JP5399011B2 - Method for detecting tissue injury or cell proliferative disorder - Google Patents

Description

  The present invention relates to a method for detecting tissue injury or cell proliferative disease. More specifically, the present invention detects tissue injury or cellularity by detecting free microRNA released with the disease in serum or plasma collected from a subject suspected of having tissue injury or cell proliferative disease. The present invention relates to a method for detecting a proliferative disease. Furthermore, the present invention relates to a method for measuring the amount of specific microRNA in serum or plasma collected from the subject a plurality of times and detecting the tissue injury or cell proliferative disease state of the subject based on the measurement result.

MicroRNA (hereinafter sometimes referred to as “miRNA”) is non-coding RNA that is not translated into protein, and is a single-stranded RNA having a length of about 22 nucleotides. The existence of many types of miRNAs has been reported in animals and plants, and about 500 types of human miRNAs are currently known (miRBase: Sanger database registration).
miRNA is transcribed by RNA polymerase II, and primary-miRNA (hereinafter referred to as “pri-miRNA”) is biosynthesized. Pri-miRNA usually has a long sequence of several hundred to a thousand nucleotides. The pri-miRNA usually has a structure having a cap structure at the 5 ′ end and a polyadenine added at the 3 ′ end, and has a structure similar to a messenger RNA encoding a protein (hereinafter referred to as “mRNA”).

  The pri-miRNA forms a hairpin-like stem-loop secondary structure and binds to a 500-650 kDa microprocessor complex. The microprocessor complex is composed of Drosha, which is an RNase III endonuclease, and DGCR8 / Pasha, which is a protein having a double-stranded RNA binding domain. The pri-miRNA is processed and a 60-70 nucleotide pre-miRNA is biosynthesized.

  The pre-miRNA is transferred to the cytoplasm by Exportin-5 (hereinafter referred to as “Exp5”) belonging to the Ran transport receptor family. In the cytoplasm, pre-miRNA is processed by Dicer, a second Rnase III endonuclease, to form short double-stranded miRNA: miRNA * duplex.

Finally, miRNA: miRNA * duplex is denatured into a single strand by helicase to become mature miRNA and miRNA *. Mature miRNA is incorporated into an RNA-induced silencing complex (hereinafter referred to as “RISC”). As a RISC complex, miRNAs act on mRNA degradation and post-transcriptional repression and control gene expression. Mature miRNA * is rapidly degraded.
miRNA is known to function to regulate gene expression by mRNA cleavage, translational repression, or mRNA degradation, and mRNA cleavage and translational repression are performed by the same mechanism.

RISC cleaves the target mRNA when the nucleotide sequence of the miRNA in RISC binds completely or nearly completely to the protein coding region or open reading frame sequence of the target mRNA. On the other hand, if the nucleotide sequence of miRNA in RISC binds incompletely with the sequence on the 3'-untranslated region of the target mRNA, RISC inhibits the target mRNA at the translation stage and suppresses gene expression. It is known.
It is known that polyadenine at the 3 ′ end of mRNA (hereinafter referred to as “poly (A)”) enhances the stability of mRNA and avoids RNA degradation. It has been clarified that miRNA is involved in mRNA degradation by directly acting on the depoly (A) reaction.

  In general, a phenomenon in which normal proliferation, differentiation, and apoptosis of cells have failed and cells proliferate randomly is considered canceration. It has been clarified that miRNA regulates cell proliferation and apoptosis (Non-patent Documents 1 and 2), and studies have been conducted on the possibility of association with cancer. Specifically, it will be described in detail below.

Human chronic lymphocytic leukemia (hereinafter referred to as “CLL”) is the most common adult leukemia in the West. Deletion of the long arm (13q14) region of chromosome 13 is known, and deletion of the 13q14 region has been observed in the majority of cases of B cell CLL (hereinafter referred to as “B-CLL”). miR-15a gene and miR-16a gene are located in the 13q14 region, and miR-15a and miR-16a expression levels are zero or decreased in about 68% of CLL patients (Non-patent literature) 3). Expression profiling using B-CLL patient specimens as a material and a microarray has also revealed a reduction in miR-15a and miR-16a expression levels (Non-Patent Documents 4, 5 and 6).
Furthermore, a patent application has been filed for a gene therapy vector based on miR-15a gene and miR-16a gene expression (Patent Document 1).

Lung cancer is one of the most common cancers in adults and has the highest cancer mortality in developed countries. It has been suggested that one type of miRNA, let-7, may be involved in lung cancer progression, and at least an important factor in the pathogenesis of lung cancer. Regardless of the stage of lung cancer, it is related to the decrease in let-7 expression in cancer tissues and short postoperative survival, and overexpression of let-7 It has been clarified that proliferation is inhibited (Non-patent Document 7). RAS and MYC are known to be lung cancer proto-oncogenes along with p53. There is a let-7 complementary sequence on the 3'-untranslated region of RAS and MYC, and let-7 inhibits RAS and MYC gene expression negatively by inhibiting it at the translational stage. It has been confirmed. In lung cancer tissues, an increase in the amount of let-7 protein has been confirmed as compared with normal lung tissues, and it has been suggested that the control of RAS by let-7 is involved in lung cancer induction (Non-patent Document 8).
Further, a patent application has been filed for a lung cancer prognosis determination method by measuring let-7 expression level and a gene therapy vector by let-7 gene expression (Patent Document 2).

