JPWO2019220759A1 - Cancer analysis methods, cancer analysis systems, programs and computer-readable recording media - Google Patents

Abstract

An extraction step of extracting exosomes from a urine sample, a step of quantifying miR-103, miR-21 and / or miR-19 among the microRNAs present in the exosomes, and miR-103 and miR-21. And / or includes an analysis step that analyzes the cancer status based on the amount of miR-19 microRNA.

Description

The present invention relates to cancer analysis methods, cancer analysis systems, programs and computer-readable recording media.

Cancer is the leading cause of death in 2016 vital statistics published by the Ministry of Health, Labor and Welfare. In addition, since cancer cells have extremely high proliferative capacity, early detection is important.

Currently, PET examinations, X-ray examinations, tumor marker examinations, etc. are performed as cancer examinations, but they cannot be detected unless the size of the cancer is 5 mm or more, while the size of the cancer is about 0.1 mm. Has already been reported on the possibility of metastasis.

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 at the time of the test such as blood collection.

On the other hand, cancer screening using microRNA (microRNA) secreted into blood is being investigated. This trial is based on 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, Taking advantage of the fact that there are 2 to 10 types of microRNAs unique to each of breast cancer, sarcoma, and glioma), cancer tests are performed by analyzing the microRNAs in the blood of patients (). For example, see Non-Patent Document 1).

In addition, tests for predicting the risk of cancer by measuring the amino acid concentration in blood are also performed (see, for example, Patent Documents 1 to 3). This test is called AminoIndex® Cancer Risk Screening, which measures amino acid levels in the blood and statistically analyzes the difference in the balance of amino acid levels between healthy and cancerous people. To predict the risk of cancer.

However, these tests are tests using blood and are not non-invasive tests that do not cause a burden on the living body.

International Publication No. 2008/07562 International Publication No. 2008/075663 International Publication No. 2008/075664

"Clinical research for early detection of 13 types of cancer with one drop of blood", [online], August 20, 2017, Nihon Keizai Shimbun, [Search on May 9, 2018] <URL: https: // www .nikkei.com/article/DGXLASDG19H4L_Q7A820C1CR8000/>

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 capable of detecting cancer at an early stage without being invasive.

As a result of diligent 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 sample, and have completed the present invention.

That is, according to the present invention.
(1) An extraction step of extracting exosomes from a urine sample, a step of quantifying miR-103, miR-21 and / or miR-19 among the microRNAs present in the exosomes, and miR-103. Methods for analyzing cancer, including an analysis step that analyzes the state of the cancer based on the amount of microRNA in miR-21 and / or miR-19.
(2) The cancer analysis method according to (1), wherein the extraction step is performed using a filter paper treated so as to trap the exosomes.
(3) The cancer analysis method according to (2), which includes a step of collecting the urine sample on the filter paper and transporting the urine sample to a laboratory.
(4) Among the microRNAs present in the exosomes extracted from the urine sample, the measurement unit for quantifying the amount of microRNA of miR-103 and miR-21 and / or miR-19, and the measurement unit quantified the amount. A measuring device including a miR-103 and a transmitting unit that transmits the microRNA amount of miR-21 and / or miR-19 to an analyzer, a receiving unit that receives the microRNA amount transmitted from the measuring device, and the above. A cancer analysis system that includes an analyzer with an analysis unit that analyzes the state of the cancer based on the amount of microRNA,
(5) A means for obtaining data on the amount of microRNA obtained by quantifying miR-103 and miR-21 and / or miR-19 among the microRNAs present in exosomes extracted from a urine sample by a computer. And a program to function as a means of analyzing cancer status based on data on the amount of microRNAs of miR-103 and / or miR-19.
(6) A means for obtaining data on the amount of microRNA obtained by quantifying miR-103 and miR-21 and / or miR-19 among the microRNAs present in exosomes extracted from a urine sample by a computer. And provided are computer-readable recording media recording programs for functioning as a means of analyzing cancer status based on the microRNA amount data of miR-103 and / or miR-19. Will be done.

INDUSTRIAL APPLICABILITY The present invention provides a cancer analysis method, a cancer analysis system, a program, and a computer-readable recording medium capable of detecting cancer at an early stage without being invasive.

It is a block diagram which shows the system structure of the cancer analysis system which concerns on embodiment of this 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 amount of miR-103 of the amount of miR-21 obtained by the quantitative PCR in the Example of this invention. It is a graph which shows the value with respect to the amount of miR-103 of the amount of miR-19 obtained by the quantitative PCR in the Example of this invention.

Hereinafter, the cancer analysis method and the cancer analysis system of the present invention will be described with reference to the drawings.

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, miR-21 and / or miR-19 among the microRNAs present in the exosomes. And an analytical step that analyzes the cancer status based on the amount of microRNAs of miR-103 and / or miR-19.

