WO2019220759A1 - Cancer analysis method, cancer analysis system, program, and computer readable recording medium - Google Patents

Cancer analysis method, cancer analysis system, program, and computer readable recording medium Download PDF

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WO2019220759A1
WO2019220759A1 PCT/JP2019/010735 JP2019010735W WO2019220759A1 WO 2019220759 A1 WO2019220759 A1 WO 2019220759A1 JP 2019010735 W JP2019010735 W JP 2019010735W WO 2019220759 A1 WO2019220759 A1 WO 2019220759A1
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cancer
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松岡　孝明
克昭 團
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Ｓｔｅｍｃｅｌｌ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M1/00—Apparatus for enzymology or microbiology
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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  • the present invention relates to a cancer analysis method, a cancer analysis system, a program, and a computer-readable recording medium.
  • Cancer is the number one cause of death in the 2016 demographic statistics published by the Ministry of Health, Labor and Welfare. In addition, since cancer cells have extremely high proliferation ability, early detection is important.
  • tumor marker tests mainly detect proteins released when cancer cells die, and are difficult to detect unless the cancer has progressed to some extent.
  • this tumor marker test is mainly a blood test, which causes a burden on the living body such as blood sampling.
  • cancer tests using microRNA secreted into the blood are being studied.
  • This trial has 13 types of cancer (stomach cancer, esophageal cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, colon cancer, ovarian cancer, prostate cancer, bladder cancer, Using the characteristic that there are 2 to 10 types of microRNAs unique to each of breast cancer, sarcoma, and glioma), cancer tests are performed by analyzing microRNAs in the blood of patients ( For example, refer nonpatent literature 1).
  • An object of the present invention is to provide a cancer analysis method, a cancer analysis system, a program, and a computer-readable recording medium that are non-invasive and capable of early detection of cancer.
  • the present inventors have found that the above object can be achieved by using an exosome derived from a urine specimen, and have completed the present invention.
  • an extraction step of extracting exosomes from a urine sample a step of quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in the exosomes, miR-103, a method of analyzing cancer, comprising an analysis step of analyzing cancer status based on the amount of miR-21 and / or miR-19 microRNA,
  • the method for analyzing cancer according to (1) wherein the extraction step is performed using filter paper that has been treated so that the exosome can be trapped.
  • the method for analyzing cancer according to (2) comprising a step of collecting the urine sample on the filter paper and transporting the sample to a testing institution.
  • a measurement unit for quantifying the amount of microRNAs of miR-103 and miR-21 and / or miR-19, and the measurement unit quantified a measuring device comprising miR-103 and a transmitting unit that transmits the microRNA amount of miR-21 and / or miR-19 to the analyzer; a receiving unit that receives the microRNA amount transmitted from the measuring device; a cancer analysis system including an analysis device including an analysis unit that analyzes a cancer state based on a microRNA amount; (5) Means for obtaining microRNA data obtained by quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in exosomes extracted from urine samples by a computer And a program for functioning as a means of analyzing cancer status based on miR-103 and miR-21 and / or miR-19 microRNA content data, (6) Means for obtaining microRNA data obtained by quantifying miR-103 and miR-21 and / or miR-19
  • a cancer analysis method a cancer analysis system, a program, and a computer-readable recording medium that are non-invasive and capable of early detection of cancer.
  • FIG. 1 is a block diagram showing a system configuration of a cancer analysis system according to an embodiment of the present invention. It is a flowchart which shows the cancer analysis method using the cancer analysis system which concerns on embodiment of this invention. It is a graph which shows the value with respect to the quantity of miR-103 of the quantity of miR-21 obtained by quantitative PCR in the Example of this invention. It is a graph which shows the value with respect to the quantity of miR-103 of the quantity of miR-19 obtained by quantitative PCR in the Example of this invention.
  • the cancer analysis method of the present invention includes an extraction step of extracting exosomes from a urine sample, and a step of quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in the exosomes And miR-103 and an analysis step of analyzing the cancer state based on the amount of miR-21 and / or miR-19 microRNA.
  • the cancer analysis system of the present invention is a measurement unit that quantifies the amount of microRNAs of miR-103 and miR-21 and / or miR-19 among microRNAs present in exosomes extracted from urine samples.
  • a measuring device comprising: a miR-103 quantified by the measuring unit; and a transmitting unit that transmits the amount of miR-21 and / or miR-19 microRNA to the analyzer; and the microRNA transmitted from the measuring device
  • An analyzer including a receiving unit that receives the amount and an analyzing unit that analyzes a cancer state based on the microRNA amount.
  • FIG. 1 is a block diagram showing a system configuration of a cancer analysis system according to an embodiment of the present invention.
  • the cancer analysis system 2 includes a quantitative PCR device 4 as a measurement device and a PC (personal computer) 6 as an analysis device.
  • the quantitative PCR device 4 includes a control unit 8 that comprehensively controls each unit of the quantitative PCR device 4, and the control unit 8 has an input unit 10 that receives input operations such as measurement conditions and measurement start, A measurement unit 12 that amplifies and quantifies using primers, miR-103 measured by the measurement unit 12, and a transmission unit 14 that transmits miR-21 and / or miR-19 microRNA data to the PC 6 are connected. Yes.
  • the PC 6 includes a CPU 16.
  • the CPU 16 receives the miR-103 and miR-21 and / or miR-19 microRNA amount data transmitted from the quantitative PCR device 4, and the microRNA amount data.
  • exosomes are extracted from the urine specimen (step S1).
  • exosome extraction methods include ultracentrifugation, ultrafiltration, gel filtration, HPLC, and methods using extraction reagents.
  • filter paper examples include FTA micro card (manufactured by GE Healthcare) and the like (for example, see US Pat. Nos. 5,496,562, 5,756,126, 5,807,527, 5,972,386, and 5,985,327).
  • This FTA microcard is a cellulose-based paper, a weak base such as trishydroxymethylmethane, a chelating agent such as ethylenediaminetetraacetate, an anionic surfactant such as sodium dodecyl sulfate, a free radical trap such as uric acid or urate.
  • a filter paper having adsorbed or incorporated therein a composition comprising
  • the filter paper constituting the FTA microcard is designed to trap the nucleic acid entangled, but according to the study by the present inventors, when a urine sample is poured or attached to the FTA microcard, the exosome Was found trapped.
  • the urine specimen-attached filter paper When transporting the urine specimen-attached filter paper, it may be transported in a predetermined bag from the viewpoint of deodorization.
  • exosomes are extracted from the urine specimen, and then microRNA is taken out from the exosomes (step S2).
  • the method for extracting microRNA from the exosome is not particularly limited, and various commercially available extraction kits can be used.
  • miRCURY RNA Isolation Kit Product # 300110; EXIQON
  • EXIQON EXIQON
  • step S3 the amount of the extracted microRNA is quantified using the quantitative PCR device 4 (step S3).
  • the microRNA extracted in step S2 is set in the measurement unit 12.
  • the control unit 8 starts quantification of the microRNA set in the measurement unit 12.
