JP2018093759A - ASSESSMENT OF CANCER BY MICRO RNA DETECTION IN Ago2 PROTEIN COMPLEX - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a detection system using stable endogenous control, identify tumor-specific circulating miRNA, and by increasing the usefulness and precision of assessment, reduce the amount a sample collected.SOLUTION: The assessment method of cancer is characterized by measuring the expression level of microRNA (Ago2-miRNA) formed by Ago2 and a complex in a blood sample obtained from a subject. Specifically, when measuring the expression level of Ago2-miR-21 and/or Ago2-miR-200c in a sample obtained from a subject, the expression level is compensated by Ago2-miR-451 in the same sample, and foreign matter is removed from the sample by the preclear method.SELECTED DRAWING: None

Description

  The present invention relates to determination of cancer. Specifically, the present invention relates to determination of cancer by measuring the expression level of microRNA forming a complex with Ago2 in a sample obtained from a subject.

  Currently, markers that screen cancer from blood are collectively referred to as tumor markers. Tumor markers refer to biological factors that increase with the progression of cancer, and many of these biological factors that are released into the blood are detected by antibodies. Many tumor markers are present in the blood even in healthy people, so few tumor markers alone can diagnose the presence of cancer. Regularly examining tumor markers in cancer patients is not sure but useful to know if there is a recurrence, disease state, cancer that has not been removed by surgery, or microscopic cancer that cannot be seen by diagnostic imaging It is a simple method. However, tumor markers are usually used to understand the dynamics of advanced cancer and are not tests that can be used for early diagnosis. Accordingly, attempts have been made to detect gene mutations occurring in the tumor itself rather than these biological factors, but no clinical application has been achieved so far. Currently, detection of tumor-derived gene mutations in the blood is collectively referred to as “Liquid Biopsy”, and is an area of active research all over the world. Specifically, a method for detecting gene mutation itself (Non-patent Document 1), a method for detecting methylation of cytosine that is supposed to occur specifically in tumors, a method for detecting microRNA (miRNA) (Non-patent Document 1) Reference 2), a method of detecting endoplasmic reticulum produced by a tumor, and the like are being developed and applied for clinical application (Non-patent Document 3).

  A microRNA (miRNA) is a functional nucleic acid that is encoded on the genome and finally becomes a microRNA having a length of 20 to 25 bases through a multi-step production process. This short-length miRNA is classified as a functional ncRNA (non-coding RNA: a general term for RNA that is not translated into protein), and regulates its expression by complementary binding to messenger RNA. (Non-patent Documents 4 and 5). In recent years, it has been shown that miRNA stably exists in blood (Non-patent Document 6), and that the distribution of miRNA (circulating miRNA) in the blood shows a different distribution depending on organs and diseases. (Non-Patent Documents 7 and 8). It has also been suggested that colorectal cancer screening is possible by detecting specific miRNA in blood (Non-patent Document 2).

S. Misale et al., Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer.Nature 486, 532 (Jun 28, 2012) Y. Toiyama et al., Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer.Journal of the National Cancer Institute 105, 849 (Jun 19, 2013) Y. Yoshioka et al., Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen.Nature communications 5, 3591 (2014) R. C. Lee, V. Ambros, An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862 (Oct 26, 2001) V. Ambros, The functions of animal microRNAs.Nature 431, 350 (Sep 16, 2004) J. Lu et al., MicroRNA expression profiles classify human cancers.Nature 435, 834 (Jun 9, 2005) X. Chen et al., Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.Cell research 18, 997 (Oct, 2008) A. Turchinovich, L. Weiz, A. Langheinz, B. Burwinkel, Characterization of extracellular circulating microRNA. Nucleic acids research 39, 7223 (Sep 1, 2011)

  However, there are problems to be solved in order to clinically apply a technique for detecting a specific circulating miRNA. One is to establish a detection system using stable endogenous controls. The second is to identify tumor specific circulating miRNAs. The third is to reduce the amount of sample when performing detection. In addition, in the reports up to now, there is also a problem that the detection is not performed in consideration of the following concept when circulating miRNA is detected. Circulating miRNAs are said to be stable to RNases. It is thought to be mainly due to two characteristic forms. One is the presence (EV-miRNA) in the form of enveloped vesicles called extracellular vesicles such as exosomes (EV-miRNA), and the other is the formation of a complex with the RNA-binding protein algorithm family. (Especially a complex with Ago2 protein, Ago2-miRNA). The findings obtained to date have reported the phenomenon that EV-miRNA is actively released from live cancer cells and Ago2-miRNA is passively released from dead cancer cells (H. Valadi et al., Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nature cell biology 9, 654 (Jun, 2007) and SA Melo et al., Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer cell 26, 707 (Nov 10, 2014)). This difference in secretory form is considered very important. This is because, for example, in blood sampling during chemotherapy, the presence of EV-miRNA in plasma means the presence of living cancer cells remaining in the body, and the presence of Ago2-miRNA in plasma This is because it may reflect the death of cells, that is, the degree of success of chemotherapy.

  In order to solve the above-mentioned problems, the present inventors first examined EV-miRNA and Ago2-miRNA, and attempted to identify a miRNA derived from a live cancer cell and a miRNA derived from a dead cancer cell. In addition, we also developed a technology that can detect a small amount of plasma.

  The present inventors have conducted intensive studies and found that not only EV-miRNA but also Ago2-miRNA that is easy to detect and measure can be used as a cancer marker. Ago2-miR-21 and Ago2-miR-200c were selected and a cancer determination system using Ago2-miR-451 as a control was successfully developed. In addition, the present inventors previously removed non-specific antibodies and proteins in the sample using protein A / G agarose, so that a very small amount of sample (10-60 μL plasma sample) was obtained. Also showed that cancer can be determined. From the above, the present inventors have completed the present invention.

