WO2011125245A1 - Méthode de prédiction de pronostic de cancer du poumon à petites cellules, méthode de traitement du cancer du poumon à petites cellules, méthode d'amélioration du pronostic du cancer du poumon à petites cellules et méthode de criblage pour la recherche d'un agent thérapeutique pour le cancer du poumon à petites cellules, chacun utilisant les miarn - Google Patents

Méthode de prédiction de pronostic de cancer du poumon à petites cellules, méthode de traitement du cancer du poumon à petites cellules, méthode d'amélioration du pronostic du cancer du poumon à petites cellules et méthode de criblage pour la recherche d'un agent thérapeutique pour le cancer du poumon à petites cellules, chacun utilisant les miarn Download PDF

Info

Publication number
WO2011125245A1
WO2011125245A1 PCT/JP2010/067432 JP2010067432W WO2011125245A1 WO 2011125245 A1 WO2011125245 A1 WO 2011125245A1 JP 2010067432 W JP2010067432 W JP 2010067432W WO 2011125245 A1 WO2011125245 A1 WO 2011125245A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
lung cancer
small cell
precursor
cell lung
Prior art date
Application number
PCT/JP2010/067432
Other languages
English (en)
Japanese (ja)
Inventor
石川　雄一
岳彦 大場
Original Assignee
財団法人癌研究会
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 財団法人癌研究会 filed Critical 財団法人癌研究会
Priority to JP2012509270A priority Critical patent/JPWO2011125245A1/ja
Publication of WO2011125245A1 publication Critical patent/WO2011125245A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the fields of small cell lung cancer prognosis prediction, examination and diagnosis method using miRNA, small cell lung cancer treatment method, small cell lung cancer prognosis improvement method, and small cell lung cancer therapeutic agent screening method.
  • SCLC small cell lung cancer
  • LCNEC large cell neuroendocrine cancer
  • microRNA is a non-coding RNA with a small nucleotide of about 20 to 25 that is not translated into protein.
  • miRNA a primary transcript (pri-miRNA) of about several hundred bases is first transcribed from a DNA region that does not encode a protein. The primary transcript is processed into a secondary transcript (pre-miRNA) by Drosha with the complex. Subsequently, after being transported to the cytoplasm, it is processed by Dicer and becomes a double-stranded mature miRNA of about 20-25 nucleotides.
  • one of the two miRNAs forms a complex with RISC and binds to the 3 ′ untranslated region of the target mRNA to cause translational repression.
  • Many miRNAs are known to exist in organisms including humans. Recently, the relationship with diseases, particularly cancer, has attracted attention, and miRNA expression patterns are used for cancer diagnosis and miRNA expression is controlled. It has been proposed to be used for cancer treatment (Patent Document 1). For example, the expression of the let-7 family is decreased in lung cancer, and the decreased expression is a poor prognostic factor, and when miR-17-92 clusters that are overexpressed in lung cancer are overexpressed, a growth promoting effect is observed.
  • Non-patent Document 1 suppression of expression with antisense oligos has been shown to cause proliferation suppression and cell death induction specifically in lung cancer cell lines that strongly express miR-17-92 clusters.
  • Non-patent Document 3 In colorectal cancer, the expression levels of miR-143 and miR-145 decreased, and miR-143 was shown to exhibit antitumor effects when administered to model animals as an agonist.
  • Non-patent Document 3 In lung adenocarcinoma and lung squamous cell carcinoma, expression levels of hsa-mir-126, has-mir-205 and has-mir-21 are increased or decreased, and expression of hsa-mir-155 is high or hsa.
  • the present invention has been made in view of the above situation, and an object of the present invention is to provide a method for analyzing a miRNA related to SCLC and examining the prognosis of an SCLC patient using the miRNA.
  • miR-153, miR-196a, miR-203 or miR-216a is related to the prognosis of SCLC, and has completed the present invention. It was.
  • the present invention is as follows: [1] Measuring at least one expression level selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof in a biological sample derived from a patient with small cell lung cancer, A test method for determining prognosis in a patient with cell lung cancer. [2] In the above method, when the expression level of miR-153, miR-216a or a precursor thereof is higher than the expression level in a control biological sample, the prognosis is determined to be poor. ] The method of description.
  • a method for treating small cell lung cancer (1) In a biological sample derived from a patient with small cell lung cancer, when the expression level of at least one of miR-196a, miR-203 or a precursor thereof is decreased compared to a control biological sample, the decrease Administering at least one miRNA, a precursor thereof, or a vector that expresses them; and / or (2) In a biological sample derived from a patient with small cell lung cancer, if the expression level of at least one of miR-153, miR-216a or a precursor thereof is increased compared to the control biological sample, the miRNA or Administering at least one compound that inhibits the function of their precursors;
  • a method of treating small cell lung cancer comprising: [6] A method for improving the prognosis of small cell lung cancer, (1) In a biological sample derived from a patient with small cell lung cancer, when the expression level of at least one of miR-196a, miR-203 or a precursor thereof is decreased compared to a control biological sample, the decrease Ad
  • a therapeutic agent for small cell lung cancer comprising at least one of miR-196a, miR-203, a precursor thereof, or a vector expressing them.
  • a therapeutic agent for small cell lung cancer comprising a compound that inhibits at least one function of miR-153, miR-216a or a precursor thereof.
  • An agent for improving the prognosis of small cell lung cancer comprising at least one of miR-196a, miR-203, a precursor thereof, or a vector that expresses them.
  • An agent for improving the prognosis of small cell lung cancer comprising a compound that inhibits at least one function of miR-153, miR-216a or a precursor thereof.
  • the change in function is inhibition of expression of miR-153, miR-216a or a precursor thereof.
  • the change in function is promotion of binding of miR-196a, miR-203 or a precursor thereof to a base sequence targeted by miR-196a, miR-203 or a precursor thereof, [12 ] The method of description. [16] The change in function is inhibition of binding of miR-153, miR-216a or a precursor thereof to a base sequence targeted by miR-153, miR-216a or a precursor thereof. ] The method of description. Is to provide.
  • the present invention provides [1] Measuring at least one expression level selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof in a biological sample derived from a patient with small cell lung cancer, A method for predicting, examining and / or diagnosing prognosis in patients with cell lung cancer. [2] In the above method, when the expression level of miR-153, miR-216a or a precursor thereof is higher than the expression level in a control biological sample, the prognosis is diagnosed or determined as [1] ] The method of description.
  • a method of treating small cell lung cancer comprising: (1) When the expression level of at least one miR-196a, miR-203 or a precursor thereof is decreased, the reduced at least one miRNA, a precursor thereof, or the expression thereof is expressed Administering a vector; or / and (2) When the expression level of at least one of miR-153, miR-216a or a precursor thereof is increased, at least one compound that inhibits the function of the miRNA or a precursor thereof is administered thing;
  • a method of treating small cell lung cancer comprising: [6] A method of treating small cell lung cancer, (1) measuring at least one expression level selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof in a biological sample derived from a patient with small cell lung cancer; (2) if the expression level is reduced compared to the control biological sample, administering the reduced at least one miRNA, a precursor thereof, or a vector expressing them; and / or (3) If the expression
  • a therapeutic agent for small cell lung cancer or a prognosis improving agent for small cell lung cancer comprising at least one of miR-196a, miR-203, a precursor thereof, or a vector that expresses them.
  • a therapeutic agent for small cell lung cancer or a prognosis improving agent for small cell lung cancer comprising a compound that inhibits at least one function of miR-153, miR-216a or a precursor thereof.
  • At least one expression level selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof in a biological sample of a small cell lung cancer patient is compared with a control biological sample
  • a therapeutic agent for treating small cell lung cancer that is increasing or decreasing, (1) when the expression level of miR-196a, miR-203 or a precursor thereof is decreased, the decreased at least one miRNA, a precursor thereof, or a vector expressing them; or / as well as, (2) when the expression level of miR-153, miR-216a or a precursor thereof is increased, at least one compound that inhibits the function of the miRNA or a precursor thereof;
  • a therapeutic agent for treating small cell lung cancer that is increasing or decreasing, (1) when the expression level of miR-196a, miR-203 or a precursor thereof is decreased, the decreased at least one miRNA, a precursor thereof, or a vector expressing them; or / as well as, (2) when the expression level of miR-153, miR
  • a biological sample of a patient with small cell lung cancer at least one expression level selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof is compared with the control biological sample
  • a prognostic improver for improving the prognosis of small cell lung cancer that is increasing or decreasing, (1) when the expression level of miR-196a, miR-203 or a precursor thereof is decreased, the decreased at least one miRNA, a precursor thereof, or a vector expressing them; or / as well as, (2) when the expression level of miR-153, miR-216a or a precursor thereof is increased, at least one compound that inhibits the function of the miRNA or a precursor thereof;
  • a prognosis improving agent for small cell lung cancer comprising: [15] The prognosis improving agent according to [14], wherein the
  • the present invention provides an SCLC therapeutic method, an SCLC prognostic improvement method, an SCLC therapeutic agent, and an SCLC therapeutic screening method using miRNA.
  • the present invention contributes to simple and accurate SCLC prognosis determination.
  • FIG. 1 shows the classification of 600 miRNA expression by hierarchical clustering. Rows and columns indicate the miRNA and case, respectively. For each miRNA, the color on the “7.0” side (original data red) indicates high expression, and the color on the “ ⁇ 7.0” side (original data blue) indicates low expression.
  • the lower bar shows small cell lung cancer (SCLC) (“1”, original data blue), large cell neuroendocrine cancer (LCNEC) (“2”, original data light blue), adenocarcinoma (Ad) (“3”, Original data pink), squamous cell carcinoma (Sq) (“4”, original data brown), normal lung (NL) (“5”, original data yellow) were shown.