  Furthermore, the miR-17-92 cluster (miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b, miR-92) composed of seven types of microRNAs ( It is suggested that the “miR-17-92 cluster” may be involved in lung cancer. In lung cancer, particularly small cell lung cancer, the expression level of miR-17-92 cluster is remarkably increased, and it has been revealed that proliferation of lung cancer cells is enhanced (Non-patent Document 9). In silico analysis predicts that the miR-17-92 cluster acts on tumor suppressor genes PTEN and RB2 (Non-patent Document 10). In small cell lung cancer, it has been reported that miR-17-92 cluster and / or proto-oncogene c-myc are either gene amplified or overexpressed (Non-patent Document 11). It has been reported that the overexpression of miR-17-92 cluster without gene amplification correlates with the overexpression of one or more genes in the myc gene family (Non-patent Document 9). Expression suggests that the miR-17-92 cluster is overexpressed.

  Breast cancer is one of the most important cancers in adult women. As a result of examining the expression levels of 76 breast cancer tissues and 10 normal tissues using miRNA microarray analysis, the expression levels of miR-125b, miR-145, miR-21, and miR-155 were higher in breast cancer tissues than in normal tissues. It was found that there was a significant decrease. In addition, miR-125b, miR-145, miR-21, and miR-155 expression levels are related to features such as breast cancer stage, growth index, estrogen receptor and progesterone receptor expression levels, and blood vessel invasion level. (Non-patent Document 12).

  Polymorphic glioblastoma is the most frequently occurring and highly malignant primary brain tumor and is one of the most invasive and active cancers that cannot be treated. As a result of examining the expression level of polymorphic glioblastoma and normal brain tissue, miR-221 expression level was significantly increased in glioblastoma compared to normal brain, miR-181a, miR-181b, and miR-181c It was found that the expression level was reduced (Non-patent Document 13). In addition, it has been reported that miR-21 expression level is significantly increased in glioblastoma tissue, glioblastoma primary culture, and glioblastoma-derived subculture cells with extremely high malignancy (non-patented). Reference 14). miR-21 knockdown experiments showed that miR-21 acts as an anti-apoptotic factor in glioblastoma-derived subcultured cells. From this, it is speculated that the abnormal expression increase of miR-21 inhibits the expression of an important apoptosis-related gene and causes a serious brain tumor (Non-patent Document 14).

  The BIC gene is associated with several types of cancer, and BIC activation is known to accelerate the development of leukemia and lymphoma. BIC expression is increased in Hodgkin lymphoma and childhood Burkitt lymphoma compared to normal lymphoid tissue. These findings suggest that BIC is a proto-oncogene such as Hodgkin lymphoma and Burkitt lymphoma in children, but the molecular mechanism of cancer development associated with BIC is unknown. miR-155 sits in a phylogenetically conserved region within the BIC gene, and miR-155 is a large number of B-cell-derived lymphomas, particularly diffuse large B-cell lymphoma (hereinafter referred to as “DLBCL”) It was found to be overexpressed in highly active B cell tumors such as In DLBCL with high activity, miR-155 expression level is increased about 10-60 times compared to CLL with low activity (Non-patent Document 16). In addition to miR-155, miR-15a is down-regulated in DLBCL (Non-patent Document 15), and miR-17-92 clusters are overexpressed in many types of lymphoma. (Non-Patent Document 16).

  Papillary thyroid cancer (hereinafter referred to as “PTC”) accounts for about 80% of thyroid cancer and is the most aggressive. Although PTC is known to be associated with changes in the RET / PTC-RAS-BRAF signaling pathway, the detailed molecular mechanism is unknown. As a result of examining the expression level of PTC and normal thyroid tissue, it was found that the expression level of miR-221, miR-222, and miR-146 was significantly increased in PTC compared to normal thyroid tissue (non- Patent Document 17). It was also found that the amount of mRNA and protein in the proto-oncogene KIT decreased as the expression level of miR-221, miR-222, and miR-146 increased in thyroid cancer. The involvement of miR-221, miR-222, and miR-146 in regulation was suggested. From in silico analysis, KIT is predicted to be a target of miR-221, miR-222, and miR-146 (Non-patent Documents 18, 19, and 20).

  MiR-143 and miR-145 expression levels in colon nematodes are lower than in normal mucosa (Non-patent Document 21), testicular germ cell tumors (hereinafter referred to as “TGCTs”), miR-372, and There is a report that miR-373 is overexpressed (Non-patent Document 22). In addition, miR-18 and miR-224 expression levels increased significantly in hepatocellular carcinoma (hereinafter referred to as “HCC”), miR-199a *, miR-195, miR-199a, miR-200a, and miR- There is a report that the expression level of 125a is decreased (Non-patent Document 23).

  As mentioned above, it is suggested that miRNAs are involved in cancer progression by regulating cell proliferation and apoptosis, and are involved in cancer by acting on proto-oncogenes and tumor suppressor genes . This shows the possibility that miRNA can be applied as a marker for cancer diagnosis. However, since all the reports on miRNA expression measurement described above use cancer tissue as a material, they are not easily collected for specimen testing and are currently used as tumor markers used in clinical testing. It was difficult to do this, and there was a need for a simpler inspection method.

  Samples that are easier to collect than cancer tissues include cells derived from cancer tissues that float in body fluids such as blood, lymph, bone marrow, and cerebrospinal fluid (hereinafter referred to as “floating cells”). Suspended cells are collected using a cell fractionator such as Fluorescence activated cell sorting (hereinafter referred to as “FACS”). Using floating cells in the blood as a test sample, prostate cancer detection method by miR-15 expression level measurement, kidney cancer detection method by miR-35 expression level measurement, brain tumor, liver by miR-16 expression level measurement A method for detecting lung cancer and a method for detecting pancreatic cancer by measuring the expression level of miR-375 have been reported (Patent Document 3).