The present invention relates to:
(1) A method for determining cancer, comprising measuring the expression level of microRNA (Ago2-miRNA) forming a complex with Ago2 in a blood sample obtained from a subject.
(2) Ago2-miRNA is
Ago2-miRNA that is passively released into the blood by cell death, and / or Ago2-miRNA that is actively released by living cells, and / or A passively released into the blood by cell death And Ago2-miRNA actively released by living cells
The method according to (1).
(3) The method according to (1) or (2), wherein the Ago2-miRNA is Ago2-miR-21 and / or Ago2-miR-200c.
(4) The method according to any one of (1) to (3), wherein the expression level of Ago2-miRNA in the blood sample is corrected with the expression level of Ago2-miR-451 in the sample. .
(5) Before measuring the expression level of Ago2-miRNA, non-specific antibodies and proteins in the sample are removed using protein A / G agarose, and any of (1) to (4) Or the method described.
(6) The expression level of Ago2-miR-21 is measured, the presence of cancer including recurrence in the subject is determined, the effect of anticancer drug treatment is predicted, or the type and dose of the anticancer drug are determined ( The method according to any one of 1) to (5).
(7) The method according to (6), wherein early detection of cancer or prediction of early recurrence in a subject is performed.
(8) The expression level of Ago2-miR-200c is measured, and prediction of cancer metastasis, prediction of the effect of anticancer drug treatment on metastatic lesions, or determination of the type and dose of anticancer drug is performed (1) to The method according to any one of (7).
(9) A kit for determining cancer, comprising means for measuring the expression level of Ago2-miRNA in a blood sample.
(10) The kit according to (9), wherein the Ago2-miRNA is Ago2-miR-21 and / or Ago2-miR-200c.
(11) The kit according to (9) or (10), further comprising means for measuring the expression level of Ago2-miR-451 in the blood sample.
(12) The kit according to any one of (9) to (11), further comprising protein A / G agarose.

  According to the present invention, cancer can be determined with a very small amount of sample (10-60 μL for plasma). In the present invention, unlike the conventional findings, it was shown that two forms of Ago2-miRNA present in blood are observed. That is, it was shown that there are two types, one that is passively released into the blood by cell death and one that is actively released by living cells. Therefore, it became possible to determine cancer using only Ago2-miRNA that is easy to detect and measure. In the present invention, when measuring the expression level of Ago2-miRNA, miR-451 is used as an endogenous control, whereby cancer can be accurately determined. In the present invention, it was found that Ago2-miR-21 has the above two forms. Therefore, measuring Ago2-miR-21 is useful not only for predicting the effect of anticancer drug treatment but also for predicting recurrence and diagnosing cancer. In the present invention, Ago2-miR-200c is also useful as a cancer marker. Since Ago2-miR-200c is highly expressed especially in metastatic lesions, it is useful as a marker for predicting the effect of anticancer drug treatment on metastatic lesions.