  • SCLC small cell lung cancer
  • LCDNEC large cell neuroendocrine cancer
  • Ad adenocarcinoma
  • Sq squamous cell carcinoma
  • FIG. 2 shows the survival curves of the SCLC of group 1 (SCLC 1) and the SCLC of group 2 (SCLC 2).
  • FIG. 3 (A) shows normal lung tissue samples (NL), lung adenocarcinoma tissue samples and lung squamous cell carcinoma tissue samples (Ad + Sq), good prognosis group (SCLC2), and poor prognosis group for each miRNA expression level. A comparison between (SCLC1) is shown. *: P ⁇ 0.05, **: p ⁇ 0.01, ***: p ⁇ 0.001, NS: p ⁇ 0.05 (vs.
  • FIG. 3 shows the correlation between the expression level of each miRNA and the quantitative RT-PCR.
  • FIG. 4 shows the relationship between each miRNA and prognosis.
  • FIG. 5 shows classification by hierarchical clustering using 3 miRNA expressions. Rows and columns indicate the miRNA and case, respectively. The color on the “2.4” side (original data red) indicates high expression, and the color on the “ ⁇ 2.4” side (original data blue) indicates low expression.
  • the second bar from the bottom shows three groups classified by clustering. The bottom bar represents a poor prognosis group (SCLC1 group) and a good prognosis group (SCLC2 group) by clustering with 600 miRNAs.
  • FIG. 6 shows classification by hierarchical clustering using 4 miRNA expressions. Rows and columns indicate the miRNA and case, respectively. The color on the “3.2” side (original data red) indicates high expression, and the color on the “ ⁇ 3.2” side (original data blue) indicates low expression.
  • the second bar from the bottom shows two groups classified by clustering. The bottom bar represents a poor prognosis group (SCLC1 group) and a good prognosis group (SCLC2 group) by clustering with 600 miRNAs.
  • FIG. 7 shows the survival curves of the SCLC high-risk group and low-risk group classified by the prognosis prediction model using miR-153, miR-203, and miR-216a.
  • the prognosis prediction model using these three miRNAs can also classify small cell lung cancer into a good prognosis group and a poor prognosis group.
  • FIG. 8 shows tissue sections of cancer tissues of the small cell lung cancer high-risk group (A) and low-risk group (B) classified by the prognosis prediction model using miR-153, miR-203, and miR-216a.
  • FIG. 9 shows the results of evaluating the validity of the prognosis prediction model using three miRNAs (miR-153, miR-203, and miR-216a). The test set was classified based on the classification model built on the randomized learning set. The operation was repeated 1,000 times. (A) The result of survival analysis is shown as a histogram of p-value (by log rank test).
  • FIG. 10 shows the growth suppression of small cell lung cancer cells by miR-153 inhibitor and miR-203 mimetic (miR-203 mimic).
  • Panels (A) and (C) show RT-PCR analysis of miR-153 and miR-203 expression levels in poor prognosis group (SCLC1 group), good prognosis group (SCLC2 group), and various SCLC cell lines, respectively. Shows the results.
  • Panel (B) shows the results of transfecting SCLC cell line DMS 53 with miR-153 inhibitor
  • Panel (D) shows the results of transfecting SCLC cell line SCB5 with miR-203 (miR-203-mimic). Show.
  • As a result in both cases, it was confirmed that cell proliferation was suppressed as compared with the negative control. The decrease in the number of cells at 96 hr due to the introduction of miR-203 mimic when compared with the control was statistically significant.
  • Prognosis means a medical outlook or the patient's life expectancy about the course of the patient's cancer after some treatment (eg, chemotherapy, radiation therapy, surgical resection). Poor prognosis includes, for example, shorter survival after treatment, worsening or rapid deterioration of clinical features, progression of stage or rapid progression, and recurrence Or it means that the period until recurrence is short.
  • a good prognosis is, for example, a longer survival after treatment, a clinical feature improving or faster to improve, a stage not progressing or slow to progress, or a recurrence Or it means that the period until recurrence is long.
  • a biological sample derived from a cancer patient means, for example, a cancer tissue (eg, a small cell lung cancer tissue) collected from a cancer patient, a lymph node adjacent to the cancer tissue, a body fluid containing blood such as plasma and serum, and cells. It is not limited. In particular, cancer tissue collected from cancer patients is preferred. The cancer tissue can be collected with an endoscope or obtained by surgery.
  • the cells may be cells contained in body fluids such as blood, serum, plasma, urine, synovial fluid, cerebrospinal fluid, cerebrospinal fluid, semen, or lymph fluid, or cells contained in cancer tissue. It may be a circulating cancer cell ("CTC").
  • CTC circulating cancer cell
  • Examples of a method for obtaining circulating cancer cells include a method using immunomagnetic beads.
  • Immunomagnetic beads have antibodies against antigens selectively recognized on the surface of cancer cells such as epithelial cell adhesion molecule (EpCAM), cytokeratin-8, 19, etc., and are recognized on the surface of cancer cells. Some of them have antibodies against CD45 expressed on the surface of blood cells.
  • circulating cancer cells can be isolated using EASYSEP (registered trademark) human EpCAM positive kit (Stemcell Technologies) and EasySep (registered trademark) Human CD45 Deletion kit (Stemcell Technologies).
  • Other methods for isolating circulating cancer cells include the ISET method (Vona G et al., 2000, Am J Pathol. 2000 156: 57-63).
  • the control biological sample may be, for example, a healthy subject, a cancer patient other than small cell lung cancer, for example, a biological sample of a lung adenocarcinoma or lung squamous cell carcinoma patient, or a biological sample of a patient who has been judged to have a good prognosis. Although it means, it is not limited to these, What is necessary is just to select an optimal thing suitably according to a biological sample, miRNA to measure, etc.
  • a biological sample derived from the same site as the biological sample to be examined for example, the same tissue, organ, cell, for example, lung tissue
  • a biological sample of a healthy subject a lung adenocarcinoma, a lung squamous cell carcinoma patient, or a small cell lung cancer patient who has been judged to have a good prognosis
  • miR-196a a biological sample of a patient who has small cell lung cancer but a good prognosis can be used as a control biological sample.
  • the target to be measured in the test method for determining the prognosis of the present invention and the method for predicting, testing and / or diagnosing the prognosis is a specific miRNA or a precursor thereof.
  • the precursor means pri-miRNA that is a primary transcript or pre-miRNA that is a secondary transcript.
  • MiR-153 is a miRNA whose mature sequence is represented by UUGCAUAGUCACAAAAGUGAUC (SEQ ID NO: 1).
  • miR-153 precursor such as mir-153-1 (SEQ ID NO 2: CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGCAGCUAGUAUUCACUCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGCAGGUGUGGC), mir-153-2 (SEQ ID NO 3: AGCGGUGGCCAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAUAUGAGCCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGAAACUGUG), and the like.
  • MiR-196a is a miRNA whose mature sequence is represented by UAGGUAGUUUCAUGUUGUUGGG (SEQ ID NO: 4).
  • miR-196a precursor e.g. mir-196a-1 (SEQ ID NO 5: GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGAACACAACAACAUUAAACCACCCGAUUCAC), mir-196a-2 (SEQ ID NO 6: UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUUUCAUGUUGUUGGGAUUGAGUUUUGAACUCGGCAACAAGAAACUGCCUGAGUUACAUCAGUCGGUUUUCGUCGAGGGC), and the like.
  • MiR-203 is a miRNA whose mature sequence is represented by GUGAAAAUGUUUAGGACCACAUAG (SEQ ID NO: 7).
  • Examples of the miR-203 precursor include mir-203 (SEQ ID NO: 8: GUGUUGGGGGACUGCGCGCGUGGGGUCCAGUGGUUCUUAACAGUUCACAGUUCUGUGACGCAAUUGUGUAGAGCAGGACGACUGAGCGCG
  • MiR-216a is a miRNA whose mature sequence is represented by UAAUCUCAGCUGGCAACUGUGA (SEQ ID NO: 9).
  • mir-216a SEQ ID NO: 10: GAUGGCUGUGUGUGUGGGCUUAAUUCUCAGCUGGCAACUGUGGAGAUGUGUCAUGAUCAUCUCUCACAGUGUGAUCUUGUGAUCUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAUGUUGAU
  • miRNAs and their precursors can be obtained, for example, by isolating them from natural products, chemically synthesizing them or using genetic recombination techniques, using conventionally known methods. it can.
  • the miRNA or precursor thereof used in the test method for determining the prognosis and the method for predicting, testing and / or diagnosing the prognosis may be used alone or in combination of two or more. However, it is preferable to use a combination of two or more in terms of improving accuracy of prediction, inspection and / or diagnosis.
  • miRNA and its precursor may be combined, or two or more different miRNAs may be combined.
  • a combination containing at least miRNA-153 and miRNA-216a is suitable in the present invention. Further, it may be a combination containing at least miRNA-153 and miR-203, or a combination containing at least miRNA-216a and miR-203. In the present invention, a combination containing at least miR-153, miR-203, and miR-216a is particularly preferred. Also preferred are combinations comprising miR-153, miR-203, miR-216a, and miR-196a.
  • total RNA is first extracted from a biological sample.
  • the extraction method include known methods such as guanidine ultracentrifugation (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P.et al., Anal). Biochem. (1987) 162, 156-159).
  • the expression level of miRNA or a precursor thereof according to the present invention can be measured using total RNA obtained from a biological sample.
  • Any known method can be used for quantitative measurement of miRNA or a precursor thereof.
  • the RT-PCR method or a modified method thereof, the Northern blot method, the in situ hybridization method, or a method using a microarray can be mentioned.
  • the expression level can be accurately measured by a quantitative RT-PCR (qRT-PCR) method.
  • the expression level of the miRNA or the precursor thereof according to the present invention in a biological sample derived from a patient to be examined to determine the prognosis, and in a biological sample derived from a patient to be predicted, examined and / or diagnosed. Can be compared with the expression level of the miRNA according to the present invention or a precursor thereof in a control biological sample.
  • miR-153, miR-216a or a precursor thereof in a biological sample derived from a patient to be examined to determine a prognosis a biological sample derived from a patient to be predicted, examined and / or diagnosed
  • the expression level of is increased compared to the expression level of miR-153, miR-216a or their precursors in the control biological sample, it can be determined that the prognosis is poor.
  • judging that the prognosis is poor means that if not (if it is not rising), it is judged that the prognosis is not bad (or good). Good.
  • Provided in the present invention are methods that include any of them, and methods that include both.