However, it is difficult to think that floating cells are always present uniformly in the body fluid. Therefore, it has been difficult to reliably acquire cells derived from cancer tissue by collecting a part of the body fluid.
As a new test material, free nucleic acid floating in body fluid (hereinafter referred to as “free nucleic acid”) is considered. It is known that there is a trace amount of free nucleic acid in the body fluid, but free DNA and free mRNA are detected. Free nucleic acids are classified according to the type of body fluid, and free nucleic acids present in circulating body fluids such as whole blood, serum, plasma, and lymph fluid are circulating nucleic acids, urine, sputum, ejaculate, semen, tears, sweat, Body fluids such as saliva, bronchial lavage fluid, pleural effusion, peritoneal fluid, meningeal fluid, amniotic fluid, glandular fluid, fine needle aspirate, nipple aspirate, cerebrospinal fluid, vaginal fluid, duodenal fluid, pancreatic fluid, bile and cerebrospinal fluid The free nucleic acid present in is called extracellular nucleic acid or cell-free nucleic acid.

  Although the mechanism by which free nucleic acids are released into body fluids is unknown, necrosis, apoptosis, active release, and the like are considered. As for the form of free DNA, the free DNA is not in a “bare” state, but in a state where it is complexed with other proteins, etc., or is bound to the cell surface via a protein that specifically binds to a nucleic acid. It is thought that it will be released inside. In addition, there are reports of the state of binding to the cell membrane of erythrocytes and leukocytes, the state of being included in apoptotic vesicles, and the state of forming a complex with p53 protein.

  As for the form of free mRNA, it has been reported that it is complexed with protein lipids and contained in apoptotic vesicles, and free mRNA is protected from RNase and is thought to exist in blood for a certain period of time. Yes.

  However, it has not been clarified at all whether miRNA exists free in body fluids such as serum and plasma. Furthermore, it has not been clarified at all whether it is possible to detect tissue damage or cell proliferative diseases by identifying the amount of microRNA that is free and present in body fluids such as serum and plasma. No attempt has been made to detect the presence of tissue damage or cell proliferative diseases by detecting an increase in the amount of microRNA in body fluids such as plasma.

Cheng, A.M. et al., 2005. Nucleic Acids Res. 33, 1290-1297. Tanno, B. at al., 2005. Cell Death Differ. 12, 213-223. Calin, G.A. et al., 2002. Proc. Natl. Acad. Sci. U. S. A. 99, 11755-11760. Iorio, M.V. et al., 2005. Cancer Res. 65, 7065-7070. Calin, G.A. et al., 2004. Proc. Natl. Acad. Sci. U. S. A. 101, 15524-15529. Calin, G.A. et al., 2004. Proc. Natl. Acad. Sci. U. S. A. 101, 2999-3004. Takamizawa, J. et al., 2004. Cancer Res. 64, 3753-3756. Johnson, S.M. et al., 2005.Cell 120, 635-647. Hayashita, Y. et al., 2005. Cancer Res. 65, 9628-9632. Lewis, B.P., 2003. Cell 115, 787-798. O'Donnell, K.A. et al., 2005. Nature 435, 839-843. Iorio, M.V. et al., 2005. Cancer Res. 65, 7065-7070. Ciafre, S.A. et al., 2005. Biochem. Biophys. Res. Commun. 334, 1351-1358 Chan, J.A. et al., 2005.Cancer Res. 65, 6029-6033. Eis, P.S. et al., 2005. Proc. Natl. Acad. Sci. U. S. A. 102, 3627-3632. He, L. et al., 2005. Nature 435, 828-833. He, H.L. et al. 2005. Proc. Natl. Acad. Sci. U. S. A. 102, 19075-19080. John, B. et al., 2004. PLOS Biol. 2, 1862-1879. Krek, A. et al., 2005. Nat. Genet. 37, 495-500. Rehmsmeier M. et al., RNA-a Publication of the RNA Society. 10, 1507-1517. Michael, M.Z. et al., 2003. Mol. Cancer Res. 1, 882-891. Voorhoeve, P.M. et al., 2006.Cell 124, 1169-1181. Murakami, Y. et al., 2006. Oncogene 25, 2537-2545. Special Table 2006-506469 JP 2005-192484 A US Patent Publication 20070054287

  The problem to be solved by the present invention is to provide a method for reliably detecting tissue injury or cell proliferative disease by a simpler method. Another problem to be solved by the present invention is to provide a method for detecting a plurality of tissue injuries or cell proliferative diseases in a subject and monitoring the state of the tissue injuries or cell proliferative diseases.

  As a result of diligent studies to achieve the above-mentioned problems, the present inventors have found that serum and plasma collected from patients with tissue injury or cell proliferation are rich in many types of free miRNAs. It was. Furthermore, by comparing the amount of free miRNA in the serum and plasma with that of healthy subjects, it was found that some specific free miRNAs were clearly increased compared to healthy subjects. In addition, the present inventors have found that the state of tissue injury or cell proliferative disease can be monitored by detecting the increase or decrease in the amount of free miRNA in the serum or plasma multiple times and comparing the results. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) Tissue injury or cell proliferative activity characterized by detecting free microRNA released in association with the disease in serum or plasma collected from a subject suspected of having tissue injury or cell proliferative disease Disease detection method.
(2) The amount of specific microRNA in serum or plasma collected from a subject suspected of having a tissue injury or cell proliferative disorder is detected by free microRNA. The amount of microRNA in serum or plasma collected from a healthy person (1) The method according to (1), characterized in that more is used as an index.
(3) Detection of tissue injury or cell proliferative disease can be performed multiple times with free microRNA released with the disease in serum or plasma collected from a subject suspected of having tissue injury or cell proliferative disease. The method according to (1) or (2), wherein the detection is performed across the body, and the tissue injury or cell proliferative disease state of the subject is monitored from the detection result.

