JP2013067508 W JP 2013067508W WO 2014003053 A1 WO2014003053 A1 WO 2014003053A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
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- the present invention relates to a method for detecting pancreatic cancer, which comprises measuring microRNA specific to pancreatic cancer.
- MicroRNA (hereinafter sometimes referred to as “miRNA”) is one of the small non-coding RNAs in cells composed of about 22 bases, and is an essential factor for the control of individual development and cell differentiation. It is. MiRNA dysfunction is found in many human diseases. In particular, abnormal expression of miRNA in cancer is observed in most cancer types, and it is considered that abnormal function of miRNA and carcinogenesis are strongly linked. In recent years, it has been clarified that miRNA is released (secreted) out of the cell in the form of being embedded in an exosome that is an inner membrane vesicle. In addition to elucidating the molecular mechanisms of cancer development, miRNA research is expected to be applied to therapeutic and diagnostic agents. The present inventors have found a colon cancer-specific miRNA that can serve as a test marker for colorectal cancer (Patent Document 1).
- Pancreatic cancer is a disease that requires a method for early detection.
- various types of diagnostic imaging and blood tests that detect tumor markers have been used for pancreatic cancer testing.
- diagnostic imaging requires extensive equipment and expensive equipment and is widely used. I can't say.
- tumor markers used so far cannot be said to have sufficient sensitivity and specificity, and none of them are suitable for early detection because many do not increase unless cancer progresses to some extent.
- Pancreatic cancer is an intractable cancer that is difficult to detect early because it has few initial symptoms, and has a fast progression and poor prognosis. Early detection is very important for reducing pancreatic cancer mortality because pancreatic cancer has a lower chance of being cured.
- An object of the present invention is to provide a method for detecting pancreatic cancer or a risk of pancreatic cancer that is simple and has high sensitivity and specificity.
- the present inventors have conducted research to solve the above problems, created a profile of exosome miRNA secreted from pancreatic cancer cell lines, and identified miRNA specific to pancreatic cancer. In addition, using clinical blood samples, it was confirmed that the profile was significantly different from the colorectal cancer profile. Furthermore, it has been found that some of the miRNAs identified in the cell lines are detected at significantly higher levels from the blood samples of pancreatic cancer patients, and the present invention has been completed.
- the present invention [1] A method for detecting pancreatic cancer in a subject, comprising the step of measuring at least one miRNA selected from the following (i) and (ii) in a sample derived from the subject: Detection method: (i) a miRNA having a sequence represented by any one of SEQ ID NOs: 1 to 120; and (ii) a miRNA that hybridizes under stringent conditions with a nucleic acid complementary to the miRNA of any of (i).
- the detection method according to the above [1] which has a sequence represented by any one of -64, 70-73, 78, 80, 81, 82, and 85-120; [3] When the measured value of the miRNA concentration is statistically significantly higher than the corresponding measured value of a healthy person, the subject is determined to have pancreatic cancer, [ [1] The detection method according to any one of [2].
- the sample derived from the subject is prepared by a method comprising a step of concentrating an exosome fraction from plasma collected from the subject and a step of extracting miRNA from the exosome fraction.
- the detection method according to any one of [1] to [3] above; [5] A kit for performing the detection method according to any one of [1] to [4], A kit comprising a solid phase carrier on which DNA that hybridizes with the miRNA selected from (i) and (ii) is immobilized; [6] A kit for performing the detection method according to any one of [1] to [4], A kit comprising a primer set necessary for amplifying the miRNA selected from (i) and (ii) above or a nucleic acid complementary thereto by PCR; [7] A method for detecting colorectal cancer in a subject, comprising a step of measuring the miRNA represented by SEQ ID NO: 47 in a sample derived from the subject; [8] The sample derived from the subject is prepared by a method comprising a step
- kits for performing the detection method according to [7] or [8] above A kit comprising a solid phase carrier on which a DNA hybridizing with the miRNA represented by SEQ ID NO: 47 is immobilized; and [10] a kit for performing the detection method according to [7] or [8] above.