  • the quantitative PCR apparatus 4 it is preferable to perform measurement by real-time PCR in which amplification by polymerase chain reaction (PCR) is measured over time.
  • microRNAs to be quantified are miR-103, miR-21 and / or miR-19.
  • miR-103 is stably expressed in serum / plasma, urine and cerebrospinal fluid exosomes (for example, http://www.exiqon.com/ls/Documents/Scientific/microRNA -See Table 6 (page 25) of -serum-plasma-guidelines.pdf).
  • miR-21 is a disease-specific molecule contained in exosomes extracted from serum or cerebrospinal fluid, etc., as ovarian cancer, colon cancer, pancreatic cancer, cervical cancer, liver cancer, pharyngeal flatness It is a microRNA found in a wide variety of cancers such as epithelial cancer and glioma. That is, it is a kind of microRNA that is secreted by a wide variety of cancer cells.
  • microRNAs that are stably expressed in serum / plasma, urine, and cerebrospinal fluid exosomes, such as miR-103 described above, and microRNAs that are not stably expressed (same as above)
  • microRNAs that are not stably expressed (same as above)
  • Table 6 page 25 of http://www.exiqon.com/ls/Documents/Scientific/microRNA-serum-plasma-guidelines.pdf. Therefore, just because it is expressed as a type of microRNA contained in an exosome extracted from serum or cerebrospinal fluid, it cannot be expressed as a type of microRNA contained in an exosome extracted from urine. That is, a complete correlation is not observed between the type of microRNA contained in exosomes extracted from serum or cerebrospinal fluid and the type of microRNA contained in exosomes extracted from urine.
  • MiR-19 is a kind of microRNA whose secretion is suppressed from a wide range of cancer cells, and its expression level is low as a kind of microRNA contained in exosomes extracted from serum or cerebrospinal fluid of cancer patients. Become.
  • the control unit 8 of the quantitative PCR device 4 transmits the microRNA amount data obtained by quantification to the PC 6 via the transmission unit 14 (step S4), and the PC 6 is obtained by quantification via the reception unit 18.
  • the microRNA amount data is received (step S5).
  • the communication between the transmission unit 14 of the quantitative PCR device 4 and the reception unit 18 of the PC 6 may be performed by wired communication such as a wired LAN, or by wireless communication such as a wireless LAN or Bluetooth (registered trademark). May be.
  • the CPU 16 of the PC 6 stores the microRNA amount data received through the receiving unit 18 in the storage unit 20 (step S6).
  • the storage unit 20 stores the microRNA amount data in association with, for example, the specimen ID, the measurement date and time, the measurement number, and the like.
  • the CPU 16 causes the analysis unit 22 to analyze the cancer state (step S7).
  • the CPU 16 reads out the data designated as the analysis target out of the microRNA amount data stored in the storage unit 20, and analyzes the cancer state in the analysis unit 22. To do.
  • the amount of microRNA quantified using the quantitative PCR device 4 can be analyzed by statistical processing, but it is preferable to statistically analyze data obtained by the comparative Ct method. For example, by continuously quantifying miR-103 and miR-21 and / or miR-19 in exosomes extracted from a specific person's urine sample, normal (ie when non-cancerous) Data can be collected and analyzed for cancer when miR-21 expression is increased and / or when miR-19 expression is decreased.
  • the amount of miR-21 and the amount of miR-19 values compared with the amount of miR-103 are used. As will be described later in the examples, these values were found to be values that fluctuate with a statistically significant difference between the two groups of cancer and non-cancer.
  • the amount of miR-103 in the amount of miR-21 than the continuously collected data is analyzed as a cancerous state.
  • the analysis when analyzing the cancer state, the analysis may be performed using a predetermined threshold.
  • a predetermined threshold In this case, the value of miR-21 relative to the amount of miR-103, and / or the value of miR-19 from all data or randomly extracted data of the amount of microRNA stored in storage unit 20
  • a threshold value for analyzing a state of cancer in advance is calculated by statistically processing a value of the amount relative to the amount of miR-103. Then, by setting this threshold value as a threshold value when analyzing the cancer state, it is possible to analyze whether or not specific data is in a cancerous state.
  • data such as outliers and missing values may be removed to analyze the cancer state.
  • cancer can be found non-invasively and at an early stage.
  • the PC 6 is described as an example of the analysis device.
  • a tablet terminal or the like may be used as the analysis device.
  • the cancer analysis system 2 has been described by taking as an example a configuration including two devices, the quantitative PCR device 4 and the PC 6, but for example, by incorporating the function of the analysis device into the quantitative PCR device 4, one device can be used.
  • An analysis system can also be configured.
  • the microRNA amount data is transmitted from the quantitative PCR device 4 serving as a measurement device to the PC 6 serving as an analysis device.
  • communication between the quantitative PCR device 4 and the PC 6 is not possible.
  • the amount of microRNA data may be transferred from the quantitative PCR device 4 to the PC 6 via a recording medium such as a USB memory or an SD card.
  • the configuration has been described in which the PC 6 analyzes the cancer state when the analysis target data is specified via the operation unit 24 and an analysis start instruction is input.
  • Analysis of cancer status based on microRNA content data obtained by quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in exosomes extracted from By downloading the program via a network such as the Internet and incorporating the program into the computer, the computer may function so that the above-described analysis processing can be performed.
  • this analysis program reads miR-103 and miR-21 and / or miR-19 microRNA content data stored on a network or on a computer and uses it to analyze cancer status. Also good.
  • the program may be recorded on a computer-readable recording medium such as a flexible disk, a CD-ROM, or a DVD. That is, by reading from a computer-readable recording medium and incorporating it in a computer, the computer may function so that the above-described cancer state analysis can be performed.
  • a computer-readable recording medium such as a flexible disk, a CD-ROM, or a DVD. That is, by reading from a computer-readable recording medium and incorporating it in a computer, the computer may function so that the above-described cancer state analysis can be performed.
  • microRNA was extracted from each exosome using a microRNA extraction kit (miRCURY RNA Isolation Kit (Product # 300110; EXIQON)). Then, the obtained microRNA is dispersed or dissolved in ultrapure water to a predetermined concentration, and then reverse transcription reaction is performed from each specimen by reverse transcriptase to synthesize cDNA, and the cDNA is used to miR -103, and miR-21 and miR-19 expression were analyzed by quantitative PCR.
  • a microRNA extraction kit miRCURY RNA Isolation Kit (Product # 300110; EXIQON)
  • the specific procedure for the quantitative PCR method was as follows.
  • the cDNA obtained from the microRNA was reverse transcribed into cDNA according to the method of PrimeScript RT Master Mix (Takara) and amplified with SYBR Premix EX Taq II (Takara).
  • the PCR reaction solution was 50 ⁇ L (25 ⁇ L SYBR Green Mix (2x), 1 ⁇ L cDNA, 2 ⁇ L primer pair mix (5 pmol / ⁇ L each primer), 22 ⁇ L H 2 O), and the reaction was carried out at 95 ° C. 30 cycles for 30 seconds and 95 cycles for 5 seconds and 60 ° C for 30 seconds under 50 cycles. Relative quantification was performed by the comparative Ct method ( ⁇ Ct method).