FIG. 1-A shows cell proliferation and cell biological activity measured using the WST assay. 5-FU was added at 48 hours of culture. FIG. 1-B is a diagram showing the results of quantitative reverse transcription PCR evaluating the relative amounts of miR-31 and miR-31 in the medium of the HT29 cell line. Based on the change in the threshold cycle value (ΔCt) obtained 24 hours after seeding, the expression ratio is expressed as the change ratio. 5-FU was added at 48 hours of culture. FIG. 1-C is a diagram showing the results of calculating the relative expression index for the control by dividing the expression ratio by the ratio of WST absorbance. 5-FU was added at 48 hours of culture. FIG. 1-D is a graph showing the results of calculating the relative expression index for 5-FU-treated cells by dividing the expression ratio by the ratio of WST absorbance. 5-FU was added at 48 hours of culture. FIG. 2 is a diagram showing miRNA analysis procedures in a medium by Ago2-immunoprecipitation and EV isolation. FIG. 3-A is a diagram showing the results of examining the relationship between the expression states of each miRNA using the Ago2-immunoprecipitation method and the EV isolation method. FIG. 3-B is a scatter diagram of ΔCt values of Ago2-miRNA and EV-miRNA isolated from 100 μl of plasma. For all samples, added cel-miR-39 was used as a normalization control. FIG. 3-C is a table showing the 10 most abundant miRNAs detected by Ago2-immunoprecipitation alone, EV isolation alone, and both methods. FIG. 4-A is a diagram showing the results of investigating the relationship between the global miRNA expression status of the primary tumor and the corresponding liver metastasis site using an RNA microarray. CT1, CT2 and LT1, LT2 indicate CRC (colon cancer) and liver metastasis specimens obtained from patients 1 and 2, respectively. It is a figure which shows the result of a cluster analysis of 59 types of selected miRNA. miRNA expression was more than 2-fold higher in tumor tissue than in normal tissue. The human map shows the expression ratio between tumor tissue and normal tissue. CN1, CN2 and LN1, LN2 represent normal large intestine mucosa tissue and normal liver tissue obtained from patients 1 and 2, respectively. FIG. 4-C shows the results of examining the expression state of miR-200c in the medium of the HT29 cell line by quantitative reverse transcription PCR. The expression ratio based on the ΔCt value obtained 24 hours after seeding is expressed as the rate of change. FIG. 4-D is a diagram showing a relative expression index for miR-200c in a culture medium (expression ratio divided by WST absorbance ratio). The relative expression index of the control is taken as the total amount of miRNA from live cells, miRNA from 5-FU treated cells, and miRNA from cell lysis after 5-FU treatment. The 5-FU treatment period was 48 hours after seeding. FIG. 5 is a diagram showing the procedure of the pre-clear method. (A) Incubate plasma with protein A / G agarose beads. (B) Preclear lysate bound non-specifically to beads. (C) Precleared lysate. (D) Add Ago2 antibodies and incubate together to form antigen-antibody complexes, then add protein A / G agarose beads to immunoprecipitate these complexes. (E) miRNA is separated from the bead-Ago2 complex and analyzed by reverse transcription PCR. FIG. 6A is a diagram showing the effect of the preclear method in detecting circulating Ago2-miRNA. Ago2-miR-451 was recovered from Ago2-immunoprecipitate by quantitative PCR. For all samples, added cel-mi-39 was used as a normalization control. Data are shown as relative fold change in miRNA levels. FIG. 6-B shows the expression levels of miR-16, miR-21, and miR-451 recovered from the first Ago2-immunoprecipitation and the second Ago2-immunoprecipitation obtained from the flow-through. is there. Anti-β-actin antibody was used as a negative control. The figure shows the mean and standard deviation of three independent experiments. FIG. 6-C shows scatter plots and correlation coefficients for miR-451 recovered from Ago2-immunoprecipitation, and plasma volume. Three independent experiments were carried out floating in each volume. Data are shown as relative fold change in miRNA levels. FIG. 6-D is a scatter diagram of the Ct value of each miRNA obtained by Ago2-immunoprecipitation method and EV isolation method using 30 μl of plasma. Each plot shows the average value of three independent experiments. FIG. 7A is an SDS-PAGE showing evaluation of antibodies in plasma and non-specific proteins that bind to protein A / G agarose gels. From left to right, untreated plasma, Ago2 antibody only, untreated protein A / G agarose, precleared plasma, protein A / G agarose after preclearing step, and Ago2-immunoprecipitate (first and second round) is there. Ago2-immunoprecipitation flow-through was used. FIG. 7B is an SDS-PAGE after performing a preclear method on various plasma volumes. Upper panel is immunoblotting of human IgG. The lower panel is a DSD-PAGE to assess plasma volume appropriate for Ago2-immunoprecipitation. A total of 20 μl protein A / G agarose gel was incubated with 10-100 μl plasma. FIG. 8A is a diagram showing Ct values of Ago2-miR-451, -21 and -200c in plasma obtained from control subjects (CS, n = 20) and colon cancer patients (CRC, n = 39). . FIG. 8B is a diagram showing Ago2-miR-21 ΔCt values (ΔCt (miR-451) values) for miR-451 in plasma obtained from CS and CRC of stages I, II, III, and IV. The vertical axis represents the ΔCt value of Ago2-miRNA normalized to Ago2-miR-451. The box in the figure indicates the interquartile region, and the horizontal line drawn in each box indicates the median value. Statistical significance was determined using the Wilcoxson test. NS indicates not statistically significant. The dotted line shows the cutoff value for each Ago2-miRNA. A ΔCt (miR-451) value below the dotted line is regarded as an abnormal value. FIG. 8C shows the ΔCt (miR-451) value of Ago2-miR-21 in plasma obtained 11 patients before CRC (Pre) and 1 year after primary tumor resection (Post). . FIG. 8D shows the Ago2-miR-200c ΔCt value (ΔCt (miR-451) value) for miR-451 in plasma obtained from CS and CRC of stages I, II, III, and IV. The vertical axis represents the ΔCt value of Ago2-miRNA normalized to Ago2-miR-451. The box in the figure indicates the interquartile region, and the horizontal line drawn in each box indicates the median value. Statistical significance was determined using the Wilcoxson test. NS indicates not statistically significant. The dotted line shows the cutoff value for each Ago2-miRNA. A ΔCt (miR-451) value below the dotted line is regarded as an abnormal value. FIG. 8E shows the ΔCt (miR-451) value of Ago2-miR-200c in plasma obtained before surgery (Pre) and 1 year after primary tumor resection (Post) of 11 CRCs. . FIG. 9A is a diagram showing the expression levels of Ago2-miR21 and Ago2-miR-200c in the course of treatment for patients undergoing chemotherapy for unresectable liver metastases. FIG. 9B is a diagram showing the expression levels of Ago2-miR21 and Ago2-miR-200c in the course of treatment for adjuvant chemotherapy patients after curative resection of colorectal cancer. FIG. 9C is a diagram showing the expression levels of Ago2-miR21 and Ago2-miR-200c in the course of treatment of patients with unresectable liver metastases that have undergone little success in primary therapy. FIG. 9D is a diagram showing the expression levels of Ago2-miR21 and Ago2-miR-200c in the course of treatment for patients who received chemotherapy for unresectable liver metastases. The number in ○ is the course number for chemotherapy. Ago2-miR21 and Ago2-miR-200c on the vertical axis indicate the ΔCt value of Ago2s-miRNA normalized to the value of ΔCt of Ago2-miR-451. The arrow below the number in the circles (other than the arrow in the seventh course in FIG. 9D) indicates the time when the blood sample was collected. The shaded part in the figure indicates that it is in a reduction period. Beva is bevacizumab, FOLFOX6 is modified FOLFOX6 therapy, and SOX is TS1 with oxaliplatin therapy. FIG. 10 shows ΔCt values of Ago2-miR-21 against miR-451 in plasma of various cancer patients. The box in the figure indicates the interquartile region, and the horizontal line drawn in each box indicates the median value.

  In one aspect, the present invention relates to a method for determining cancer, which comprises measuring the expression level of microRNA forming a complex with Ago2 protein in a sample obtained from a subject. The subject is not particularly limited as long as it is a human. For example, a human who has cancer or a human who may have cancer may be used.

  The determination of cancer refers to examining the state of cancer in the subject, and includes, for example, examining the presence or absence of cancer in the subject, predicting the effect of anticancer drug treatment, and predicting recurrence of cancer.

  The Ago2 protein is known and belongs to the algorithm family that is an RNA binding protein. Many types of microRNA (miRNA, miR) are known. A complex of Ago2 protein and microRNA is also known, and it is known that circulating miRNA is stabilized by forming a complex. In the present specification, a complex of Ago2 protein and microRNA is denoted as Ago2-miR or Ago2-miRNA. For example, a complex of Ago2 protein and miR-21 is denoted as Ago2-miR-21.

  Traditionally, it has been reported that EV-miRNA is actively released from living cancer cells and Ago2-miRNA is passively released from dead cancer cells (H. Valadi et al., Nature cell). biology 9, 654 (Jun, 2007), SA Melo et al., Cancer cell 26, 707 (Nov 10, 2014)). However, in the present invention, in addition to (1) Ago2-miRNA that is passively released into the blood by cell death, (2) Ago2-miRNA that is actively released by living cells, (1) and (2 ) Ago2-miRNA having both properties was found in the blood.

  The Ago2-miRNA having the property (1) has a ΔCt value that increases with time in a culture solution in which cells are cultured by adding a cytotoxic agent. The Ago2-miRNA having the property (2) has a ΔCt value that increases over time in a culture medium in which cells are cultured without adding a cytotoxic agent. Ago2-miRNA having the properties of both (1) and (2) is present both in a culture medium in which cells are cultured with addition of a cytotoxic agent and in a culture medium in which cells are cultured without addition of a cytotoxic agent. The ΔCt value increases.