- pancreatic cancer or pancreatic cancer risk with high sensitivity and specificity can be detected by a simple method of measuring a predetermined miRNA in a sample of a subject. .
- FIG. 1 shows that small RNAs are concentrated in the exosome fraction obtained from a pancreatic cancer cell line.
- FIG. 2 is a profile of endogenous miRNA and exosome miRNA derived from pancreatic cancer cell line and immortalized pancreatic duct epithelial cell line by microarray analysis.
- FIG. 3 is a representative example of a result of creating a profile of exosome miRNA derived from a plasma specimen of a pancreatic cancer patient with a microarray and comparing it with analysis data of a plasma specimen of a healthy person.
- FIG. 2 is a profile of endogenous miRNA and exosome miRNA derived from pancreatic cancer cell line and immortalized pancreatic duct epithelial cell line by microarray analysis.
- FIG. 3 is a representative example of a result of creating a profile of exosome miRNA derived from a plasma specimen of a pancreatic cancer patient with a microarray and comparing it with analysis data of a plasma specimen of a healthy
- FIG. 4 is a representative example of a result of creating a profile of exosome miRNA derived from a plasma specimen of a pancreatic cancer patient with a microarray and comparing it with analysis data of a plasma specimen of a healthy person.
- FIG. 5 shows the result of creating a microarray profile of exosome miRNA derived from plasma specimens of colorectal cancer patients, pancreatic cancer patients, and healthy individuals.
- FIG. 6 shows the results of measurement of miR-23a-3p expression in exosomes derived from plasma specimens of colorectal cancer patients and healthy individuals.
- FIG. 7 shows the results of measurement of miR-23a-3p expression in exosomes derived from plasma specimens before and after surgery for colorectal cancer patients.
- One aspect of the method for detecting pancreatic cancer according to the present invention includes a step of measuring at least one miRNA selected from the following (i) and (ii). (i) an miRNA having a sequence represented by any one of SEQ ID NOs: 1 to 120; and (ii) an miRNA that hybridizes under stringent conditions with a nucleic acid complementary to the miRNA of any of (i). MiRNA which is 17-30 bases in length.
- the “miRNA having a sequence represented by any one of SEQ ID NOs: 1 to 120” described in (i) above is commonly used in six types of pancreatic cancer cell lines, as shown in the Examples described later.
- Secreted miRNA or miRNA detected at a significantly higher value in plasma specimens from pancreatic cancer patients than in healthy controls.
- the following 65 miRNAs were detected in plasma samples derived from pancreatic cancer patients at significantly higher values than those in healthy controls.
- high sequence identity means 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more at the nucleic acid level.
- the miRNA of (ii) includes, for example, one in which one or two bases are deleted, added or substituted in any sequence of the miRNA of (i).
- the site to be deleted, added or substituted may be the 5 ′ end or 3 ′ end of the miRNA of (i), or may be a portion other than the end.
- the miRNA of (ii) may be, for example, 17 to 25 bases long, and preferably has the same base length as the corresponding miRNA of (i).
- stringent conditions include, for example, 5% Denhardt's Solution (containing 0.1% Ficoll (Pharmacia), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 0.5% SDS and 100 ⁇ g / ml salmon. This refers to the conditions of washing at 65 ° C. in a 6 ⁇ SSC solution (1 ⁇ SSC is 0.15 M NaCl, 15 mM sodium citrate) containing sperm DNA. Stringency can be controlled by salt concentration (ionic strength), temperature, and the like. Under higher stringency conditions, i.e., lower salt concentration and higher temperature, washing removes the background due to non-specific hybridization, leaving only the specifically hybridized nucleic acid. A person skilled in the art can appropriately select stringent conditions by adjusting the temperature and the salt concentration.
