WO2013038737A1 - 膀胱癌細胞の検出方法、膀胱癌細胞の検出方法に用いるプライマー及び膀胱癌マーカ - Google Patents
膀胱癌細胞の検出方法、膀胱癌細胞の検出方法に用いるプライマー及び膀胱癌マーカ Download PDFInfo
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Definitions
- the present invention relates to a method for detecting cancer of a subject from a sample obtained from the subject, particularly a method for detecting bladder cancer cells, a primer used in the method, and a substance that serves as a bladder cancer marker.
- urine cytology is most widely used for detection of bladder cancer.
- bladder cells that have been detached from the bladder and dropped into the urine are collected from the urine, and their shape is observed with a microscope to detect cancer cells.
- microRNA which is a short RNA that performs gene regulation on tissues
- microRNA may be used interchangeably with “miRNA”, “miR”, and “hsa-miRNA”.
- Research has been conducted on a method for detecting cancer tissue by detecting the expression level of.
- Patent Document 1 discloses a cancer detection method for detecting canceration of a specimen using a decrease in gene expression of miRNA containing miR-9 and miR-137 as an index in the specimen, and cancer suppression by expressing them. Methods and cancer inhibitors are disclosed.
- Patent Document 2 discloses a method of measuring the level of a miRNA gene product in a test sample from a subject, and detecting whether or not there is a risk of developing breast cancer.
- Patent Document 3 discloses a method for increasing the efficacy of an anti-cancer treatment of a BCL2-related cancer, comprising administering at least one anti-cancer treatment to a subject and at least one miR gene product in the BCL2 gene transcript.
- methods for diagnosis and therapy of BCL2-related cancers by administering a miR gene product comprising a nucleotide sequence that is complementary to the nucleotide sequence of.
- the bladder has been described as a cancer tissue that may have reduced miRNA expression.
- miR-137 and miR-9 are exemplified as miRNA that can be used for diagnosis and therapy of BCL2-related cancer.
- Patent Document 4 discloses a method for determining gynecological cancer using microRNA containing miR-137 as a biomarker for gynecological cancer. As a method for determining gynecological cancer, a method for directly detecting the expression level of miRNA is described.
- Patent Document 5 discloses a method for predicting survival after treatment of a cancer patient, wherein the expression level of a microRNA containing hsa-miR137 in a cancer patient who has been treated is detected, and based on the expression level of the microRNA.
- a method for predicting survival after treatment of a cancer patient comprising calculating a patient's risk score and determining a prospect of post-treatment survival based on the value of the risk score.
- cancers to be predicted include lung cancer, leukemia, breast cancer, pancreatic cancer, adenocarcinoma, squamous cell carcinoma, colon cancer or hepatocellular carcinoma.
- Patent Document 6 discloses a method of measuring the level of at least one miR gene product and determining whether the subject has solid cancer or is at risk of developing it.
- Non-Patent Document 1 describes the expression of miR-137 in colorectal cancer.
- Non-Patent Document 2 describes the expression of miRNA containing miR-137 in oral cancer.
- Non-Patent Document 3 describes the expression of miR-137 in colorectal cancer.
- urine cytology widely used for detection of bladder cancer has a problem in the sensitivity of detection of bladder cancer.
- bladder cancer cells can be specifically detected if cells that can be clearly recognized as tumor cells can be detected from the shape, but low malignant tumors that cannot be detected from the shape It cannot be detected for cells. For this reason, it has been difficult to detect tumors with low malignancy.
- Patent Document 1 describes a method for detecting cancer by detecting miRNA, a method for suppressing cancer by expressing them, and a cancer suppressant.
- it is disclosed for oral squamous cell carcinoma. The bladder cancer tissue has not been verified.
- Patent Document 2 describes a method and a composition for detecting breast cancer or the risk of developing breast cancer, but the tissue of bladder cancer has not been verified.
- the bladder is included in one of many cancer tissues exemplified that the expression of miRNA may generally be reduced, but a gene whose expression is decreased in bladder cancer is specifically described. However, there is no evidence that it can be applied to bladder cancer.
- miR-137 and miR-9 are mentioned as miRNA genes to be administered, the expression of BCL2 gene is theoretically reduced under the hypothesis that reducing BCL2 gene expression leads to cancer treatment. It is only exemplified as one of many possible miRNA candidates, and it has not been verified whether these actually reduce BCL2 gene expression. In addition, this method is limited to the possibility of treating cancer associated with overexpression of the BCL2 gene and / or gene product, and does not verify whether administration of miR-137 and miR-9 actually leads to cancer treatment. Not done.
- Patent Document 4 describes a determination method and determination kit for so-called gynecological cancer, but does not describe bladder cancer. Furthermore, it has been confirmed that miR-137 expression is increased in endometrial cancer.
- Patent Document 5 discloses a method for predicting survival after treatment of a cancer patient, but does not describe a method for detecting whether or not the patient is a cancer patient. In addition, bladder cancer is not described for cancer.
- miR-137 is not changed in 6 types of cancer (milk, large intestine, lung, pancreas, prostate, stomach). miR-137 is listed as a gene that is not selected as a gene to be measured (paragraph [0037]), and is particularly listed as one that is not selected for each of the six types of cancer described above.
- Non-Patent Documents 1 to 3 describe the relationship between the expression of miRNA including miR-137 and the onset of colorectal cancer, oral cancer, and invasion of colorectal cancer, but bladder cancer has not been verified. .
- an object of the present invention is to provide a method for detecting a bladder cancer cell having high detection sensitivity and capable of detecting a bladder cancer tissue having high specificity and low malignancy, a primer used for this method, and a bladder cancer marker. It is to provide.
- the method for detecting bladder cancer cells of the present invention comprises detecting the expression level of a bladder cancer marker comprising one or more miRNAs selected from miR-124, miR-9 and miR-137 from a subject sample collected from the subject. including.
- bladder cancer tissue miRNAs that are specifically expressed differently than normal tissues are detected as bladder cancer markers.
- the presence or amount of miRNA in the tissue can be quantitatively detected quickly and accurately by means such as real-time reverse-transcription PCR (real-time reverse transcription PCR, real-time RT-PCR, quantitative RT-PCR). Therefore, bladder cancer cells can be specifically detected by detecting the expression level of one or more miRNAs selected from miR-124, miR-9 and miR-137, and low-grade bladder cancer tissue Even it can be detected.
- the detection of the expression level of the bladder cancer marker is preferably performed by detecting a decrease in the expression level of the bladder cancer marker by detecting methylation (methylated cytosine) of the bladder cancer marker gene.