  When the cytotoxic agent is 5-fluorouracil (5-FU), an Ago2-miRNA having the property (1) may be defined as an Ago2-miRNA whose relative expression index increases exponentially. Ago2-miRNA having the property (2) has not been confirmed so far, but is an Ago2-miRNA whose relative expression index increases exponentially under normal culture conditions in which no cytotoxic agent is administered. Ago2-miRNA having both the properties of (1) and (2) is an Ago2-miRNA whose relative expression index increases exponentially both in the presence of a cytotoxic agent and in normal culture conditions in which no cytotoxic agent is administered. May be defined.

  As described above, since Ago2-miRNA and EV-miRNA have different secretory forms, by analyzing their expression, for example, the presence of living cancer cells in vivo in blood sampling during chemotherapy By analyzing the death of cancer cells, it is considered that the determination of cancer can be performed in many ways and in detail. However, EV-miRNA has a problem that it is difficult to detect and measure.

  In the present invention, it was confirmed that Ago2-miRNA having only the above property (1) and Ago2-miRNA having both the above properties (1) and (2). Moreover, Ago2-miRNA is easier to detect and measure than EV-miRNA present in a form encapsulated in membrane vesicles. Therefore, in the present invention, it has become possible to make multifaceted and detailed cancer determinations using only Ago2-miRNA that is easy to detect and measure.

  Ago2-miRNA having the property (1) is important, for example, in predicting the effects of anticancer drug treatment. The plateau state of Ago2-miRNA having only the property (1) during anticancer drug treatment suggests refractory and can be determined before confirming the progression of disease state on an image. For example, when Ago2-miRNA having the property (2) is present in a body fluid such as blood exceeding the normal range, it also indicates the presence of living cancer cells. That is, the Ago2-miRNA having the property (2) is useful for predicting recurrence at an earlier stage observed in an image and diagnosing early cancer. Ago2-miRNA having both the properties of (1) and (2) is useful for predicting the effects of anticancer drug treatment, predicting recurrence at the previous stage observed in images, diagnosing early cancer, etc. is there.

  In the cancer determination method and kit of the present invention, Ago2-miRNA having the property (1) may be used, or Ago2-miRNA having the property (2) may be used. (1) and (2) Ago2-miRNA having both of these properties may be used. These Ago2-miRNAs may be used alone or in combination.

  Cancer can be determined by using together Ago2-miRNA having the property (1) and Ago2-miRNA having the property (2). Moreover, cancer can also be determined using Ago2-miRNA having both the properties of (1) and (2). Alternatively, cancer can be determined by combining Ago2-miRNA having both the properties (1) and (2) and Ago2-miRNA having the property (1). Alternatively, cancer can be determined by combining Ago2-miRNA having both the properties (1) and (2) and Ago2-miRNA having the property (2). Thus, by using Ago2-miRNA having different properties in combination, cancer can be determined from various, detailed and accurate.

  Preferred Ago2-miRNA used in the method of the present invention includes Ago2-miR-21 having both the properties of (1) and (2), and Ago2-miR-200c having the properties of (1). Is not limited to these Ago2-miRNAs.

  Since Ago2-miR-21 has both the properties (1) and (2) above, it is a very useful marker for knowing the presence of cancer. By measuring the expression level of Ago2-miR-21, it is possible to determine the presence (including recurrence) of cancer in a subject, predict the effect of anticancer drug treatment, or determine the type and dose of an anticancer drug. By measuring the expression level of Ago2-miR-21, it is possible to detect early cancer or predict early recurrence in a subject.

  As described above, Ago2-miR-21 also has the property (2). This also suggests the presence of live cancer cells when Ago2-miR-21 is present in the blood beyond the normal range. That is, Ago2-miR-21 is useful for predicting recurrence at the previous stage observed in an image and diagnosing early cancer.

  Ago2-miR-200c is particularly expressed in metastatic lesions and is useful as a marker for predicting cancer metastasis, predicting the effect of anticancer drug treatment on metastatic lesions, or determining the type and dosage of anticancer agents. is there.

  Ago2-miR-21 may be used alone, Ago2-miR-200c may be used alone, or Ago2-miR-21 and Ago2-miR-200c may be used in combination.

  What is used as an endogenous control in the method of the present invention is important. It is preferable to use Ago2-miRNA stably present in the body fluid as an endogenous control. In the present invention, Ago2-miR-451 is particularly preferably used as an endogenous control, but is not limited thereto.

  The types of cancer determined by the method of the present invention are, for example, esophageal cancer, stomach cancer, colon cancer, liver cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, renal cell cancer, gastrointestinal stromal tumor, mesothelioma Tumor, brain tumor, head and neck cancer, laryngeal cancer, oral cancer, oral cavity cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, sinus cancer, maxillary sinus cancer, frontal sinus cancer, ethmoid sinus cancer, butterfly Bone sinus cancer, thyroid cancer, lung cancer, osteosarcoma, bladder cancer, prostate cancer, testicular cancer, testicular cancer, penile cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, skin cancer, Examples include, but are not limited to, rhabdomyosarcoma, leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and the like.

  Samples subjected to the method of the present invention include, but are not limited to, body fluids such as blood, lymph, and cerebrospinal fluid. A preferred sample subjected to the method of the present invention is blood, more preferably plasma. In the present invention, before measuring the expression level of Ago2-miRNA, by removing non-specific antibodies and proteins in the sample using protein A / G agarose, the amount of sample used for analysis can be reduced. (Referred to as “pre-clear method”) If the pre-clear method is used, the expression level of Ago2-miRNA in about 10 μl of plasma can be measured.

  In a further aspect, the present invention relates to a kit for determining cancer, comprising a means for measuring the expression level of Ago2-miRNA in a blood sample. The Ago2-miRNA and the sample are as described above. Preferred Ago2-miRNA is Ago2-miR-21 and / or Ago2-miR-200c. Examples of means for measuring the expression level of Ago2-miRNA include antibodies specific for Ago2, materials for performing quantitative reverse transcription PCR, and the like. These antibodies and materials can be easily selected, obtained or prepared by those skilled in the art. Usually, an instruction manual is attached to the kit of the present invention.

  The kit of the present invention may include a means for measuring the expression level of Ago2-miR-451 as a control in a blood sample.