(4) The tissue injury or cell proliferative disease is a brain tissue injury or cell proliferative disease, and free microRNA is released in association with the brain tissue injury or cell proliferative disease (1) to The method according to any one of (3).
(5) The method according to (4), wherein the free microRNA is at least one selected from miR-9 and miR-124a.
(6) The tissue injury or cell proliferative disease is a liver tissue injury or cell proliferative disease, and free microRNA is released in association with the liver tissue injury or cell proliferative disease (1) to The method according to any one of (3).

(7) The method according to (6), wherein the free microRNA is miR-122a.
(8) The detection of free microRNA is performed by any of the reverse transcription reaction real-time PCR method, the Northern blot method, the microarray method, and the bead array method. the method of.
(9) A detection kit for performing the method according to any one of (1) to (8), comprising at least free microRNA extraction means and specific microRNA detection means.

  According to the present invention, serum or plasma obtained by a low invasive collection method is used as a test material, and noninvasiveness is detected with respect to tissue injury or cell proliferative disease by detecting an increase in the amount of free microRNA in serum or plasma. A simple and reliable detection method is provided.

Hereinafter, the present invention will be described in detail.
The present invention comprises detecting tissue micro-organism or cell proliferation characterized by detecting free microRNA released in association with the disease in serum or plasma collected from a subject suspected of having tissue injuries or cell proliferative diseases. This is a method for detecting sex diseases.

  In the present invention, the subject suspected of having a tissue injury or a cellular disease is not limited as long as the disease can be detected and has a necessity, and humans, monkeys, rabbits, mice, Examples thereof include mammals such as rats. Again, serum or plasma can be obtained from blood collected from a subject by an appropriate method known per se.

  The mature miRNA is a non-coding RNA that is not translated into a protein, and means a miRNA that is a single-stranded RNA having a length of about 22 nucleotides. In the present invention, free miRNA includes pri-miRNA, pre-miRNA, miRNA: miRNA * duplex, and mature miRNA * that are precursors in the biosynthetic process in addition to mature miRNA.

  The type of disease to be detected is not particularly limited as long as it is a tissue damage or cell proliferative disease in the organ in the subject, but the esophagus, thyroid gland, uterus, lymph node, placenta, breast, pancreas, liver, brain And tissue damage or cell proliferative diseases in the thymus, heart, lung, spleen, testis, ovary, kidney, skeletal muscle, small intestine, colon, rectum, prostate, bladder, adrenal gland, stomach and the like. Among them, particularly preferable examples include tissue damage or cell proliferative diseases in the liver or brain.

  MiRNA released with these tissue injury or cell proliferative disease is detectable in the serum or plasma of patients with the tissue injury or cell proliferative disease compared to that of healthy subjects It means something that is too much.

  Specifically, miR-9, miR-124a and the like can be used for detection of brain tissue injury or cell proliferative disease (eg, brain tumor). One of these may be detected, or a plurality may be combined to make a comprehensive determination. Examples of detection of liver tissue injury or cell proliferative disease (eg, liver cancer) include miR-122a.

  As a method for extracting miRNA from serum or plasma collected from a subject, methods known to those skilled in the art can be used. Specifically, for example, AGPC (acid guanidine phenol chloroform) method, mirVana PARIS kit manufactured by Ambion, and the like can be used.

  The method for measuring the target miRNA contained in the miRNA extracted as described above is not particularly limited as long as it can detect the expression level of the miRNA detected in the present invention. Specific examples include Northern blotting, reverse transcription reaction real-time PCR, microarray, and bead array. According to the reverse transcription reaction real-time PCR method, using a primer for reverse transcription reaction complementary to miRNA to be detected in the present invention, and a primer for PCR, for example, via a fluorescently labeled probe such as a complementary TaqMan probe, The miRNA detected in the present invention can be detected quantitatively.

  About 500 types of human miRNA are currently known, and their nucleotide sequences are also known (miRBase: Sanger database registration). Those skilled in the art can design and synthesize base sequences of reverse transcription reaction primer and PCR primer complementary to miRNA to be detected in the present invention based on these known base sequences. Primers attached to can also be used. As a commercially available kit, for example, TaqMan MicroRNA Assays (Applied Biosystems) and the like are preferably used.

  According to the microarray method or the bead array method, a person skilled in the art can design a base sequence of an oligonucleotide that hybridizes complementary to the miRNA to be detected in the present invention based on a known base sequence, and a glass slide substrate. A material fixed on a plastic slide substrate or magnetic beads may be used. A commercially available kit can also be used for this. As a commercially available kit, for example, miRCURY Array (Exiqon), FlexmiR microRNA (Luminex) and the like are preferably used.

  The specific amount of miRNA thus measured is compared with the same amount of miRNA in the serum or plasma of a healthy person as a control. If the miRNA is measured more significantly than the value (control value) of a healthy person, it can be determined that the subject has a specific tissue injury or cell proliferative disease.