- At least one miRNA selected from the above (i) and (ii) may be measured. That is, the determination of pancreatic cancer or pancreatic cancer risk may be performed by measuring a certain miRNA, or by measuring two or more miRNAs. When two or more miRNAs are combined, any number can be combined. For example, it is also preferable to select and combine two or more of 65 miRNAs shown in Table 1.
- the “step of measuring miRNA” may be a step of accurately measuring the concentration of miRNA in the sample, and it can be confirmed that the concentration in the sample is significantly higher than the level of a healthy person. As long as the concentration is not accurately measured, it may be a step of simply detecting whether miRNA is contained in the sample. In particular, for miRNA that is hardly detected from a sample of a healthy person, as a step of measuring miRNA, it is only necessary to confirm whether or not these miRNAs are present in the sample of the subject.
- the concentration of each miRNA may be obtained, or the expression profile (expression pattern) of a plurality of miRNAs contained in a sample and the expression profile of the miRNA in a healthy person as a whole You may carry out by comparing and evaluating the similarity.
- the method for measuring miRNA is not particularly limited, and any method capable of detecting and measuring RNA having a specific sequence from a sample can be used. Examples of such methods include Northern hybridization, RNase protection assay, quantitative PCR such as RT-PCR and real-time PCR, and methods using DNA microarrays.
- the Northern hybridization method was selected from the above (i) and (ii) after the extracted miRNA was developed on a gel by electrophoresis such as agarose electrophoresis and transferred to a membrane such as a nitrocellulose membrane or a nylon membrane.
- a target miRNA is detected using a probe that hybridizes with miRNA (target miRNA).
- radioactive isotopes such as 32 P, digoxigenin (DIG) and antibodies can be used.
- an RNA probe labeled with a radioisotope or DIG is hybridized with a target miRNA, and then the region of the probe that has not hybridized with the target miRNA using a single strand-specific RNase. After digestion, the probe portion protected by hybridization is detected by electrophoresis or the like.
- cDNA is obtained from miRNA in a sample using reverse transcriptase, PCR is performed using this cDNA as a template and appropriate primers are used, and amplification products are quantified by electrophoresis or the like. This is a method for determining the amount of RNA.
- Real-time PCR is a method for detecting PCR amplification products by fluorescence.
- cDNA can be obtained from miRNA in a sample with reverse transcriptase and used as a template.
- the method using a DNA microarray is a method of detecting a target miRNA hybridized to a probe by immobilizing a DNA probe that hybridizes with all or a part of the target miRNA on a solid phase carrier and bringing it into contact with a sample. It is suitable for comprehensive measurement of miRNA expression profiles in samples. Design a part of the miRNA to hybridize to the probe immobilized on the solid support, and detect the target miRNA by hybridizing the remaining part of the miRNA to the labeled second DNA probe. You can also.
- the sample derived from the subject is not particularly limited, but is preferably a blood sample, particularly serum or plasma.
- a blood sample particularly serum or plasma.
- the concentration method is not limited to the ultracentrifugation method, and may be performed by a membrane preparation method, an antibody preparation method, or a method using a reagent that specifically concentrates exosomes.
- pancreatic cancer is used in a normal sense, and includes pancreatic cancer of any state regardless of pathological classification, morphology, depth of advance, stage indicated by progression, and the like.
- detection means to examine a sample collected from a subject in order to obtain information necessary for diagnosis, and the detection method of the present invention can be carried out, for example, by an inspection company. Further, in the present invention, “detection” includes checking whether or not the patient is susceptible to pancreatic cancer in addition to confirming whether or not the patient is susceptible to pancreatic cancer; Screening for possible cancer, or after finding out that you have pancreatic cancer, to investigate cancer trends such as its progress, prognosis, therapeutic effects, and likelihood of recurrence Also included.
- the detection method according to the present invention may be combined with an inspection method using a tumor marker or an image diagnosis that has been conventionally used for pancreatic cancer inspection.