  The kit of the present invention may contain protein A / G agarose in order to remove non-specific antibodies and proteins in the sample before measuring the expression level of Ago2-miRNA.

  The present invention will be described in more detail and specifically with reference to the following examples, but the examples are not intended to limit the present invention.

I. Experimental methods and materials The main methods and materials used in the experiments are described below.
(1) Blood collection and plasma preparation Peripheral blood was collected using an EDTA collection tube, and plasma was isolated immediately thereafter. Whole blood was centrifuged at 3000 rp for 10 minutes at 4 ° C. Plasma was separated from whole blood and centrifuged at 3000 rp for 10 minutes at 4 ° C. to obtain a plasma supernatant. Cell debris or large extracellular vesicles were removed from the sample by further centrifugation at 14400 rpm for 10 minutes, followed by filtration through a 0.22 μm filter (Merck Millipore, Merck KGaA, Darmstadt, Germany).

(2) Cell Culture Assay Human colon cancer cell line HT29 was purchased from American Type Culture Collection (Manassas, VA, USA) and supplemented with exosome-depleted-10% fetal calf serum (System Biosciences, CA, USA) at 37 ° C. under 5% CO ₂ . One million cells were seeded per 10 ml of medium in the dish, and 750 μl of cell culture supernatant was collected every 24 hours. In order to remove cell debris, the collected supernatant was centrifuged at 14400 rpm for 10 minutes and filtered through a 0.22 μm filter. 48 hours after collection, 1 ml of a medium containing a 5-FU solution was added to each dish so that the 5-FU concentration was 0.5 μM. 100 μl of medium containing 100,000 cells is seeded in a 96-well plate, 10 μl of Cell Proliferation Reagent WST-1 (Roche Diagnostics) is added after 0, 24, 48, 72 and 96 hours, and the absorbance is measured after 1 hour. did. After the measurement at 48 hours, 1 ml of a medium containing a 5-FU solution was added so that the 5-FU concentration was 0.5 μM.

(3) Immunoprecipitation
Extracellular miRNA was immunoprecipitated with anti-human Ago2 monoclonal antibody (WAKO, Osaka Japan, 4G8; Cat. 015-22031) using Pierce Classic IP kit (Thermo Scientific). Briefly, PBS was added to a plasma sample (10-100 μl) to a volume of 200 μl, Pierce Protein A / G (20 μl) not bound to antibody was added and incubated overnight with rotation ( Pre-clear process). After collecting the flow-through by centrifugation (1000 rpm for 1 minute), the precleared plasma sample is incubated with 1 μg (1 μl) of anti-human Ago2 monoclonal antibody for 1 hour at room temperature while rotating. did. As a negative control, 1 μg of anti-β-actin monoclonal antibody (Sigma-Aldrich) was used. In the conditioned medium assay, 200 μl of medium was incubated with anti-human Ago2 antibody without the preclear step. To capture immune complexes, samples were incubated with fresh 20 μl Pierce Protein A / G agarose for 1 hour at room temperature with rotation. After centrifugation, the flow-through was discarded or set aside for a second Ago2 immunoprecipitation. The resin was washed 3 times with 200 μl of TBST, 200 μl of Qiazol reagent (QIAGEN) was added and left for 5 minutes. After collecting the eluate by centrifugation, 500 μl of Qiazol reagent was further added to make the total volume 700 μl.

(4) Isolation of extracellular vesicles (EV)
Extracellular vesicles were isolated from 200 μl of plasma or medium using the exoRNeasy Serum / Plasma Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.

(5) miRNA isolation and reverse transcription
Total miRNA was isolated from Ago2 immunoprecipitates and EV isolates using miRNeasy Serum / Plasma kit (Qiagen) according to the manufacturer's instructions. 3.5 μl cel-miR-39 (1.6 × 10 ⁸ copies / μl) as normalization control and 2 μl yeast RNA (0.1 μg / μl) as carrier RNA were added to the sample, followed by chloroform. CDNA was synthesized using 4 μl of extracted RNA template using miScript II RT Kit (Qiagen) according to the manufacturer's instructions.

(6) Quantitative real-time PCR
The expression level of each miRNA was analyzed using miScript Primer Assays (Qiagen). Quantitative real-time PCR was performed using gene specific primers, miScript SYBR Green PCR Kit (Qiagen) and LightCycler 480 (Roche Diagnostics). The quantitative value of the miRNA sample was calculated by subtracting the cycle threshold (Ct) value (external reference) of cel-miR-39. The Ct values obtained from the two-system reaction were averaged. ΔCt was calculated by subtracting the Ct value of the added cel-miR-39 control from the Ct value of the target miRNA. ΔΔCt was calculated by subtracting the ΔCt value of the reference sample from the ΔCt value of the control sample. The fold change for each miRNA was determined.

(7) Comprehensive analysis of global expression of Ago2-miRNA and global expression of EV-miRNA in plasma by miRNA PCR array
The global expression ratio of Ago2-miRNA and EV-miRNA was evaluated using miScript miRNA PCR Arrays (Qiagen). In order to maintain a uniform sample volume, 100 μl of plasma was divided into 25 μl aliquots and subjected to Ago2 immunoprecipitation four times. In the elution step, 175 μl of Qiazaol reagent was added to each A / G agarose resin and the eluate was mixed. MiRNA isolation was performed by adding the added cel-miR-39 as a control, and reverse transcription was performed using 1.5 μl of the extracted RNA template. The preamplification step was performed using miScript PreAMP PCR Kit in combination with miScript PreAMP Human miRNome Primer Mix (Qiagen). The miScript miRNA PCR Array Human miRNome was performed using LightCycler 480, and ΔCt was calculated by subtracting the Ct value of the added cel-miR-39 control from the Ct of the target miRNA. The Ct value of miRNA that was not detected was set to 45 or more.