  The present invention also detects, in a time series, a plurality of free microRNAs released in association with a disease in serum or plasma collected from a subject suspected of having a tissue injury or a cell proliferative disease. Also included is a method of monitoring the tissue injury or cell proliferative disease state of the subject from the detection result.

  The monitoring of the tissue injury or cell proliferative disease state of a subject is, for example, cancer surgical treatment, chemotherapy, radiotherapy, immunotherapy, or arterial embolization, etc. It means checking the condition of the tissue after surgery and confirming the therapeutic effect.

  Furthermore, according to the present invention, there is provided a detection kit for carrying out the above-described method for detecting tissue injury or cell proliferative disease according to the present invention, comprising at least free microRNA extraction means and specific microRNA detection means.

  Examples of the free microRNA extraction means include a reagent for performing the AGPC (acid guanidine phenol chloroform) method. In addition, specific microRNA detection methods include primers specific to the microRNA used to detect specific microRNAs by reverse transcription reaction real-time PCR, and reagents for performing reverse transcription reaction real-time PCR (reversal). Photoenzyme, DNA polymerase, deoxynucleotide triphosphate, etc.).

  The following examples further illustrate the present invention. However, the technical scope of the present invention is not limited by these examples.

Example 1 Measurement of concentration level of free microRNA present in plasma [Materials and Methods]
Blood Collection and Plasma Separation Blood was collected from a healthy volunteer who had consented to participate in this study using 7 mL (Terumo) of Venoject II vacuum blood collection tube EDTA-2Na. The blood collection tube was centrifuged (2,400 × g, 20 ° C. for 10 minutes) to fractionate the plasma, and transferred to another 15 mL centrifuge tube. The 15 mL centrifuge tube into which the plasma was transferred was centrifuged (20,000 × g, 20 ° C. for 10 minutes), and the plasma fraction was collected so as not to aspirate the precipitate. The collected plasma was dispensed in 500 μL aliquots and stored frozen at −80 ° C. until Small RNA extraction.

Extraction of Small RNA in Plasma Using 500 μL of plasma as a starting material, Small RNA was extracted using mirVana PARIS Kit (Ambion) according to the Enrichment Procedure for Small RNAs described in the attached operation procedure. After extraction, sterilized distilled water was added to adjust the liquid volume to 125 μL. The small RNA extract was stored frozen at −80 ° C. until measurement of the amount of microRNA.

Measurement of microRNA amount
Using 5 μL of Small RNA extract as a material, TaqMan MicroRNA Assays (Applied Biosystems) was used, and 157 types of microRNA were measured by reverse transcription reaction real-time PCR according to the attached operation procedure manual. For the measurement, Sequence Detection System 7700 (Applied Biosystems) was used. A 240 ^-Ct value was calculated from the number of rising cycles (Ct) in the real-time PCR method, and a quantitative value was shown as an apparent copy number. When the Ct value was 36 or more, ^240-Ct value was calculated, but it was handled as below the limit of quantification.

〔result〕
The measurement results of 157 types of microRNA present in 2 μL of plasma of one healthy volunteer are shown in FIG. In the graph, the vertical axis is the apparent copy number (scale is logarithmic), the horizontal axis is the type of microRNA, and the graph is displayed in descending order of copy number.
Of the 157 types examined, 98 were microRNAs with 100 or more copies present in 2 μL of plasma. There were 31 types of microRNAs with more than 10,000 copies, of which there were 3 types exceeding 100,000.

  From the above findings, it became clear for the first time that many microRNAs are abundant in plasma. Plasma was cell-free and no nucleic acid was present, suggesting that microRNA was released from the tissue and was present in plasma.

Example 2 Analysis of microRNA expression level and expression pattern in human tissues [Materials and Methods]
Human tissue total RNA
Human tissue-derived total RNA (esophagus, thyroid, uterus, breast, liver, lung, testis, ovary, kidney, small intestine, colon, prostate, bladder, adrenal gland, stomach) commercially available from Ambion was used. For brain-derived total RNA, three types of brain-derived total RNA from different donors commercially available from Ambion, BioChain, and Stratagene were used. For pancreatic total RNA, two types of total RNA with different donors commercially available from Ambion and Stratagene were used. Regarding total RNA derived from blood cells, blood was collected from healthy volunteers using PAXgene Blood Tube (Becton-Dickinson), and then extracted using PAXgene Blood RNA Kit (QIAGEN). Finally, the total RNA concentration was adjusted to 2 ng / μL.

Measurement of microRNA amount Using 5 μL (total equivalent to 10 ng) of total RNA extract derived from human tissue, TaqMan MicroRNA Assays were used, and 157 types of microRNA were measured by reverse transcription reaction real-time PCR according to the attached operation procedure. . A Sequence Detection System 7700 was used for the measurement. The 240 ^-Ct value was calculated from the ^Ct value, and the quantitative value was displayed as the apparent copy number. When the Ct value was 36 or more, a ^240-Ct value was calculated, but it was handled as being below the limit of quantification.

Hierarchical cluster analysis was performed using analysis software (TIGR) and analysis using Euclidean distance and average linkage, using 157 types of microRNA measurement values (Ct values) in human tissues used for cluster analysis studies. .

〔result〕
2. MicroRNA expression level in human tissue FIG. 2 shows the measurement results of 157 types of microRNA per 10 ng of total RNA derived from human tissues. In the graph, the value of each tissue was plotted as the apparent copy number (scale is logarithmic) on the vertical axis. The horizontal axis is the type of microRNA, and the median expression copy number in each tissue is displayed in descending order.