  • predetermined primers were used as the primers for various targets (miR-103, miR-21 and miR-19) used for RT-PCR (real-time PCR).
  • One Step-RT-PCR Kit that can perform this RT reaction and quantitative PCR in one test tube based on microRNA can be used.
  • RT reaction and quantitative PCR in one test tube based on microRNA can be used.
  • ladder Universal One-step Reaction Mix New England BioLabs
  • FIG. 3 shows the value of miR-21 obtained by quantitative PCR with respect to the amount of miR-103
  • FIG. 4 shows the value of miR-19 with respect to the amount of miR-103.
  • the portion surrounded by a dotted line is a range that can be analyzed as being a cancer state
  • the portion surrounded by a solid line is a range that can be analyzed as being a non-cancer state, and is surrounded by a broken line. This is the range that can be analyzed with the border state or threshold value.

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Abstract

The present invention comprises: an extraction step for extracting an exosome from a urine sample; a step for quantitatively analyzing the miR-103 and the miR-21 and/or the miR-19 from among the microRNAs present in the exosome; and an analysis step for analyzing the cancer condition on the basis of the quantities of the microRNAs, i.e., the quantities of the miR-103 and of the miR-21 and/or the miR-19.

Description

がんの分析方法、がんの分析システム、プログラムおよびコンピュータ読み取り可能な記録媒体Cancer analysis method, cancer analysis system, program, and computer-readable recording medium
　本発明は、がんの分析方法、がんの分析システム、プログラムおよびコンピュータ読み取り可能な記録媒体に関するものである。 The present invention relates to a cancer analysis method, a cancer analysis system, a program, and a computer-readable recording medium.
　がんは、厚生労働省により発表された平成２８年（２０１６）人口動態統計の死因において第１位となっている。また、がん細胞は増殖能力が極めて高いため、早期発見が重要となっている。 Cancer is the number one cause of death in the 2016 demographic statistics published by the Ministry of Health, Labor and Welfare. In addition, since cancer cells have extremely high proliferation ability, early detection is important.
　現在、がんの検査としてＰＥＴ検査、Ｘ線検査、腫瘍マーカー検査等が行われているが、がんの大きさが５ｍｍ以上とならないと検出できない一方、がんの大きさが０．１ｍｍ程度からすでに転移が生じる可能性についても報告されている。 Currently, PET tests, X-ray tests, tumor marker tests, etc. are performed as cancer tests, but they cannot be detected unless the cancer size is 5 mm or more, while the cancer size is about 0.1 mm. The possibility of metastasis has already been reported.
　例えば、腫瘍マーカー検査は主にがん細胞が死ぬ際に放出されるたんぱく質を検出するものであり、がんがある程度進行した状態でなければ発見が困難である。また、この腫瘍マーカー検査は血液検査が主体の検査であり、血液採取等、検査時の生体への負担も生じるものである。 For example, tumor marker tests mainly detect proteins released when cancer cells die, and are difficult to detect unless the cancer has progressed to some extent. In addition, this tumor marker test is mainly a blood test, which causes a burden on the living body such as blood sampling.
　他方で、血中に分泌するmicroRNA（マイクロRNA）を用いるがん検査が検討されている。この試みは、１３種類のがん（胃がん、食道がん、肺がん、肝臓がん、胆道がん、膵臓（すいぞう）がん、大腸がん、卵巣がん、前立腺がん、ぼうこうがん、乳がん、肉腫、神経膠腫）のそれぞれに特有のmicroRNAが２～１０種類存在するという特徴を利用して、患者の血液中のmicroRNAを分析することにより、がん検査を行うというものである（例えば非特許文献１参照）。 On the other hand, cancer tests using microRNA secreted into the blood are being studied. This trial has 13 types of cancer (stomach cancer, esophageal cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, colon cancer, ovarian cancer, prostate cancer, bladder cancer, Using the characteristic that there are 2 to 10 types of microRNAs unique to each of breast cancer, sarcoma, and glioma), cancer tests are performed by analyzing microRNAs in the blood of patients ( For example, refer nonpatent literature 1).
　また、血液中のアミノ酸濃度を測定して、がんのリスクを予測する検査も行われている（例えば、特許文献１～３参照）。この検査はアミノインデックス（登録商標）がんリスクスクリーニングと呼ばれるものであり、血液中のアミノ酸濃度を測定し、健康な人とがんである人のアミノ酸濃度のバランスの違いを統計的に解析することにより、がんのリスクの予測を行っている。 Also, a test for predicting cancer risk by measuring the amino acid concentration in blood has been carried out (for example, see Patent Documents 1 to 3). This test is called AminoIndex (R) cancer risk screening, which measures amino acid levels in the blood and statistically analyzes the difference in amino acid levels between healthy and cancerous people To predict cancer risk.
　しかし、これらの検査は血液を用いる検査であり、生体への負担を生じない非侵襲のものではなかった。 However, these tests are tests using blood, and are not non-invasive that do not cause a burden on the living body.
国際公開第２００８／０７５６６２号International Publication No. 2008/077562 国際公開第２００８／０７５６６３号International Publication No. 2008/077563 国際公開第２００８／０７５６６４号International Publication No. 2008/077564
　本発明の目的は、非侵襲かつ、早期にがんの発見が可能ながんの分析方法、がんの分析システム、プログラムおよびコンピュータ読み取り可能な記録媒体を提供することである。 An object of the present invention is to provide a cancer analysis method, a cancer analysis system, a program, and a computer-readable recording medium that are non-invasive and capable of early detection of cancer.
　本発明者らは、上記課題を解決するために鋭意検討の結果、尿検体由来のエクソソームを用いることにより上記目的を達成できることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that the above object can be achieved by using an exosome derived from a urine specimen, and have completed the present invention.
　即ち、本発明によれば、
（１）　尿検体からエクソソームを抽出する抽出工程と、前記エクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とを定量する工程と、miR-103と、miR-21および／またはmiR-19のmicroRNA量に基づいてがんの状態を分析する分析工程とを含むがんの分析方法、
（２）　前記抽出工程は、前記エクソソームをトラップできるように処理されたろ紙を用いて行う（１）記載のがんの分析方法、
（３）　前記尿検体を前記ろ紙上に採取して検査機関に輸送する工程を含む（２）記載のがんの分析方法、
（４）　尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とのmicroRNA量を定量する測定部と、前記測定部により定量されたmiR-103と、miR-21および／またはmiR-19のmicroRNA量を分析装置に送信する送信部とを備える測定装置と、前記測定装置から送信された前記microRNA量を受信する受信部と、前記microRNA量に基づいてがんの状態を分析する分析する分析部とを備える分析装置とを含むがんの分析システム、
（５）　コンピュータを尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とを定量して得られたmicroRNA量のデータを取得する手段と、miR-103と、miR-21および／またはmiR-19のmicroRNA量のデータに基づいてがんの状態を分析する手段として機能させるためのプログラム、
（６）　コンピュータを尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とを定量して得られたmicroRNA量のデータを取得する手段と、miR-103と、miR-21および／またはmiR-19の前記microRNA量のデータに基づいてがんの状態を分析する手段として機能させるためのプログラムを記録したコンピュータ読み取り可能な記録媒体
が提供される。 That is, according to the present invention,
(1) an extraction step of extracting exosomes from a urine sample, a step of quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in the exosomes, miR-103, a method of analyzing cancer, comprising an analysis step of analyzing cancer status based on the amount of miR-21 and / or miR-19 microRNA,
(2) The method for analyzing cancer according to (1), wherein the extraction step is performed using filter paper that has been treated so that the exosome can be trapped.