  • the present invention is such that the expression level of miR-153, miR-216a or a precursor thereof in the biological sample derived from the patient is compared with the expression level of miR-153, miR-216a or a precursor thereof in the control biological sample. It relates to a method for predicting, judging, diagnosing, testing and / or determining that the prognosis is poor if it is elevated and / or otherwise the prognosis is good (and / or not bad).
  • the expression level of miR-153, miR-216a or a precursor thereof in a biological sample derived from a patient with a poor prognosis is equal to the expression level of miR-153, miR-216a or a precursor thereof in a control biological sample. 2 times, preferably 3 times, more preferably 5 times, more preferably 10 times, more preferably 20 times, more preferably 50 times, more preferably 100 times, more preferably 1000 times, most preferably 10000 times It has risen above each value.
  • the expression level of miR-153, miR-216a, miR-203, or a precursor thereof in a biological sample derived from a patient whose prognosis is not bad (good) is the same as the expression level in a control biological sample.
  • the ratio of the sample with high expression / the sample with low expression is less than 2 times, less than 1.8 times, less than 1.5 times, or less than 1.3 times.
  • the expression level of miR-203 or a precursor thereof in a biological sample derived from a patient for which the prognosis is predicted, judged, diagnosed, examined, and / or determined is the expression level of miR-203 or a precursor thereof in a control biological sample.
  • the prognosis can be judged as poor. Further, when the decrease is made, it is judged that the prognosis is poor. If not (if it is not reduced), it means that the prognosis is judged not bad (or good). Good. Methods are included in the present invention that include any of them, and methods that include both. That is, the present invention has a poor prognosis when the expression level of miR-203 or a precursor thereof in a biological sample derived from the patient is reduced compared to the expression level of miR-203 or a precursor thereof in a control biological sample.
  • the expression level of miR-203 or a precursor thereof in a biological sample derived from a patient with a poor prognosis is 1/2 times the expression level of miR-203 or a precursor thereof in a control biological sample, preferably 1 / 3 times, more preferably 1/5 times, more preferably 1/10 times, more preferably 1/20 times, more preferably 1/50 times, further preferably 1/100 times, more preferably 1/1000 times
  • the most preferable value is 1 / 10,000 times or less.
  • the expression level of miR-196a or a precursor thereof in a biological sample derived from a patient for which the prognosis is predicted, judged, diagnosed, examined and / or determined is determined in a biological sample derived from a small cell lung cancer patient having a good prognosis. If the expression level of miR-196a or its precursor is decreased, it can be determined that the prognosis is poor. Further, when the decrease is made, it is judged that the prognosis is poor. If not (if it is not reduced), it means that the prognosis is judged not bad (or good). Good.
  • Provided in the present invention are methods that include any of them, and methods that include both.
  • the present invention shows that the expression level of miR-196a or a precursor thereof in a biological sample derived from the patient is higher than the expression level of miR-196a or a precursor thereof in a biological sample derived from a small cell lung cancer patient having a good prognosis. It relates to a method for predicting, judging, diagnosing, testing and / or determining that the prognosis is poor if it is decreasing and / or that the prognosis is good (and / or not bad) otherwise.
  • the expression level of miR-196a or a precursor thereof in a biological sample derived from a patient with a poor prognosis is the expression level of miR-196a or a precursor thereof in a biological sample derived from a small cell lung cancer patient with a good prognosis.
  • the value is reduced to 100 times, more preferably 1/1000 times, most preferably 1 / 10,000 times or less.
  • a biological sample derived from the patient for whom the prognosis is predicted judged, diagnosed, examined and / or judged If the expression level of miR-196a or its precursor in the sample is higher than the expression level of miR-196a or its precursor in the control biological sample, it can be judged that the prognosis is not poor (good), etc. If not, it can be determined that the prognosis is poor.
  • the present invention has a poor prognosis when the expression level of miR-196a or a precursor thereof in the biological sample derived from the patient is not increased compared to the expression level of miR-196a or a precursor thereof in the control biological sample. It relates to a method for predicting, judging, diagnosing, examining and / or determining that the prognosis is good (and / or not bad) when it is elevated.
  • the expression level of miR-196a or a precursor thereof in a biological sample derived from a patient whose prognosis is not poor (good) is, for example, that of a healthy subject, lung adenocarcinoma, or lung squamous cell carcinoma 2 times, preferably 3 times, more preferably 5 times, more preferably 10 times, more preferably 20 times, more preferably 50 times, more preferably 100 times, even more preferably compared to the same expression level in the control biological sample.
  • the expression level of miR-196a or a precursor thereof in a biological sample derived from a patient with a poor prognosis is increased by 1000 times, most preferably 10,000 times or more, for example, a healthy subject, lung adenocarcinoma, or The expression level is similar to that in the control biological sample of a patient determined to be squamous cell carcinoma of the lung. Specifically, for example, the ratio of the high expression / low sample is not doubled. , Less than 1.8-fold, less than 1.5, or less than 1.3 times.
  • one control biological sample may be used, but a plurality of biological samples may be used, for example, a plurality of healthy human biological samples may be used, or a healthy biological sample and a cancer patient biological sample may be used in combination. Also good.
  • the prognosis of a patient determined or diagnosed as small cell lung cancer is treated with chemotherapy, radiation, and the like. Predict, judge, determine, test and / or diagnose before therapy or surgery. Therefore, the treatment to be performed thereafter can be optimized. It is also possible to confirm the therapeutic effect by using the method for predicting, judging, judging, examining and / or diagnosing the prognosis according to the present invention at any time during chemotherapy and radiation therapy.
  • the prognosis according to the present invention can be predicted, judged, judged, examined and / or diagnosed after surgery by using the method for predicting, judging, judging, examining and / or diagnosing the prognosis of the present invention. is there.
  • the prediction and inspection method of the present invention can be implemented separately from the diagnosis. For example, even if a diagnosis is performed by a doctor, a test can be performed by a person who is not a doctor (for example, an external organization) and the doctor can make a diagnosis based on the test result.
  • the inspection method of the present invention can be implemented separately from the diagnostic method. For example, a biological sample may be converted into an ID.
  • the test method of the present invention is also useful in situations where the prognostic distribution of patients with small cell lung cancer is statistically investigated, such as when it is not finally linked to patient diagnosis.
  • the present invention also detects a method for obtaining an intermediate result for the diagnostic method of the present invention, a test method for determining a prognosis in a patient with small cell lung cancer, and a prognostic marker or indicator in a patient with small cell lung cancer.
  • a method for examining the prognosis of a biological sample derived from a patient with small cell lung cancer, a test method for determining or diagnosing the prognosis in a patient with small cell lung cancer, and in a patient with small cell lung cancer It also relates to non-diagnostic methods for examining prognosis. The steps of each method may be performed according to the above description, and in the final step, information on prognosis is provided as an intermediate or non-diagnostic result rather than a diagnosis.
  • the present invention also relates to at least one selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or their precursors in the examination, prediction, judgment, determination and diagnosis of the present invention. It relates to the use of probes, primers, and nucleic acids that hybridize to the at least one.
  • the probe, primer, and nucleic acid are useful as a drug for performing the test, prediction, judgment, determination, and diagnosis of the present invention, that is, a test agent, a diagnostic agent, and the like.
  • the present invention also relates to the diagnostic agent and the diagnostic agent comprising the probe, primer and nucleic acid, and the use of the probe, primer and nucleic acid in the production of the diagnostic agent or the diagnostic agent.
  • the probe, primer, and nucleic acid are preferably those that specifically hybridize with miR-153, miR-196a, miR-203, miR-216a or their precursors.
  • stringent conditions specifically , 1 x SSC (1 x SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 37 ° C, or 1 x SSC is 40 ° C, 45 ° C, 50 ° C, or 55 ° C, preferably 37 It may be a nucleic acid that hybridizes at 37 ° C., 40 ° C., 45 ° C., or 50 ° C.
  • one of the nucleic acids to be hybridized is labeled, the other is immobilized on a membrane, and both are incubated.
  • Hybridization conditions are, for example, 5xSSC, 7% (W / V) SDS, 100 ⁇ g / ml denatured salmon sperm DNA, 5x Denhardt solution (1x Denhardt solution is 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficoll) May be performed in a solution containing After incubating for a sufficient period of time (eg, 3, 4, 5 or 6 hours or more), washing is performed under the above conditions, and by detecting whether the labeled nucleic acid is hybridized, the nucleic acid is hybridized under the condition. Or not.
  • the present invention also relates to a method for treating small cell lung cancer and a method for improving the prognosis of small cell lung cancer.
  • the diagnostic method and test of the present invention it is the same as the description of the method.
  • the expression level in a biological sample derived from a small cell lung cancer patient with a good prognosis can be used as a comparison target.
  • the expression level in a biological sample derived from a small cell lung cancer patient with a good prognosis can be used as a comparison target.
  • the expression level in a biological sample derived from a small cell lung cancer patient with a good prognosis can be used as a comparison target.
  • the expression level in a biological sample derived from a small cell lung cancer patient with a good prognosis can be used as a comparison target.
  • miR-196a, miR-203 or their precursors for example, miR-196a, miR-203 or their This can be done by administering a precursor.