The results of the cluster analysis based on microRNA expression in microRNA expression patterns Human each tissue in human tissues shown in Fig. The level of expression was expressed in gray shades, and the statistically similar between tissues were displayed as dendrograms.
In the cluster analysis, three types of donor brains and two types of pancreas were classified into the closest class. In addition, the small intestine and colon, which are embryologically similar, were classified into the closest class. The brain and blood cells classified as the farthest class in the cluster analysis showed significant differences in expression patterns. From these findings, it was found that the expression pattern of microRNA reflects the similarity in embryology and the specificity of each tissue.

Example 3 Search for tissue-specific microRNA [Method]
Comparison of microRNA expression level in each human tissue (acquired data in Example 2), “The expression level in the tissue with the highest expression level is 50,000 copies or more, and the expression ratio with the second tissue is 10 times or more. Was defined as a tissue-specific microRNA, and the corresponding microRNA was searched.

〔result〕
As a result, it was found that miR-9 and miR-124a are brain-specific, and miR-122a is liver-specific. These tissue-specific expression patterns are shown in FIGS. MiR-9 found as brain-specific microRNA has an expression level (apparent copy number) of brain 1 (71,220), brain 2 (158,048), brain 3 (124,864), and the uterine with the second highest expression level ( The expression ratio with 1,017) was 70 times. Similarly, miR-124a, which is brain-specific, is brain 1 (115,698), brain 2 (100,721), brain 3 (84,111), and its expression ratio to the kidney (175) with the second highest expression level is 661 times there were. MiR-122a found as liver-specific microRNA had an expression level of (102,837) and an expression ratio of 3,116 times that of the second highest expression level of thyroid (33).

Example 4 Measurement of miR-9, miR-124a, miR-122a levels in healthy human plasma [Method]
The amount of miR-9, miR-124a, miR-122a was examined using the amount of free microRNA present in plasma (data obtained in Example 1).
〔result〕
FIG. 7 shows the amounts of miR-9, miR-124a and miR-122a in 2 μL of plasma from one healthy volunteer. The apparent number of copies was 63 copies of miR-9 in 2 μL of healthy human plasma, the amount of miR-124a was below the limit of quantification (1 copy), and the amount of miR-122a was 385 copies. The amount of the three types of microRNAs in the plasma of healthy persons was at a low level.

Example 5 Measurement of miR-9 level in brain tumor patient serum [Materials and Methods]
Brain tumor patient serum Ten commercially available brain tumor patient serum (ProMedDx) were used.

Healthy person control Prepared by the same method as in Example 1.

Extraction of Small RNA in Serum Using 500 μL of serum as a starting material, Small RNA was extracted using mirVana PARIS Kit according to the Enrichment Procedure for Small RNAs described in the attached operation procedure. After extraction, sterilized distilled water was added to adjust the liquid volume to 125 μL. The small RNA extract was stored frozen at −80 ° C. until measurement of the amount of microRNA.

Measurement of microRNA amount
The amount of miR-9 was measured by reverse transcription reaction real-time PCR using TaqMan MicroRNA Assays (Applied Biosystems) using 5 μL of the Small RNA extract as a material and according to the attached operation manual. A Sequence Detection System 7700 was used for the measurement. The 240 ^-Ct value was calculated from the ^Ct value, and the quantitative value was displayed as the apparent copy number. When the Ct value was 36 or more, a ^240-Ct value was calculated, but it was handled as being below the limit of quantification.

〔result〕
FIG. 8 shows the measurement results of miR-9 per commercially available brain tumor patient serum and 2 μL of serum from one healthy volunteer. The amount of miR-9 in healthy human serum is 19 copies in terms of apparent copy number, whereas the amount of miR-9 in brain patient serum is B01 (10 copies), B02 (below the limit of quantification; 4 copies) , B03 (below the quantitation limit; 3 copies), B04 (206 copies), B05 (below the quantitation limit; 8 copies), B06 (below the quantitation limit; 4 copies), B07 (below the quantitation limit; 8 copies), B08 (quantitative) Below the limit; 6 copies), B09 (below the limit of quantification; 15 copies), and B10 (below the limit of quantification; 6 copies). Among 10 cases, the amount of miR-9 was significantly increased in 1 case of B04.

Example 6 Measurement of miR-122a level in liver cancer patient serum [Materials and Methods]
Liver Cancer Patient Serum Five commercially available liver cancer patient sera (ProMedDx) were used.

Healthy person control Prepared by the same method as in Example 1.

Extraction of Small RNA in Serum Using 500 μL of serum as a starting material, Small RNA was extracted using mirVana PARIS Kit according to the Enrichment Procedure for Small RNAs described in the attached operation procedure. After extraction, sterilized distilled water was added to adjust the liquid volume to 125 μL. The small RNA extract was stored frozen at −80 ° C. until measurement of the amount of microRNA.

Measurement of microRNA amount
The amount of miR-122a was measured by reverse transcription reaction-real-time PCR method using TaqMan MicroRNA Assays (Applied Biosystems) using 5 μL of Small RNA extract as a material, according to the attached operation manual. A Sequence Detection System 7700 was used for the measurement. The 240 ^-Ct value was calculated from the ^Ct value, and the quantitative value was displayed as the apparent copy number. When the Ct value was 36 or more, a ^240-Ct value was calculated, but it was handled as being below the limit of quantification.