- “statistically significant” includes, for example, a case where the risk factor (significance level) of the obtained value is smaller than 0.05, 0.01, or 0.001. Therefore, “statistically significantly higher” for the measured value means that there is a significant difference between the two when statistically processing the quantitative difference of miRNA obtained from each of the subject and the healthy person. Yes, and the amount of the miRNA of the subject is relatively high compared to that of a healthy person.
- the test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t test or multiple comparison test can be used.
- the present invention also includes a kit for performing the detection method according to the present invention described above.
- One embodiment of the detection kit according to the present invention includes a solid phase carrier on which a DNA that hybridizes with the miRNA selected from the above (i) and (ii) is immobilized.
- the solid phase carrier is not particularly limited as long as it can immobilize DNA, and examples thereof include glass, metallic, resin microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, and the like. It is done. DNA can be immobilized on these solid phase carriers by a known method.
- the kit may further include, for example, various reaction / detection reagents, buffers, instruction manuals, and the like.
- the detection kit according to the present invention includes a primer set necessary for amplifying miRNA selected from (i) and (ii) above or a nucleic acid complementary thereto by PCR.
- the detection kit according to the present invention may include a primer set capable of amplifying the target miRNA or a DNA complementary thereto.
- a primer set can be appropriately designed by those skilled in the art based on the sequence of the target miRNA.
- Such a kit may further include, for example, a reverse transcriptase, various reaction / detection reagents, a buffer, an instruction manual, and the like.
- the present invention also includes a method for diagnosing pancreatic cancer, comprising a step of measuring at least one miRNA selected from the above (i) and (ii) in a blood sample derived from a subject.
- diagnosis means that the medical practitioner determines whether the subject is suffering from a specific disease based on the detection result or the like.
- the terms used for the method for diagnosing pancreatic cancer according to the present invention have the same meaning, and the description thereof is omitted here.
- the present application relates to a miRNA having a sequence represented by SEQ ID NO: 47 in a blood sample derived from a subject, or a miRNA that hybridizes with a nucleic acid having a sequence complementary to the miRNA under stringent conditions.
- a method for detecting colorectal cancer including a step of measuring miRNA having a length of 17 to 30 bases is also included.
- the miRNA represented by SEQ ID NO: 47 is referred to as hsa-miR-23a-3p.
- the sample is preferably a blood sample such as serum or plasma.
- this miRNA is hardly contained in a sample of a healthy person, but it is often contained in a high concentration even in a case of colorectal cancer patients when the stage is low.
- concentration is significantly decreased after surgery as compared with that before surgery for colorectal cancer, it is also useful for follow-up after surgery.
- hsa-miR-23a-3p can be used alone as a useful colorectal cancer marker, or for example, may be used in combination with at least one of the miRNAs disclosed in Patent Document 1.
- the present invention also includes a colorectal cancer detection kit including a solid phase carrier on which DNA that hybridizes with miRNA having the sequence represented by SEQ ID NO: 47 is immobilized.
- the solid phase carrier is not particularly limited as long as it can immobilize DNA, and examples thereof include glass, metallic, resin microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, and the like. It is done. DNA can be immobilized on these solid phase carriers by a known method.
- the kit may further include, for example, various reaction / detection reagents, buffers, instruction manuals, and the like.
- the present invention also includes a colorectal cancer detection kit including a primer set necessary for amplifying miRNA having the sequence represented by SEQ ID NO: 47 or a nucleic acid complementary thereto by PCR.
- a primer set can be appropriately designed by those skilled in the art based on the sequence of the target miRNA.
- Such a kit may further include, for example, a reverse transcriptase, various reaction / detection reagents, a buffer, an instruction manual, and the like.
- the present invention measures miRNA having a sequence represented by SEQ ID NO: 47 or miRNA hybridized under stringent conditions with a nucleic acid having a sequence complementary to the miRNA in a sample derived from a subject. It also includes a method for diagnosing colorectal cancer, including a step.