- methylation methylated cytosine
- the expression of miR-124, miR-9, miR-137, etc. is suppressed by methylation on the genome gene to which each is encoded, so miR-124 is miR-124-2
- the detection of methylation in the miR-124-3 gene, miR-9-3 gene for miR-9, miR-137 gene for miR137, and miR-137 gene for miR137 decreased the expression level of these miRNAs in bladder cancer cells and bladder cancer Cells can be detected.
- cancer tissue When cancer tissue is included in normal tissue that expresses a large amount of the target miRNA, expression in the normal tissue is detected by means of directly detecting the expression level, so the target miRNA in the cancer tissue is detected. In some cases, it is difficult to detect the presence of phenoline, but by detecting a decrease in the expression level as a positive signal of methylation, its presence can be clearly and reliably detected.
- the method for detecting bladder cancer cells comprises the level of methylation of one or more genes selected from the miR-137 gene, miR-124-2 gene, miR-124-3 gene, and miR-9-3 gene. Detecting from a subject sample taken from the subject. In this detection method, it is preferable to compare the methylation level with a threshold value. The above-mentioned gene in the tissue to be detected with respect to this threshold value, using the methylation level of the above-mentioned gene in another tissue or other site tissue that has been found not to be cancerous tissue as a threshold value By comparing the levels of methylation, bladder cancer cells can be detected. When the level of methylation in the subject sample is higher than the threshold value, it can be determined that the subject sample is a cancer tissue.
- the detection of methylation is preferably performed by the bisulfite pyrosequencing method. According to this method, since the target miRNA can be detected precisely and quantitatively, the tissue of bladder cancer cells can be reliably detected.
- the bladder cancer marker includes at least miR-137. Since miR-137 has a markedly different expression level in bladder cancer cells compared to normal tissues, miR-137 can detect bladder cancer cells with the highest certainty.
- a cancer tissue can be detected by comparing the expression level of miRNA of another tissue that is known not to be a cancer tissue or a tissue of another site as a threshold. When the expression level of the bladder cancer marker in the subject sample is lower than the threshold value, it can be determined that the subject sample is a cancer tissue.
- the threshold value is preferably the expression level of the bladder cancer marker in a control sample collected from a normal tissue. Since expression of miR-124, miR-9, miR-137 and the like is suppressed in bladder cancer cells, bladder cancer cells can be detected by a decrease in the expression level compared to normal tissues.
- the threshold value is preferably the expression level of the bladder cancer marker in a control sample collected at different times from the subject or collected from different tissues of the subject.
- bladder cancer cells can be detected by comparison with when the cancer does not develop or when the cancer is excised. Since temporal data on the expression of bladder cancer cells can be obtained, the occurrence of cancer and the outcome of treatment can be determined.
- bladder cancer cells can be detected by comparison with a tissue that has not developed cancer. By collecting a sample for each site, the site of the tissue in which cancer has occurred can be detected.
- the subject sample is a urine sample.
- a urine sample can be collected frequently, safely, simply and quickly without invasiveness regardless of surgery.
- the amount of miRNA contained is smaller than that in the blood sample or excised sample.
- the bisulfite pyrosequencing method is used. Since methylation is detected by, for example, detection with sufficiently high specificity and sensitivity is possible with a urine sample.
- the primer used for the bladder cancer cell detection method is represented by SEQ ID NO: 1 as the primer sequence used for the amplification of the miR-137 gene by the bisulfite pyrosequencing method.
- the forward primer (GGGTTTTAGYGAGTAGTAAGAGTTTTG) and the reverse primer (CCCCCTACCRCTCATAACTACTCCTCTC) represented by SEQ ID NO: 2 are preferable, and the sequence of the primer used for the sequencing reaction is preferably GGATTTTTGGGTGGAATAAT represented by SEQ ID NO: 3.
- the primer used for the bladder cancer cell detection method has the sequence of the primer used for amplification of the miR-124-2 gene by the bisulfite pyrosequencing method as SEQ ID NO: 4 It is preferable that the forward primer (GTTGGGATTGTTATAGAGAGATTATTTG) and the reverse primer (ACTACRAAAATCCAAAAAAAAATACATAC) shown in SEQ ID NO: 5 are preferable, and that the primer sequence used in the sequencing reaction is YGTTTTTTTGTTTGTTTTTGTTT, which is preferably shown in SEQ ID NO: 6.
- the primer used for the detection method of the bladder cancer cell is the sequence of the primer used for amplification of the miR-124-3 gene by the bisulfite pyrosequencing method. It is preferable that it is a forward primer (AAAAGAGAGAYGAGTTTTTATTTTTTGAGTAT) shown by (5) and a reverse primer (TCCTCCRCCAACTACTCTCCCCTA) shown by SEQ ID NO: 8, and it is preferable that the sequence of the primer used for the sequence reaction is GAGATTYGTTTTTTATA shown by SEQ ID NO: 9.
- the primer used for the detection method of the bladder cancer cell is the sequence of the primer used for amplification of the miR-9-3 gene by the bisulfite pyrosequencing method.
- the reverse primer (TCTCRAAAACTCACRTAAAACACCC) represented by SEQ ID NO: 11 and the primer sequence used in the sequence reaction are preferably TGGATTGAYGTTTTTTT represented by SEQ ID NO: 12.
- the forward primer represented by (GATTTTGAATGGGAGTTTGTATTGT) and the reverse primer represented by SEQ ID NO: 11 are preferred.
- MSP method methylation-specific PCR method
- the primer used in the bladder cancer cell detection method is a forward primer in which the primer sequence used for detection of miR-137 gene methylation is represented by SEQ ID NO: 13 as a methylation allele-specific primer.
- SEQ ID NO: 13 a methylation allele-specific primer.
- GTAGCGGGTAGTAGCGGGTAGCGGT and reverse primer (GCTAATACTCTCCTCGAACTACCGCG) shown in SEQ ID NO: 14, forward primer (TGGTTAGTGGGTAGTAGTGTAGTGGT) and reverse primer shown in SEQ ID NO: 16 Is preferred.
- the primer used for the bladder cancer cell detection method has the sequence of the primer used for detection of the methylation of the miR-124-2 gene represented by SEQ ID NO: 17 as a methylation allele-specific primer.
- Forward primer AGGGGGCGTATTTTGGGGTTTTTGC
- reverse primer shown in SEQ ID NO: 18
- forward primer shown in SEQ ID NO: 19 as an unmethylated allele specific primer TTTAGGGGTGTTTGTCATCAT reverse
- the primer used for the bladder cancer cell detection method has the sequence of the primer used for detection of the methylation of the miR-124-3 gene represented by SEQ ID NO: 21 as a methylation allele-specific primer.