(8) SDS-PAGE and immunoblotting The A / G agarose resin, Ago2 immunoprecipitate subjected to the preclear step, and the second immunoprecipitate were washed 3 times with 200 μl TBST, and 50 μl lane marker loading buffer (300 mM Tris -HCl, pH 6.8; 1% SDS; 10% glycerol; 20 mM DTT) for 10 minutes at 100 ° C. 5 μl of plasma, 1 μl of Ago2 antibody or 10 μl of precleared plasma were then eluted separately in lane marker loading buffer. The protein was developed on an SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. The gel was then incubated with Oriole Fluorescent Stain (Bio Rad) and then the membrane was incubated with human IgG-Fc antibody (Bethyl Laboratories, Inc) for 1 hour at room temperature. After incubation, the membrane was incubated with HRP-conjugated secondary antibody. ECL was used for blot color development.

(9) Comprehensive analysis by miRNA microarray of miRNA expression in primary tumors, liver metastasis sites, and corresponding normal tissues Total miRNA was isolated from frozen specimens using miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol, and Agilent 2100 Analysis was performed with Bioanalyzer (Agilent Technologies). SurePrint G3 Human miRNA Microarray Kit Release 21.0 (Agilent Technologies) contained 2549 human microRNA probes. The expression level of each probe was the sum of the raw intensities minus the background for 20 spots. The expression level was set to 0.1 for the target miRNA that was not detected as a spot. Data were normalized to 90 ^th percentile, it was excluded target miRNA was not detected in all samples.

(10) Statistical analysis Statistical analysis was performed using JMP software (version 10.0; SAS Institute Inc. Cary, NC, USA). miRNA expression levels were expressed as Ct and ΔCt. The relative expression ratio was expressed as an average value ± standard deviation (SD). The Pearson product-moment correlation coefficient (r ² ) was determined, and the correlation between ΔCt and plasma volume for Ago2-miR-451 was examined. Hierarchical cluster analysis for miRNA microarrays was performed using the Ward method. In addition to comparing the difference between the Ct and ΔCt values of Ago2-miRNA in plasma samples, the Wilcoxon signed-rank test was used to compare Ago2-miR-21 and Ago2-mir- in paired plasma samples. The expression of 200c was compared. All P-values were the result of a two-sided test, and P-values less than 0.05 were considered statistically significant.

  Other experimental methods and procedures used were known to those skilled in the art.

II. Experimental results Evaluation of extracellular miRNA release mechanism in the presence of cytotoxic agent First, it was examined whether two forms of circulating miRNA could be detected from the extracellular fluid using HT29 CRC cell line. MiR-21 and miR-31 were used as biomarkers. The HT29 CRC cell line has abundant miR-21 and miR-31 in the cytoplasm, and 5FU was added to the 5-fluorouracil (5-FU) -treated group 48 hours after seeding. Evaluation of HT29 cell proliferation was performed using a water-soluble tetrazolium salt (WST) assay (FIG. 1A).

  Cell culture fluid is collected every 24, 48, 72, and 96 hours after culturing, and Ago2-miRNAs therein are obtained by a method using immunoprecipitation (Ago2-immunoprecipitation) using Ago2 antibody. After purification using exoRNeasy Serum / Plasma kit, each was measured by quantitative reverse transcription PCR (qRT-PCR) (FIG. 2).

  The expression levels of miR-21 and miR-31 after 48, 72 and 96 hours in the cell culture medium are relative ratios based on the expression levels in the cell culture medium collected 24 hours after seeding (hold changes based on A comparative study was performed using the change in threshold cycle value [ΔCt]) (FIG. 1B). In order to examine whether Ago2-miRNA and EV-miRNAs are actively released from living cells, relative expression index (Ago2-miRNA expression relative ratio or EV-miRNA expression relative ratio is expressed as WST). The value divided by the relative ratio of absorbance was calculated (FIG. 1C).

  In the control group, the relative expression index represents the amount of miRNA that is actively released from living cells as it is. In the 5-FU treatment group, the relative expression index reflects the amount of miRNA that is passively released to the outside due to cell death. In the 5-FU treatment group, the relative expression index (REI) in miR-31 increased with time in both Ago2 and EV, and both Ago2-miRNA and EV-miRNA were released to the outside by cell death. It was shown that. In contrast, in the control group, only Ago2-miR-21 REI showed an increasing trend in the extracellular medium over time, EV-miR-21 showed a slight increase, Ago2-miR-31, EV -The REI of miR-31 showed no increase throughout the course of the experiment. An increase in REI of Ago2-miR-21 in this control group suggests that it was actively released from living cells.

  From these results, it was suggested that the mechanism of miRNA release outside the cell may vary depending on the type of miRNA or the type of miRNA complex. And for colorectal cancer, Ago2-miR-21 is not only passively released by cell death but also actively released from living cells and may be important as a biomarker Was suggested.

2. Classification of circulating miRNA Prior to detecting different forms of circulating miRNA from plasma, a comprehensive study of Ago2-miRNA and EV-miRNA in healthy human plasma was performed using miRNA PCR arrays. In addition, the method of FIG. 2 was used for the analysis of Ago2-miRNA and EV-miRNA. By this analysis, it was possible to clearly classify plasma miRNA into three groups. (I) Ago2-miRNA (captured only by Ago2 immunoprecipitation method and can be ignored by EV isolation), (ii) EV-miRNA (captured only by EV isolation, Ago2 immunoprecipitation method) (Iii) can be ignored), (iii) both forms (detected by both methods) (Figure 3).

  This experiment showed that miR-451 exists as an Ago2-miR-451 complex and very little exists as EV-miR-451. In contrast, most of miR-let-7d was detected as EV-miRNA (FIG. 3C).

3. Identification of miRNA associated with metastasis Next, in order to examine whether Ago2-miRNA or EV-miRNA can be a biomarker for determining drug effect during chemotherapy (anticancer agent), first, colon cancer metastasis Attempts were made to identify miRNAs that are characteristically expressed in. For this purpose, a miRNA microarray was used to comprehensively analyze miRNA expression in primary and liver metastatic tissues.

  miRNA expression analysis by miRNA microarray was performed using RNA obtained from primary lesion tissue, normal large intestine mucosa tissue, liver metastasis tissue, and normal liver tissue of stage IV great face patients with liver metastasis. The miRNA microarray used in this study is capable of comprehensive analysis of 2549 human miRNAs. This analysis revealed a strong correlation between miRNA expression in the primary lesion and liver metastases (FIG. 4A). Among 2549 human miRNAs, 59 miRNAs were more than twice as expressed in cancer tissues (primary foci / liver metastases) than normal tissues. When cluster analysis was performed using these 59 types of miRNA, it was shown that miR-200c is highly expressed in liver metastases (FIG. 4B). Therefore, miR-200c was selected as a biomarker candidate, and the extracellular release mechanism of Ago2-miR-200c and EV-miR-200c was examined using the HT29 cell line (FIG. 4C). In the 5-FU treatment group, the concentrations of Ago2- and EV-miR-200c in the cell culture medium increased with time, and in the control group, Ago2- and EV-miR-200c showed no increase over time. . This suggested that both Ago2-miR-200c and EV-miR-200c are released extracellularly only by passive release due to cell death.