- RNA having a length of about 20 to 30 bases was concentrated in the exosome fraction concentrated by ultracentrifugation.
- the results of microarray analysis using this RNA sample are shown in FIG.
- the profile of exosome miRNA secreted by pancreatic cancer cells is significantly different from the expression profile of endogenous miRNA, so that miRNA secreted by pancreatic cancer cells may be selective.
- the endogenous expression profile cannot distinguish between cancer cell lines and immortalized cells (underlined in the figure) (left panel), but the exosome miRNA profile can distinguish between the two. all right. This result suggests that changes in the profile of exosome miRNA may be induced by malignant transformation (carcinogenesis) of cells.
- miRNAs secreted by 6 types of pancreatic cancer cell lines were selected.
- Microarray data was normalized by cell number, miR-923 (ribosomal RNA), and total RNA. As shown in the table below, 85 types of miRNAs expressed in pancreatic cancer cell lines were identified.
- SEQ ID Nos: 1 to 120 represent miRNA sequences.
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Abstract
Description
本発明者らは、これまでに大腸がんの検査マーカーとなりうる大腸がん特異的なmiRNAを見出している(特許文献1)。
膵臓がんは、初期症状がほとんどないため早期発見が困難であり、進行が早く予後も悪い難治性がんである。膵臓がんはステージが低いほど治癒の可能性は高くなるので、膵臓がんによる死亡率を低下させるために早期発見は非常に重要である。
即ち本発明は、
〔1〕被検者の膵臓がんを検出する方法であって、被検者に由来する試料中における以下の(i)及び(ii)から選択される少なくとも1つのmiRNAを測定する工程を含む、検出方法:
(i) 配列番号:1~120のいずれかで表される配列を有するmiRNA;及び
(ii) (i)のいずれかのmiRNAと相補的な配列の核酸とストリンジェントな条件でハイブリダイズするmiRNAであって、17~30塩基長であるmiRNA;
〔2〕前記(i)から選択されるmiRNAが、配列番号:1~3、5、7、12、13、19~21、26、27、30、32、35、36、42、50、62~64、70~73、78、80、81、82、及び85~120のいずれかで表される配列を有する、上記〔1〕に記載の検出方法;
〔3〕前記miRNAの濃度の測定値が健常人の対応する測定値と比較して統計学的に有意に高いとき、前記被検者が膵臓がんに罹患していると決定する、上記〔1〕又は〔2〕のいずれかに記載の検出方法。
〔4〕前記被検者に由来する試料は、被検者から採取された血漿からエクソソーム画分を濃縮する工程と、前記エクソソーム画分からmiRNAを抽出する工程と、を含む方法によって調製される、上記〔1〕から〔3〕のいずれかに記載の検出方法;