〔result〕
The measurement results of miR-122a per commercially available liver cancer patient serum and 2 μL of serum from one healthy volunteer are shown in FIG. The amount of miR-122a in healthy human serum is 185 copies as an apparent copy number, whereas the amount of miR-122a in liver cancer patient serum is L01 (3,558 copies), L02 (175 copies), L03 (3,835 copies), L04 (521 copies), and L05 (56 copies), and the amount of miR-122a was significantly increased in 2 cases of L01 and L03.

Example 7 Measurement of the amount of miR-122a in patient serum and comparison with ALT (alanine aminotransferase) and CRP (C-reactive protein) in an example of arterial embolization for a liver cancer patient (first example) [ Material and method]
Case
Hepatoma associated with chronic hepatitis B and C
Positive for both hepatitis B virus and hepatitis C virus

Blood samples were collected over time and day before and after performing serum sampling arterial embolization, and serum was separated immediately after coagulation reaction. The collected serum was stored frozen at −80 ° C. until extraction of Small RNA.

Extraction of Small RNA in Serum Using 500 μL of serum as a starting material, Small RNA was extracted using mirVana PARIS Kit according to the attached operation procedure Enrichment Procedure for Small RNAs. After extraction, sterilized distilled water was added to adjust the liquid volume to 125 μL. The small RNA extract was stored frozen at −80 ° C. until measurement of the amount of microRNA.

Measurement of microRNA amount
The amount of miR-122a was measured by reverse transcription reaction-real-time PCR method using TaqMan MicroRNA Assays (Applied Biosystems) using 5 μL of Small RNA extract as a material, according to the attached operation manual. A Sequence Detection System 7700 was used for the measurement. The 240 ^-Ct value was calculated from the ^Ct value, and the quantitative value was displayed as the apparent copy number. When the Ct value was 36 or more, a ^240-Ct value was calculated, but it was handled as being below the limit of quantification.

ALT and CRP amount
ALT was measured by the UV method (JSCC compliant method), and the unit was IU / I / 37 ° C. CRP was measured by latex agglutination turbidimetry, and the unit was expressed in mg / dL.

〔result〕
FIG. 10 shows the measurement results of miR-122a per 2 μL of serum in the serum of one healthy volunteer and in the case of arterial embolization for a liver cancer patient (first example). The amount of miR-122a in the serum of healthy volunteers is 233 as an apparent copy number, whereas the amount of miR-122a in liver cancer patient serum is just before (3,841 copies), immediately after (3,662 copies), 3 hours later (5,692 copies), 7 hours later (3,278 copies), 10 hours later (9,953 copies), 1 day later (28,143 copies), 2 days later (3,515 copies), 4 days later (1,450 copies), 5 days later (2,908 copies) ), 6 days later (1,573 copies), and 7 days later (2,660 copies).

  FIG. 11 shows the measurement results of the serum ALT level in an arterial embolization example (first example) for a liver cancer patient. The reference value of ALT level (in normal human serum) is 5 to 45 IU / U / 37 ° C, whereas the ALT level in liver cancer patient serum is just before (102.0), immediately after (100.0), and 3 hours later (95.0), 7 hours later (97.7), 10 hours later (95.9), 1 day later (150.0), 2 days later (173.7), 4 days later (105.4), 5 days later (105.9), 6 days later (102.6), 7 days later (98.6) (unit omitted).

  FIG. 12 shows the measurement results of serum CRP levels in the case of arterial embolization for liver cancer patients (first example). The standard value of CRP amount (in normal human serum) is 0.30 mg / dL or less, whereas the CRP amount in liver cancer patient serum is just before (2.140), just after (2.070), and 3 hours later (1.990) 7 hours (2.343), 10 hours (2.606), 1 day (3.604), 2 days (8.975), 4 days (13.596), 5 days (14.538), 6 days (13.024), 7 days (12.798) (Unit omitted).

  From this result, it was found that the post-operative progress can be monitored by the amount of miRNA in serum in the case of arterial embolization for liver cancer patients (1st case). It was found to be equivalent to the image analysis of the tissue.

Example 8 Measurement of miR-122a in patient serum and comparison with ALT (alanine aminotransferase) and CRP (C-reactive protein) in an example of arterial embolization for a liver cancer patient (second example) [ Material and method]
Case
Hepatoma associated with chronic hepatitis B and C
Positive for both hepatitis B virus and hepatitis C virus

Serum collection The procedure was the same as in Example 7.

Extraction of Small RNA in Serum The same method as in Example 7 was performed.
Measurement of microRNA amount The same method as in Example 7 was used.
Measurement of ALT and CRP amounts The same method as in Example 7 was used.

〔result〕
FIG. 13 shows the measurement results of miR-122a per 2 μL of serum obtained from the serum of one healthy volunteer and the case of arterial embolization performed on a liver cancer patient (second example). The amount of miR-122a in the serum of healthy volunteers was 313 copies in apparent copy number, whereas the amount of miR-122a in the serum of patients with liver cancer was 1 day before the operation (160 copies), 2 hours later (1,060 Copy), 8 hours later (1,010 copies), 17 hours later (43,238 copies), 2 days later (2,402 copies), 3 days later (744 copies), 5 days later (63 copies), 8 days later (82 copies).

  FIG. 14 shows the measurement results of the serum ALT level in an arterial embolization example (second example) for a liver cancer patient. The standard value of ALT level (in normal human serum) is 5 to 45 IU / U / 37 ° C, whereas the ALT level in liver cancer patient serum is the day before (97), 2 hours (91), After 8 hours (103), 17 hours (323), 2 days (947), 3 days (668), 5 days (271), and 8 days (125) (unit omitted).