  From the above series of research results, we focused on Ago2-miRNA and aimed to develop a detection technique for clinical application. In addition, it was decided to investigate using Ago2-miR-21 and Ago2-miR-200c for colorectal cancer.

4). Development of a new method for detecting circulating Ago2-miRNA Next, an attempt was made to develop a technique for detecting stable Ago2-miRNA that is reproducible in clinical practice. It is speculated that antibodies and proteins present in the plasma prevent the Ago2-miRNA from binding to the A / G agarose beads as a result. In order to reduce the adverse effects of antibodies and proteins present in these plasmas, before performing immunoprecipitation (Ago2-IP) with Ago2 antibody, nonspecific antibodies and proteins are removed with A / G agarose beads, An attempt was made to improve the efficiency of Ago2-IP (pre-clear method, FIG. 5).

  In order to optimize this pre-clear method, miR-451 was targeted and plasma was obtained from healthy individuals. The preclear method improves the relative expression level of mir-451 in Ago2-IP and clearly reduces miR-451 in the flow-through compared to the conventional Ago2-IP method (referred to as “non-preclear method”). (FIG. 6A). Next, an experiment was conducted with the amount of plasma used being 10 to 60 μL, and the amount of plasma used in clinical samples was examined (FIG. 6B).

These results were also confirmed by human IgG immunoblot and SDS-PAGE (FIG. 7). A strong correlation was observed between the relative expression level of miR-451 and plasma volume up to 30 μl of plasma (r ² = 0.903) (FIG. 6C). Comparison of detection of Ago2-miRNA by plasma pre-clear method in 30 μl of plasma and detection of EV-miRNA by normal method (plasma of 200 μl) showed that miR-451 was found in Ago2, similar to the miRNA microarray results. (FIG. 6D). Therefore, 30 μl of plasma usage in clinical samples was considered sufficient. Reports to date have reported that the amount of plasma (serum) required to detect circulating miRNA is 80 to 400 μl. This method can be detected with less than half of the minimum amount reported in the past. In addition, when detecting Ago2-miRNA in plasma, Ago2-miR-451 in plasma was stably present and was considered to be optimal as an endogenous control. Therefore, in subsequent experiments, Ago2-miR Analysis proceeded with -451 as an endogenous control.

5. Prediction of colorectal cancer patients using Ago2-miRNA in blood sample Before the treatment of 40 colorectal cancer patients, whether Ago2-miR-21 and -200c are increased in the blood of colorectal cancer patients Analysis was performed using the obtained blood and the blood of 20 control patients. The control patient here refers to a patient in a colorectal cancer patient who has undergone curative resection and in which no recurrence is observed on an image. When the Ago2-miR-451 amplification state was used as a control related to the blood state, amplification of Ago2-miR-451 was poor in one of 40 cases. For this reason, this one example was excluded from the following analysis. FIG. 8A shows measured Ct values. The average value of Ago2-miR-451 was not significantly different between 39 colorectal cancer patients and 20 control patients (average Ct values of control patients and colorectal cancer patients were 29.9 (95% confidence interval, respectively). (CI) was 28.4-31.4) and 30.5 (95% CI was 29.5-31.5).

  In contrast, the average value of Ago2-miR-21 was clearly lower in 39 colon cancer patients than in 20 controls (the average Ct values of colon cancer patients and control patients were 31.4 (95% CI was 31.0-31.9) and 33.0 (95% CI was 32.0-34.0, P = 0.0097). In addition, the average value of Ago2-miR-200c was not significantly different between 39 colorectal cancer patients and 20 controls (average Ct values were 39.9 (95% CI was 39.5-40. 2) and 41.6 (95% CI is 40.1-43.1)).

  Next, ΔCt of Ago2-miR-451, -21, -200c is calculated using cel-miR-39, and is further corrected by ΔCt of Ago2-miR-451 to obtain ΔCt (miR-451 ). ΔCt (miR-451) of Ago2-miR-21 and -200c was calculated. As shown in FIGS. 8B and 8D, ΔCt (miR-451) of Ago2-miR-21 and -200c clearly showed a decrease in colorectal cancer patients. That is, the amounts of Ago2-miR-21 and -200c in plasma obtained from colon cancer patients increased compared to control patients. In addition, Ago2-miR-21 and -200c ΔCt (miR-451) were examined in blood before and after surgery in 11 colon cancer patients (FIGS. 8C and 8E). The amount of Ago2-miR-21 and -200c present in the blood was clearly high before the operation and tended to decrease after the operation.

  The average value of ΔCt (miR-451) of Ago2-miR-21 of the control patient is 3.12 (95% CI is 1.81 to 4.42), and the average value of ΔCt (miR-451) of the colon cancer patient Since the value was 0.96 (95% CI was 0.27 to 1.65), in this study, ΔCt (miR-451) of Ago2-miR-21 was 2.00 or more as a normal value . Similarly, the normal value of ΔCt (miR-451) of Ago2-miR-200c was set to 10.00 or more. This is because the average value of ΔCt (miR-451) of Ago2-miR-200c of control patients is 11.67 (95% CI is 9.72 to 13.62), and ΔCt (miR-451 of colon cancer patients). ) Has an average value of 9.38 (95% CI is 8.51-10.2).

6). Trend of Ago2-miRNA in plasma obtained from colorectal cancer patients undergoing chemotherapy treatment Blood of four colorectal cancer patients undergoing chemotherapy treatment with Ago2-miR-21 and -200c (before each course of chemotherapy) The plasma was obtained from blood collected in (1).