In addition, the cancer analysis system of the present invention is a measuring unit that quantifies the amount of microRNAs of miR-103 and miR-21 and / or miR-19 among the microRNAs present in exosomes extracted from urine samples. A measuring device including a miR-103 quantified by the measuring unit, a transmitting unit for transmitting the amount of microRNA of miR-21 and / or miR-19 to the analyzer, and the microRNA transmitted from the measuring device. It includes an analyzer including a receiving unit that receives the amount and an analysis unit that analyzes the state of cancer based on the amount of microRNA.

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 measuring device and a PC (personal computer) 6 as an analysis device.

Further, the quantitative PCR device 4 includes a control unit 8 that comprehensively controls each part of the quantitative PCR device 4, and the control unit 8 is provided with an input unit 10 and microRNA that accept input operations such as measurement conditions and measurement start. A measurement unit 12 that amplifies and quantifies using a primer, a miR-103 measured by the measurement unit 12, and a transmission unit 14 that transmits data on the amount of microRNA of miR-21 and / or miR-19 to PC6 are connected. There is.

Further, the PC 6 includes a CPU 16, and the CPU 16 includes miR-103 transmitted from the quantitative PCR device 4, a receiving unit 18 for receiving microRNA amount data of miR-21 and / or miR-19, and microRNA amount data. Storage unit 20 that stores data, analysis unit 22 that analyzes the cancer status based on microRNA amount data, operation unit that specifies data to be used for analysis of the cancer status and inputs instructions for starting cancer analysis, etc. 24 are connected.

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.

First, in the present invention, urine is used as a sample, and exosomes are extracted from this urine sample (step S1). Examples of the method for extracting exosomes include ultracentrifugation, ultrafiltration, gel filtration, HPLC, and methods using extraction reagents. It is possible to use filter paper treated so that exosomes can be trapped. preferable.

Examples of such filter paper include FTA microcards (manufactured by GE Healthcare) and the like (see, for example, US Pat. Nos. 5,495,562, 5807527, 5972386 and 5985327, etc.). This FTA microcard is a cellulose-based paper with a weak base such as trishydroxymethylmethane, a chelating agent such as ethylenediaminetetraacetate, an anionic surfactant such as sodium dodecyl sulfate, and a free radical trap such as uric acid or urate. It has a filter paper on which a composition containing the above is adsorbed or incorporated.

The filter paper constituting the FTA microcard is designed to entangle and trap nucleic acids, but according to the study by the present inventors, when a urine sample is poured or attached to the FTA microcard, an exosome is used. Was found to be trapped.

Therefore, this state (hereinafter, may be referred to as "urine sample adhering filter paper") after adhering or adsorbing the urine sample by pouring a urine sample or the like onto a filter paper treated so as to trap exosomes such as FTA microcards. Can be easily transported.

When exosomes are extracted by ultracentrifugation, ultrafiltration, gel filtration, HPLC or a method using extraction reagents, urine is collected and then liquidized by a urine collection tube, etc. It is necessary to transport it to the laboratory, but if filter paper processed so that it can trap exosomes is used, it can be transported to the inspection institution / laboratory using cards such as FTA microcards or paper as a medium, so at room temperature. It is preferable in terms of ease of transportation such as being able to be transported and transportation cost.

When transporting the urine sample-attached filter paper, it may be transported in a predetermined bag or the like from the viewpoint of deodorization and the like.

Next, in the extraction step, exosomes are extracted from the urine sample, and then microRNA is extracted from the exosomes (step S2). The method for extracting microRNA from exosomes 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.

Next, the amount of extracted microRNA is quantified using the quantitative PCR device 4 (step S3). When performing quantification, first, the microRNA taken out in step S2 is set in the measuring unit 12. Then, when the operator inputs an instruction to start quantification from the input unit 10, the control unit 8 starts quantification of the microRNA set in the measurement unit 12. Here, in the quantitative PCR device 4, it is preferable to perform the measurement by real-time PCR, which measures the amplification by the polymerase chain reaction (PCR) over time.

In the present invention, the microRNAs to be quantified are miR-103 and miR-21 and / or miR-19.

Here, miR-103 is stably expressed in serum / plasma, urine, and cerebrospinal fluid exosomes (eg, http://www.exiqon.com/ls/Documents/Scientific/microRNA). -See Table 6 (page 25) of serum-plasma-guidelines.pdf).

In addition, miR-21 is a disease-specific molecule contained in exosomes extracted from serum or cerebrospinal fluid, etc., such as ovarian cancer, colon cancer, pancreatic cancer, cervical cancer, liver cancer, and pharyngeal flattened. It is a microRNA found in a wide variety of cancers, including epithelial cancer and glioma. That is, it is a type of microRNA that is secreted in large quantities from a wide variety of cancer cells.