  • the miRNA to be administered or a precursor thereof may be partially modified to improve or stabilize the resistance to nuclease.
  • the 2′-OH of the ribose of pyrimidine nucleotides may be fluorinated or methylated.
  • the nucleotides that make up the nucleic acid may be natural nucleotides, modified nucleotides, artificial nucleotides, or combinations thereof.
  • the nucleic acid may be composed of RNA, may be RNA / DNA chimera, may contain other nucleic acid analogs, and may contain any combination thereof.
  • Nucleic acids include not only those linked by phosphodiester bonds but also those having amide bonds or other backbones (such as peptide nucleic acids (PNA)). Nucleic acids include, for example, natural and artificial nucleic acids, and may be nucleic acid derivatives, nucleic acid derivatives, nucleic acid analogs, and the like. Such nucleic acid analogs are well known in the art and include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, peptide nucleic acids (PNA), and the like. Not.
  • the backbone of PNA may include a backbone consisting of aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfinamide, polysulfonamide, or combinations thereof (Krutzfeldt, J. et al., Nucleic Acids Res. 35: 2885-2892; Davis, S. et al., 2006, Nucleic Acids Res. 34: 2294-2304; Boutla, A. et al., 2003), Nucleic Acids Res. 31: 4973-4980; Hutvagner, G. et al., 2004, PLoS Biol. 2: E98; Chan, JA et al., 2005, Cancer Res. 65: 6029-6033; Esau, C. et al., 2004, J. Biol. Chem. 279: 52361- 52365; Esau, C. et al., 2006, Cell Metab. 3: 87-98).
  • Nucleic acid modifications are specifically 2 ′ or 3 ′ sugar modifications such as 2′-O-methyl (2′-O-Me) ylated nucleotides or 2′-deoxynucleotides, or 2′-fluoro, difluorotoluyl.
  • DFT difluorotoluyl modification, for example, substitution of 2,4-difluorotoluyluracil or guanidine to inosine may be performed.
  • the nucleic acid may contain a conjugate at the end.
  • the conjugate include lipophilic substances, terpenes, protein binding substances, vitamins, carbohydrates, retinoids, peptides, and the like.
  • examples include cholesterol, dU-cholesterol, alkyl chains, aryl groups, heterocyclic complexes, and modified sugars (D-ribose, deoxyribose, glucose, etc.).
  • the conjugate and the nucleic acid can be bound via, for example, an arbitrary linker, and specific examples include a pyrrolidine linker, serinol linker, aminooxy, or hydroxyprolinol linker.
  • a cell permeation signal can be appropriately added to the nucleic acid.
  • many cell-permeable peptides for introducing nucleic acids into cells are known (WO2008 / 082885).
  • arginine-rich peptides such as polyarginine, such as HIV-1 Tat (48-60), HIV-1 (Rev (34-50), FHV Coat (35-49), BMV Gag ( 7-25), HTLV-II Rex (4-16), a partial peptide thereof, or an inverso or retro-inverso thereof.
  • amino acid d-form may be used as appropriate.
  • a cell-penetrating peptide or the like may be bound to a nucleic acid by a known linker.
  • modified nucleic acids, nucleic acid derivatives, nucleic acid derivatives, nucleic acid analogs, and the like are also included in the miRNA or precursor thereof of the present invention.
  • MiR-196a and miR-203 mimics mimicking natural miR-196a and miR-203 are already commercially available (eg MISSION TM microRNA Mimics, Sigma-Aldrich Corporation; miRIDIAN miRNA Mimic, Dharmacon, Lafayette, LA). These can be appropriately used as miR-196a and miR-203, respectively.
  • the method for administering miRNA or a precursor thereof to a patient is not particularly limited.
  • the miRNA according to the present invention or a precursor thereof may be administered as it is, and liposomes, lipofectins, cellfectins, polycations, nanoparticles, etc. May be administered in combination.
  • the DNA encoding the miRNA according to the present invention or a precursor thereof can be inserted into an expression vector for administration.
  • expression vectors include pME18S (Med. Immunol. 20: 27-32 (1990)), pEF-BOS (Nucleic Acids Res. 18: 5322 (1990)), pCDM8 (Nature 329: 840-842 (1987)).
  • the promoter for transcription of RNA is not particularly limited, and a Pol I promoter, Pol II promoter, Pol III promoter, a bacteriophage promoter (RNA polymerase recognition sequence of T4 phage or T7 phage), and the like can be used.
  • Examples of the polymerase II (Pol II) promoter include CMV promoter and ⁇ -globin promoter.
  • a polymerase III (Pol III) promoter that can be expressed in a higher amount than Pol II.
  • Pol III promoters include U6 promoter, H1 promoter, tRNA promoter, 7SK promoter, 7SL promoter, Y3 promoter, 5S rRNA promoter, Ad2 VAI and VAII promoter (Das, G. et al., 1988).
  • miRNA which concerns on this invention, or its precursor can also insert the nucleic acid which codes them in a well-known viral vector, and can also administer it.
  • a well-known viral vector for example, the following various viral vectors for gene therapy are known (see Adolph K.W. ed., Viral Genome Methods, CRC Press, Florida (1996)). Retrovirus vector adenovirus vector vaccinia virus vector poxvirus vector adeno-associated virus vector HVJ vector etc.
  • Administration may be oral administration or parenteral administration (eg, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, cutaneous administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to cancer tissue) But you can.
  • parenteral administration eg, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, cutaneous administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to cancer tissue
  • a known therapeutic agent for small cell lung cancer may be administered together with the miRNA according to the present invention or a precursor thereof, or radiation therapy may be performed.
  • miR-196a, miR-203 or their Doing by administering the precursor As the administration method, the same method as the method for treating small cell lung cancer according to the present invention can be used.
  • the compound, nucleic acid, vector, and other conditions to be used can also refer to the description of the method for treating small cell lung cancer according to the present invention, and any combination of the description and the description of the improvement method of the present invention , Disclosed herein.
  • miR-153, miR-216a or a precursor thereof is applied to a patient having increased expression of miR-153, miR-216a or a precursor thereof. Inhibits the function of Specifically, for example, it can be carried out by administering at least one compound that reduces the function of miR-153, miR-216a or a precursor thereof.
  • the compound that reduces the function is not particularly limited.
  • double-stranded RNA including RNAi constructs such as siRNA
  • antisense oligonucleotides, ribozymes, or expression vectors that express them can be used.
  • antisense oligonucleotides, ribozymes, or expression vectors that express them can be used.
  • These compounds only need to reduce the function of the miRNA or precursor thereof according to the present invention.
  • all or part of the compound binds to at least a part of the miRNA or precursor thereof according to the present invention, What inhibits this miRNA or its precursor binding
  • the compound may be RNA or DNA as long as it reduces the function of miRNA or a precursor thereof according to the present invention, may be a chimeric type containing DNA and RNA in the same strand, and one strand is DNA.
  • a hybrid type in which the other strand is RNA can also be used.
  • the compound that reduces the function of miRNA or a precursor thereof may be a nucleic acid (including a nucleic acid analog) that acts as an antagonist or inhibitor on the miRNA or a precursor thereof.
  • a nucleic acid including a nucleic acid analog
  • an antisense nucleic acid that binds to the miRNA or a precursor thereof an RNAi construct for the miRNA or a precursor thereof (including a double-stranded RNA such as siRNA), a ribozyme that cleaves the miRNA or a precursor thereof, and an miRNA or a thereof Nucleic acids that inhibit precursors.
  • These nucleic acids contain a base sequence that is complementary to at least a part or all of the base sequence of the miRNA or a precursor thereof in order to recognize a miRNA or a precursor thereof that has a reduced function.
  • MiRNA recognizes a target base sequence consisting of several bases present in the 3'-untranslated region of the target gene.
  • This target base sequence is, for example, a base sequence complementary to the 1st to 8th base sequences at the 5 'end of miRNA (Nature 433: 769-773 (2005)).
  • miRNA can be effectively inhibited by RNA having a sequence complementary to the base sequence at the 5 ′ end of miRNA that lowers the function.
  • Such RNA may be completely complementary to the above 5 ′ base sequence of miRNA (base 1 to 8, 2 to 7, or 3 to 8 from the 5 ′ end of miRNA). Or there may be 1-2 gaps.
  • this base sequence is completely complementary, and continuously contains at least 8 bases, more preferably 9 bases, more preferably 10 bases of complementary bases for miRNA. More preferably, it contains a total of 11 bases or more, more preferably 12 bases or more, and more preferably 13 bases or more complementary bases for miRNA.
  • Complementary bases may be contiguous or may include one or more (2, 3, 4) gaps. Mismatch with miRNA can suppress cleavage by RISC and increase the inhibitory activity of miRNA. Thus, it need not be completely complementary to the miRNA and can also be designed to contain a bulge.
  • the compound is at least 85% or more, preferably 90% or more, based on at least a part (for example, 6, 7, 8, 9, 10, 15 to 30 bases) or all of the miRNA or a precursor thereof according to the present invention More preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100% sequence homology (identity).
  • the compound to be administered may be partially modified to improve or stabilize the resistance to nuclease.
  • the 2′-OH of the ribose of pyrimidine nucleotides may be fluorinated or methylated, Steroids, bile acids, polyethylene glycols, etc. may be added.
  • an antisense nucleic acid that binds to miRNA or a precursor thereof may contain about 15-30 bases or more.
  • Antisense nucleic acids may also contain one or more modified backbones and / or base moieties.
  • antisense nucleic acids include natural oligonucleotides and modified oligonucleotides such as phosphorothioates, phosphorodithioates, methylphosphonates, phosphoramidates, H-phosphonate types, triesters, alpha- Anomers, peptide nucleic acids, other artificial nucleic acids, and nucleic acid modifying compounds are included.
  • Antisense nucleic acids may also contain 2′-O-alkylated ribonucleotides.
  • the antisense nucleic acid may contain a base sequence complementary to the base sequence of miRNA, for example, 15 bases or more, 16, 17, 18, 19, 20, 25, 30, 35, 40 bases or more.
  • Complementary sequences may be contiguous or may include one or more (2, 3, 4) gaps (Schmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense NA Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad.
  • the antisense nucleic acid is, for example, at least 85% or more, preferably 90% with respect to at least a part (for example, 6, 7, 8, 9, 10, 15 to 30 bases) or all of the miRNA or a precursor thereof according to the present invention. % Or more, more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100% sequence homology (identity).