(3) The method for analyzing cancer according to (2), comprising a step of collecting the urine sample on the filter paper and transporting the sample to a testing institution.
(4) Among the microRNAs present in exosomes extracted from urine specimens, a measurement unit for quantifying the amount of microRNAs of miR-103 and miR-21 and / or miR-19, and the measurement unit quantified a measuring device comprising miR-103 and a transmitting unit that transmits the microRNA amount of miR-21 and / or miR-19 to the analyzer; a receiving unit that receives the microRNA amount transmitted from the measuring device; a cancer analysis system including an analysis device including an analysis unit that analyzes a cancer state based on a microRNA amount;
(5) Means for obtaining microRNA data obtained by quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in exosomes extracted from urine samples by a computer And a program for functioning as a means of analyzing cancer status based on miR-103 and miR-21 and / or miR-19 microRNA content data,
(6) Means for obtaining microRNA data obtained by quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in exosomes extracted from urine samples by a computer And a computer-readable recording medium on which miR-103 and a program for functioning as a means for analyzing the cancer state based on the microRNA data of miR-21 and / or miR-19 are recorded Is done.
　本発明によれば、非侵襲かつ、早期にがんの発見が可能ながんの分析方法、がんの分析システム、プログラムおよびコンピュータ読み取り可能な記録媒体が提供される。 According to the present invention, there are provided a cancer analysis method, a cancer analysis system, a program, and a computer-readable recording medium that are non-invasive and capable of early detection of cancer.
本発明の実施の形態に係るがんの分析システムのシステム構成を示すブロック図である。1 is a block diagram showing a system configuration of a cancer analysis system according to an embodiment of the present invention. 本発明の実施の形態に係るがんの分析システムを用いたがんの分析方法を示すフローチャートである。It is a flowchart which shows the cancer analysis method using the cancer analysis system which concerns on embodiment of this invention. 本発明の実施例における定量ＰＣＲにより得られたmiR-21の量のmiR-103の量に対する値を示すグラフである。It is a graph which shows the value with respect to the quantity of miR-103 of the quantity of miR-21 obtained by quantitative PCR in the Example of this invention. 本発明の実施例における定量ＰＣＲにより得られたmiR-19の量のmiR-103の量に対する値を示すグラフである。It is a graph which shows the value with respect to the quantity of miR-103 of the quantity of miR-19 obtained by quantitative PCR in the Example of this invention.
　以下、図面を参照して本発明のがんの分析方法およびがんの分析システムについて説明する。 Hereinafter, the cancer analysis method and cancer analysis system of the present invention will be described with reference to the drawings.
　本発明のがんの分析方法は、尿検体からエクソソームを抽出する抽出工程と、前記エクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とを定量する工程と、miR-103と、miR-21および／またはmiR-19のmicroRNA量に基づいてがんの状態を分析する分析工程とを含む。 The cancer analysis method of the present invention includes an extraction step of extracting exosomes from a urine sample, and a step of quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in the exosomes And miR-103 and an analysis step of analyzing the cancer state based on the amount of miR-21 and / or miR-19 microRNA.
　また、本発明のがんの分析システムは、尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とのmicroRNA量を定量する測定部と、前記測定部により定量されたmiR-103と、miR-21および／またはmiR-19のmicroRNA量を分析装置に送信する送信部とを備える測定装置と、前記測定装置から送信された前記microRNA量を受信する受信部と、前記microRNA量に基づいてがんの状態を分析する分析部とを備える分析装置とを含む。 In addition, the cancer analysis system of the present invention is a measurement unit that quantifies the amount of microRNAs of miR-103 and miR-21 and / or miR-19 among microRNAs present in exosomes extracted from urine samples. A measuring device comprising: a miR-103 quantified by the measuring unit; and a transmitting unit that transmits the amount of miR-21 and / or miR-19 microRNA to the analyzer; and the microRNA transmitted from the measuring device An analyzer including a receiving unit that receives the amount and an analyzing unit that analyzes a cancer state based on the microRNA amount.
　図１は、本発明の実施の形態に係るがんの分析システムのシステム構成を示すブロック図である。図１に示すようにがんの分析システム２は、測定装置としての定量ＰＣＲ装置４、分析装置としてのＰＣ（パーソナルコンピュータ）６を備えている。 FIG. 1 is a block diagram showing a system configuration of a cancer analysis system according to an embodiment of the present invention. As shown in FIG. 1, the cancer analysis system 2 includes a quantitative PCR device 4 as a measurement device and a PC (personal computer) 6 as an analysis device.
　また、定量ＰＣＲ装置４は、定量ＰＣＲ装置４の各部を統括的に制御する制御部８を備え、制御部８には測定条件や測定開始等の入力操作を受け付ける入力部１０、microRNAを所定のプライマーを用いて増幅し、定量する測定部１２、測定部１２により測定したmiR-103と、miR-21および／またはmiR-19のmicroRNA量のデータをＰＣ６へ送信する送信部１４が接続されている。 The quantitative PCR device 4 includes a control unit 8 that comprehensively controls each unit of the quantitative PCR device 4, and the control unit 8 has an input unit 10 that receives input operations such as measurement conditions and measurement start, A measurement unit 12 that amplifies and quantifies using primers, miR-103 measured by the measurement unit 12, and a transmission unit 14 that transmits miR-21 and / or miR-19 microRNA data to the PC 6 are connected. Yes.
　また、ＰＣ６は、ＣＰＵ１６を備え、ＣＰＵ１６には定量ＰＣＲ装置４から送信されたmiR-103と、miR-21および／またはmiR-19のmicroRNA量のデータを受信する受信部１８、microRNA量のデータを記憶する記憶部２０、microRNA量のデータに基づいてがんの状態を分析する分析部２２、がんの状態の分析に用いるデータの指定およびがんの分析開始指示等の入力を行う操作部２４が接続されている。 The PC 6 includes a CPU 16. The CPU 16 receives the miR-103 and miR-21 and / or miR-19 microRNA amount data transmitted from the quantitative PCR device 4, and the microRNA amount data. A storage unit 20 for storing the data, an analysis unit 22 for analyzing the cancer state based on the microRNA amount data, an operation unit for inputting data used for analyzing the cancer state, an instruction to start cancer analysis, and the like 24 is connected.