- Forward primer (GTTTTAGGTGATATCGGTCTCGTACTC) and reverse primer shown in SEQ ID NO: 22 (TCTCACGAAATCCACGCTCAAAACG), forward primer shown in SEQ ID NO: 23 as an unmethylated allele specific primer
- GTTTTAGGTGATATCGGTCTCGTACTC forward primer shown in SEQ ID NO: 22
- SEQ ID NO: 23 as an unmethylated allele specific primer
- the primer used for the bladder cancer cell detection method has the sequence of the primer used for detection of the methylation of the miR-9-3 gene represented by SEQ ID NO: 25 as a methylation allele-specific primer.
- Forward primer GATGGACGTTATTTTTTCGCGGGGC
- reverse primer CGAAACTCACGTAAAACACCCCGCG
- forward primer TTGATTGATCGTACTGCTCCTGCTGATCGATCGATCGATCGATCGACTGCG
- the bladder cancer marker of the present invention contains one or more miRNAs selected from miR-124, miR-9, and miR-137.
- miRNAs that specifically differ in expression level from the normal tissue are detected as bladder cancer markers in the bladder cancer tissue. Since the expression level of miRNA in the tissue and the methylation level of the gene can be quantitatively detected by the means such as real-time PCR and bisulfite pyrosequencing method, the presence or absence and the amount thereof can be quantitatively detected.
- the expression level of one or more miRNAs selected from miR-124, miR-9 and miR-137 and the methylation level of the gene bladder cancer cells can be specifically detected, and the grade of malignancy Even bladder cancer tissues with low levels can be detected.
- the bladder cancer marker of the present invention is used in a method for detecting bladder cancer cells, which includes detecting the expression level of a bladder cancer marker from a subject sample collected from the subject.
- the detection of the expression level of the bladder cancer marker is a method for detecting bladder cancer cells, comprising detecting a decrease in the expression level of the bladder cancer marker by detecting methylation of a genomic gene encoding the bladder cancer marker. Use.
- detection is performed from a subject sample collected from a subject having a depth of penetration pTa or a variant G1 / G2.
- the nucleic acid molecule of the present invention comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.
- SEQ ID NO: 12 SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 23 It has the nucleotide sequence of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. These molecules can be used in a method for detecting bladder cancer cells.
- miRNA having a specific expression level different from that in a normal tissue is detected as a bladder cancer marker in a bladder cancer tissue.
- the expression level of miRNA in the tissue and the methylation level of the gene can be rapidly and accurately quantitatively detected by means such as real-time PCR or bisulfite pyrosequencing, so miR-124, miR-9
- bladder cancer cells can be specifically detected by detecting the expression level of one or more miRNAs selected from miR-137 and the methylation level of the gene, and low-grade bladder cancer tissue. Can be detected.
- FIG. 3 is a graph showing the results of analyzing the methylation status of miR-9-1, miR-9-3, miR-10b, and miR-34b among the miRNAs of FIG. 1 by the bisulfite pyrosequencing method.
- 2 is a graph showing the results of analyzing the methylation status of miR-124-1, miR-124-2, miR-124-3, and miR-137 of the miRNA of FIG. 1 by the bisulfite pyrosequencing method.
- 2 is a graph showing the results of analyzing the methylation status of miR-200b, miR-203, miR-409, and miR-675 of the miRNAs of FIG. 1 by the bisulfite pyrosequencing method.
- 2 is a graph showing the results of analysis of miR-137 miRNA expression level (a) and miR-137 gene methylation (b) for each bladder cancer cell line. It is a graph which shows the result of having analyzed the expression of miR-137 in a cancer part tissue (T) and the tissue (DN) considered to be normal.
- the graph which shows the result of analyzing methylation analysis by the bisulfite pyrosequencing method and its ROC curve of NMIBC (non-invasive, superficial), MIBC (invasive), and both cases for the miR-9-3 gene It is. It is a graph which shows the result of having analyzed the methylation (a) of the miR-137 gene of a urine sample, and its ROC curve (b) before and after cancer tissue removal surgery. It is a graph which shows the result of having analyzed the methylation of the miR-137 gene in the urine sample before and after the operation of cancer tissue extraction surgery (a), and before and after (b) non-cancer patients.
- Bladder cancer in the present embodiment refers to cancer that occurs in the bladder, such as urothelial cancer (transitional cell carcinoma), squamous cell carcinoma or adenocarcinoma.
- one or more miRNA selected from one or more miR-124, miR-9, and miR-137 is used as a bladder cancer marker.
- the miRNA is a short single-stranded RNA of 10 base pairs or more having the sequence described below, and has a 5'-phosphate, 3'-OH structure, and the 3 'end may protrude by 2 bases. In the present embodiment, it refers to either artificially chemically synthesized or synthesized in a living organism.
- miR-137 (5′-uuauugcuuaagaauacccguag-3 ′) (MIMAT000029) (SEQ ID NO: 29) miR-124 (5′-uaagggacggcggugaaugcc-3 ′) (MIMAT000022) (SEQ ID NO: 30) miR-9 (5′-ucuuugguuuaucucucuugauga-3 ′) (MIMAT0000441) (SEQ ID NO: 31) miR-137 gene (5'-ggtcctctactactctctctggtgacgggtatttttggggtgataatacggattacgttgttattgcttagagaatacgcgttagcgaggagacgaccagc
- miR-137 among bladder cancers can be a clear indicator of bladder cancer cells in bladder cancer.
- a plurality of the above-described miRNAs may be detected and the results may be compared and verified. By comparing and verifying a plurality of miRNAs, detection with higher specificity and sensitivity becomes possible.
- the expression level of the miRNA in the subject sample collected from the subject who is the target for detecting bladder cancer cells is detected.
- the subject sample can be obtained by collecting a bladder cancer tissue or a biological sample containing the tissue, and can be detected from a urine sample, a blood sample, a sample excised and collected by an endoscope, or the like. In this embodiment, a sample excised by an endoscope or a sample collected from a total cystectomy specimen is used.
- the target expression level of the bladder cancer marker is detected by comparing the expression level of the bladder cancer marker in a subject sample collected from a subject who is a target for detecting bladder cancer cells with a threshold value.
- the tissue of the subject sample can be determined as bladder cancer.
- the threshold is set for a control sample collected from normal tissue, and the expression level is compared.
- the control sample may be a normal tissue of a normal subject different from a subject suspected of having bladder cancer, or the same subject taken when the subject suspected of having bladder cancer is healthy or from a healthy tissue It may be.