  • the RNAi construct for the miRNA or a precursor thereof includes, for example, a base sequence complementary to at least a part of the target miRNA or the precursor thereof, and reduces the function of the miRNA (decreases the expression level of the miRNA, or RNA having an activity that inhibits the binding of the miRNA to a target gene or increases the expression level of the gene, and may be about 14 to 50 nucleotides, preferably 19 to 30 nucleotides, or The above may be included.
  • the double stranded portion of the RNAi construct is about 21-23 nucleotides in length.
  • RNAi construct may be siRNA (including hairpin type single-stranded RNA), or may be RNA (for example, double-stranded RNA) that generates siRNA by processing with Dicer in the cell.
  • RNAi constructs such as siRNA and double stranded RNA may contain DNA.
  • TT may be included at the 3 'end.
  • the target miRNA can be effectively inhibited by oligonucleotides such as 2′-O methyl (2′-OMe) RNA, locked nucleic acid (LNA), and antagomir, etc.
  • oligonucleotides such as 2′-O methyl (2′-OMe) RNA, locked nucleic acid (LNA), and antagomir, etc.
  • Methods for inhibiting target miRNAs are also known (Hutvagner, G. et al. (2004) PLoS Biol, 2, E98; Meister, G. et al. (2004) Rna, 10, 544-550; Orom, UA et al (2006) Gene, 372, 137-141; Krutzfeldt, J. et al. (2005) Nature, 438, 685-689; Ebert, MS et al. (2007) Nat Methods, 4, 721-726).
  • ribozymes that cleave miRNA or its precursor include ribozymes that cleave target miRNA or its precursor (WO 90/11364; US Pat. No. 5,093,246; Sarver ; et al., Science 247: 1222-). 1225).
  • the ribozyme includes any nucleic acid enzyme, and may be, for example, an RNA-type ribozyme, a DNA-type ribozyme (deoxyribozyme), or a DNA-RNA chimeric ribozyme.
  • a ribozyme can be appropriately produced using techniques well known to those skilled in the art (Haselof and Gerlach, 1988, Nature, 334: 585-591; Zaug et al., 1984, Science, 224: 574-578; Zaug and Cech, 1986, Science, 231: 470-475; Zaug et al., 1986, Nature, 324: 429-433; WO88 / 04300; Been and Cech , 1986, Cell, 47: 207-216).
  • the nucleic acid that inhibits miRNA or its precursor is preferably a nucleic acid that hybridizes with the miRNA sequence under physiological conditions.
  • physiological conditions include 1 ⁇ SSC (1 ⁇ SSC is 150 ⁇ M NaCl, 15 ⁇ M sodium citrate, pH 7.0), 37 ° C. More preferably, it may be a nucleic acid that hybridizes with miRNA under stringent conditions.
  • the stringent conditions are, for example, 1 ⁇ SSC or 0.5 ⁇ SSC, 42 ° C., more preferably 1 ⁇ SSC or 0.5 ⁇ SSC, 45 ° C., more preferably 1 ⁇ SSC or 0.5 ⁇ .
  • the conditions are SSC and 50 ° C.
  • Hybridization for example, either RNA containing a miRNA sequence or nucleic acid that inhibits miRNA is labeled, the other is immobilized on a membrane, and both are hybridized.
  • Hybridization conditions are, for example, 5xSSC, 7% (W / V) SDS, 100 ⁇ g / ml denatured salmon sperm DNA, 5x Denhardt solution (1x Denhardt solution is 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficoll) For example, at 37 ° C., 45 ° C., or 50 ° C.
  • the nucleic acid may be RNA or DNA, may be a chimeric type containing DNA and RNA in the same strand, or a hybrid type in which one strand is DNA and the other strand is RNA.
  • Double-stranded RNA may be administered as they are, but may be administered by a known method such as using an expression vector.
  • an expression vector for example, those described in the present specification can be used.
  • miRNA inhibitors An efficient method for producing an miRNA inhibitor is already known (Vermeulen A et al. RNA 13, 723-730 (2007)).
  • miR-153 inhibitors and miR-216a inhibitors are already commercially available (eg Synthetic® human® miRNA® inhibitor oligonucleotide, gene Genepopoia, Inc., Rockville, MD; miRIDIAN® microRNA® Hairpin® Inhibitor, Dharmacon, Lafayette, LA). These can be used as miRNA inhibitors as appropriate.
  • miR-153, miR-216a or their precursors are increased in patients with increased expression of miR-153, miR-216a or their precursors. It is performed by administering double-stranded RNA (including siRNA), antisense oligonucleotide, ribozyme, etc. to the precursor.
  • double-stranded RNA including siRNA
  • antisense oligonucleotide ribozyme, etc.
  • the administration method the same method as the method for treating small cell lung cancer according to the present invention can be used.
  • the compound, nucleic acid, vector, and other conditions to be used the above description regarding the method for treating small cell lung cancer according to the present invention can be referred to, and any of these descriptions and the description of the improvement method of the present invention can be referred to. Combinations of these are disclosed herein.
  • the therapeutic agent and prognosis improving agent according to the present invention include miR-196a, miR-203, a precursor thereof or a vector expressing them, miR-153, miR-216a or a precursor thereof according to the present invention as an active ingredient. Containing at least one kind of compound that inhibits the function of pharmacologically, and optionally adding pharmaceutically acceptable excipients, tonicity agents, solubilizers, stabilizers, preservatives, soothing agents, etc. It can be prepared as a pharmaceutical composition such as tablets, powders, granules, capsules, liposome capsules, injections, liquids, nasal drops and the like, and can also be lyophilized. These can be prepared according to conventional methods.
  • the therapeutic agent and prognosis improving agent according to the present invention can be appropriately adjusted according to the patient's condition and used in preferable amounts.
  • it can be administered in the range of 0.001 to 100 mg / kg, preferably 0.1 to 10 mg / kg, but is not particularly limited. Further, for example, such a dose may be divided into several times, and such a dose may be administered several times.
  • the therapeutic agent and prognosis improving agent according to the present invention can be subjected to pharmacological evaluation in, for example, the following in vitro or in vivo system.
  • Examples of the in vitro pharmacological evaluation include a cell growth inhibition assay and a colony formation inhibition assay.
  • the cell growth inhibition assay for example, the therapeutic agent or prognosis improving agent according to the present invention is brought into contact with a cancer cell in which the miRNA according to the present invention or a precursor thereof is expressed, and a 3 H-thymidine uptake assay after a predetermined period of time,
  • the cell growth inhibitory activity can be evaluated by MTT assay or the like.
  • the therapeutic agent or prognosis improving agent according to the present invention is contacted with cancer cells expressing the miRNA according to the present invention or a precursor thereof, followed by Giemsa staining after culture, and the number of resistant colonies is determined. It can be evaluated by counting.
  • cancer cells expressing miRNA or a precursor thereof according to the present invention animal cells or yeast cells directly introduced with the nucleic acid encoding the miRNA or precursor thereof, or the miRNA or precursor thereof are encoded.
  • a transformed cell obtained by introducing a vector expressing a nucleic acid into a host cell such as an animal cell or a yeast cell can be used.
  • miRNA for example, miR-153, miR-216a or a precursor thereof can be used, but is not limited thereto.
  • Pharmacological evaluation in vivo can be performed by administering an appropriate amount of the therapeutic agent or prognosis improving agent according to the present invention to a cancer animal model an appropriate number of times.
  • the size of the tumor can be used as an index for the evaluation.
  • a cancer animal model for example, a cancer-bearing animal model in which a cancer cell expressing the miRNA according to the present invention or a precursor thereof is transplanted can be used, but it is not particularly limited thereto.
  • the present invention also provides miR-153, miR-196a, miR-203, miR-216a, their precursors, their expression vectors, and compounds that inhibit the function of the miRNA or its precursors (including expression vectors) At least one selected from the group consisting of: a use for the treatment of small cell lung cancer, a use for improving the prognosis of the lung cancer, a use in the manufacture of a medicament or a medicament for the treatment of the lung cancer, and the lung cancer It relates to the use in the manufacture of a medicament or medicament for improving the prognosis of The present invention also provides miR-153, miR-196a, miR-203, miR-216a, their precursors, their expression vectors, and compounds that inhibit the function of the miRNA or its precursors (including expression vectors) At least one selected from the group consisting of: a use for inhibiting the growth of small cell lung cancer cells; and a use for producing a medicament or a medicament for inhibiting the growth of the cells.
  • the present invention also relates to a therapeutic agent for small cell lung cancer and a prognosis improving agent for small cell lung cancer, comprising at least one selected from the group.
  • the present invention also relates to a growth inhibitor of small cell lung cancer cells, comprising at least one selected from the group.
  • the present invention also includes a composition for inhibiting the growth of small cell lung cancer cells, a composition for the treatment of small cell lung cancer, comprising at least one selected from the group and a pharmaceutically acceptable carrier, And a composition for improving the prognosis of small cell lung cancer.
  • the pharmaceutically acceptable carrier include distilled water, phosphate buffered saline (PBS), sodium chloride solution, Ringer's solution, culture solution and the like.
  • vegetable oil, suspension agent, surfactant, stabilizer, biocide, etc. may be contained.
  • the present invention also provides miR-153, miR-196a, miR-203, miR-216a, their precursors, their expression vectors, and compounds that inhibit the function of the miRNA or its precursors (including expression vectors) At least one selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof in at least one biological sample derived from a patient with small cell lung cancer selected from the group consisting of The present invention relates to a use for treating small cell lung cancer in which the expression level is changed (increased or decreased) compared to a control biological sample, and a use for improving the prognosis of the small cell lung cancer.
  • the present invention is selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof in at least one biological sample derived from a patient with small cell lung cancer selected from the group.
  • the treatment or the prognosis improvement is (1) When the expression level of at least one of miR-196a, miR-203, or a precursor thereof is decreased, the decreased at least one miRNA, the precursor thereof, or the expression thereof is expressed Or / and (2) When the expression level of at least one of miR-153, miR-216a or a precursor thereof is increased, at least one compound that inhibits the function of the miRNA or a precursor thereof is administered May be included.