　次に、図２に示すフローチャートを参照して本発明の実施の形態に係るがんの分析システムを用いたがんの分析方法について説明する。 Next, a cancer analysis method using the cancer analysis system according to the embodiment of the present invention will be described with reference to the flowchart shown in FIG.
　まず、本発明においては、検体として尿を用い、この尿検体からエクソソームを抽出する（ステップＳ１）。エクソソームの抽出方法としては、超遠心分離法、限外濾過法、ゲル濾過法、ＨＰＬＣや抽出用試薬を用いた方法等が挙げられるが、エクソソームをトラップできるように処理されたろ紙を用いることが好ましい。 First, in the present invention, urine is used as a specimen, and exosomes are extracted from the urine specimen (step S1). Examples of exosome extraction methods include ultracentrifugation, ultrafiltration, gel filtration, HPLC, and methods using extraction reagents. Use of filter paper that has been treated to trap exosomes. preferable.
　このようなろ紙としては例えば、ＦＴＡマイクロカード（ＧＥヘルスケア社製）等が挙げられる（たとえば、米国特許第５４９６５６２号、第５７５６１２６号、第５８０７５２７号、第５９７２３８６号および第５９８５３２７号等参照）。このＦＴＡマイクロカードは、セルロースベースの紙に、トリスヒドロキシメチルメタン等の弱塩基、エチレンジアミン四酢酸塩等のキレート剤、ドデシル硫酸ナトリウム等のアニオン性界面活性剤、尿酸または尿酸塩等のフリーラジカルトラップを含んでなる組成物を吸着又は組み込ませたろ紙を有する。 Examples of such filter paper include FTA micro card (manufactured by GE Healthcare) and the like (for example, see US Pat. Nos. 5,496,562, 5,756,126, 5,807,527, 5,972,386, and 5,985,327). This FTA microcard is a cellulose-based paper, a weak base such as trishydroxymethylmethane, a chelating agent such as ethylenediaminetetraacetate, an anionic surfactant such as sodium dodecyl sulfate, a free radical trap such as uric acid or urate. A filter paper having adsorbed or incorporated therein a composition comprising
　なお、ＦＴＡマイクロカードを構成するろ紙は、核酸を絡めてトラップするように設計されているが、本発明者らの検討によれば、ＦＴＡマイクロカードに尿検体を注ぐ、又は付着させると、エクソソームがトラップされることが分かった。 The filter paper constituting the FTA microcard is designed to trap the nucleic acid entangled, but according to the study by the present inventors, when a urine sample is poured or attached to the FTA microcard, the exosome Was found trapped.
　したがって、ＦＴＡマイクロカード等のエクソソームをトラップできるように処理されたろ紙に尿検体を注ぐ等することにより付着又は吸着させた後に、この状態（以下、「尿検体付着ろ紙」ということがある。）で簡便に運搬することができる。 Therefore, after being attached or adsorbed by pouring the urine sample on a filter paper treated so as to trap exosomes such as FTA microcards, this state (hereinafter sometimes referred to as “urine sample-attached filter paper”). Can be transported easily.
　エクソソームの抽出方法として超遠心分離法、限外濾過法、ゲル濾過法、ＨＰＬＣや抽出用試薬を用いた方法を用いた場合には、採尿してから採尿管等により液体の状態で検査機関・検査室まで運搬する必要があるが、エクソソームをトラップできるように処理されたろ紙を用いると、ＦＴＡマイクロカード等のカードや紙を媒体として検査機関・検査室まで運搬することができるため、室温で運搬できるなどの輸送の容易さの点、輸送コストの点で好ましい。 When using an ultracentrifugation method, ultrafiltration method, gel filtration method, HPLC or a method using an extraction reagent as an exosome extraction method, in the liquid state with a urine collection tube after collecting urine, It is necessary to transport to the laboratory, but if filter paper that has been processed so that exosomes can be trapped is used, it can be transported to a laboratory or laboratory using a card or paper such as an FTA microcard as a medium. It is preferable in terms of ease of transportation such as being transportable and transportation cost.
　なお、尿検体付着ろ紙を運搬する際には、防臭等の観点から所定の袋等に入れて運搬してもよい。 When transporting the urine specimen-attached filter paper, it may be transported in a predetermined bag from the viewpoint of deodorization.
　次に抽出工程において尿検体からエクソソームを抽出した後、エクソソームからmicroRNAを取り出す（ステップＳ２）。エクソソームからmicroRNAを取り出す方法としては、特に限定されず、市販の種々の抽出用キットを用いることができる。例えば、miRCURY RNA Isolation Kit (Product# 300110; EXIQON社)などが使用できる。 Next, in the extraction step, exosomes are extracted from the urine specimen, and then microRNA is taken out from the exosomes (step S2). The method for extracting microRNA from the exosome is not particularly limited, and various commercially available extraction kits can be used. For example, miRCURY RNA Isolation Kit (Product # 300110; EXIQON) can be used.
　次に、抽出したmicroRNAの量を、定量ＰＣＲ装置４を用いて定量する（ステップＳ３）。定量を行う際には、まず、ステップＳ２にて取り出したmicroRNAを測定部１２にセットする。そして、操作者により入力部１０から定量開始の指示が入力されると、制御部８は測定部１２にセットされたmicroRNAの定量を開始させる。ここで、定量ＰＣＲ装置４においてはポリメラーゼ連鎖反応（ＰＣＲ）による増幅を経時的に測定するリアルタイムＰＣＲにより測定を行うことが好ましい。 Next, the amount of the extracted microRNA is quantified using the quantitative PCR device 4 (step S3). When quantification is performed, first, the microRNA extracted in step S2 is set in the measurement unit 12. Then, when an instruction to start quantification is input from the input unit 10 by the operator, the control unit 8 starts quantification of the microRNA set in the measurement unit 12. Here, in the quantitative PCR apparatus 4, it is preferable to perform measurement by real-time PCR in which amplification by polymerase chain reaction (PCR) is measured over time.
　本発明において、定量する対象となるmicroRNAは、miR-103と、miR-21および／またはmiR-19である。 In the present invention, microRNAs to be quantified are miR-103, miR-21 and / or miR-19.
　ここで、miR-103は血清／血漿中、尿中および脳脊髄液のエクソソーム中において安定して発現するものである（例えば、http://www.exiqon.com/ls/Documents/Scientific/microRNA-serum-plasma-guidelines.pdfのTable6（25頁）参照）。 Here, miR-103 is stably expressed in serum / plasma, urine and cerebrospinal fluid exosomes (for example, http://www.exiqon.com/ls/Documents/Scientific/microRNA -See Table 6 (page 25) of -serum-plasma-guidelines.pdf).