- a normal tissue that is sufficiently separated from a tissue that is likely to have cancer in the same subject is used as a control sample.
- the expression level of the target miRNA is decreased. Therefore, when the frequency of methylation of the genomic sequence encoding the target miRNA in the subject sample is high, the presence of bladder cancer cells can be determined. According to the detection of methylation, detection can be performed with high accuracy regardless of the background of the miRNA expression level in the tissue.
- methylation detection means can be used for the detection of methylation, including bisulfite sequencing, methylation-specific PCR (MSP), quantitative MSP, COBRA (cobra), and bisulfite pyro.
- a sequence method or the like can be used. Any of these methods may be used alone, or two or more of these methods may be used in combination.
- the bisulfite pyrosequencing method is preferably used in that it can accurately and quantitatively detect the target miRNA, and the methylation-specific PCR method (MSP method) is quick and simple.
- MSP method methylation-specific PCR method
- it is preferably used because methylation can be detected from a small amount of DNA sample.
- MSP method is Methods Mol Med. 2005; 113: 279-91 and Taku Suzuki, Minoru Toyoda, Kozo Imai: Bisulfite PCR Method New Genetic Engineering Handbook Revision 4th Edition (Masamatsu Muramatsu and Masaru Yamamoto), Yodosha, pp 99-106, 2003, Bisulfite Sequence The method is described in Methods. 2002 Jun; 27 (2): 101-7 and Taku Suzuki, Minoru Toyoda, Kozo Imai: bisulfite PCR method. New Genetic Engineering Handbook Revised 4th Edition (Masamatsu Muramatsu, Masaru Yamamoto), Yodosha, pp99-106, 2003, Bisulfite pyrosequencing method is described in Nat Protoc.
- the cobra method is Methods Mol Biol. 2002; 200: 71-85 and Taku Suzuki, Minoru Toyota, Kozo Imai: bisulfite PCR method.
- New 4th edition of Genetic Engineering Handbook (Masamatsu Muramatsu, Masaru Yamamoto), Yodosha, pp99-106, 2003, Methylite method is Methods. 2001 Dec; 25 (4): 456-62, etc., or these can be used with appropriate modifications.
- the bisulfite pyrosequencing method is generally sometimes abbreviated as the pyrosequencing method, and the pyrosequencing method in this case and the bisulfite pyrosequencing method in this specification indicate the same method.
- the gene expression level can also be detected by directly detecting the miRNA expression level.
- miRNA detection means such as real-time RT-PCR method or Northern blot method can be appropriately used. Any of these methods may be used alone, or two or more of these methods may be used in combination. Among the above, detection by the real-time RT-PCR method is preferable from the viewpoint of simplicity and sensitivity.
- Bladder cancer cells can be detected using either the direct detection of the target miRNA expression level or the detection of methylation of the genomic sequence encoding the target miRNA, or a combination of both. You can also.
- FIG. 27, FIG. 28, and FIG. 29 show the primers used in the above and the sequences that are the basis thereof.
- the figure shows the sequences upstream of the miR-137 gene, miR-124-2 gene, miR-124-3 gene, miR-9-3 gene (SEQ ID NOs: 36, 38, 40, 42), after bisulfite conversion, respectively.
- Sequence (underlined indicates the sequence used as a primer) (SEQ ID NO: 37, 39, 41, 43), forward primer and reverse primer used for PCR in simultaneous amplification of methylated allele and unmethylated allele, and An example of a combination of a primer used for a sequence reaction after PCT amplification, a methylated allele-specific forward primer and a reverse primer, and an unmethylated allele-specific forward primer and a reverse primer used for detection by the MSP method is shown.
- the forward primer is GGGTTTAGYGAGTAGTAGATAGTTTG
- the reverse primer is CCCCCTACCRCTATAATCTCTCTCTC.
- GGTATTTTTGGGTGGAATAAT is used as a primer used for the sequencing reaction.
- a primer used for PCR amplification by the bisulfite pyrosequencing method GTTGGAGTGTATAGAAGGATTATTTG is used as a forward primer, and ACTACRAAAATCCAAAAAAAAATACATAC is used as a reverse primer.
- YGTTTTTATTGTTTTAGTTTT is used as a primer for the sequencing reaction.
- AAAAGAGAYGAGGTTTTATTTTTTGAGTAT is used as a forward primer and TCCTCCRCAACTACTTCTCCCTA is used as a reverse primer as primers for PCR amplification by the bisulfite pyrosequencing method.
- GAGATTYGTTTTTTAAT is used as a primer used for the sequence reaction.
- GATTTTAAATTGGAGTTGTGTATTGTGT is used as a forward primer and TCCCRAAACTCACRTAAAACACCC is used as a reverse primer.
- TTGGATTGAYGTTTATTT is used as a primer for the sequencing reaction.
- the primer sequences used for detection of miR-137 are as follows: GTAGCGGGTAGTAGCGGGTAGCGGT as a forward primer as a methylated allele specific primer, GCTAATACTCTCCTCGATACCGCGCT as a reverse primer, TGGTAGCTAGTAGCTTAGTAGTTAG
- sequences of the primers used for detection of miR-124-2 are as follows: AGGGGCGGTATTTTTGGGGTTTTTGC as a methylated allele-specific primer, CCCCTTACGACGTAATCGTACCCG as a non-methylated allele-specific primer, .
- sequences of the primers used for detection of miR-124-3 are as follows. .
- the primer sequences used for detection of miR-9-3 are: GATTGACGGTTTATTTTTTCGCGGGGC as a forward primer as a methylated allele-specific primer, CGAAAACTCACGTAAAACACCCGCG as a reverse primer, and TGGATCATTGTATGTATGTATGTATGTATGTATGTATGTATGTATGTATGTATTTATGTATGTT .
- the miR-137 gene, the miR-124-2 gene, the miR-124-3 gene, and the miR-9-3 gene have primers such as bisulfite shown in FIGS. You may select the other arrangement
- the subject sample is a urine sample collected from the subject.
- a urine sample after treatment for bladder cancer, for example, excision of the bladder is used.
- the expression level of miRNA is detected by detecting methylation as in the above embodiment.
- Other points are the same as in the above-described embodiment.
- control sample is less methylated than the subject sample, it can be determined that the bladder cancer tissue has been removed by treatment.
- a urine sample is desirable because it is not invasive to the subject and suffers minimal pain and can be collected very easily. Since the urinary epithelial cells peeled off in the urine are detected in the urine sample, the amount of miRNA contained is smaller than that of the blood sample or the excised sample.
- the bisulfite pyrosequencing method is used. Detection of methylation by means of urine samples enables detection with sufficiently high specificity and sensitivity.