  • the present invention also relates to the use of at least one selected from the group consisting of miR-196a, miR-203, their precursors, and their expression vectors for treating small cell lung cancer, and prognosis of small cell lung cancer.
  • the invention relates to use for improving, use in the manufacture of a medicament or medicament for treating small cell lung cancer, and use in the manufacture of a medicament or medicament for improving the prognosis of small cell lung cancer.
  • the invention relates to use, in the manufacture of a medicament or medicament for treating the small cell lung cancer, and in use in the manufacture of a medicament or medicament for improving the prognosis of the small cell lung cancer.
  • the present invention also relates to the use of a compound (including an expression vector) that inhibits the function of at least one of miR-153, miR-216a or a precursor thereof for treating small cell lung cancer, prognosis of small cell lung cancer
  • the present invention relates to a use for improving the prognosis, a use in the manufacture of a medicament or medicament for treating small cell lung cancer, and a use in the manufacture of a medicament or medicament for improving the prognosis of small cell lung cancer.
  • the invention relates to use, in the manufacture of a medicament or medicament for treating the small cell lung cancer, and in use in the manufacture of a medicament or medicament for improving the prognosis of the small cell lung cancer.
  • the present invention is also selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a, their precursors, and compounds (including expression vectors) that inhibit the function of the miRNA or its precursors.
  • At least one use for treating small cell lung cancer, use for improving the prognosis of small cell lung cancer, and use in the treatment or manufacture of a medicament or medicament for improving the prognosis The treatment or the prognostic improvement is (1) measuring at least one expression level selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a or a precursor thereof in a biological material derived from a small cell lung cancer patient; (2) if the expression level is decreased compared to the control biological sample, administering the decreased at least one miRNA or a precursor thereof, or a vector expressing them; or / and , (3) a step of administering at least one compound (including an expression vector) that inhibits the function of the miRNA or a precursor thereof when the expression level is increased
  • the present invention also relates to use of at least one selected from the group consisting of miR-196a, miR-203, their precursors, and their expression vectors for treating small cell lung cancer, prognosis of small cell lung cancer Use in the manufacture of a medicament or medicament for treating small cell lung cancer, and use in the manufacture of a medicament or medicament for improving the prognosis of small cell lung cancer comprising the treatment or the prognosis Improvement of (1) In a biological material derived from a patient with small cell lung cancer, at least one expression level selected from the group consisting of miR-196a, miR-203, or a precursor thereof is measured, (2) When the expression level is reduced as compared to the control biological sample, administering the reduced at least one miRNA or a precursor thereof, or a vector expressing them, Concerning use, including The present invention also relates to the use of a compound (including an expression vector) that inhibits at least one function of miR-153, miR-216a, or a precursor thereof for treating small cell lung cancer, and prognos
  • the improvement is (1) measuring at least one expression level selected from the group consisting of miR-153, miR-216a, or a precursor thereof, in a biological material derived from a small cell lung cancer patient; (2) a step of administering at least one compound (including an expression vector) that inhibits the function of the miRNA or a precursor thereof when the expression level is increased compared to a control biological sample; Concerning use, including
  • a biological sample derived from a small cell lung cancer patient, a control biological sample, and measurement and comparison of the expression level are the same as those described for the diagnostic method and test method of the present invention.
  • the expression level in a biological sample derived from a small cell lung cancer patient with a good prognosis can be used as a comparison target.
  • the present invention also provides a therapeutic agent for small cell lung cancer, comprising as an index at least one function change selected from the group consisting of miR-153, miR-196a, miR-203, miR-216a, or a precursor thereof.
  • the present invention relates to a prognosis improving agent for cell lung cancer and a screening method for those candidate compounds.
  • the screening method according to the present invention is based on the finding that the expression level of a specific miRNA is associated with the prognosis in a biological sample derived from a patient with small cell lung cancer.
  • the therapeutic agent for small cell lung cancer, the prognosis improving agent for small cell lung cancer, and the candidate compound screening method thereof according to the present invention include miR-153, miR-196a, miR-203, miR-216a or their precursors.
  • “change in function” includes, for example, a case where the expression level of miRNA or a precursor thereof according to the present invention changes.
  • the expression level of the miRNA or precursor thereof according to the present invention does not change, but the expression level of the gene having the target base sequence of the miRNA or precursor thereof is changed by modifying the miRNA or precursor thereof.
  • the expression level of the miRNA according to the present invention or its precursor does not change, but the expression or activity of a factor that changes the expression level of the gene having the target base sequence of the miRNA or its precursor changes.
  • the case where the expression level of the gene having the target base sequence of the miRNA or a precursor thereof changes depending on circumstances is also included.
  • the first screening method can be performed using, for example, an increase in the expression level of miR-196a, miR-203 or a precursor thereof as an indicator. More specifically, for example, a screening method including the following steps (A) to (C) can be mentioned. (A) contacting a test compound with a cell expressing a nucleic acid encoding miR-196a, miR-203 or a precursor thereof; (B) measuring the expression level of a nucleic acid encoding miR-196a, miR-203 or a precursor thereof, (C) a step of selecting a test compound in which the expression level of a nucleic acid encoding miR-196a, miR-203 or a precursor thereof is increased compared to when no test compound is added.
  • the second screening method can be performed using, for example, a decrease in the expression level of miR-153, miR-216a or a precursor thereof as an indicator. More specifically, for example, a screening method including the following steps (A) to (C) can be mentioned. (A) contacting a test compound with a cell expressing a nucleic acid encoding miR-153, miR-216a or a precursor thereof; (B) measuring the expression level of a nucleic acid encoding miR-153, miR-216a or a precursor thereof, (C) A step of selecting a test compound in which the expression level of a nucleic acid encoding miR-153, miR-216a or a precursor thereof is reduced compared to when no test compound is added.
  • the third screening method is carried out, for example, by promoting the binding of miR-196a, miR-203 or their precursors to the base sequence targeted by miR-196a, miR-203 or their precursors. can do.
  • the binding of miRNA to the target base sequence may detect physical binding, or may detect binding functionally using the biological effect of binding as an index. More specifically, for example, a screening method including the following steps (A) to (C) can be mentioned.
  • A contacting a test compound with a cell expressing a nucleic acid encoding miR-196a, miR-203 or a precursor thereof;
  • B measuring the expression level of a gene (target gene) having a base sequence targeted by miR-196a, miR-203 or a precursor thereof;
  • C A test compound in which the expression level of a gene (target gene) having a base sequence targeted by miR-196a, miR-203 or a precursor thereof is reduced compared to the case where no test compound is added.
  • the fourth screening method is performed using, for example, inhibition of binding of miR-153, miR-216a or a precursor thereof to a base sequence targeted by miR-153, miR-216a or a precursor thereof as an indicator. can do. More specifically, for example, a screening method including the following steps (A) to (C) can be mentioned.
  • A contacting a test compound with a cell expressing a nucleic acid encoding miR-153, miR-216a or a precursor thereof;
  • B measuring the expression level of a gene (target gene) having a base sequence targeted by miR-153, miR-216a or a precursor thereof;
  • C A test compound in which the expression level of a gene (target gene) having a base sequence targeted by miR-153, miR-216a or a precursor thereof is increased as compared with the case where no test compound is added.
  • test compound in the step (A) according to the present invention is not particularly limited.
  • the nucleic acid may be modified and may contain a conjugate. Specific examples thereof are as described in this specification.
  • the test compound can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label.
  • limiting in particular as "a some test compound” For example, in addition to the said test compound, the mixture which mixed multiple types of these test compounds is also contained.
  • contact may mean that the test compound comes into contact outside and / or inside the cell, and can be performed, for example, by adding the test compound to the cell culture medium. .
  • a transfection reagent, a virus vector, etc. can be utilized suitably.
  • Cells expressing miR-153, miR-196a, miR-203, miR-216a or their precursors and / or genes having the target nucleotide sequence include cells that naturally express them, artificial In addition, cells expressing them can be used.
  • examples of the cell in which the nucleic acid encoding the miRNA or the precursor thereof according to the present invention is expressed include, for example, cancer cells and animal cells into which the nucleic acid encoding the miRNA or a precursor thereof is directly introduced.
  • miR-153, miR-196a, miR-203, miR- Cells expressing 216a or their precursors can be expressed exogenously, for example, with an expression vector or the like.
  • cells expressing miR-153, miR-196a, miR-203, miR-216a or their precursors are preferably naturally Cells expressing them are used.
  • any known measurement method is used for measuring the expression level of the nucleic acid encoding the miRNA or its precursor in the step (B) according to the present invention and the gene having the target base sequence of the miRNA or its precursor. It can. Specifically, for example, RT-PCR, a modified method thereof, a Northern blot method, an in situ hybridization method, and a method using a microarray can be mentioned. Further, the target base sequence of the miRNA or a precursor thereof is inserted into a known appropriate reporter gene expression vector, the vector is introduced into a suitable host cell, the cell is contacted with a test substance, and the reporter gene Screening according to the present invention can also be performed using expression as an indicator.