　また、miR-21は、血清または脳脊髄液等から抽出されたエクソソームに含まれる疾患特異的分子として、卵巣がん、大腸がん、膵臓がん、子宮頸がん、肝臓がん、咽頭扁平上皮がんおよび神経膠腫といった幅広い種類のがんにみられるmicroRNAである。即ち、幅広い種類のがん細胞から多く分泌される種類のmicroRNAである。 In addition, miR-21 is a disease-specific molecule contained in exosomes extracted from serum or cerebrospinal fluid, etc., as ovarian cancer, colon cancer, pancreatic cancer, cervical cancer, liver cancer, pharyngeal flatness It is a microRNA found in a wide variety of cancers such as epithelial cancer and glioma. That is, it is a kind of microRNA that is secreted by a wide variety of cancer cells.
　ここで、上述のmiR-103のように血清／血漿中、尿中および脳脊髄液のエクソソームで安定して発現する種類のmicroRNAもあれば、安定して発現しないmicroRNAも存在する（上記と同様に、例えば、http://www.exiqon.com/ls/Documents/Scientific/microRNA-serum-plasma-guidelines.pdfのTable6（25頁）参照）。したがって、血清や脳脊髄液等から抽出されたエクソソームに含まれる種類のmicroRNAとして発現するからといって、尿から抽出されたエクソソームに含まれる種類のmicroRNAとして発現するとはいえないものである。すなわち、血清や脳脊髄液等から抽出されたエクソソームに含まれるmicroRNAの種類と、尿から抽出されたエクソソームに含まれるmicroRNAの種類との間に完全な相関が認められるものではない。 Here, there are microRNAs that are stably expressed in serum / plasma, urine, and cerebrospinal fluid exosomes, such as miR-103 described above, and microRNAs that are not stably expressed (same as above) For example, see Table 6 (page 25) of http://www.exiqon.com/ls/Documents/Scientific/microRNA-serum-plasma-guidelines.pdf). Therefore, just because it is expressed as a type of microRNA contained in an exosome extracted from serum or cerebrospinal fluid, it cannot be expressed as a type of microRNA contained in an exosome extracted from urine. That is, a complete correlation is not observed between the type of microRNA contained in exosomes extracted from serum or cerebrospinal fluid and the type of microRNA contained in exosomes extracted from urine.
　また、miR-19は、幅広いがん細胞から分泌が抑制される種類のmicroRNAであり、がん患者の血清または脳脊髄液等から抽出されたエクソソームに含まれる種類のmicroRNAとしては発現量が少なくなる。 MiR-19 is a kind of microRNA whose secretion is suppressed from a wide range of cancer cells, and its expression level is low as a kind of microRNA contained in exosomes extracted from serum or cerebrospinal fluid of cancer patients. Become.
　次に、定量ＰＣＲ装置４の制御部８は送信部１４を介して、定量によって得られたmicroRNA量のデータをＰＣ６へ送信し（ステップＳ４）、ＰＣ６は受信部１８を介して定量によって得られたmicroRNA量のデータを受信する（ステップＳ５）。ここで、定量ＰＣＲ装置４の送信部１４とＰＣ６の受信部１８との間の通信は有線ＬＡＮ等の有線通信により行ってもよいし、無線ＬＡＮやBluetooth（登録商標）等の無線通信により行ってもよい。 Next, the control unit 8 of the quantitative PCR device 4 transmits the microRNA amount data obtained by quantification to the PC 6 via the transmission unit 14 (step S4), and the PC 6 is obtained by quantification via the reception unit 18. The microRNA amount data is received (step S5). Here, the communication between the transmission unit 14 of the quantitative PCR device 4 and the reception unit 18 of the PC 6 may be performed by wired communication such as a wired LAN, or by wireless communication such as a wireless LAN or Bluetooth (registered trademark). May be.
　次に、ＰＣ６のＣＰＵ１６は、受信部１８を介して受信したmicroRNA量のデータを記憶部２０に記憶させる（ステップＳ６）。ここで、記憶部２０においては、microRNA量のデータを例えば、検体ＩＤや測定日時、測定番号などと対応させて記憶する。 Next, the CPU 16 of the PC 6 stores the microRNA amount data received through the receiving unit 18 in the storage unit 20 (step S6). Here, the storage unit 20 stores the microRNA amount data in association with, for example, the specimen ID, the measurement date and time, the measurement number, and the like.
　そして、操作部２４を介して分析対象のデータを指定し、分析開始の指示が入力されると、ＣＰＵ１６は分析部２２にがんの状態の分析を行わせる（ステップＳ７）。なお、がんの状態の分析を行う際には、ＣＰＵ１６は記憶部２０に記憶されたmicroRNA量のデータのうち、分析対象として指定されたデータを読み出し、分析部２２にがんの状態の分析を行わせる。 Then, when data to be analyzed is designated via the operation unit 24 and an analysis start instruction is input, the CPU 16 causes the analysis unit 22 to analyze the cancer state (step S7). When analyzing the cancer state, the CPU 16 reads out the data designated as the analysis target out of the microRNA amount data stored in the storage unit 20, and analyzes the cancer state in the analysis unit 22. To do.
　ここで、定量ＰＣＲ装置４を用いて定量されたmicroRNA量は、統計処理により分析することができるが、比較Ｃｔ法により得られたデータを統計処理して分析することが好ましい。たとえば、特定の人の尿検体から抽出したエクソソームに含まれるmiR-103と、miR-21および／またはmiR-19を継続的に定量することにより、平常時（すなわち、非がんであるとき）のデータを収集し、これに対してmiR-21の発現量が多くなった時、および／またはmiR-19の発現量が少なくなった時にがんと分析することができる。ここで、miR-21の量およびmiR-19の量は、miR-103の量と比較した値を用いる。これらの値は、実施例においても後述するように、がんと非がんとの２群間で統計的有意差をもって変動する値であることが、本発明者らの検討により判明した。 Here, the amount of microRNA quantified using the quantitative PCR device 4 can be analyzed by statistical processing, but it is preferable to statistically analyze data obtained by the comparative Ct method. For example, by continuously quantifying miR-103 and miR-21 and / or miR-19 in exosomes extracted from a specific person's urine sample, normal (ie when non-cancerous) Data can be collected and analyzed for cancer when miR-21 expression is increased and / or when miR-19 expression is decreased. Here, as the amount of miR-21 and the amount of miR-19, values compared with the amount of miR-103 are used. As will be described later in the examples, these values were found to be values that fluctuate with a statistically significant difference between the two groups of cancer and non-cancer.
　分析部２２によるがんの状態の分析においては、たとえば、特定の個人ごとに継続的に収集したデータを用いることにより、継続的に収集したデータよりもmiR-21の量のmiR-103の量に対する値が多くなった時、および／または、miR-19の量のmiR-103の量に対する値が少なくなった時にがんである状態と分析することができる。 In the analysis of the cancer state by the analysis unit 22, for example, by using the data continuously collected for each specific individual, the amount of miR-103 in the amount of miR-21 than the continuously collected data. When the value for is increased and / or when the value for the amount of miR-19 is decreased, the amount of miR-19 can be analyzed as a cancerous state.