- Bladder cancer inhibitor Another embodiment of the present invention is a bladder cancer inhibitor containing one or more miRNA selected from miR-124, miR-9, and miR-137.
- the present inventors have found that bladder cancer can be suppressed by administering or expressing these genes whose expression is suppressed in cancer cells of bladder cancer to cancer tissues.
- a bladder cancer suppressor contains the above-described miRNA as an active ingredient, and this active ingredient is blended into a base used as a gene therapy agent.
- a buffer solution or an amino acid or other nutrient can be added to form a solution or powdered injection. Or it can mix
- the above-described miRNA can be introduced into cells and used as a drug for expression in cancer cells.
- a drug for encapsulating the above-described miRNA in a liposome or the like to introduce a nucleic acid molecule into a tissue a drug for a method for introducing a nucleic acid molecule into a cell by a microinjection method, a cell administered to a living body via a viral vector It can be set as the chemical
- miRNAs whose expression increased after drug treatment were found in common in the two cell lines T24 and UM-UC-3.
- CpG islands were present in the region within 5 kb upstream of pre-miRNA, and were the following 23 types (registered in the above-mentioned miR BASE). Since the expression of these genes is increased when methylation is inhibited, the expression of these genes may be suppressed by methylation in these cell lines.
- hsa-miR-10b hsa-miR-124, hsa-miR-132, hsa-miR-137, hsa-miR-147b, hsa-miR-148a, hsa-miR-152, hsa-miR-185a, hsa- miR-193a-5p, hsa-miR-200b, hsa-miR-200b *, hsa-miR-203, hsa-miR-22, hsa-miR-330-5p, hsa-miR-34c-5p, hsa-miR -409-3p, hsa-miR-409-5p, hsa-miR-449b, hsa-miR-545 *, hsa-miR-636, hsa-miR-639, hsa-
- FIG. 2 The results of further analysis of the methylation status of these genes by the bisulfite pyrosequencing method are shown in FIG. 2, FIG. 3, and FIG.
- Primary Bladder Cancer Tissue is DNA derived from surgically removed bladder cancer tissue
- Blade Cancer Cell Lines is DNA from bladder cancer tissue cell lines (T24, UM-UC3, HT1197, HT-1376, SW780)
- SV-U 1 shows the result of analysis of DNA derived from a normal urothelial cell line
- Normal Urothelium analyzed for DNA derived from normal urothelium purchased from BioChain.
- FIG. 5 shows real-time RT-PCR analysis using PCR (Applied Biosystems). This result indicates that miR-137 has low expression in HT1197 and SW780.
- FIG. 5 (b) shows the results of analyzing the methylation of the miR-137 gene by bisulfite pyrosequencing for these bladder cancer cell lines. This result indicates that the miR-137 gene is methylated in UM-UC-3, HT1197, HT-1376, and SW780.
- the results in FIGS. 5 (a) and 5 (b) indicate that miR-137 expression is decreased in bladder cancer cell lines, and that the decrease may be due to methylation.
- RNA is extracted from the cancer tissue (T) and the tissue (DN: Distant Normal-appearing tissue) that is sufficiently separated from the cancer and obtained at the time of cancer removal, and the expression level of miR-137 is determined in real-time RT -Results of PCR analysis are shown in FIG. In this result, a tendency that the expression level is low in the cancerous part (T) is recognized. Although there is no difference that can be compared at the level of expression level, it is considered that it is difficult to detect a gene having a decreased expression level because T also contains normal tissue.
- FIG. 7 (b) shows the result of analyzing the methylation of the miR-137 gene by the bisulfite pyrosequencing method. From this result, it is recognized that there is an inverse correlation between the expression level of miRNA and the methylation in the encoded genomic gene, that is, the expression level of miRNA is decreased by methylation of the gene CpG island on the genome. From these results, it was shown that by analyzing the methylation of the miR-137 gene, it is possible to detect a decrease in the expression level, that is, whether or not it is a cancer cell.
- FIG. 8 shows the results of analyzing the methylation of the miR-137 gene for DN by the bisulfite pyrosequencing method. The results from the same individual are connected by a line.
- ROC receiver operating characteristic
- FIG. 14 shows a bisulfite pyro before and after surgery for cancer tissue removal surgery (a), and before and after surgery for a non-cancerous subject (example that was found not to be cancer after removal).
- the result of the analysis of the methylation of miR-137 gene by the sequencing method is shown. The results from the same individual are connected by a line.
- the miR-137 gene methylation was reduced by excision of the cancer tissue, and when it was not cancer, there was no change, which correlated with the result in the urine sample shown in FIG. From these results, it is considered that methylation of miR-137 gene in urine samples is useful as a diagnostic marker for bladder cancer cells, and analysis of methylation in urine can be applied to the examination of the presence of cancer tissue. Indicated.
- FIG. 16 shows the results before and after surgery for cancer tissue removal surgery (a), and before and after surgery for surgery for a non-cancer subject. The results from the same individual are connected by a line.
- the miR-124-2 gene methylation was reduced by the removal of cancer tissue, and when it was not cancer, there was no change, correlating with the result in the urine sample shown in FIG. 15 (a). From these results, it is considered that methylation of miR-124-2 gene in urine specimens is useful as a diagnostic marker for bladder cancer, and analysis of methylation in urine can be applied to the presence or absence of cancer tissue It has been shown.
- FIG. 18 shows the results before and after the operation for removing cancer tissue (a), and before and after the operation for removing non-cancer subjects (b).
- the results from the same individual are connected by a line.
- the miR-124-3 gene methylation is reduced by the removal of cancer tissue, and there is no change when it is not cancer, which correlates with the result in the urine sample shown in FIG. 17 (a). From these results, it is considered that methylation of miR-124-3 gene in urine specimens is useful as a diagnostic marker for bladder cancer, and analysis of methylation in urine can be applied to the presence or absence of cancer tissue It has been shown.
- FIG. 20 shows the results before and after the surgical operation for removing cancer tissue (a), and before and after the surgical operation for a non-cancer subject (b).
- the results from the same individual are connected by a line.
- the methylation of the miR-9-3 gene is reduced by the removal of the cancer tissue, and there is no change when it is not cancer, which correlates with the result in the urine sample shown in FIG. 19 (a). From these results, it is considered that methylation of miR-9-3 gene in urine specimens is useful as a diagnostic marker for bladder cancer, and analysis of methylation in urine can be applied to the presence or absence of cancer tissue It has been shown.
- bladder cancer using a urine sample by combining the methylation detection results of four types of genes, miR-137 gene, miR-124-2 gene, miR-124-3 gene and miR-9-3 gene We examined and analyzed the diagnostic method.