  FIG. 9A shows the course of treatment of patients undergoing chemotherapy with unresectable liver metastases and the course of carcinoembryonic antigen (CEA) levels, Ago2-miR-21 and -200c in plasma. This patient is a patient who became resistant to treatment after successful first-line chemotherapy for the first 4 months. The course of treatment is classified into three categories according to the change in CEA: contraction phase (Day 1 to Day 48), stationary phase (Days 49 to 113), and progression phase (Days 113 to 168) ). In the shrinking phase, chemotherapy has been successful. The ΔCt (miR-451) of Ago2-miR-200c is lower than the cut-off value of 10.00 (abnormal value) in the stationary phase (from day 49 to day 113), and ΔCt (miR−) of Ago2-miR-21 451) decreased from the normal value (ie, abnormal value) after 2 courses of administration, indicating a response to chemotherapy. In the stationary phase, CEA indicates that the disease is stable, but ΔCt of Ago2-miRNA indicates that the fourth dose is responsive to treatment, but after the fifth dose, ΔCt showed an increase, suggesting that it was refractory to the treatment regimen. In the advanced stage, CEA increased, whereas ΔCt of Ago2-miRNA hardly changed. That is, it was suggested that cancer cells were not destroyed by the chemotherapy.

  FIG. 9B shows the course of treatment and CEA values, plasma levels of Ago2-miR-21 and -200c in adjuvant chemotherapy patients after curative resection of colorectal cancer. After the first dose, ΔCt (miR-451) of Ago2-miRNAs decreased (ie, the amount of Ago2-miRNAs in plasma increased, not visible on the image, but some cancer cells were present, and chemotherapy Suggested collapsed). However, at the second dose, Ago2-miRNA ΔCt increased (ie, the amount of Ago2-miRNAs in plasma decreased). These suggested the possibility that there were no cancer cells or that they acquired resistance. Actually, liver metastasis was confirmed on the 29th day by CT. For this reason, the possibility of refractory to FOLFOX6 was suggested for the second time. Subsequent introduction of bevacizumab again reduced CEA and decreased the ΔCt of Ago2-miRNA (ie, the amount of Ago2-miRNAs in plasma increased, not visible on the image, but some cancer cells Was suggested to have been disrupted by chemotherapy). These suggested that the chemotherapy was still successful.

  FIG. 9C shows the treatment course and CEA values, Ago2-miR-21 and -200c in plasma of patients with unresectable liver metastases that have passed little success with primary therapy. This patient is a patient who became resistant to treatment after successful first-line chemotherapy for the first 4 months. The course of treatment can be divided into two according to the CEA value: stationary phase (from day 1 to day 84) and advanced phase (from day 85 to day 126). ΔCt (miR-451) of Ago2-miR-21 shows an abnormal value immediately before the first administration. This Ago2-miR-21 is suggested to be the result of active excretion. After chemotherapy, the abnormal value boundary was shown. On the other hand, ΔCt of Ago2-miR-200c only showed an abnormal value after the first administration, and thereafter maintained a normal value, and only the first few chemotherapys were successful against cancer. It has been suggested.

  FIG. 9D shows the course of treatment for patients undergoing chemotherapy with unresectable liver metastases and the course of carcinoembryonic antigen (CEA) levels, Ago2-miR-21 and -200c in plasma. Although this patient showed an increase in CEA after resection of the primary lesion, this patient succeeded immediately after the introduction of chemotherapy. Ago2-miR-21 and -200c ΔCt (miR-451) did not differ between primary excision and chemotherapy initiation, but Ago2-miR-21 ΔCt (miR-451) after chemotherapy initiation. ) Showed an abnormal value only once, but then it remained normal. The ΔCt (miR-451) of Ago2-miR-200c remained normal during the observation period, suggesting that Ago2-miR-21 and -200c are not suitable as monitoring markers in this case. .

  The course of 3 cases with different situations was presented. A certain tendency is observed in ΔCt of Ago2-miRNAs. That is, it was recognized that ΔCt of Ago2-miRNA showed a decrease in a state where chemotherapy was successful, and that ΔCt of Ago2-miRNA showed a plateau or an increase if it was refractory to the chemotherapy.

  The blood of cancer patients other than colorectal cancer was analyzed (FIG. 10). As a result, Ago2-miR-21 showed an increasing tendency even in the blood of cancer patients other than colorectal cancer, indicating that it is an important cancer marker.

  The present invention is useful in the fields of cancer diagnostic agents and cancer research reagents.

Claims (12)

  A method for determining cancer, comprising measuring the expression level of microRNA (Ago2-miRNA) forming a complex with Ago2 in a blood sample obtained from a subject. Ago2-miRNA is
(1) Ago2-miRNA passively released into the blood by cell death, and / or (2) Ago2-miRNA actively released by living cells, and / or (3) Passive by cell death Ago2-miRNA released into the blood and actively released by living cells
The method according to claim 1.   The method according to claim 1 or 2, wherein the Ago2-miRNA is Ago2-miR-21 and / or Ago2-miR-200c.   The method according to any one of claims 1 to 3, wherein the expression level of Ago2-miRNA in the blood sample is corrected by the expression level of Ago2-miR-451 in the sample.   The non-specific antibody and protein in a sample are removed using protein A / G agarose before measuring the expression level of Ago2-miRNA, according to any one of claims 1 to 4. Method.   The expression level of Ago2-miR-21 is measured, and the presence of cancer including recurrence in the subject is determined, the effect of anticancer drug treatment is predicted, or the type and dose of the anticancer drug are determined. 6. The method according to any one of 5 above.   The method according to claim 6, comprising detecting early cancer or predicting early recurrence in a subject.   The expression level of Ago2-miR-200c is measured, and prediction of cancer metastasis, prediction of the effect of anticancer drug treatment on metastatic lesions, or determination of the type and dose of anticancer drug is performed. The method according to claim 1.   A kit for determining cancer, comprising a means for measuring the expression level of Ago2-miRNA in a blood sample.   The kit according to claim 9, wherein the Ago2-miRNA is Ago2-miR-21 and / or Ago2-miR-200c.   The kit according to claim 9 or 10, further comprising means for measuring the expression level of Ago2-miR-451 in the blood sample.   The kit according to any one of claims 9 to 11, further comprising protein A / G agarose.