  FIG. 15 shows the measurement results of serum CRP levels in the case of arterial embolization for liver cancer patients (second example). The standard value of CRP level (in normal human serum) is 0.30mg / dL or less, whereas the CRP level in liver cancer patient serum is the day before the administration (0.4), 2 hours later (0.37), 8 hours later (0.76), 17 hours later (1.59), 2 days later (9.49), 3 days later (17.53), 5 days later (15.45), 8 days later (12.69) (unit omitted).

  The results of the example of arterial embolization (second example) were reproduced as the first example. This confirms that post-operative progress can be monitored using the amount of serum microRNA as an index.

Example 9 Measurement of microRNA amount showing non-specific expression of liver in patient's serum in case of arterial embolization for liver cancer patient (second case) Because cell necrosis occurs when arterial embolization is performed It is possible that various microRNAs contained in the cells are released and detected in serum. In addition, various organs are secondarily stimulated by arterial embolization, and inflammation may occur in some areas. Therefore, the amounts of 16 types of microRNA per 2 μL of serum were measured using ID01 and ID04 shown in Table 4 of Example 8 above. In addition, Table 5 shows the types of tissues in which each of the 16 types of microRNAs is expressed by 50,000 copies or more. In particular, regarding expression in liver tissue, an expression level of 50,000 copies or more is indicated by +, and an expression level of 50,000 copies or less is indicated by-.

〔result〕
FIG. 16 shows the measurement results of 16 types of microRNA in the patient serum on the day before and 17 hours after the execution of arterial embolization for liver cancer patients (second example). There are five types of miRNA-99a, miR-135a, miR-204, miR-214, and miR-296 that have a significant increase in microRNA in serum collected 17 hours after administration, such as miR-122a. 11 types did not increase. This indicates that the amount of serum microRNA is not increased by any arterial embolization. From this, it is considered that the mechanism by which microRNA is released by necrosis of cells, or the mechanism of existence after the release is different for each type of microRNA.

In addition, five types of miR-99a, miR-135a, miR-204, miR-214, and miR-296 increased markedly 17 hours after administration, but were not expressed specifically in the liver, It is difficult to determine if it was caused by tissue injury in the liver. Although there are many unclear areas where miR-99a, miR-135a, miR-204, miR-214, and miR-296 have increased, some organs are secondarily stimulated by arterial embolization, It is also speculated that this is due to inflammation.
From the results of Examples 7, 8, and 9, it was shown that tissue damage in the liver can be suitably detected by miR-122a showing liver-specific expression.

The measurement results of 157 types of microRNA present in 2 μL of plasma from one healthy volunteer are shown. The measurement results of 157 types of microRNA per 10 ng of total RNA derived from human tissues are shown. The result of the cluster analysis based on the microRNA expression level in each human tissue is shown. The result of the expression pattern according to the tissue of miR-9 is shown. The result of the expression pattern according to the tissue of miR-124a is shown. The result of the expression pattern according to tissue of miR-122a is shown. The result of the concentration level of miR-9, miR-124a, miR-122a in 2 microliters plasma of 1 healthy volunteer is shown. The measurement result of miR-9 per 2 microliters serum of commercially available brain tumor patient serum and 1 healthy volunteer is shown. The measurement result of miR-122a per commercially available liver cancer patient serum and 2 μL of serum of a healthy volunteer is shown. The measurement result of miR-122a per 2 μL of serum in an arterial embolization example (first example) for a liver cancer patient is shown. The measurement result of serum ALT in an arterial embolization operation example (1st example) to a liver cancer patient is shown. The measurement result of serum CRP in an arterial embolization operation example (1st example) to a liver cancer patient is shown. The measurement result of miR-122a per 2 μL of serum in an arterial embolization example (second example) for a liver cancer patient is shown. The measurement result of serum ALT in an arterial embolization operation example (2nd example) to a liver cancer patient is shown. The measurement result of serum CRP in an arterial embolization operation example (second example) for a liver cancer patient is shown. The measurement result of 16 types of microRNA per 2 μL of serum in an example of performing arterial embolization for a liver cancer patient (second example) is shown.

Claims (5)

Detecting free miR-9 or miR-124a released with the disease in serum or plasma collected from a subject suspected of having brain tissue injury or brain cell proliferative disease A method for assisting in the detection of brain tissue injury or brain cell proliferative disease. Detection of free miR-9 or miR-124a indicates that the amount of miR-9 or miR-124a in serum or plasma collected from subjects suspected of having brain tissue injury or brain cell proliferative disease 2. The method according to claim 1, wherein the amount is greater than the amount of miR-9 or miR-124a in the collected serum or plasma. Free the detection of tissue injury or cell proliferative disorders of the brain brain, the serum or plasma tissue is injured or collected from a subject suspected of cell proliferative disorders of the brain of the brain, it is released in association with the disease 3. The miR-9 or miR-124a is detected a plurality of times, and the state of tissue damage or brain cell proliferative disease in the subject is monitored from the detection result. The method described in 1. The detection of free miR-9 or miR-124a is performed by any of the reverse transcription reaction real-time PCR method, the Northern blot method, the microarray method, or the bead array method. The method described in 1. The detection kit for performing the method according to any one of claims 1 to 4, comprising at least free microRNA extraction means and detection means for miR-9 or miR-124a.