- the panel detection method is shown in FIG. 21 and FIG.
- the numerical values that provide the best sensitivity (sensitivity, Sens) and specificity (specificity, Spec) are used as the cutoff values, and 5.2% for the miR-137 gene and miR-124- Set 2% for 2 genes, 12% for miR-124-3 gene, and 7.2% for miR-9-3 gene. If this value is met, add 1 point for each gene. (Summing the number of “Yes”). This was designated as miRscore, and an ROC curve was created at each point. As a result, the sensitivity was 94% and the specificity was 64% between 0-1 points. Between 1-2 points, the sensitivity was 81% and the specificity was 89%.
- the sensitivity was 65% and the specificity was 97%. Moreover, this was made into the training set (Training Set) shown in FIG. 21, and it examined again as a test set (Test Set) shown in FIG. 22 using another urine sample. As a result, the sensitivity was 94% and the specificity was 64% between 0-1 points. Between 1-2 points, the sensitivity was 82% and the specificity was 91%. The sensitivity was 71% and the specificity was 91% between 2-3 points. From this result, reproducibility was confirmed for this detection method.
- the detection method based on the Tree diagram is shown in FIG. 23 and FIG.
- the methylation of the miR-137 gene which has the highest sensitivity at a specificity of 100%, is divided by a cutoff value of 9.8% (sensitivity is 57%). If this is satisfied, classify as category 3. If this is not satisfied, the methylation of miR-9-3 gene is further divided by a cutoff value of 6.7%.
- the methylation of the miR-137 gene was 9.8% or less, ROC curves were prepared for each of the miR-124-2, miR-124-3, and miR-9-3 genes. In this case, the values with the highest sensitivity and specificity were 87% and 75%, respectively.
- category 2 When this is satisfied, it is classified as category 2, and when it is not satisfied, it is classified as category 1. In addition, this was used as a training set shown in FIG. 23, and the test set shown in FIG. 24 was examined again using another urine sample. As a result, specificity for category 3 was 100% and sensitivity was 68%, and in classifications of categories 1 and 2, specificity was 55% and sensitivity was 91%. From this result, reproducibility was confirmed for this detection method. Note that category 3 has a specificity of 100%, and if it is classified into this category, it is determined that it is cancer with a considerable probability. If it is classified into category 1, it is determined that it is unlikely that it is cancer, and if it is classified into category 2, it is determined that it is probably cancer.
- Table 2 shows the results of urine cytology in the above urine specimen.
- FIG. 25 shows the results of miR-137 transiently introduced into bladder cancer cells (SW780) and forced expression, and comparison of cell viability with miR-cont (which expresses a random sequence). Viability in the figure indicates cell viability. Since the expression level of miR-137 decreases for a long time due to the introduction of the transient, analysis was performed every 24 hours up to 72 hours. The result was that the expression of miR-137 decreased the cell viability of cancer cells. From these results, it was shown that miR-137 can be used as an inhibitor of bladder cancer.
- Table 3 shows the results of extracting only the data of patients whose depth of penetration pTa and atypical degree G1 / G2 classified as early cancers from Table 1.
- FIG. 31 shows a detection method in which ROC curves are generated by combining the results of these genes and analyzing with the same panel detection method and tree diagram as in FIG. 21-24. From these results, it was revealed that the analysis of methylation of each gene can detect early bladder cancer in patients with pTa and G1 / G2 by using each gene alone or in combination.
- Table 4 shows the results of urine cytology in the urine specimen of the above-mentioned early cancer patient.
- the present invention can be widely applied in the medical and pharmaceutical fields by cancer treatment and prevention.
Abstract
Description