  • the gene having the target base sequence of step (B) according to the present invention may be a natural target gene of the miRNA, or a reporter gene prepared by incorporating the target base sequence of the miRNA.
  • the target base sequence can be incorporated into a 3′-UTR of a reporter gene (such as GFP or luciferase gene).
  • a sequence that hybridizes with miRNA can be used as appropriate, and even if it is not completely complementary to miRNA or has a bulge, it can be a target.
  • the target gene can be constructed by inserting a plurality of copies of the target base sequence, for example, in tandem.
  • a specific reporter gene can be prepared according to a known method (Vermeulen A et al. RNA 13, 723-730 (2007); Ebert, MS et al., Nat. Methods 4, 721-726 (2007) ).
  • the base sequence targeted by miRNA or its precursor is the number recognized by miRNA or its precursor existing in the 3′-untranslated region of mRNA that causes translational suppression of miRNA or its precursor.
  • the target base sequence is the target base sequence (Nature 433: 769-773 (2005)), and such a sequence is converted into a 3′-untranslated region.
  • the gene contained in is a target gene.
  • the target gene can be obtained, for example, by a method for searching a gene DATA base using a target base sequence, the HITS-CLIP method (Nature 460: 479-486 (2009)).
  • the patient background is shown in Table 1. Most were combined with chemotherapy before or after surgery.
  • Total RNA Extraction and MicroRNA Analysis A frozen tissue section was sliced and subjected to HE staining, and a specimen in which the proportion of tumor cells in the section was 70% or more was used for Total RNA extraction.
  • Total RNA was extracted with mirVana miRNA Isolation Kit (Applied Biosystems). The quality of the extracted RNA was evaluated with an Agilent 2100 bioanalyzer (manufactured by Agilent).
  • Total RNA labeled with Cy3 was hybridized to Human miRNA Microarray (manufactured by Agilent) loaded with 866 human miRNAs registered in miRBase database v12.0.
  • Microarray data was analyzed using GeneSpring GX10 software (manufactured by Agilent). Normalization was performed with a 75% tile value of the miRNA expression level of each sample, and analysis was performed with 600 miRNAs having a raw signal value of 5 or more. Extraction of miRNA having a difference in expression was performed by a signal ratio (2.0 or more), and ANOVA and Tukey post hoc test (p ⁇ 0.05). Benjamini-Hochberg correction was used for multiple test correction. Hierarchical clustering was performed using the Pearson coefficient as a measure of similarity.
  • Quantitative RT-PCR CDNAs of miR-153, miR-196a, miR-203, and miR-216a were synthesized using Taqman microRNA kit (Applied Biosystem). Quantitative RT-PCR was performed using Taqman microRNA assays (Applied Biosystem) containing Taqman probes and primers compatible with the miRNA. U6 was used as an endogenous control for miRNA expression level correction.
  • Group 1 consisted of only neuroendocrine cancers (SCLC 20 cases, LCNEC 3 cases), whereas group 2 classified neuroendocrine cancers (SCLC 15 cases, LCNEC 8 cases) and non-neuroendocrine cancers (4 cases of squamous cell carcinoma) It was done. The remaining non-neuroendocrine cancers were also classified in Group 2.
  • the classification by mRNA expression profiling performed previously by the present inventors (Jones MH et al., Lancet, 2004, 363: 775-781) and the classification by miRNA expression profiling were almost the same.
  • FIG. 2 A comparison of survival curves and clinicopathological background of group 1 SCLC (SCLC 1) and group 2 SCLC (SCLC 2) is shown in FIG. 2 and Table 2, respectively.
  • the table represents a comparison of clinical features between SCLC subgroups identified by unsupervised hierarchical clustering with 600 miRNAs.
  • SCLC 1 showed 6 successful cases / invariant 3 cases
  • SCLC 2 had 2 successful cases / invariant 1 case. There was no obvious difference in sensitivity.
  • the proGRP value is also affected by the tumor volume, but in this study, the difference in stage between the two groups is not clear, and the difference in value may reflect the difference in proGRP production ability It was. From these results, it is speculated that neuroendocrine activity is involved in the prognosis of SCLC.
  • miR-153, miR-216a decreased expression group, miR-196a, miR-203 increased expression group tend to have a low serum proGRP value that increases in neuroendocrine cancer, and the positive rate of neuroendocrine markers (immunostaining) Tended to be low.
  • SCLC prognosis and miRNA expression Among the SCLC patient tissue samples used in the analysis in Example 1, 10 SCLC tissue samples that survived without recurrence for 3 years or more and 14 SCLC patients who died of cancer in less than 3 years was selected, and miRNAs that differed in expression between the two were searched. Extraction of miRNA having a difference in expression was performed by a signal ratio (2.0 or more), and ANOVA and Tukey post hoc test (p ⁇ 0.05).
  • miR-153, miR-196a, miR-203, or miR-216a was found as miRNA having a difference in expression between the two.
  • Normal lung tissue sample (NL), lung adenocarcinoma tissue sample (Ad), lung squamous cell carcinoma tissue sample (Sq), and SCLC good prognosis group (SCLC2) and poor prognosis group (SCLC1) classified by hierarchical clustering ) MiRNA expression levels were compared (FIG. 3A).
  • miR-153, miR-203, and miR-216a have altered expression only in the SCLC poor prognosis group (SCLC1), and the expression in the SCLC good prognosis group (SCLC2) is normal lung or lung cancer other than SCLC It was the same.
  • the expression levels of miR-153 and miR-216a were higher in any group with poor prognosis than in any group, and the expression levels of miR-203 were lower than in any group.
  • the expression level of miR-196a was increased in the good prognosis group compared to the poor prognosis group.
  • Hierarchical clustering suggests that the miRNA expression profile of SCLC with good prognosis is close to that of normal lung and non-neuroendocrine cancers, so these miR-153, miR-216a, and miR-203 may be particularly relevant for prognosis It was considered expensive. Therefore, the expression levels of these three were also measured by quantitative RT-PCR. Microarray data and quantitative RT-PCR results correlated well (FIG. 3B). Compared with the poor prognosis group (SCLC1), the good prognosis group (SCLC2) had significantly decreased miR-153 and miR-216a expression and significantly increased miR-203 and miR-196a expression.
  • Example 1 Thirty-five SCLC patients used in the analysis in Example 1 were classified into a decrease-increase group and an increase-in-expression group with the median as the cut-off value for each of miR-153, miR-196a, miR-203, or miR-216a. .
  • the clinical characteristics and survival curves of both groups are shown in Table 3 and FIG.
  • the miR-153, miR-216a expression decreased group, and miR-196a expression increased group had significantly better prognosis.
  • the miR-203 expression increased group also showed a favorable prognosis.
  • FIG. 5 shows the results of classifying 35 SCLC patients used in the analysis in Example 1 and the survival curve of each group by hierarchical clustering using three of miR-203, miR-153, and miR-216a.
  • FIG. 6 shows the classification by the same clustering using four of miR-203, miR-196a, miR-196a, and miR-216a and the survival curve of each group. All SCLC patients could be classified into good prognosis group and poor prognosis group.
  • the SCLC-specific survival curves of the classified poor prognosis group (high risk) and good prognosis group (low risk) are shown in FIG. 7, and the clinical characteristics of patients are shown in Table 4.
  • MS1 and DMS53 are used.
  • MS1 is cultured in RPMI medium supplemented with 10% FBS
  • DMS53 is cultured in DMEM medium supplemented with 10% FBS.
  • miRIDIAN microRNA Hairin Inhibitors / Mimic (Dharmacon, Lafayette, Co, USA) inhibits miR-153 and miR-216a and enhances miR-203 and miR-196a.
  • Introduction into MS1 cells is performed by electroporation method using Nucleofector II Device (Amaxa).
  • Introduction into DMS53 cells is performed using Lipofectamine TM 2000 (Invitrogen). Cells introduced with miRIDIAN microRNA Hairpin Inhibitors / Mic negative control are used as controls.
  • the therapeutic effect of miRNA function control is detected by the proliferation curve of the introduced cells.
  • cell blocks are prepared from the introduced cells, immunostaining of neuroendocrine markers (Synaptophysin, Chromogranin A, NCAM) is performed, proteins are extracted from the introduced cells, and neuroendocrine markers (Synaptophysin, Chromogranin A, NCAM) are quantified. Evaluate the neuroendocrine character of the introduced cells.
  • DMS53 (ATCC CRL-2062) and SBC-5 (JCRB NO: JCRB0819, Mitsuhashi, Y. et al., Cancer, 70: 2540) are adhesion-adapted SCLC cell lines of small cell lung cancer cells with miRNA and miRNA inhibitors -2546, 1992).
  • miR-153 is highly expressed
  • SBC-5 miR-203 is lowly expressed (FIGS. 10 (A) and (C)).
  • DMS53 and SBC-5 were cultured in DMEM medium supplemented with 10% FBS.
  • miRIDIAN miR-153 inhibitor and miRIDIAN miR-203 mimic were prepared by miRIDIAN microRNA Hairpin Inhibitors / Mimic (Dharmacon, Lafayette, Co, USA), and miR-153 function inhibition and miR-203 function enhancement were performed.
  • oligos (miR-153 inhibitor and negative control * 1 ) were similarly diluted to 60 pmol / 3 ⁇ L, added to 3 ⁇ L at a ratio of 150 ⁇ L of OPTI-MEM, and lightly vortexed and spun down. These were added and allowed to stand at room temperature for 20 minutes, and 306 ⁇ L each was dispensed into the above-mentioned dish containing cells and culture solution and mixed well. Growth was confirmed by culturing for several days in a 37 ° C., 5% CO 2 incubator. The number of cells was counted at 0 hr, 48 hrs, and 122 hrs (confluent) for comparison. (* 1: miRIDIAN microRNA Hairpin Inhibitor Negative Control # 1 IN-001005-01-05 (catalog item))
  • miRIDIAN miR-153 inhibitor inhibits the proliferation of DMS-53.
  • MiRIDIAN miR-203 mimic also reduced SBC-5 proliferation (FIG. 10). Therefore, it has been clarified that an inhibitor of miR-153 may be used as a therapeutic agent for small cell lung cancer, particularly in patients where miR-153 is highly expressed.
  • miR-203 mimic could be used as a therapeutic agent for small cell lung cancer, particularly in patients with reduced miR-203 expression.
  • miR-216a inhibitor and miR-196a mimic could also be used as therapeutic agents for small cell lung cancer.
  • the present invention provides a method for predicting, examining, and diagnosing the prognosis of SCLC using miRNA.
  • the present invention provides an SCLC treatment method, an SCLC prognosis improvement method, an SCLC treatment agent, an SCLC prognosis improvement agent, and an SCLC treatment / prognosis improvement screening method using miRNA.