　また、がんの状態を分析する際に、所定の閾値を用いて分析を行ってもよい。この場合には、記憶部２０に記憶されたmicroRNA量のうちの全部のデータや無作為に抽出されたデータからmiR-21の量のmiR-103の量に対する値、および／またはmiR-19の量のmiR-103の量に対する値を統計処理することにより、予めがんである状態と分析するための閾値を算出する。そして、この閾値をがんの状態を分析する際の閾値として設定することにより、特定のデータががんである状態であるか否かの分析を行うことができる。 Moreover, when analyzing the cancer state, the analysis may be performed using a predetermined threshold. In this case, the value of miR-21 relative to the amount of miR-103, and / or the value of miR-19 from all data or randomly extracted data of the amount of microRNA stored in storage unit 20 A threshold value for analyzing a state of cancer in advance is calculated by statistically processing a value of the amount relative to the amount of miR-103. Then, by setting this threshold value as a threshold value when analyzing the cancer state, it is possible to analyze whether or not specific data is in a cancerous state.
　なお、上述のがんの状態の分析においては外れ値や欠損値等のデータを除去して、がんの状態の分析を行ってもよい。 In the above-described analysis of the cancer state, data such as outliers and missing values may be removed to analyze the cancer state.
　本発明の実施の形態に係るがんの分析方法およびがんの分析システムによれば、非侵襲かつ、早期にがんを発見することができる。 According to the cancer analysis method and the cancer analysis system according to the embodiment of the present invention, cancer can be found non-invasively and at an early stage.
　なお、上述の実施の形態においては、分析装置としてＰＣ６を例に説明したが、分析装置としてタブレット端末等を用いてもよい。また、がんの分析システム２として定量ＰＣＲ装置４およびＰＣ６の２台の装置を備える構成を例に説明したが、例えば、定量ＰＣＲ装置４に分析装置の機能を組み込むことにより１台の装置により分析システムを構成することもできる。 In the above-described embodiment, the PC 6 is described as an example of the analysis device. However, a tablet terminal or the like may be used as the analysis device. Further, the cancer analysis system 2 has been described by taking as an example a configuration including two devices, the quantitative PCR device 4 and the PC 6, but for example, by incorporating the function of the analysis device into the quantitative PCR device 4, one device can be used. An analysis system can also be configured.
　また、上述の実施の形態において、測定装置である定量ＰＣＲ装置４から分析装置であるＰＣ６へmicroRNA量のデータを送信する構成としたが、定量ＰＣＲ装置４とＰＣ６との間での通信が不可能である場合等に、ＵＳＢメモリやＳＤカード等の記録媒体を介して定量ＰＣＲ装置４からＰＣ６へmicroRNA量のデータを移行してもよい。 In the above-described embodiment, the microRNA amount data is transmitted from the quantitative PCR device 4 serving as a measurement device to the PC 6 serving as an analysis device. However, communication between the quantitative PCR device 4 and the PC 6 is not possible. If possible, the amount of microRNA data may be transferred from the quantitative PCR device 4 to the PC 6 via a recording medium such as a USB memory or an SD card.
　また、上述の実施の形態において、操作部２４を介して分析対象のデータを指定し、分析開始の指示が入力されると、ＰＣ６によりがんの状態を分析する構成として説明したが、尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とを定量して得られたmicroRNA量のデータに基づいてがんの状態を分析する分析プログラムをインターネット等のネットワークを介してダウンロードし、コンピュータに組み込むことによって、上述の分析処理が行うことができるようにコンピュータを機能させるようにしてもよい。ここで、この分析プログラムは、ネットワーク上またはコンピュータ上に記憶されているmiR-103と、miR-21および／またはmiR-19のmicroRNA量のデータを読み出して、がんの状態の分析に用いてもよい。 Further, in the above-described embodiment, the configuration has been described in which the PC 6 analyzes the cancer state when the analysis target data is specified via the operation unit 24 and an analysis start instruction is input. Analysis of cancer status based on microRNA content data obtained by quantifying miR-103 and miR-21 and / or miR-19 among microRNAs present in exosomes extracted from By downloading the program via a network such as the Internet and incorporating the program into the computer, the computer may function so that the above-described analysis processing can be performed. Here, this analysis program reads miR-103 and miR-21 and / or miR-19 microRNA content data stored on a network or on a computer and uses it to analyze cancer status. Also good.
　また、当該プログラムはフレキシブルディスク、ＣＤ－ＲＯＭ、ＤＶＤ等のコンピュータ読み取り可能な記録媒体に記録されていてもよい。即ち、コンピュータ読み取り可能な記録媒体から読み取り、コンピュータに組み込むことによって、上述のがんの状態の分析を行うことができるようにコンピュータを機能させるようにしてもよい。 In addition, the program may be recorded on a computer-readable recording medium such as a flexible disk, a CD-ROM, or a DVD. That is, by reading from a computer-readable recording medium and incorporating it in a computer, the computer may function so that the above-described cancer state analysis can be performed.
　以下に、実施例を挙げて本発明を説明するが、本発明はこれに限定されるものではない。なお、本実施例における部および％は、特記しない限り重量基準である。 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto. In the examples, parts and% are based on weight unless otherwise specified.
　がん患者（ｎ＝６）および非がん患者（ｎ＝６）のそれぞれについて尿検体をＦＴＡマイクロカードに付着または吸着させた。これにより尿検体からエクソソームを抽出した。 A urine sample was adhered to or adsorbed to an FTA microcard for each of cancer patients (n = 6) and non-cancer patients (n = 6). As a result, exosomes were extracted from the urine specimen.
　次にmicroRNA抽出用キット（miRCURY RNA Isolation Kit (Product# 300110; EXIQON社)）を用いてそれぞれのエクソソームからmicroRNAを抽出した。そして、得られたmicroRNAをそれぞれ超純水により分散または溶解させて所定の濃度とし、次にそれぞれの検体から逆転写酵素によって逆転写反応させて、cDNAを合成させ、そのcDNAを用いて、miR-103と、miR-21およびmiR-19の発現を定量ＰＣＲで解析した。 Next, microRNA was extracted from each exosome using a microRNA extraction kit (miRCURY RNA Isolation Kit (Product # 300110; EXIQON)). Then, the obtained microRNA is dispersed or dissolved in ultrapure water to a predetermined concentration, and then reverse transcription reaction is performed from each specimen by reverse transcriptase to synthesize cDNA, and the cDNA is used to miR -103, and miR-21 and miR-19 expression were analyzed by quantitative PCR.