〔5〕上記〔1〕から〔4〕のいずれかに記載の検出方法を行うためのキットであって、
前記(i)及び(ii)から選択されるmiRNAとハイブリダイズするDNAが固定された固相担体を含む、キット;
〔6〕上記〔1〕から〔4〕のいずれかに記載の検出方法を行うためのキットであって、
前記(i)及び(ii)から選択されるmiRNAまたはこれに相補的な核酸をPCR法で増幅するのに必要なプライマーセットを含む、キット;
〔7〕被検者の大腸がんを検出する方法であって、被検者に由来する試料中における配列番号:47で表されるmiRNAを測定する工程を含む、方法;
〔8〕前記被検者に由来する試料は、被検者から採取された血清からエクソソーム画分を濃縮する工程と、前記エクソソーム画分からmiRNAを抽出する工程と、を含む方法によって調製される、上記〔7〕に記載の方法;
〔9〕上記〔7〕または〔8〕に記載の検出方法を行うためのキットであって、
配列番号:47で表されるmiRNAとハイブリダイズするDNAが固定された固相担体を含む、キット;及び
〔10〕上記〔7〕または〔8〕に記載の検出方法を行うためのキットであって、
配列番号:47で表されるmiRNAまたはこれに相補的な核酸をPCR法で増幅するのに必要なプライマーセットを含む、キット;
に関する。
本発明に係る膵臓がんの検出方法の一態様は、以下の(i)及び(ii)から選択される少なくとも1つのmiRNAを測定する工程を含む。
(i) 配列番号:1~120のいずれかで表される配列を有するmiRNA;及び
(ii) (i)のいずれかのmiRNAと相補的な配列の核酸とストリンジェントな条件でハイブリダイズするmiRNAであって、17~30塩基長であるmiRNA。
このうち、以下の65種類のmiRNAは、膵臓がん患者由来の血漿検体において、コントロールの健常人と比べて有意に高い値で検出された。
(ii)のmiRNAとしては、例えば、(i)のmiRNAのいずれかの配列において、1個または2個の塩基が欠失、付加または置換したものを含む。欠失、付加または置換される部位は、(i)のmiRNAの5’末端または3’末端でもよいし、末端以外の部分であってもよい。(ii)のmiRNAは、例えば17~25塩基長のものでもよく、対応する(i)のmiRNAと同じ塩基長であることも好ましい。
2つ以上のmiRNAを組み合わせる場合、組み合わせる個数はいくつでもよい。例えば、表1に示される65種のmiRNAの中から2つ以上を選択して組み合わせることも好ましい。
特に、健常人の試料からはほとんど検出されないmiRNAについては、miRNAを測定する工程として、被検者の試料中にこれらのmiRNAが存在するか否かの確認を行うだけでもよい。
また、miRNAを測定する工程では、各miRNAの濃度をそれぞれ求めてもよいし、試料中に含まれる複数のmiRNAの発現プロファイル(発現パターン)と、健常人における当該miRNAの発現プロファイルとを全体として比較し、その類似性を評価することによって行ってもよい。
また、上述したmiRNAを測定する工程を行う前に、血清または血漿からエクソソーム画分を濃縮する工程を行ってもよい。濃縮する方法は、超遠心法に限られず、膜で調製する方法、抗体で調製する方法、エクソソームを特異的に濃縮する試薬による方法により行ってもよい。さらに、エクソソーム画分からmiRNAを抽出する工程を行ってもよい。これらの工程は、当業者が常法にしたがって行うことができる。
また、本発明において、「検出」は、膵臓がんに罹患しているか否かを確認することに加え、膵臓がんに罹患しやすいかどうかのリスクを調べること、症状が現れない段階で膵臓がんの可能性をスクリーニングすること、膵臓がんに罹患していることが明らかになった後で、その進行度、予後、治療効果、再発の可能性などの癌の趨勢を調べることのいずれも含んでいる。
本発明は、上述した本発明に係る検出方法を行うためのキットも包含する。
本発明に係る検出用キットの一態様は、上記(i)及び(ii)から選択されるmiRNAとハイブリダイズするDNAが固定された固相担体を含む。ここで固相担体は、DNAを固定できる担体であれば特に限定されず、ガラス製、金属性、樹脂製等のマイクロタイタープレート、基板、ビーズ、ニトロセルロースメンブレン、ナイロンメンブレン、PVDFメンブレン等が挙げられる。DNAは、これらの固相担体に公知の方法で固定することが可能である。