Here, there are microRNAs that are stably expressed in serum / plasma, urinary, and cerebrospinal fluid exosomes, such as miR-103 described above, and microRNAs that are not stably expressed (similar to the above). See, for example, Table 6 (page 25) of http://www.exiqon.com/ls/Documents/Scientific/microRNA-serum-plasma-guidelines.pdf). Therefore, even if it is expressed as a type of microRNA contained in exosomes extracted from serum or cerebrospinal fluid, it cannot be said that it is expressed as a type of microRNA contained in exosomes extracted from urine. That is, no complete correlation is found between the type of microRNA contained in exosomes extracted from serum, cerebrospinal fluid, etc. and the type of microRNA contained in exosomes extracted from urine.

In addition, miR-19 is a type of microRNA whose secretion is suppressed from a wide range of cancer cells, and its expression level is low as a type of microRNA contained in exosomes extracted from the serum or cerebrospinal fluid of cancer patients. Become.

Next, the control unit 8 of the quantitative PCR device 4 transmits the data of the amount of microRNA obtained by the quantification to the PC 6 via the transmitting unit 14 (step S4), and the PC 6 is obtained by the quantification via the receiving unit 18. Receive the data on the amount of microRNA (step S5). Here, the communication between the transmitting unit 14 of the quantitative PCR device 4 and the receiving unit 18 of the PC 6 may be performed by wired communication such as a wired LAN, or by wireless communication such as wireless LAN or Bluetooth (registered trademark). You may.

Next, the CPU 16 of the PC 6 stores the data of the amount of microRNA received via the receiving unit 18 in the storage unit 20 (step S6). Here, in the storage unit 20, the data of the amount of microRNA is stored in association with, for example, the sample ID, the measurement date and time, the measurement number, and the like.

Then, when the data to be analyzed is designated via the operation unit 24 and the instruction to start the analysis 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 from the data of the amount of microRNA stored in the storage unit 20, and the analysis unit 22 analyzes the cancer state. To do.

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 the data obtained by the comparative Ct method. For example, by continuously quantifying miR-103 and miR-21 and / or miR-19 contained in exosomes extracted from a specific person's urine sample, in normal times (ie, when non-cancerous). Data can be collected and analyzed as cancer when miR-21 expression is high and / or miR-19 expression is low. Here, as the amount of miR-21 and the amount of miR-19, the values compared with the amount of miR-103 are used. As will be described later in the examples, it was found by the examination by the present inventors that these values fluctuate with a statistically significant difference between the two groups of cancer and non-cancer.

In the analysis of the cancer status by the analysis unit 22, for example, by using the data continuously collected for each specific individual, the amount of miR-21 is larger than the amount of miR-103 continuously collected. It can be analyzed as a cancer state when the value for miR-19 increases and / or when the value for miR-19 amount decreases.

Further, when analyzing the state of cancer, the analysis may be performed using a predetermined threshold value. In this case, the amount of miR-21 relative to the amount of miR-103 and / or miR-19 from all data of the amount of microRNA stored in the storage unit 20 or randomly sampled data. By statistically processing the value of the amount of miR-103 with respect to the amount, the threshold value for analyzing the state of cancer is calculated in advance. Then, by setting this threshold value as a threshold value when analyzing the state of cancer, it is possible to analyze whether or not specific data is in the state of cancer.

In the above-mentioned analysis of the cancer state, data such as outliers and missing values may be removed to analyze the cancer state.

According to the cancer analysis method and the cancer analysis system according to the embodiment of the present invention, cancer can be detected non-invasively and at an early stage.

In the above-described embodiment, the PC 6 has been described as an example of the analyzer, but a tablet terminal or the like may be used as the analyzer. Further, the configuration in which two devices of the quantitative PCR device 4 and the PC 6 are provided as the cancer analysis system 2 has been described as an example. For example, by incorporating the function of the analyzer into the quantitative PCR device 4, one device can be used. An analysis system can also be configured.

Further, in the above-described embodiment, the data of the amount of microRNA is transmitted from the quantitative PCR device 4 which is a measuring device to the PC 6 which is an analyzer, but the communication between the quantitative PCR device 4 and the PC 6 is not possible. When possible, data on the amount of microRNA 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.

Further, in the above-described embodiment, when the data to be analyzed is specified via the operation unit 24 and the instruction to start the analysis is input, the configuration of analyzing the cancer state by the PC 6 has been described, but the urine sample has been described. Analysis to analyze cancer status based on microRNA amount data obtained by quantifying miR-103 and miR-21 and / or miR-19 among the microRNAs present in exosomes extracted from By downloading the program via a network such as the Internet and incorporating it into the computer, the computer may be made to function so that the above-mentioned analysis process can be performed. Here, this analysis program reads out the microRNA amount data of miR-103 and miR-21 and / or miR-19 stored on the network or computer and uses it for the analysis of cancer status. May be good.