　定量ＰＣＲ法の具体的な手順としては以下のように行った。上記microRNAから得たcDNAを用いてPrimeScript RT Master Mix (Takara)の方法に準じ、cDNAに逆転写し、SYBR Premix EX Taq II (Takara)により増幅した。なお、PCR反応液としては、５０μＬ（２５μＬ SYBR Green Mix (2x), １μＬ cDNA, ２μＬ primer pair mix (５ｐｍｏｌ／μＬ each primer), ２２μＬ Ｈ２Ｏ）のＰＣＲ反応液を用い、反応は９５℃にて３０秒を１サイクル、さらに９５℃にて５秒および６０℃にて３０秒のサイクルを５０サイクルの条件にて行った。比較Ct法（ΔΔCt法）より相対定量を行った。なお、RT-PCR（リアルタイムＰＣＲ）に用いた各種ターゲット（miR-103と、miR-21およびmiR-19）のPrimerとしてはそれぞれ所定のものを用いた。 The specific procedure for the quantitative PCR method was as follows. The cDNA obtained from the microRNA was reverse transcribed into cDNA according to the method of PrimeScript RT Master Mix (Takara) and amplified with SYBR Premix EX Taq II (Takara). The PCR reaction solution was 50 μL (25 μL SYBR Green Mix (2x), 1 μL cDNA, 2 μL primer pair mix (5 pmol / μL each primer), 22 μL H 2 O), and the reaction was carried out at 95 ° C. 30 cycles for 30 seconds and 95 cycles for 5 seconds and 60 ° C for 30 seconds under 50 cycles. Relative quantification was performed by the comparative Ct method (ΔΔCt method). In addition, as the primers for various targets (miR-103, miR-21 and miR-19) used for RT-PCR (real-time PCR), predetermined primers were used.
　またmicroRNAを元にして、このRT反応と定量PCRを１つの試験管内で行えるOne Step-RT-PCR Kitを用いることも出来る。例えば、Luna Universal One-step Reaction Mix (New England BioLabs社)も使用できる。 Also, One Step-RT-PCR Kit that can perform this RT reaction and quantitative PCR in one test tube based on microRNA can be used. For example, Luna Universal One-step Reaction Mix (New England BioLabs) can also be used.
　そして、定量ＰＣＲにより得られたmiR-21の量のmiR-103の量に対する値を図３に、およびmiR-19の量のmiR-103の量に対する値を図４にそれぞれ示す。 FIG. 3 shows the value of miR-21 obtained by quantitative PCR with respect to the amount of miR-103, and FIG. 4 shows the value of miR-19 with respect to the amount of miR-103.
　図３および図４から、miR-21の量のmiR-103の量に対する値が大きいほどがんである状態と分析することができ、miR-19の量のmiR-103の量に対する値が小さいほど、がんである状態と分析することができることが分かった。 3 and 4, it can be analyzed that the larger the value of miR-21 relative to the amount of miR-103, the more cancerous state, and the smaller the amount of miR-19 relative to the amount of miR-103, the smaller the value. It was found that the cancer can be analyzed.
　なお、図３および図４において点線で囲った部分ががんの状態であると分析できる範囲であり、実線で囲った部分が非がんの状態であると分析できる範囲であり、破線で囲った部分がボーダーの状態、または、閾値と分析できる範囲である。また、図３のmiR-21の値で小さいほうに外れている検体が１点あるが、この検体は図４のmiR-19においてもはずれており、miR-21、miR-19が共通してはずれているため、正常では無いと判断できる。
 
  In FIGS. 3 and 4, the portion surrounded by a dotted line is a range that can be analyzed as being a cancer state, and the portion surrounded by a solid line is a range that can be analyzed as being a non-cancer state, and is surrounded by a broken line. This is the range that can be analyzed with the border state or threshold value. In addition, there is one sample that is out of the smaller value of miR-21 in FIG. 3, but this sample is also out of miR-19 in FIG. 4, and miR-21 and miR-19 are common. Since it is off, it can be determined that it is not normal.

Claims (6)

  1. 　尿検体からエクソソームを抽出する抽出工程と、
    　前記エクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21および／またはmiR-19とを定量する工程と、
    　miR-103と、miR-21および／またはmiR-19のmicroRNA量に基づいてがんの状態を分析する分析工程と
    を含むがんの分析方法。     An extraction process for extracting exosomes from urine samples;
    Quantifying miR-103 and miR-21 and / or miR-19 among the microRNAs present in the exosome;
    A method for analyzing cancer, comprising miR-103 and an analysis step of analyzing a cancer state based on the amount of miR-21 and / or miR-19 microRNA.
  2. 　前記抽出工程は、前記エクソソームをトラップできるように処理されたろ紙を用いて行う請求項１記載のがんの分析方法。 The method for analyzing cancer according to claim 1, wherein the extraction step is performed using filter paper that has been treated so that the exosome can be trapped.
  3. 　前記尿検体を前記ろ紙上に採取して検査機関に輸送する工程を含む請求項２記載のがんの分析方法。 3. The method for analyzing cancer according to claim 2, comprising a step of collecting the urine sample on the filter paper and transporting the sample to a laboratory.
  4. 　尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21
    および／またはmiR-19とのmicroRNA量を定量する測定部と、
    　前記測定部により定量されたmiR-103と、miR-21および／またはmiR-19のmicroRNA量を分析装置に送信する送信部と
    を備える測定装置と、
    　前記測定装置から送信された前記microRNA量を受信する受信部と、
    　前記microRNA量に基づいてがんの状態を分析する分析する分析部と
    を備える分析装置と
    を含むがんの分析システム。     Of the microRNAs present in exosomes extracted from urine samples, miR-103 and miR-21
    And / or a measurement unit for quantifying the amount of microRNA with miR-19,
    A measurement device comprising miR-103 quantified by the measurement unit, and a transmission unit that transmits the amount of miR-21 and / or miR-19 microRNA to the analyzer;
    A receiving unit for receiving the microRNA amount transmitted from the measuring device;
    A cancer analysis system comprising: an analysis device including an analysis unit that analyzes a cancer state based on the amount of microRNA.
  5. 　コンピュータを
    　尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21
    および／またはmiR-19とを定量して得られたmicroRNA量のデータを取得する手段と、
    　miR-103と、miR-21および／またはmiR-19のmicroRNA量のデータに基づいてがんの状態を分析する手段
    として機能させるためのプログラム。     Among the microRNAs present in exosomes extracted from urine samples, miR-103 and miR-21
    And / or means for obtaining data on the amount of microRNA obtained by quantifying miR-19;
    A program for functioning as a means of analyzing cancer status based on miR-103 and miR-21 and / or miR-19 microRNA data.
  6. 　コンピュータを
    　尿検体から抽出されたエクソソーム中に存在するmicroRNAのうち、miR-103と、miR-21
    および／またはmiR-19とを定量して得られたmicroRNA量のデータを取得する手段と、
    　miR-103と、miR-21および／またはmiR-19の前記microRNA量のデータに基づいてがんの状態を分析する手段
    として機能させるためのプログラムを記録したコンピュータ読み取り可能な記録媒体。     Among the microRNAs present in exosomes extracted from urine samples, miR-103 and miR-21
    And / or means for obtaining data on the amount of microRNA obtained by quantifying miR-19;
    A computer-readable recording medium recording a program for causing miR-103 and miR-21 and / or miR-19 to function as a means for analyzing a cancer state based on the microRNA amount data.
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MATSUZAKI K. ET AL.: "MiR-21-5p in urinary extracellular vesicles is a novel biomarker of urothelial carcinom a", ONCOTARGET, vol. 8, no. 15, 2017, pages 24668 - 24678, XP055575028, DOI: 10.18632/oncotarget.14969 *

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