本実施形態における膀胱癌とは、尿路上皮癌(移行上皮癌)、扁平上皮癌又は腺癌といった癌で膀胱に発生するものを指す。
miR-137(5’-uuauugcuuaagaauacgcguag-3’)(MIMAT0000429)(配列番号29)
miR-124(5’-uaaggcacgcggugaaugcc-3’)(MIMAT0000422)(配列番号30)
miR-9(5’-ucuuugguuaucuagcuguauga-3’)(MIMAT0000441)(配列番号31)
miR-137遺伝子(5’-ggtcctctgactctcttcggtgacgggtattcttgggtggataatacggattacgttgttattgcttaagaatacgcgtagtcgaggagagtaccagcggca-3’
) (MI0000454)(配列番号32)
miR-124-2遺伝子(5’-atcaagattagaggctctgctctccgtgttcacagcggaccttgatttaatgtcatacaattaaggcacgcggtgaatgccaagagcggagcctacggctgcacttgaa-3’ ) (MI0000444)(配列番号33)
miR-124-3遺伝子(5’-tgagggcccctctgcgtgttcacagcggaccttgatttaatgtctatacaattaaggcacgcggtgaatgccaagagaggcgcctcc-3’ ) (MI0000445)(配列番号34)
miR-9-3遺伝子(5’-ggaggcccgtttctctctttggttatctagctgtatgagtgccacagagccgtcataaagctagataaccgaaagtagaaatgattctca-3’ ) (MI0000468)(配列番号35)
本発明の他の実施形態では、被験者サンプルは被験者から採取した尿サンプルである。コントロールサンプルとしては、膀胱癌に対する治療、例えば膀胱の切除手術を行った後の尿サンプルを用いる。miRNAの発現量の検出は、上述の実施形態同様のメチル化の検出により行う。その他の点は上述の実施形態と同様である。
本発明の他の実施形態は、miR-124、miR-9及びmiR-137から選ばれる1以上のmiRNAを含有する膀胱癌抑制剤である。本発明者らは、膀胱癌の癌細胞において発現が抑制されているこれらの遺伝子を癌組織に対して投与、又は発現させることで、膀胱癌を抑制することができることを見出している。
膀胱癌細胞株(T24及びUM-UC-3)をDNAメチル化酵素阻害剤5-aza-2’-deoxycytidine (5-aza-dC)及びHDAC阻害剤4-phenylbutyric acid (4-PBA)で処理した。処理後の試料と処理していないコントロールの発現プロファイルを、TaqMan miRNA Low Density Array System (アプライドバイオシステム)を用いてそれぞれ解析した。処理後の試料でコントロールよりも発現が上昇しているmiRNAをスクリーニングした。
上述のmiRNAのうちmiR-137について、膀胱癌細胞株であるT24、UM-UC-3、HT1197、HT-1376、SW780、正常尿路上皮細胞株SV-HUC-1における発現量についてTaqMan RT-PCR(Applied Biosystems社)を用いてリアルタイムRT-PCR解析したものを図5(a)に示す。この結果は、miR-137がHT1197、SW780において発現が低いことを示している。
癌部組織(T)及び癌摘出の際に得られた、癌から充分に離れ正常と思われる組織(DN;Distant Normal-appearing tissue)からRNAを抽出し、miR-137の発現量をリアルタイムRT-PCR解析した結果を図6に示す。この結果では、癌部(T)で発現量が低い傾向が認められる。発現量のレベルでの比較が可能なほどの差は認められないが、これはTについて正常組織も混入するので発現量が低下した遺伝子を検出するのが困難なためと考えられる。
被験者の癌組織摘出手術の術前(Pre-OP)及び癌組織摘出後(Post-OP)の尿検体について、バイサルファイト・パイロシークエンス法によるmiR-137遺伝子のメチル化の解析を行った。その結果(a)とROC曲線(b)を図13に示す。なお、Post-OPについては、開腹手術の場合は膀胱を全摘するので内視鏡切除例の尿検体しか得られないため、N数が少なくなっている。術後でmiR-137遺伝子のメチル化が減少している傾向が見られる。
被験者の癌組織摘出手術の術前(Pre-OP)及び癌組織摘出後(Post-OP)の尿検体について、バイサルファイト・パイロシークエンス法によるmiR-124-2遺伝子のメチル化の解析を行った。その結果(a)とROC曲線(b)を図15に示す。術後でmiR-124-2遺伝子のメチル化が減少している傾向が見られる。
図16に、癌組織の摘出手術の術前及び術後(a)、癌でない被験者の摘出手術の術前及び術後(b)の結果を示す。なお、同一個体からの結果を線で結んでいる。癌組織の摘出によりmiR-124-2遺伝子のメチル化は減少し、癌でなかった場合は変化がなく、図15(a)に示す尿検体における結果と相関している。これらの結果より、尿検体の中のmiR-124-2遺伝子のメチル化は膀胱癌の診断マーカとして有用であると考えられ、尿中のメチル化の解析が癌組織の有無の検査に応用できることが示された。
被験者の癌組織摘出手術の術前(Pre-OP)及び癌組織摘出後(Post-OP)の尿検体について、バイサルファイト・パイロシークエンス法によるmiR-124-3遺伝子のメチル化の解析を行った。その結果(a)とROC曲線(b)を図17に示す。術後でmiR-124-3遺伝子のメチル化が減少している傾向が見られる。
被験者の癌組織摘出手術の術前(Pre-OP)及び癌組織摘出後(Post-OP)の尿検体について、バイサルファイト・パイロシークエンス法によるmiR-9-3遺伝子のメチル化の解析を行った。その結果(a)とROC曲線(b)を図19に示す。術後でmiR-9-3遺伝子のメチル化が減少している傾向が見られる。
miR-137が膀胱癌において発現が低下していることから、miR-137を強制発現させることで、癌を制御する因子として働くかを解析した。膀胱癌細胞(SW780)に対してmiR-137をトランジエントで導入し強制発現し、miR-cont(ランダム配列を発現するもの)に対して細胞生存性を比較した結果を図25に示す。図中のviabilityが細胞生存性を示している。トランジエント導入のため長時間ではmiR-137の発現量が低下するため、72時間までの24時間ごとに解析した。miR-137の発現により、癌細胞の細胞生存性が低下しているという結果が得られた。この結果から、miR-137は膀胱癌の抑制剤として使用できる可能性が示された。
表1より、初期癌に分類される深達度pTa、異型度G1/G2であった患者のデータのみを抽出した結果を、表3に示す。
Claims (26)
- miR-124、miR-9及びmiR-137から選ばれる1つ以上のmiRNAからなる膀胱癌マーカの発現量を被験者から採取された被験者サンプルから検出することを含む膀胱癌細胞の検出方法。
- 前記膀胱癌マーカの発現量の検出は、前記膀胱癌マーカがコードされているゲノム遺伝子のメチル化の検出により前記膀胱癌マーカの発現量の低下を検出することを特徴とする請求項1に記載の膀胱癌細胞の検出方法。
- 前記メチル化の検出はバイサルファイト・パイロシークエンス法により行うことを特徴とする請求項2に記載の膀胱癌細胞の検出方法。
- 前記膀胱癌マーカが少なくともmiR-137を含むことを特徴とする請求項1から3のいずれか1項に記載の膀胱癌細胞の検出方法。
- 前記被験者サンプルにおける前記膀胱癌マーカの発現量をしきい値と比較することを特徴とする請求項1から4のいずれか1項に記載の膀胱癌細胞の検出方法。
- 前記しきい値は、正常組織から採取されたコントロールサンプルにおける前記膀胱癌マーカの発現量であることを特徴とする請求項1から5のいずれか1項に記載の膀胱癌細胞の検出方法。
- 前記しきい値は、被験者から異なる時に採取された又は被験者の異なる組織から採取されたコントロールサンプルにおける前記膀胱癌マーカの発現量であることを特徴とする請求項1から5のいずれか1項に記載の膀胱癌細胞の検出方法。
- 前記被験者サンプルが尿サンプルであることを特徴とする請求項1から7のいずれか1項に記載の膀胱癌細胞の検出方法。