Further, the program may be recorded on a computer-readable recording medium such as a flexible disc, a CD-ROM, or a DVD. That is, the computer may be made to function so that the above-mentioned analysis of the cancer state can be performed by reading from a computer-readable recording medium and incorporating the computer into the computer.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto. Unless otherwise specified, parts and% in this embodiment are based on weight.

Urine specimens were attached or adsorbed to FTA microcards for each of cancer patients (n = 6) and non-cancer patients (n = 6). As a result, exosomes were extracted from the urine sample.

Next, microRNA was extracted from each exosome using a microRNA extraction kit (miRCURY RNA Isolation Kit (Product # 300110; EXIQON)). Then, each of the obtained microRNAs is dispersed or dissolved with ultra-pure water to obtain a predetermined concentration, and then reverse transcription reaction is carried out from each sample with reverse transcriptase to synthesize cDNA, and miR is used using the cDNA. Expression of -103 and miR-21 and miR-19 was analyzed by quantitative PCR.

The specific procedure of the quantitative PCR method was as follows. Using the cDNA obtained from the above microRNA, it was reverse transcribed into cDNA according to the method of PrimeScript RT Master Mix (Takara) and amplified by SYBR Premix EX Taq II (Takara). As the PCR reaction solution, a PCR reaction solution of 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) was used, and the reaction was carried out at 95 ° C. A cycle of 30 seconds was performed for one cycle, and a cycle of 5 seconds at 95 ° C. and a cycle of 30 seconds at 60 ° C. was performed under the conditions of 50 cycles. Relative quantification was performed by the comparative Ct method (ΔΔCt method). As the Primers of various targets (miR-103, miR-21 and miR-19) used for RT-PCR (real-time PCR), predetermined ones were used.

It is also possible to use the One Step-RT-PCR Kit, which can perform this RT reaction and quantitative PCR based on microRNA in one test tube. For example, Luna Universal One-step Reaction Mix (New England Biolabs) can also be used.

Then, the value of the amount of miR-21 obtained by quantitative PCR with respect to the amount of miR-103 is shown in FIG. 3, and the value of the amount of miR-19 with respect to the amount of miR-103 is shown in FIG. 4, respectively.

From FIGS. 3 and 4, it can be analyzed that the larger the value of the amount of miR-21 with respect to the amount of miR-103, the more cancerous the condition is, and the smaller the value of the amount of miR-19 with respect to the amount of miR-103, the smaller the value. It turns out that it can be analyzed as a cancerous condition.

The part surrounded by the dotted line in FIGS. 3 and 4 is the range that can be analyzed as being in a cancerous state, and the part surrounded by the solid line is the range that can be analyzed as being in a non-cancerous state, and is surrounded by a broken line. The part is the border state or the range that can be analyzed as the threshold value. In addition, there is one sample that deviates from the smaller value of miR-21 in Fig. 3, but this sample also deviates from miR-19 in Fig. 4, and miR-21 and miR-19 are common. Since it is out of alignment, it can be judged that it is not normal.

Claims (6)

Extraction process to extract exosomes from urine samples,
A step of quantifying miR-103 and miR-21 and / or miR-19 among the microRNAs present in the exosome.
A method for analyzing cancer that comprises miR-103 and an analytical step that analyzes the state of the cancer based on the amount of microRNA in miR-21 and / or miR-19. The method for analyzing cancer according to claim 1, wherein the extraction step is performed using a filter paper treated so that the exosomes can be trapped. The cancer analysis method according to claim 2, further comprising a step of collecting the urine sample on the filter paper and transporting the urine sample to a laboratory. Among the microRNAs present in exosomes extracted from urine samples, miR-103 and miR-21
And / or a measuring unit that quantifies the amount of microRNA with miR-19,
A measuring device including a miR-103 quantified by the measuring unit, a transmitting unit that transmits the amount of microRNA of miR-21 and / or miR-19 to the analyzer, and a measuring device.
A receiver that receives the amount of microRNA transmitted from the measuring device, and
A cancer analysis system including an analyzer including an analysis unit that analyzes the state of cancer based on the amount of microRNA. Among the microRNAs present in exosomes extracted from urine samples by computer, miR-103 and miR-21
And / or a means to obtain data on the amount of microRNA obtained by quantifying miR-19,
A program to function as a means of analyzing cancer status based on miR-103 and miR-21 and / or miR-19 microRNA content data. Among the microRNAs present in exosomes extracted from urine samples by computer, miR-103 and miR-21
And / or a means to obtain data on the amount of microRNA obtained by quantifying miR-19,
A computer-readable recording medium on which a program is recorded to function as a means for analyzing the state of cancer based on the data of the amount of microRNA of miR-103 and / or miR-19.

Images