キットは、例えば、各種反応・検出用試薬、緩衝液、取扱説明書等をさらに備えていてもよい。
このようなキットは、例えば、逆転写酵素、各種反応・検出試薬、緩衝液、取扱説明書等をさらに備えていてもよい。
なお、本発明は、被検者に由来する血液試料中における、上記(i)及び(ii)から選択される少なくとも1つのmiRNAを測定する工程を含む、膵臓がんの診断方法も含む。
ここで「診断」は、医療行為者が、検出結果等に基づいて、被検者が特定の疾患に罹患しているかどうか判断することを意味する。
本発明に係る膵臓がんの診断方法について用いられる用語のうち、前述の本発明に係る検出方法で用いられた用語はそれと同義であり、ここでは説明を省略する。
本出願は、被検者に由来する血液試料中における配列番号:47で表される配列を有するmiRNA、または当該miRNAと相補的な配列の核酸とストリンジェントな条件でハイブリダイズするmiRNAであって、17~30塩基長であるmiRNAを測定する工程を含む大腸がんの検出方法も包む。配列番号:47で表されるmiRNAは、hsa-miR-23a-3pと称される。
大腸がんの検出方法について用いられる用語のうち、上述した膵臓がんの検出方法でも用いられたものはそれと同義である。
試料としては、血液試料、例えば血清または血漿が好ましい。
hsa-miR-23a-3pは単独でも有用な大腸がんマーカーとして用いることができるし、例えば、特許文献1に開示されたmiRNAの少なくとも1つと組み合わせて用いてもよい。
100mmシャーレで10%FBSを含む培地で培養し、新しい培地に交換してから48時間後の6種類の膵臓がん細胞株(BxPC3, CAPAN1, HPAFII, Hs766T, PANC1, PSN1)及び2種類の不死化膵管上皮細胞株(HPDE4, HPDE6)の培養上清から、段階的に超遠心法(16,500 x g for 20 min、120,000 x g for 70 min)によって、エクソソーム画分を濃縮し、RNAをカラム調整し、マイクロアレイ解析の鋳型とした。16,500 x g で20 分間遠心した後、0.2 μmのナイロンメンブランで濾過したものを次の遠心の試料とした。
エクソソームmiRNAのプロファイルは、アジレント社のmiRNAマイクロアレイを用いて使用説明書に従って行い、GeneSpring Xでデータ解析を行った。
このRNAサンプルを用いて、マイクロアレイ解析を行った結果を図2に示す。
図2に示されるように、膵臓がん細胞が分泌するエクソソームmiRNAのプロファイルは、内在性のmiRNAの発現プロファイルと大きく異なることから、膵臓がん細胞が分泌するmiRNAには選択性があることが分かった。
また、興味深いことに内在性の発現プロファイルでは、がん細胞株と不死化細胞(図中、下線部分)を区別することはできないが(左パネル)、エクソソームmiRNAのプロファイルでは、両者を区別できることがわかった。
この結果は、エクソソームmiRNAのプロファイルの変化は、細胞の悪性転換(がん化)によって誘導される可能性を示唆するものである。
次に、膵臓がん患者由来の血漿検体(n=12)を用いて、エクソソームmiRNAのプロファイリングを行い、膵臓がんに特異的であるmiRNAの候補の検出が臨床の検体でも可能であるか検討を加えた。
まず、各膵臓がん患者の血漿検体(0.75~1 mL)をPBSで10倍に希釈した後、超遠心法によりエクソソーム画分を濃縮した。
具体的には健常人(healthy control)10名及び化学療法前の膵臓がん患者(pancreas cancer)12名の血漿検体のアレイデータをシグナル強度として算出し、血漿量(plasma volume)で補正後に、算出したものを規格化したシグナル強度(normalized signal intensity (AU))とした。
解析には、検出された全miRNAのシグナル強度の総和を100%とし、各miRNAのシグナル値を%として示した値を用いてT検定を行い、p値が0.05以下のものを膵がん患者血漿特異的miRNAとして選別した。結果を下表に示す。また、測定の代表例を図3及び4に示す。
また、別の膵臓がん患者由来の血漿検体(n=12)を用いて、「2.膵臓がん患者血漿を用いたエクソソームmiRNAの測定(1)」と同様の測定を行った。