Abstract

L'invention concerne une méthode de prédiction du pronostic du cancer du poumon à petites cellules, une méthode de traitement du cancer du poumon à petites cellules, une méthode d'amélioration du pronostic du cancer du poumon à petites cellules, un agent thérapeutique pour le cancer du poumon à petites cellules et une méthode de criblage pour la recherche d'un agent thérapeutique pour le cancer du poumon à petites cellules, chacun utilisant les miARN. Spécifiquement, l'invention concerne : une méthode de diagnostic du pronostic d'un patient atteint d'un cancer du poumon à petites cellules, qui utilise miR-153, miR-196a, miR-203 ou miR-216a comme mesure ; une méthode d'amélioration du pronostic du cancer du poumon à petites cellules qui utilise la substance mentionnée ci-dessus comme mesure ; une méthode de traitement de cancer du poumon à petites cellules qui cible la substance susmentionnée ; un agent thérapeutique pour le cancer du poumon à petites cellules ; et une méthode de criblage pour la recherche d'un agent thérapeutique pour un cancer du poumon à petites cellules qui utilise la substance susmentionnée.
PCT/JP2010/067432 2010-04-05 2010-10-05 Méthode de prédiction de pronostic de cancer du poumon à petites cellules, méthode de traitement du cancer du poumon à petites cellules, méthode d'amélioration du pronostic du cancer du poumon à petites cellules et méthode de criblage pour la recherche d'un agent thérapeutique pour le cancer du poumon à petites cellules, chacun utilisant les miarn WO2011125245A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2012509270A JPWO2011125245A1 (ja) 2010-04-05 2010-10-05 ｍｉＲＮＡを用いた小細胞肺癌の予後予測方法、小細胞肺癌治療方法、小細胞肺癌予後改善方法、及び小細胞肺癌治療剤のスクリーニング方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2010-087093 2010-04-05
JP2010087093 2010-04-05

Publications (1)

Publication Number Publication Date
WO2011125245A1 true WO2011125245A1 (fr) 2011-10-13

Family

ID=44762220

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/067432 WO2011125245A1 (fr) 2010-04-05 2010-10-05 Méthode de prédiction de pronostic de cancer du poumon à petites cellules, méthode de traitement du cancer du poumon à petites cellules, méthode d'amélioration du pronostic du cancer du poumon à petites cellules et méthode de criblage pour la recherche d'un agent thérapeutique pour le cancer du poumon à petites cellules, chacun utilisant les miarn

Country Status (2)

Country Link
JP (1) JPWO2011125245A1 (fr)
WO (1) WO2011125245A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126233A1 (fr) * 2013-02-15 2014-08-21 国立大学法人東京医科歯科大学 Procédé pour l'analyse de micro-arn, agent thérapeutique anticancéreux, et composition médicinale le contenant pour une thérapie anticancéreuse
CN107384921A (zh) * 2017-07-27 2017-11-24 中国人民解放军总医院 miR216a用于抑制骨肉瘤细胞的增殖、侵袭和迁移
CN112094904A (zh) * 2020-09-30 2020-12-18 中国人民解放军军事科学院军事医学研究院 急性电离辐射早期损伤检测的微生物种水平标志物及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008154333A2 (fr) * 2007-06-08 2008-12-18 Asuragen, Inc. Gènes et chemins régulés par mir-34 en tant que cibles pour une intervention thérapeutique
JP2009028036A (ja) * 2007-06-28 2009-02-12 Mitsubishi Chemical Medience Corp 組織傷害あるいは細胞増殖性疾患の検出方法
JP2009531018A (ja) * 2006-01-05 2009-09-03 ジ・オハイオ・ステイト・ユニバーシティ・リサーチ・ファウンデイション 肺癌の診断、予後診断及び治療のためのマイクロｒｎａを基盤とした方法及び組成物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009531018A (ja) * 2006-01-05 2009-09-03 ジ・オハイオ・ステイト・ユニバーシティ・リサーチ・ファウンデイション 肺癌の診断、予後診断及び治療のためのマイクロｒｎａを基盤とした方法及び組成物
WO2008154333A2 (fr) * 2007-06-08 2008-12-18 Asuragen, Inc. Gènes et chemins régulés par mir-34 en tant que cibles pour une intervention thérapeutique
JP2009028036A (ja) * 2007-06-28 2009-02-12 Mitsubishi Chemical Medience Corp 組織傷害あるいは細胞増殖性疾患の検出方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
OBA, TAKEHIKO ET AL: "Identification of subgroup with good prognosis of small-cell lung cancer by miRNA expression profiling", AACR 101ST ANNUAL MEETING 2010, 12 March 2010 (2010-03-12), Retrieved from the Internet <URL:http://www.abstractsonline.com/Plan/ViewAbstract.aspx?sKey=ab4788a7-757c-403c-a00e-404cd806d69f&cKey=7308da44-068a-4bb9-b211-7b4e3c054407&mKey=%7B0591FA3B-AFEF-49D2-8E65-55F41EE8117E%7D> [retrieved on 20101206] *
TAKEHIKO OBA ET AL.: "Hai Shosaibo Gan ni Okeru microRNA Hatsugen Profile no Kaiseki, O-117", JAPANESE JOURNAL OF LUNG CANCER, vol. 49, 5 October 2009 (2009-10-05), pages 624 *
TAKEHIKO OBA ET AL.: "MicroRNA Hatsugen Profiling ni yoru Yogo Ryoko na Hai Shosaibo Gan no Dotei, 1-E-12", PROCEEDINGS OF THE JAPANESE SOCIETY OF PATHOLOGY, vol. 99, 26 March 2010 (2010-03-26), pages 190 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126233A1 (fr) * 2013-02-15 2014-08-21 国立大学法人東京医科歯科大学 Procédé pour l'analyse de micro-arn, agent thérapeutique anticancéreux, et composition médicinale le contenant pour une thérapie anticancéreuse
US9994843B2 (en) 2013-02-15 2018-06-12 National University Corporation Tokyo Medical And Dental University Method for assaying microRNA, cancer therapeutic agent, and medicinal composition containing same for cancer therapy
US10876115B2 (en) 2013-02-15 2020-12-29 National University Corporation Tokyo Medical And Dental University Method for assaying MicroRNA, cancer therapeutic agent, and medical composition containing same for cancer therapy
CN107384921A (zh) * 2017-07-27 2017-11-24 中国人民解放军总医院 miR216a用于抑制骨肉瘤细胞的增殖、侵袭和迁移
CN107384921B (zh) * 2017-07-27 2020-09-15 中国人民解放军总医院 miR216a用于抑制骨肉瘤细胞的增殖、侵袭和迁移
CN112094904A (zh) * 2020-09-30 2020-12-18 中国人民解放军军事科学院军事医学研究院 急性电离辐射早期损伤检测的微生物种水平标志物及应用

Also Published As

Publication number Publication date
JPWO2011125245A1 (ja) 2013-07-08

Similar Documents

Publication Publication Date Title
JP5624470B2 (ja) ヒト末梢血微小胞におけるｍｉＲＮＡの発現およびその使用
JP6153932B2 (ja) 骨肉腫のためのマイクロｒｎａに基づく方法およびアッセイ
Wu et al. miR-362-5p inhibits proliferation and migration of neuroblastoma cells by targeting phosphatidylinositol 3-kinase-C2β
Zhan et al. MicroRNA-548j functions as a metastasis promoter in human breast cancer by targeting Tensin1
WO2006074367A2 (fr) Procede permettant de predire et de reduire le risque de metastases du cancer du sein dans les poumons
US20160346311A1 (en) Long non-coding rna spry4-it1 as a diagnostic and therapeutic agent
EP2683411B1 (fr) Procédés d&#39;utilisation de microarn-26a destinés à favoriser l&#39;angiogenèse
US11639531B2 (en) Composition, kit, and method for diagnosis and treatment of taxane anticancer agent-resistant cancer
US9994915B2 (en) miR-211 expression and related pathways in human melanoma
CA2879410C (fr) Agent pour le traitement du cancer
WO2011125245A1 (fr) Méthode de prédiction de pronostic de cancer du poumon à petites cellules, méthode de traitement du cancer du poumon à petites cellules, méthode d&#39;amélioration du pronostic du cancer du poumon à petites cellules et méthode de criblage pour la recherche d&#39;un agent thérapeutique pour le cancer du poumon à petites cellules, chacun utilisant les miarn
US8921333B2 (en) Therapeutic agent for tumor
KR102142791B1 (ko) miR-204 억제제의 골관절염 치료 용도
Miao et al. Exosomal circCOL1A2 from cancer cells accelerates colorectal cancer progression via regulating miR-665/LASP1 signal axis
US9631194B2 (en) Methods and compositions for use in treatment of FOXP2-related cancers
WO2010050328A1 (fr) Inhibiteur de métastase de tumeur
US10905708B2 (en) MicroRNA-based methods and assays for osteocarcinoma
US11260072B2 (en) MicroRNA-based methods and assays for osteocarcinoma
EP2199407A1 (fr) Procédés in vitro et compositions pour le diagnostic et/ou le traitement de l&#39;adénocarcinome
CN110964832A (zh) 肺癌诊断和治疗的靶基因
CN110951888A (zh) 检测及靶向al845472.2的试剂及其在肺癌诊治中的应用
Mody Investigation of Micro-RNA-based Approaches to Overcome Epithelial-Mesenchymal Transition in Pancreatic Cancer
WO2017176215A1 (fr) Biomarqueurs de cancer et procédés d&#39;utilisation
JP2012512642A (ja) Ｔｒａｉｌ誘導アポトーシスに関する遺伝子の同定方法およびその治療適用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10849485

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2012509270

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10849485

Country of ref document: EP

Kind code of ref document: A1