- 前記膀胱癌マーカの発現量の検出において、miR-137遺伝子の増幅に用いるプライマーの配列が配列番号1で示されるフォワードプライマー(GGGTTTAGYGAGTAGTAAGAGTTTTG)及び配列番号2で示されるリバースプライマー(CCCCCTACCRCTAATACTCTCCTC)であることを特徴とする請求項1から8のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記膀胱癌マーカの発現量の検出において、miR-137のシークエンス反応に用いるプライマーの配列が配列番号3で示されるGGTATTTTTGGGTGGATAATであることを特徴とする請求項1から9のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記膀胱癌マーカの発現量の検出において、miR-124-2の増幅に用いるプライマーの配列が配列番号4で示されるフォワードプライマー(GTTGGGATTGTATAGAAGGATTATTTG)及び配列番号5で示されるリバースプライマー(ACTACRAAAATCCAAAAAAAAATACATAC)であることを特徴とする請求項1から10のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記膀胱癌マーカの発現量の検出において、miR-124-2のシークエンス反応に用いるプライマーの配列が配列番号6で示されるYGTTTTTATTGTTTTAGTTTであることを特徴とする請求項1から11のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記膀胱癌マーカの発現量の検出において、miR-124-3の増幅に用いるプライマーの配列が配列番号7で示されるフォワードプライマー(AAAAGAGAYGAGTTTTTATTTTTGAGTAT)及び配列番号8で示されるリバースプライマー(TCCTCCRCAACTACCTTCCCCTA)であることを特徴とする請求項1から12のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記膀胱癌マーカの発現量の検出において、miR-124-3のシークエンス反応に用いるプライマーの配列が配列番号9で示されるGAGATTYGTTTTTTTAATであることを特徴とする請求項1から13のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記膀胱癌マーカの発現量の検出において、miR-9-3の増幅に用いるプライマーの配列が配列番号10で示されるフォワードプライマー(GATTTGAATGGGAGTTTGTGATTGGT)及び配列番号11で示されるリバースプライマー(TCCCRAAACTCACRTAAAACACCC)であることを特徴とする請求項1から14のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記膀胱癌マーカの発現量の検出において、miR-9-3のシークエンス反応に用いるプライマーの配列が配列番号12で示されるTTGGATTGAYGTTATTTTであることを特徴とする請求項1から15のいずれか1項に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記メチル化の検出はメチル化特異的PCR法により行うことを特徴とする請求項2に記載の膀胱癌細胞の検出方法。
- 前記メチル化特異的PCR法において、miR-137の検出に用いるプライマーの配列が、メチル化アレル特異的プライマーとして配列番号13で示されるフォワードプライマー(GTAGCGGTAGTAGCGGTAGCGGT)及び配列番号14で示されるリバースプライマー(GCTAATACTCTCCTCGACTACGCG)、非メチル化アレル特異的プライマーとして配列番号15で示されるフォワードプライマー(TGGTAGTGGTAGTAGTGGTAGTGGT)及び配列番号16で示されるリバースプライマー(CCACTAATACTCTCCTCAACTACACA)であることを特徴とする請求項17に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記メチル化特異的PCR法において、miR-124-2の検出に用いるプライマーの配列が、メチル化アレル特異的プライマーとして配列番号17で示されるフォワードプライマー(AGGGGCGTATTTTGGGGTTTTTGC)及び配列番号18で示されるリバースプライマー(CCCCTACGACGTAATCGACCCG)、非メチル化アレル特異的プライマーとして配列番号19で示されるフォワードプライマー(TTTAGGGGTGTATTTTGGGGTTTTTGT)及び配列番号20で示されるリバースプライマー(CATCCCCTACAACATAATCAACCCA)であることを特徴とする請求項17に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記メチル化特異的PCR法において、miR-124-3の検出に用いるプライマーの配列が、メチル化アレル特異的プライマーとして配列番号21で示されるフォワードプライマー(GTTTTAGTGATAATCGGTCGGTGTC)及び配列番号22で示されるリバースプライマー(TCCACGAAATCCACGCTACAAACG)、非メチル化アレル特異的プライマーとして配列番号23で示されるフォワードプライマー(TGTGTTTTAGTGATAATTGGTTGGTGTT)及び配列番号24で示されるリバースプライマー(ATATCCACAAAATCCACACTACAAACA)であることを特徴とする請求項17に記載の膀胱癌細胞の検出方法に用いるプライマー。
- 前記メチル化特異的PCR法において、miR-9-3の検出に用いるプライマーの配列が、メチル化アレル特異的プライマーとして配列番号25で示されるフォワードプライマー(GATTGACGTTATTTTTTCGCGGGGC)及び配列番号26で示されるリバースプライマー(CGAAACTCACGTAAAACACCCGCG)、非メチル化アレル特異的プライマーとして配列番号27で示されるフォワードプライマー(TTGGATTGATGTTATTTTTTTGTGGGGT)及び配列番号28で示されるリバースプライマー(CCCAAAACTCACATAAAACACCCACA)であることを特徴とする請求項17に記載の膀胱癌細胞の検出方法に用いるプライマー。
- miR-124、miR-9及びmiR-137から選ばれる1つ以上のmiRNAを含有する膀胱癌マーカ。
- 前記膀胱癌マーカの発現量を被験者から採取された被験者サンプルから検出することを含む膀胱癌細胞の検出方法に用いる請求項22の膀胱癌マーカ。
- 前記膀胱癌マーカの発現量の検出は、前記膀胱癌マーカがコードされているゲノム遺伝子のメチル化の検出により前記膀胱癌マーカの発現量の低下を検出することを含む膀胱癌細胞の検出方法に用いる請求項23の膀胱癌マーカ。
- 深達度pTa又は異型度G1/G2の被験者から採取された被験者サンプルから検出を行うことを特徴とする請求項2に記載の膀胱癌細胞の検出方法。
- 配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9、配列番号10、配列番号11、配列番号12、配列番号13、配列番号14、配列番号15、配列番号16、配列番号17、配列番号18、配列番号19、配列番号20、配列番号21、配列番号22、配列番号23、配列番号24、配列番号25、配列番号26、配列番号27、または配列番号28のヌクレオチド配列を有する核酸分子。
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CN108060231A (zh) * | 2018-02-24 | 2018-05-22 | 韩林志 | 用于宫颈癌基因FAM19A4、miR-124-2甲基化检测的引物对、试剂盒及方法 |
WO2019208671A1 (ja) * | 2018-04-25 | 2019-10-31 | 東レ株式会社 | 膀胱がんの検出のためのキット、デバイス及び方法 |
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CN104141009B (zh) * | 2014-07-01 | 2016-11-16 | 蔡志明 | 早期膀胱癌的多靶标检测方法 |
WO2016061465A1 (en) * | 2014-10-17 | 2016-04-21 | The Regents Of The University Of Colorado, A Body Corporate | Biomarkers for head and neck cancer and methods of their use |
KR101759382B1 (ko) | 2015-02-25 | 2017-07-19 | 충북대학교 산학협력단 | Prac 메틸화를 이용한 방광암 예후 진단방법 및 이의 용도 |
CN105999269A (zh) * | 2016-05-05 | 2016-10-12 | 温州医科大学 | miR-411作为膀胱癌的靶标及其应用 |
CN106555003B (zh) * | 2016-11-25 | 2020-01-17 | 广州中鑫基因医学科技有限公司 | 腺性膀胱炎和膀胱癌诊断区分标志物、诊断试剂或试剂盒 |
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WO2019208671A1 (ja) * | 2018-04-25 | 2019-10-31 | 東レ株式会社 | 膀胱がんの検出のためのキット、デバイス及び方法 |
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CA2848999A1 (en) | 2013-03-21 |
JPWO2013038737A1 (ja) | 2015-03-23 |
EP2757154A4 (en) | 2015-10-14 |
KR20140064899A (ko) | 2014-05-28 |
CN103857796A (zh) | 2014-06-11 |
US20150024389A1 (en) | 2015-01-22 |
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