具体的には健常人(healthy control)13名及び化学療法前の膵臓がん患者(pancreas cancer)12名の血漿検体のアレイデータをシグナル強度として算出し、血漿量(plasma volume)で補正後に、算出したものを規格化したシグナル強度(normalized signal intensity (AU))とした。健常人と膵臓がん症例群について検定(Student’s t-test)を行い、平均値が膵臓がん群で高く、p値が0.05以下のものを健常人に対して有意に高いと判定した。
さらに血漿からエクソソームmiRNAのプロファイルをマイクロアレイで作成し、健常人及び大腸がん患者の血漿の解析データと比較検討した。
エクソソーム画分の濃縮、マイクロアレイによるプロファイリングやその後のデータ解析は、上記1.と同様の方法で行った。
さらに、図5に示されるように、血漿からのエクソソームmiRNAのプロファイルは、大腸がんと膵臓がん患者では大きく異なっており、がん種によって特異的である可能性が強く示唆された。
健常人血清検体(n=11)及びTNMステージ別大腸がん患者血清検体(Stages I(n=20)、II(n=20)、IIIa(n=20)、IIIb(n=16)、IV(n=12))を用いて、エクソソームmiRNAのプロファイリングを行い、大腸がん特異的miRNAを選別した。
まず、血清検体(1 mL)をPBSで10倍に希釈した後、超遠心法によりエクソソーム画分を濃縮し、RNAを調整し、マイクロアレイ解析の鋳型とした。
血清からエクソソームmiRNAのプロファイルをマイクロアレイで作成し、健常人及び大腸がん患者血清の解析データを比較検討した。
エクソソーム画分の濃縮、マイクロアレイによるプロファイリングやその後のデータ解析は、上記1.と同様の方法で行った。
結果を図6に示す。図示されるように、miR-23a-3pについて、どのTNMステージにおいても、コントロールである健常人(n=11)と比べて有意に高い値で検出されることが判明した。また、miR-23a-3pは、健常人の血清からはほとんど検出されなかった。
大腸がん患者同一人物のがん摘出手術の術前術後の血清検体(Stages I(n=6)、II(n=5)、IIIa(n=5)、IIIb(n=5)、IV(n=3))を用いて、miR-23a-3pが術後に減少するか検討した。
血清からエクソソームmiRNAのプロファイルをマイクロアレイで作成し、術前術後の大腸がん患者血清の解析データを比較検討した。
エクソソーム画分の濃縮、マイクロアレイによるプロファイリングやその後のデータ解析は、上記1.と同様の方法で行った。
結果を図7に示す。図示されるように、術前に比べて術後では、miR-23a-3pは有意に低い値で検出されることが判明した。また、どのTNMステージにおいても術後に減少した。
Claims (5)
- 被検者の膵臓がんを検出する方法であって、
被検者に由来する試料中における以下の(i)及び(ii)から選択される少なくとも1つのmiRNAを測定する工程を含む、検出方法:
(i) 配列番号:1~120のいずれかで表される配列を有するmiRNA;及び
(ii) (i)のいずれかのmiRNAと相補的な配列の核酸とストリンジェントな条件でハイブリダイズするmiRNAであって、17~30塩基長であるmiRNA。 - 前記(i)から選択されるmiRNAが、配列番号:1~3、5、7、12、13、19~21、26、27、30、32、35、36、42、50、62~64、70~73、78、80、81、82、及び85~120のいずれかで表される配列を有する、請求項1に記載の検出方法。
- 前記被検者に由来する試料は、
被検者から採取された血漿からエクソソーム画分を濃縮する工程と、前記エクソソーム画分からmiRNAを抽出する工程と、を含む方法によって調製される、請求項1または2に記載の検出方法。 - 請求項1から3のいずれか1項に記載の検出方法を行うためのキットであって、
前記(i)及び(ii)から選択されるmiRNAとハイブリダイズするDNAが固定された固相担体を含む、キット。 - 請求項1から3のいずれか1項に記載の検出方法を行うためのキットであって、
前記(i)及び(ii)から選択されるmiRNAまたはこれに相補的な核酸をPCR法で増幅するのに必要なプライマーセットを含む、キット。
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